Section 2 Epidemiology

Histoplasma *and Histoplasmosis*

2011;**17**(3):167-171

2018;**12**:e0006802

[48] Cáceres DH, Samayoa BE, Medina NG, Tobón AM, Guzmán BJ, Mercado D, et al. Multicenter validation of commercial antigenuria reagents to diagnose progressive disseminated histoplasmosis in people living with HIV/AIDS in two Latin American countries. The Journal of Clinical Microbiology. 2018;**56**(6):JCM.01959-17. Available from: http://jcm.asm.org/ lookup/doi/10.1128/JCM.01959-17

[49] Bracca A, Tosello ME, Girardini JE,

Amigot SL, Gomez C, Serra E. Molecular detection of *Histoplasma capsulatum* var. capsulatum in human clinical samples. Journal of Clinical Microbiology. 2003;**41**(4):1753-1755

[50] Dieng T, Massaly A, Sow D, Vellaissamy S, Sylla K, Tine RC, et al. Amplification of blood smear DNA to confirm disseminated histoplasmosis.

[51] Gago S, Esteban C, Valero C, Zaragoza Ó, De La Bellacasa JP,

Buitrago MJ. A multiplex real-time PCR assay for identification of pneumocystis jirovecii, histoplasma capsulatum,and cryptococcus neoformans/cryptococcus gattii in samples from AIDS patients

Infection. 2017

for diagnosis of histoplasmosis. Clinical Infectious Diseases. 2011;**53**(5):448-454

with opportunistic pneumonia. The Journal of Clinical Microbiology. 2014;**52**(4):1168-1176. Available from: http://jcm.asm.org/content/52/4/1168. full.pdf+html http://ovidsp.ovid.com/ ovidweb.cgi?T=JS&PAGE=reference&D =emed16&NEWS=N&AN=372725898

[52] Botero Aguirre JP, Restrepo Hamid AM. Amphotericin B deoxycholate versus liposomal amphotericin B: Effects on kidney function. Cochrane Database of Systematic Reviews. 2015. Available from: http://doi.wiley.

com/10.1002/14651858.CD010481.pub2

diagnosis of fungal pneumonia. Current Opinion in Pulmonary Medicine.

[47] Nacher M, Blanchet D, Bongomin F, Chakrabarti A, Couppié P, Demar M, et al. Histoplasma capsulatum antigen detection tests as an essential diagnostic tool for patients with advanced HIV disease in low and middle income countries: A systematic review of diagnostic accuracy studies. PLoS Neglected Tropical Diseases.

[46] Hage CA, Knox KS, Davis TE, Wheat LJ. Antigen detection in bronchoalveolar lavage fluid for

**12**

**15**

**Chapter 2**

in Asia

**Abstract**

**1. Introduction**

The Review of Histoplasmosis

Endemicity and Current Status

Histoplasmosis is a common disease among immunocompromised patients and a notable endemic disease among immunocompetent patients. Disseminated histoplasmosis in acquired immunodeficiency syndrome (AIDS) patients is mostly lethal. We conducted a literature review of histoplasmosis and histoplasmin skin test in Asia. There were around 1692 cases of histoplasmosis reported from Asian countries. India (623 cases), China (611 cases) and Thailand (234 cases) are detected as endemic areas of histoplasmosis although the complete mapping of this disease is still missing in some Asian countries. However, definite diagnosis is difficult since the symptoms frequently mimicking tuberculosis (TB), tissue samples are rarely obtained, and

The first documented case of histoplasmosis in Asia was detected in a 7-year-old boy from Java, Indonesia, 26 years after Dr Samuel Darling described the first case of histoplasmosis [1, 2]. The differential diagnosis of the patient was visceral leishmaniasis, but the appendix, ileum and mesenteric lymph nodes showed histological evidences of histoplasmosis [2]. Randhawa reported 30 cases of histoplasmosis in 1970 from Asia [3]. India accounted for the highest number of 11 cases, followed by Indonesia (8 cases), Malaysia (4 cases), Thailand (2 cases), Singapore (2 cases), Vietnam (2 cases) and Japan (1 case) [3]. Together with the isolation of *Histoplasma capsulatum* from soil in Malaysia, it is implied that Malaysia is the endemic area of histoplasmosis [3]. Recent study documented 407 cases of histoplasmosis from Southeast Asian countries with Thailand as the leading country with 223 cases [4]. Human immunodeficiency virus (HIV) is a classical risk factor in histoplasmosis

although there are a growing number of reports of histoplasmosis among other immunocompromised patients as well as immunocompetent patients in Asia [5–7]. The mortality of histoplasmosis among immunocompromised patients was 27.5%; this rate is significantly higher than the rate in immunocompetent patients (10%) [5]. Histoplasmosis was also often misdiagnosed as tuberculosis (TB) as many countries in Asia are high-burden TB countries [8–10]. The complete understanding of *Histoplasma* endemicity is crucial to enhance the awareness of clinicians in Asia to diagnose histoplasmosis. We reviewed the current status of histoplasmosis in Asia

*Anna Rozaliyani and Findra Setianingrum*

lacked of serology tests in Asian countries.

through analysis of published studies.

**Keywords:** histoplasmosis, Asia, epidemiology

#### **Chapter 2**

## The Review of Histoplasmosis Endemicity and Current Status in Asia

*Anna Rozaliyani and Findra Setianingrum*

#### **Abstract**

Histoplasmosis is a common disease among immunocompromised patients and a notable endemic disease among immunocompetent patients. Disseminated histoplasmosis in acquired immunodeficiency syndrome (AIDS) patients is mostly lethal. We conducted a literature review of histoplasmosis and histoplasmin skin test in Asia. There were around 1692 cases of histoplasmosis reported from Asian countries. India (623 cases), China (611 cases) and Thailand (234 cases) are detected as endemic areas of histoplasmosis although the complete mapping of this disease is still missing in some Asian countries. However, definite diagnosis is difficult since the symptoms frequently mimicking tuberculosis (TB), tissue samples are rarely obtained, and lacked of serology tests in Asian countries.

**Keywords:** histoplasmosis, Asia, epidemiology

#### **1. Introduction**

The first documented case of histoplasmosis in Asia was detected in a 7-year-old boy from Java, Indonesia, 26 years after Dr Samuel Darling described the first case of histoplasmosis [1, 2]. The differential diagnosis of the patient was visceral leishmaniasis, but the appendix, ileum and mesenteric lymph nodes showed histological evidences of histoplasmosis [2]. Randhawa reported 30 cases of histoplasmosis in 1970 from Asia [3]. India accounted for the highest number of 11 cases, followed by Indonesia (8 cases), Malaysia (4 cases), Thailand (2 cases), Singapore (2 cases), Vietnam (2 cases) and Japan (1 case) [3]. Together with the isolation of *Histoplasma capsulatum* from soil in Malaysia, it is implied that Malaysia is the endemic area of histoplasmosis [3]. Recent study documented 407 cases of histoplasmosis from Southeast Asian countries with Thailand as the leading country with 223 cases [4].

Human immunodeficiency virus (HIV) is a classical risk factor in histoplasmosis although there are a growing number of reports of histoplasmosis among other immunocompromised patients as well as immunocompetent patients in Asia [5–7]. The mortality of histoplasmosis among immunocompromised patients was 27.5%; this rate is significantly higher than the rate in immunocompetent patients (10%) [5]. Histoplasmosis was also often misdiagnosed as tuberculosis (TB) as many countries in Asia are high-burden TB countries [8–10]. The complete understanding of *Histoplasma* endemicity is crucial to enhance the awareness of clinicians in Asia to diagnose histoplasmosis. We reviewed the current status of histoplasmosis in Asia through analysis of published studies.

### **2. Methods**

A literature search was conducted in PubMed and Google Scholar using the keywords "histoplasmosis" and "Asia", and also the combinations of "histoplasmosis" or *Histoplasma* or "histoplasmin" with the name of each country in Asia, in February 2020. The following countries were included as part of Asia: the Philippines, Singapore, Thailand, Vietnam, Cambodia, Brunei, Indonesia, Laos, Malaysia, Myanmar, India, Bangladesh, Pakistan, Sri Lanka, Nepal, China, Turkey, Iraq, Iran, Taiwan, Lebanon, Syria, Qatar, Saudi Arabia, Kuwait, Japan and Korea.

#### **2.1 Epidemiology of histoplasmosis in Asia**

Our review of the literature produced a total of 1692 cases of histoplasmosis reported in Asia from both areas known as endemic (Indian subcontinent, China, Southeast Asia) and non-endemic (Turkey, Iraq, Iran and Taiwan). India accounted for the highest number of cases with 623 cases, followed by China (611 cases), Thailand (234 cases), Malaysia (77 cases) and Indonesia (48 cases) (**Table 1**).

The countries included in the search to not produce any indigenous cases were Lebanon, Syria, Qatar, Saudi Arabia, Kuwait, Jordan, Japan, Korea and Brunei. Other countries in Asia were considered as non-endemic areas for *Histoplasma*, but there were possibilities of imported cases of histoplasmosis [11, 12]. Imported cases were revealed based on the travel history of patients to other Asian countries such as Malaysia, Indonesia, Thailand, Singapore and China [12]. Japan and Korea


**17**

**Table 2.**

therapy.

**Type of histoplasmosis**

*The Review of Histoplasmosis Endemicity and Current Status in Asia*

revealed imported cases of histoplasmosis with 100 cases and 245 cases, respectively [13–15]. There is also small portion of indigenous cases from Iraq, Iran, Turkey and Taiwan which occurred in immunocompromised patients such as patients with long-term corticosteroid therapy, history of renal transplant or

The histoplasmosis cases from India are based on the study from Randhawa and Gugnani who found a total of 426 cases (1954–2018) and 207 cases from the literature search (2018–2020, excluded studies cited in Randhawa and Gugnani) [6]. The trend of reporting histoplasmosis cases was notably increased in the past 2 years, which might be caused by rising of awareness of clinicians to diagnose histoplasmosis. However, whether this increase is caused by a real increased number of patients

**Reference HIV** 

Disseminated 0 – 29 [8, 10, 22, 23, 27, 28, 33, 38, 44,

Adrenal 0 – 54 [26, 36, 43, 50, 56] Pulmonary 1 [10] 1 [37] Gastrointestinal 3 [24, 35] 4 [35] Cutaneous 0 – 2 [34, 39] Conjunctival 1 [41] 0 – Laryngeal 0 – 1 [29] Oral 0 – 2 [32, 33] Mixed organs 6 [59] 31 [59]

**negative**

**Reference**

45, 47–49, 52, 53, 55, 88]

The regions along the Ganga river basins such as West Bengal and Uttar Pradesh in India were considered as endemic pockets of histoplasmosis [6, 82, 83]. Histoplasmosis cases also existed from other areas in India, which included Delhi, Rajasthan, Maharashtra and Haryana [6, 84–87]. The sole evidence of environmental source of *Histoplasma* in India was the isolation of this organism from the soil from building related to bat habitats near Calcutta, part of Ganga river basins [83]. The data of human immunodeficiency virus (HIV) status were available for 136 patients from 207 histoplasmosis patients in India between 2018 and 2020 (**Table 2**). Histoplasmosis mostly occurred in HIV-negative patients (91%, n = 124). Only 12 patients (9%) were HIV positive in this case series. The most common manifestation in the HIV-negative patients is adrenal histoplasmosis (44%, n = 54). Other study also found HIV-negative cases outnumbered HIV-positive cases with the rate of 64% proven histoplasmosis in a retrospective study from New Delhi [42]. Adrenal histoplasmosis (44%, n = 54) was the most common manifestation of histoplasmosis among 124 histoplasmosis cases with HIV negative. A recent study reported an adequate response of antifungal therapy in adrenal histoplasmosis with the rate of 86% with a median duration of follow-up of 2.5 years [50]. However, the mortality is high (20%, 8 out of 29 patients) even among patients with completed

*DOI: http://dx.doi.org/10.5772/intechopen.92448*

genetic immunodeficiency [16–21].

infected by *Histoplasma* in India is unclear.

**HIV positive**

Total 11 124

*HIV status from 135 patients (2017–2020) with available data.*

*2.1.1 Indian subcontinent*

**Table 1.**

*Cases of histoplasmosis in Asian countries.*

#### *The Review of Histoplasmosis Endemicity and Current Status in Asia DOI: http://dx.doi.org/10.5772/intechopen.92448*

revealed imported cases of histoplasmosis with 100 cases and 245 cases, respectively [13–15]. There is also small portion of indigenous cases from Iraq, Iran, Turkey and Taiwan which occurred in immunocompromised patients such as patients with long-term corticosteroid therapy, history of renal transplant or genetic immunodeficiency [16–21].

#### *2.1.1 Indian subcontinent*

Histoplasma *and Histoplasmosis*

**2.1 Epidemiology of histoplasmosis in Asia**

A literature search was conducted in PubMed and Google Scholar using the keywords "histoplasmosis" and "Asia", and also the combinations of "histoplasmosis" or *Histoplasma* or "histoplasmin" with the name of each country in Asia, in February 2020. The following countries were included as part of Asia: the Philippines, Singapore, Thailand, Vietnam, Cambodia, Brunei, Indonesia, Laos, Malaysia, Myanmar, India, Bangladesh, Pakistan, Sri Lanka, Nepal, China, Turkey, Iraq, Iran, Taiwan, Lebanon, Syria, Qatar, Saudi Arabia, Kuwait, Japan and Korea.

Our review of the literature produced a total of 1692 cases of histoplasmosis reported in Asia from both areas known as endemic (Indian subcontinent, China, Southeast Asia) and non-endemic (Turkey, Iraq, Iran and Taiwan). India accounted for the highest number of cases with 623 cases, followed by China (611 cases), Thailand (234 cases), Malaysia (77 cases) and Indonesia (48 cases) (**Table 1**).

The countries included in the search to not produce any indigenous cases were Lebanon, Syria, Qatar, Saudi Arabia, Kuwait, Jordan, Japan, Korea and Brunei. Other countries in Asia were considered as non-endemic areas for *Histoplasma*, but there were possibilities of imported cases of histoplasmosis [11, 12]. Imported cases were revealed based on the travel history of patients to other Asian countries such as Malaysia, Indonesia, Thailand, Singapore and China [12]. Japan and Korea

**No. Country No. of cases References** India 623 [6, 8–10, 22–59] Bangladesh 27 [6, 60] Pakistan 5 [6, 61] Sri Lanka 4 [6, 62] Nepal 5 [6] Cambodia 5 [4] China 611 [7, 63–72] Thailand 234 [4, 73] Indonesia 48 [4] Laos 1 [74] Myanmar 3 [75–77] Singapore 21 [4] Philippines 14 [4] Malaysia 77 [4, 78] Vietnam 6 [4, 79] Turkey 2 [16, 80] Iraq 2 [18] Iran 2 [19, 81] Taiwan 2 [12, 20] Total 1692

**2. Methods**

**16**

**Table 1.**

*Cases of histoplasmosis in Asian countries.*

The histoplasmosis cases from India are based on the study from Randhawa and Gugnani who found a total of 426 cases (1954–2018) and 207 cases from the literature search (2018–2020, excluded studies cited in Randhawa and Gugnani) [6]. The trend of reporting histoplasmosis cases was notably increased in the past 2 years, which might be caused by rising of awareness of clinicians to diagnose histoplasmosis. However, whether this increase is caused by a real increased number of patients infected by *Histoplasma* in India is unclear.

The regions along the Ganga river basins such as West Bengal and Uttar Pradesh in India were considered as endemic pockets of histoplasmosis [6, 82, 83]. Histoplasmosis cases also existed from other areas in India, which included Delhi, Rajasthan, Maharashtra and Haryana [6, 84–87]. The sole evidence of environmental source of *Histoplasma* in India was the isolation of this organism from the soil from building related to bat habitats near Calcutta, part of Ganga river basins [83].

The data of human immunodeficiency virus (HIV) status were available for 136 patients from 207 histoplasmosis patients in India between 2018 and 2020 (**Table 2**). Histoplasmosis mostly occurred in HIV-negative patients (91%, n = 124). Only 12 patients (9%) were HIV positive in this case series. The most common manifestation in the HIV-negative patients is adrenal histoplasmosis (44%, n = 54). Other study also found HIV-negative cases outnumbered HIV-positive cases with the rate of 64% proven histoplasmosis in a retrospective study from New Delhi [42].

Adrenal histoplasmosis (44%, n = 54) was the most common manifestation of histoplasmosis among 124 histoplasmosis cases with HIV negative. A recent study reported an adequate response of antifungal therapy in adrenal histoplasmosis with the rate of 86% with a median duration of follow-up of 2.5 years [50]. However, the mortality is high (20%, 8 out of 29 patients) even among patients with completed therapy.


#### **Table 2.**

*HIV status from 135 patients (2017–2020) with available data.*

There were no risk factors detected in 10 patients among 29 HIV-negative patients with histoplasmosis, considering this group of patients was immunocompetent [8, 10, 23, 28, 33, 44, 45, 88]. One patient had dual infections of disseminated histoplasmosis and TB with fever, lymphadenopathy, splenomegaly and pancytopenia [23]. The patient showed a partial response to antitubercular therapy. *Histoplasma* grew from a bone marrow culture of patient proved the diagnosis of disseminated histoplasmosis. Another unusual case was the occurrence of a progressive disseminated histoplasmosis in an immunocompetent patient with prolonged fever of unknown origin [8]. CT of the thorax was normal; later, the patient developed pharyngeal ulcer and papulonodular rash that the biopsies from these lesions turned to be positive for *Histoplasma*. The most common risk factor in HIV-negative patients was diabetes (41 patients) (**Table 3**).

A review of literature from Bangladesh revealed 27 cases of histoplasmosis with the first case reported from Dhaka in 1982. Dhaka is the most common city appeared in the list with 17 cases, followed by Chittagong, Gopalganj and Mymensingh with each of the area had one case of histoplasmosis. Four patients were diagnosed outside Bangladesh and three patients had unknown location. The cases from Bangladesh were all adult males, with 15% (four patients) who had acquired immunodeficiency syndrome (AIDS). The predominance of male population with histoplasmosis was also found in many case series [42, 50, 59]. The most common type of histoplasmosis was disseminated histoplasmosis. Seven cases of adrenal histoplasmosis in HIV-negative patients were observed in the recent case series from Bangladesh [60]. Weight loss, anorexia and fever were the common symptoms. Bilateral adrenal enlargement was seen in these patients. The diagnosis of histoplasmosis was obtained from CT-guided fine needle aspiration cytology. Four patients have diabetes as their risk factor. There is no travel history outside Bangladesh in all seven patients indicating Bangladesh as a potential endemic area of *Histoplasma*. Histoplasmosis was also reported in a small number of cases from Pakistan (five patients), Sri Lanka (four cases) and Nepal (five cases).

Several studies reported the result of histoplasmin tests in India [3, 89–91]. The range of positivity was estimated between 0 and 12.3% [3, 91]. The highest rate of positivity (12.3%) was identified from a survey in Delhi, particularly in the area near the Jumna River [89]. The zero rate of positivity was obtained from Kelur, Goa, Ramgarh and Kerala [3, 90]. The positivity of histoplasmin between 12.58 and


**19**

*The Review of Histoplasmosis Endemicity and Current Status in Asia*

13.75% was observed among 2,729 people from Bangladesh (formerly called East Pakistan) [92]. Meanwhile, all of the 575 people (healthy participants, schoolchildren and TB and leprosy patients) showed a zero rate of histoplasmin sensitivity in Pakistan [93]. Nepal reported histoplasmin positivity in the rate of 5.7% among

The total number of histoplasmosis cases from Southeast Asian countries was 410 cases with 178 (43%) cases were related to HIV-positive patients. Diabetes was noted as the risk factor in histoplasmosis case among HIV-negative patients in Southeast Asia [4]. Thailand ranked one in the number of cases of histoplasmosis in Southeast Asia, followed by Malaysia based on our literature search (**Table 1**). The dominant cases from these two countries may reflect the clinical awareness and presence of active case finding of histoplasmosis especially from HIV-positive

HIV was found in 85% of patients in the retrospective cohort study in Thailand

A retrospective study of oral histoplasmosis in Malaysians observed older age (>54) and serious systemic illness as common risk factors [97]. All cases were proven histopathologic findings of the yeast phase of *Histoplasma capsulatum*. TB and squamous cell carcinoma were the differential diagnoses in this report. However, there is no available information regarding the immune status in this study. Five (14%) patients later developed disseminated histoplasmosis. The clinical information was not available in the remaining patients whether the patients had disseminated disease or isolated oral histoplasmosis. Other histoplasmosis cases from Malaysia were reported as a single or small case series. Disseminated histoplasmosis was diagnosed in HIV-negative patients with diabetes and high exposure of bird droppings in Sarawak, Malaysia [98]. The Malaysian Medical Association published environmental control measures to replace tall trees with shorter trees and promoted the electricity and telephone cables underground to reduce bird droppings in Sarawak [99]. The areas nearby Sarawak, Sibu and Sarikei had many

histoplasmosis cases possibly due to abundant exposure of bird droppings.

Most of the histoplasmosis in Indonesia was also reported as a case report or small case series. Seven HIV-positive patients with disseminated histoplasmosis were diagnosed based on the histopathologic findings in Jakarta. The study showed there is no correlation between CD4 counts with the clinical morphology and distribution of lesions [100]. The mean CD4 count was 49.8 cells/mm3. Papules, plaques, hypertrophic scars, ulcers and scales were identified dominantly in the face. Touch biopsy was used to identify histoplasmosis in 27 patients with HIV

[95]. Risk factors for histoplasmosis were not detected in three patients (5%). Progressive disseminated histoplasmosis was the most common (86%) clinical manifestation. The organs involved include the lung, oral cavity, adrenal gland and heart valve. Most of the patients (69%) were located in Central Thailand. Weight loss, fever and anemia were commonly found in patients, followed by cough, lymphadenopathy, hepatomegaly and skin lesions. *Histoplasma* were present in microscopic examination at the rate of 89% from skin biopsy and scrape, while only 16.7% of fungal culture positive for this organism from skin tissue. The mortality rate is 12%, with all deaths coming from progressive disseminated histoplasmosis. The evidence of environmental sources of *Histoplasma* in Thailand was obtained from the soil contaminated with bat guano and chicken dropping from Chiang Mai [96]. The detection was carried out by nested PCR on 265 soil samples. Bat guano and chicken dropping were often used as fertilizers among local gardeners. This might play a role as one of the possible contacts of this organism to humans.

population, which lacked in most other Southeast Asian countries [4].

*DOI: http://dx.doi.org/10.5772/intechopen.92448*

1336 students and prisoners [94].

*2.1.2 Southeast Asia*

**Table 3.**

*Risk factors in HIV-negative cases from 207 histoplasmosis cases (2018–2020).*

13.75% was observed among 2,729 people from Bangladesh (formerly called East Pakistan) [92]. Meanwhile, all of the 575 people (healthy participants, schoolchildren and TB and leprosy patients) showed a zero rate of histoplasmin sensitivity in Pakistan [93]. Nepal reported histoplasmin positivity in the rate of 5.7% among 1336 students and prisoners [94].

#### *2.1.2 Southeast Asia*

Histoplasma *and Histoplasmosis*

There were no risk factors detected in 10 patients among 29 HIV-negative patients with histoplasmosis, considering this group of patients was immunocompetent [8, 10, 23, 28, 33, 44, 45, 88]. One patient had dual infections of disseminated histoplasmosis and TB with fever, lymphadenopathy, splenomegaly and pancytopenia [23]. The patient showed a partial response to antitubercular therapy. *Histoplasma* grew from a bone marrow culture of patient proved the diagnosis of disseminated histoplasmosis. Another unusual case was the occurrence of a progressive disseminated histoplasmosis in an immunocompetent patient with prolonged fever of unknown origin [8]. CT of the thorax was normal; later, the patient developed pharyngeal ulcer and papulonodular rash that the biopsies from these lesions turned to be positive for *Histoplasma*. The most common risk factor in

A review of literature from Bangladesh revealed 27 cases of histoplasmosis with the first case reported from Dhaka in 1982. Dhaka is the most common city appeared in the list with 17 cases, followed by Chittagong, Gopalganj and Mymensingh with each of the area had one case of histoplasmosis. Four patients were diagnosed outside Bangladesh and three patients had unknown location. The cases from Bangladesh were all adult males, with 15% (four patients) who had acquired immunodeficiency syndrome (AIDS). The predominance of male population with histoplasmosis was also found in many case series [42, 50, 59]. The most common type of histoplasmosis was disseminated histoplasmosis. Seven cases of adrenal histoplasmosis in HIV-negative patients were observed in the recent case series from Bangladesh [60]. Weight loss, anorexia and fever were the common symptoms. Bilateral adrenal enlargement was seen in these patients. The diagnosis of histoplasmosis was obtained from CT-guided fine needle aspiration cytology. Four patients have diabetes as their risk factor. There is no travel history outside Bangladesh in all seven patients indicating Bangladesh as a potential endemic area of *Histoplasma*. Histoplasmosis was also reported in a small number of cases from

Pakistan (five patients), Sri Lanka (four cases) and Nepal (five cases).

Total 55

*Risk factors in HIV-negative cases from 207 histoplasmosis cases (2018–2020).*

Several studies reported the result of histoplasmin tests in India [3, 89–91]. The range of positivity was estimated between 0 and 12.3% [3, 91]. The highest rate of positivity (12.3%) was identified from a survey in Delhi, particularly in the area near the Jumna River [89]. The zero rate of positivity was obtained from Kelur, Goa, Ramgarh and Kerala [3, 90]. The positivity of histoplasmin between 12.58 and

**Conditions No. of cases Reference** Diabetes 41 [30, 35, 47, 48, 53, 59] Chronic granulomatous disease 2 [52, 55] Corticosteroid therapy 3 [9, 22, 38] Other immunosuppressive therapies 2 [22, 38] Idiopathic CD4 lymphocytopenia 2 [27, 35] Kidney transplant 1 [29] TB with severe malnutrition 1 [35] Lymphoma 1 [35] Chronic liver disease 1 [59] Chronic kidney disease 1 [59]

HIV-negative patients was diabetes (41 patients) (**Table 3**).

**18**

**Table 3.**

The total number of histoplasmosis cases from Southeast Asian countries was 410 cases with 178 (43%) cases were related to HIV-positive patients. Diabetes was noted as the risk factor in histoplasmosis case among HIV-negative patients in Southeast Asia [4]. Thailand ranked one in the number of cases of histoplasmosis in Southeast Asia, followed by Malaysia based on our literature search (**Table 1**). The dominant cases from these two countries may reflect the clinical awareness and presence of active case finding of histoplasmosis especially from HIV-positive population, which lacked in most other Southeast Asian countries [4].

HIV was found in 85% of patients in the retrospective cohort study in Thailand [95]. Risk factors for histoplasmosis were not detected in three patients (5%). Progressive disseminated histoplasmosis was the most common (86%) clinical manifestation. The organs involved include the lung, oral cavity, adrenal gland and heart valve. Most of the patients (69%) were located in Central Thailand. Weight loss, fever and anemia were commonly found in patients, followed by cough, lymphadenopathy, hepatomegaly and skin lesions. *Histoplasma* were present in microscopic examination at the rate of 89% from skin biopsy and scrape, while only 16.7% of fungal culture positive for this organism from skin tissue. The mortality rate is 12%, with all deaths coming from progressive disseminated histoplasmosis.

The evidence of environmental sources of *Histoplasma* in Thailand was obtained from the soil contaminated with bat guano and chicken dropping from Chiang Mai [96]. The detection was carried out by nested PCR on 265 soil samples. Bat guano and chicken dropping were often used as fertilizers among local gardeners. This might play a role as one of the possible contacts of this organism to humans.

A retrospective study of oral histoplasmosis in Malaysians observed older age (>54) and serious systemic illness as common risk factors [97]. All cases were proven histopathologic findings of the yeast phase of *Histoplasma capsulatum*. TB and squamous cell carcinoma were the differential diagnoses in this report. However, there is no available information regarding the immune status in this study. Five (14%) patients later developed disseminated histoplasmosis. The clinical information was not available in the remaining patients whether the patients had disseminated disease or isolated oral histoplasmosis. Other histoplasmosis cases from Malaysia were reported as a single or small case series. Disseminated histoplasmosis was diagnosed in HIV-negative patients with diabetes and high exposure of bird droppings in Sarawak, Malaysia [98]. The Malaysian Medical Association published environmental control measures to replace tall trees with shorter trees and promoted the electricity and telephone cables underground to reduce bird droppings in Sarawak [99]. The areas nearby Sarawak, Sibu and Sarikei had many histoplasmosis cases possibly due to abundant exposure of bird droppings.

Most of the histoplasmosis in Indonesia was also reported as a case report or small case series. Seven HIV-positive patients with disseminated histoplasmosis were diagnosed based on the histopathologic findings in Jakarta. The study showed there is no correlation between CD4 counts with the clinical morphology and distribution of lesions [100]. The mean CD4 count was 49.8 cells/mm3. Papules, plaques, hypertrophic scars, ulcers and scales were identified dominantly in the face. Touch biopsy was used to identify histoplasmosis in 27 patients with HIV

positive in Jakarta [101]. *Histoplasma capsulatum* were detected in 10 patients (37%), suggesting this method might be suitable in the resource-limited setting to diagnose histoplasmosis.

Histoplasmin positivity in Southeast Asia varied widely across countries, ranging from 0.5% in Sarawak, Malaysia, to 86.4% in Maguee, Myanmar [4, 102–104]. Most of the studies used the induration of 5 mm or more as a sign of positive reaction. The o.5% rate was obtained from 181 schoolchildren/hospital patients, and the 86.4% rate was observed from prisoners/prison staff. Luzon Island, Philippines, reported a histoplasmin positivity rate of 26% among 143 electric company employees [104].

#### *2.1.3 China*

The number of cases from China (611 cases) was based on the recent report from Xin et al. with most of the cases reported from Yunnan province, followed by Hunan and Hubei provinces [7]. These three regions are located along the Yangtze River flows. Other provinces that are located in the Yangtze River were identified as histoplasmosis endemic regions, including Jiangsu, Sichuan and Chongqing [63]. Among the 611 cases, 34 cases from Hunan were reviewed for clinical history. It showed 13 patients were immunocompromised with most of them (8 patients) confirmed as HIV-positive cases [7]. Twenty-one patients were considered as immunocompetent. All cases were proven with tissue biopsy with the bone marrow as the most common site of biopsy. The predominant organ involved was the lungs, oral cavities and intestines.

Pan identified 300 cases of histoplasmosis in China during 1990–2011 [63]. Traveling history was present in 195 patients. It revealed that most of the patients (91%, n = 178) had no history travel outside China which indicated the endemicity of histoplasmosis in China. HIV positive comprised 22% (n = 38) of the cases among 173 patients with clear clinical information. Around 49% (n = 85) patients showed no underlying disease, with most of them (89%, n = 76) unexpectedly diagnosed with disseminated histoplasmosis.

The histoplasmin rate in normal people with no history of travel outside China revealed the highest rate is from Jiangsu (15.1%) [105]. Hunan reported a rate of 8.9%, while Xinjiang reported a rate of only 2.1%. Another study from China revealed a rate of 35% of histoplasmin positivity from Sichuan province [106].

#### **3. Diagnostic challenges of histoplasmosis in Asia**

The diagnosis of histoplasmosis in many regions in Asia is still inadequate due to the lack of awareness of this disease. Clinical symptoms are difficult to distinguish from other lung diseases, including tuberculosis and several other diseases that require different managements. The identification of *H. capsulatum* in clinical material can be determined by several techniques, including direct fungal visualization and culture, histopathology, antibody or antigen detection and molecular-based testing. These methods have different sensitivity and specificity ranges, depending upon the methodology, clinical form of the disease and host immune status.

In Asian countries with limited facilities, the diagnosis is classically carried out by direct examination of clinical specimens. The sensitivity and specificity of direct examination by using potassium hydroxide (KOH 10%) are very low. Histopathology and cytopathology with specific fungal staining is considered another important method in diagnosing histoplasmosis, but it requires invasive procedures for obtaining specimens and needs professional expertise to identify the specific fungal elements. The histopathologic examination may achieve the highest

**21**

**5. Conclusion**

*The Review of Histoplasmosis Endemicity and Current Status in Asia*

factor in the mortality of adrenal histoplasmosis [50].

yield by evaluating bone marrow biopsies and respiratory specimens from disseminated histoplasmosis patients. A touch preparation analysis of the skin or skin biopsy shows the utility in diagnosing cutaneous mycoses or skin-related systemic mycoses, including disseminated histoplasmosis [101, 107]. The test is simple and suitable for rapid presumptive diagnosis, especially in HIV-infected patients.

The histoplasmosis cases during the last 3 years in India were dominated with HIV-negative patients based on our literature search. This is in contrast with the period before 2018 that indicated HIV as the common comorbidity among histoplasmosis in India [6]. This may also reflect a decline of annual new HIV infections by 27% during 2010–2017 [108]. The endemic area of histoplasmosis in India (West Bengal and Uttar Pradesh) reported a decline of new HIV infection in 2010–2017, in the rate of 11% and 34%, respectively [108]. Meanwhile there are a limited number of studies from other regions in Asia regarding the HIV status of histoplasmosis. Diabetes is the most common risk factor detected in HIV-negative histoplasmosis in India and Southeast Asia. India is the second country with the biggest burden of diabetes in the world [109]. There is an increase of diabetes population in India from 40.9 million people in 2007 to 77 million people in 2019 [109, 110]. The growing population of diabetes might contribute to the common findings of diabetes as a risk factor in histoplasmosis. Diabetes was commonly found as a predisposing

Histoplasmosis has the most varied presentations. The mild pulmonary histoplasmosis may occasionally get complicated by pericarditis or rheumatologic manifestation. After heavy exposure to *H. capsulatum* spores in the soil contaminated with bird or bat droppings, patients may present with severe acute pulmonary histoplasmosis needing hospitalization. Patients with underlying obstructive pulmonary disease may exhibit progressive chronic pulmonary histoplasmosis. Immunosuppressed patients usually present with progressive disseminated histoplasmosis, which manifests as either a chronic or acute disease. The diagnosis of *Histoplasma* which are based on tissue histopathology should be carefully evaluated as the overlap morphology of *H. capsulatum* and *Penicillium marneffei*, particularly

A rising trend of reporting histoplasmosis was observed in many areas in Asian countries. Diabetes becomes a notable risk factor among HIV-negative patients with histoplasmosis in Asia. However, the actual number of cases is still far underdiagnosed because of the lack of awareness of the clinician, minimum laboratory facilities to diagnose and the unspecific appearance of histoplasmosis often mimicking other endemic disease in Asia such as tuberculosis. Most of the studies in Southeast Asia were from a case study; a cohort study with a large number of patients is needed to reveal the clinical characteristic and evaluate the management of histoplasmosis in this region. There are an increased number of immunocompetent cases of histoplasmosis from India and China indicating that *Histoplasma* is capable to cause a disease both in immunocompromised and immunocompetent patients. There is an urgent need of proper epidemiology study of histoplasmosis to draw the actual map of histoplasmosis in Asia in order to increase the awareness of clinicians

because *P. marneffei* also endemic in certain areas in Asia [111, 112].

and also to facilitate early diagnosis and management of patients.

*DOI: http://dx.doi.org/10.5772/intechopen.92448*

**4. Discussion**

yield by evaluating bone marrow biopsies and respiratory specimens from disseminated histoplasmosis patients. A touch preparation analysis of the skin or skin biopsy shows the utility in diagnosing cutaneous mycoses or skin-related systemic mycoses, including disseminated histoplasmosis [101, 107]. The test is simple and suitable for rapid presumptive diagnosis, especially in HIV-infected patients.

#### **4. Discussion**

Histoplasma *and Histoplasmosis*

diagnose histoplasmosis.

company employees [104].

*2.1.3 China*

positive in Jakarta [101]. *Histoplasma capsulatum* were detected in 10 patients (37%), suggesting this method might be suitable in the resource-limited setting to

Histoplasmin positivity in Southeast Asia varied widely across countries, ranging from 0.5% in Sarawak, Malaysia, to 86.4% in Maguee, Myanmar [4, 102–104]. Most of the studies used the induration of 5 mm or more as a sign of positive reaction. The o.5% rate was obtained from 181 schoolchildren/hospital patients, and the 86.4% rate was observed from prisoners/prison staff. Luzon Island, Philippines, reported a histoplasmin positivity rate of 26% among 143 electric

The number of cases from China (611 cases) was based on the recent report from Xin et al. with most of the cases reported from Yunnan province, followed by Hunan and Hubei provinces [7]. These three regions are located along the Yangtze River flows. Other provinces that are located in the Yangtze River were identified as histoplasmosis endemic regions, including Jiangsu, Sichuan and Chongqing [63]. Among the 611 cases, 34 cases from Hunan were reviewed for clinical history. It showed 13 patients were immunocompromised with most of them (8 patients) confirmed as HIV-positive cases [7]. Twenty-one patients were considered as immunocompetent. All cases were proven with tissue biopsy with the bone marrow as the most common site of biopsy.

The predominant organ involved was the lungs, oral cavities and intestines.

diagnosed with disseminated histoplasmosis.

**3. Diagnostic challenges of histoplasmosis in Asia**

Pan identified 300 cases of histoplasmosis in China during 1990–2011 [63]. Traveling history was present in 195 patients. It revealed that most of the patients (91%, n = 178) had no history travel outside China which indicated the endemicity of histoplasmosis in China. HIV positive comprised 22% (n = 38) of the cases among 173 patients with clear clinical information. Around 49% (n = 85) patients showed no underlying disease, with most of them (89%, n = 76) unexpectedly

The histoplasmin rate in normal people with no history of travel outside China

The diagnosis of histoplasmosis in many regions in Asia is still inadequate due to the lack of awareness of this disease. Clinical symptoms are difficult to distinguish from other lung diseases, including tuberculosis and several other diseases that require different managements. The identification of *H. capsulatum* in clinical material can be determined by several techniques, including direct fungal visualization and culture, histopathology, antibody or antigen detection and molecular-based testing. These methods have different sensitivity and specificity ranges, depending

revealed the highest rate is from Jiangsu (15.1%) [105]. Hunan reported a rate of 8.9%, while Xinjiang reported a rate of only 2.1%. Another study from China revealed a rate of 35% of histoplasmin positivity from Sichuan province [106].

upon the methodology, clinical form of the disease and host immune status. In Asian countries with limited facilities, the diagnosis is classically carried out by direct examination of clinical specimens. The sensitivity and specificity of direct examination by using potassium hydroxide (KOH 10%) are very low. Histopathology and cytopathology with specific fungal staining is considered another important method in diagnosing histoplasmosis, but it requires invasive procedures for obtaining specimens and needs professional expertise to identify the specific fungal elements. The histopathologic examination may achieve the highest

**20**

The histoplasmosis cases during the last 3 years in India were dominated with HIV-negative patients based on our literature search. This is in contrast with the period before 2018 that indicated HIV as the common comorbidity among histoplasmosis in India [6]. This may also reflect a decline of annual new HIV infections by 27% during 2010–2017 [108]. The endemic area of histoplasmosis in India (West Bengal and Uttar Pradesh) reported a decline of new HIV infection in 2010–2017, in the rate of 11% and 34%, respectively [108]. Meanwhile there are a limited number of studies from other regions in Asia regarding the HIV status of histoplasmosis.

Diabetes is the most common risk factor detected in HIV-negative histoplasmosis in India and Southeast Asia. India is the second country with the biggest burden of diabetes in the world [109]. There is an increase of diabetes population in India from 40.9 million people in 2007 to 77 million people in 2019 [109, 110]. The growing population of diabetes might contribute to the common findings of diabetes as a risk factor in histoplasmosis. Diabetes was commonly found as a predisposing factor in the mortality of adrenal histoplasmosis [50].

Histoplasmosis has the most varied presentations. The mild pulmonary histoplasmosis may occasionally get complicated by pericarditis or rheumatologic manifestation. After heavy exposure to *H. capsulatum* spores in the soil contaminated with bird or bat droppings, patients may present with severe acute pulmonary histoplasmosis needing hospitalization. Patients with underlying obstructive pulmonary disease may exhibit progressive chronic pulmonary histoplasmosis. Immunosuppressed patients usually present with progressive disseminated histoplasmosis, which manifests as either a chronic or acute disease. The diagnosis of *Histoplasma* which are based on tissue histopathology should be carefully evaluated as the overlap morphology of *H. capsulatum* and *Penicillium marneffei*, particularly because *P. marneffei* also endemic in certain areas in Asia [111, 112].

#### **5. Conclusion**

A rising trend of reporting histoplasmosis was observed in many areas in Asian countries. Diabetes becomes a notable risk factor among HIV-negative patients with histoplasmosis in Asia. However, the actual number of cases is still far underdiagnosed because of the lack of awareness of the clinician, minimum laboratory facilities to diagnose and the unspecific appearance of histoplasmosis often mimicking other endemic disease in Asia such as tuberculosis. Most of the studies in Southeast Asia were from a case study; a cohort study with a large number of patients is needed to reveal the clinical characteristic and evaluate the management of histoplasmosis in this region. There are an increased number of immunocompetent cases of histoplasmosis from India and China indicating that *Histoplasma* is capable to cause a disease both in immunocompromised and immunocompetent patients. There is an urgent need of proper epidemiology study of histoplasmosis to draw the actual map of histoplasmosis in Asia in order to increase the awareness of clinicians and also to facilitate early diagnosis and management of patients.

Histoplasma *and Histoplasmosis*

### **Author details**

```
Anna Rozaliyani1,2*
        †
          and Findra Setianingrum1,2†
```
1 Faculty of Medicine Universitas, Department of Parasitology, Indonesia


† These two authors contributed equally.

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**23**

2018;**7**:126-129

*The Review of Histoplasmosis Endemicity and Current Status in Asia*

nodule: An interesting clinical mimicry of pulmonary tuberculosis. Medical Journal, Armed Forces India.

[10] Mahajan VK, Raina RK, Singh S, Rashpa RS, Sood A, Chauhan PS, et al. Case report: Histoplasmosis in Himachal Pradesh (India): An emerging endemic focus. The American Journal of Tropical Medicine and Hygiene.

[11] Hung CC, Wong JM, Hsueh PR, Hsieh SM, Chen MY. Intestinal, obstruction and peritonitis resulting from gastrointestinal histoplasmosis in an AIDS patient. Journal of the Formosan Medical Association.

[12] Lai CH, Lin HH. Cases of histoplasmosis reported in Taiwan. Journal of the Formosan Medical Association. 2006;**105**:527-528

[13] Kamei K, Sano A, Kikuchi K, Makimura K, Niimi M, Suzuki K, et al. The trend of imported mycoses in Japan. Journal of Infection and Chemotherapy.

[14] Hatakeyama S, Okamoto K, Ogura K, Sugita C, Nagi M.

[15] Cho SH, Bin YY, Park JS, Yook KD, Kim YK. Epidemiological characterization of imported

Perspectives. 2018;**9**:362

2016;**18**:116-118

Histoplasmosis among HIV-infected patients in Japan: A case report and literature review. Japanese Journal of Infectious Diseases. 2019;**72**:330-333

systemic mycoses occurred in Korea. Osong Public Health and Research

[16] Turhan U, Aydoğan M, Özkısa T, Karslıoğlu Y, Gümüş S. A rare case in Turkey: Pulmonary histoplasmosis. Eurasian Journal of Pulmonology.

2019;**75**:115-118

2017;**97**:1749-1756

1998;**97**:577-580

2003;**9**:16-20

*DOI: http://dx.doi.org/10.5772/intechopen.92448*

pseudotubercles in the lungs. Journal of the American Medical Association.

[2] Müller H. Histoplasmosis in East Java. Geneeskd Tijdschr Voor Ned.

[3] Randhawa H. Occurrence of

[4] Baker J, Setianingrum F, Wahyuningsih R, Denning DW. Mapping histoplasmosis in South East Asia—Implications for diagnosis in AIDS. Emerging Microbes & Infections.

histoplasmosis in Asia. Mycopathologia.

[5] Gupta A, Ghosh A, Singh G, Xess I. A twenty-first-century perspective of disseminated histoplasmosis in India: Literature review and retrospective analysis of published and unpublished

cases at a tertiary care hospital in North India. Mycopathologia.

[6] Randhawa HS, Gugnan HC. Occurrence of histoplasmosis in the Indian sub-continent: An overview and update. Journal of Medical Research and

[7] Lv X, Meng J, Jiang M, He R, Li M. Clinical features and endemic trend of histoplasmosis in China: A retrospective analysis and literature review. The Clinical Respiratory Journal. 2019:1-7

[8] Bauddha NK, Jadon RS, Mondal S, Vikram NK, Sood R. Progressive disseminated histoplasmosis in an immunocompetent adult: A case report. Intractable & Rare Diseases Research.

[9] Dutta V, Chopra M, Kovilapu UB, Gahlot GPS. Solitary pulmonary

**References**

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1906;**XLVI**:1283-1285

1932;**72**:889-895

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2019;**8**:1139-1145

2017;**182**:1077-1093

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*The Review of Histoplasmosis Endemicity and Current Status in Asia DOI: http://dx.doi.org/10.5772/intechopen.92448*

#### **References**

Histoplasma *and Histoplasmosis*

**22**

**Author details**

Anna Rozaliyani1,2\*

†

† These two authors contributed equally.

provided the original work is properly cited.

2 Pulmonary Mycosis Centre, Jakarta, Indonesia

\*Address all correspondence to: annaroza1110@gmail.com

and Findra Setianingrum1,2†

1 Faculty of Medicine Universitas, Department of Parasitology, Indonesia

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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[15] Cho SH, Bin YY, Park JS, Yook KD, Kim YK. Epidemiological characterization of imported systemic mycoses occurred in Korea. Osong Public Health and Research Perspectives. 2018;**9**:362

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[18] Yehia MM, Abdulla ZA. Isolation of *Histoplasma capsulatum* and *Blastomyces dermatitidis* from Iraqi patients with lower respiratory tract infections. Journal of the Islamic Medical Association of North America. 2011;**43**

[19] Pourfarziani V, Taheri S. Is pulmonary histoplasmosis a risk factor for acute renal failure in renal transplant recipients? Saudi Journal of Kidney Diseases and Transplantation. 2009;**20**:643-645

[20] Lai CH, Huang CK, Chin C, Yang YT, Lin HF, Lin HH. Indigenous case of disseminated histoplasmosis, Taiwan. Emerging Infectious Diseases. 2007;**13**:127-129

[21] Chang YT, Huang SC, Hu SY, Tsan YT, Wang LM, Wang RC. Disseminated histoplasmosis presenting as haemolytic anaemia. Postgraduate Medical Journal. 2010;**86**:443-444

[22] Bhut B, Kulkarni A, Rai V, Agrawal V, Verma A, Jain M, et al. A rare case of disseminated histoplasmosis in a patient with Crohn's disease on immunosuppressive treatment. Indian Journal of Gastroenterology. 2018;**37**:472-474

[23] Choudhury AK, Mishra AK, Gautam DK, Tilak R, Tilak V, Gambhir IS, et al. Histoplasmosis accompanying disseminated tuberculosis in an immunocompetent adolescent boy. The American Journal of Tropical Medicine and Hygiene. 2019;**102**:352-354

[24] Dawra S, Mandavdhare HS, Prasad KK, Dutta U, Sharma V. Gastrointestinal: Unusual cause of ileocecal thickening in an

immunocompromised patient: A histologic surprise. Journal of Gastroenterology and Hepatology. 2018;**33**:769

[25] Gupta R, Majumdar K, Saran R, Srivastava S, Sakhuja P, Batra V. Role of endoscopic ultrasound-guided fineneedle aspiration in adrenal lesions: Analysis of 32 patients. Journal of Cytology. 2008;**5**:4-8

[26] Gupta R, Majumdar K, Srivastava S, Varakanahalli S, Saran R. Endoscopic ultrasound-guided cytodiagnosis of adrenal histoplasmosis with reversible CD4 T-lymphocytopenia and jejunal lymphangiectasia. Journal of Cytology. 2018;**5**:4-8

[27] Gupta S, Jain SK, Kumar R, Saxena R. Idiopathic CD4 lymphocytopenia presenting with progressive disseminated histoplasmosis. Medical Journal, Armed Forces India. 2018;**74**:280-283

[28] Gupta N, Vinod KS, Mittal A, Kumar APA, Kumar A, Wig N. Histoplasmosis, heart failure, hemolysis and haemophagocytic lymphohistiocytosis. The Pan African Medical Journal. 2019;**32**:1-5

[29] Jawale PM, Gulwani HV. Histoplasmosis presenting as laryngeal ulcer in a post-renal transplant male: An unusual case from India. Journal de Mycologie Medicale. 2017;**27**:573-576

[30] Kapatia G, Gupta P, Harshal M, Usha D, Rajwanshi A. Isolated lymph nodal histoplasmosis: A rare presentation. Diagnostic Cytopathology. 2019;**47**:834-836

[31] Khetarpal S, Mhapsekar T, Nagar R, Parihar A. Oral histoplasmosis masquerading as acute necrotizing ulcerative gingivitis: A rare case report. International Journal of Health Sciences (Qassim). 2019;**13**:37-40

**25**

*The Review of Histoplasmosis Endemicity and Current Status in Asia*

crisis. Indian Journal of Pathology & Microbiology. 2017;**60**:612-613

[42] Rajeshwari M, Xess I, Sharma MC, Jain D. Acid-fastness of histoplasma in surgical pathology practice. Journal of Pathology and Translational Medicine.

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mimicking tuberculosis in a patient with chronic hepatitis C. Tropical Doctor.

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[41] Pujari A, Rakheja V, Bajaj M, Sen S, Yadav B. Isolated conjunctival histoplasmosis in an elderly patient: A rare but important scenario. Canadian Journal of Ophthalmology.

2019;**54**:e15-e16

2017;**51**:482-487

2018:3-5

[43] Ramesh V, Narreddy S,

origin: Adrenal histoplasmosis

[44] Samaddar A, Sarma A,

Case Reports. 2019;**25**:49-52

*DOI: http://dx.doi.org/10.5772/intechopen.92448*

Ramesh V, Sharma S. Asymptomatic indurated plaque on the tongue in an immunocompetent man.

International Journal of Dermatology.

[33] Kumar V, Bhatia A, Madaan GB, Marwah S, Nigam AS. Role of bone marrow examination in the evaluation of infections: Clinico-hematological analysis in a tertiary care centre. Turk

Patoloji Dergisi. 2020;**36**:17-22

[34] Mahto S, Jamal A, Gupta P, Majumdar P, Grewal V, Agarwal N. A rare cause of nodular skin lesions with fever in an immunocompetent individual. Journal of Family Medicine and Primary Care. 2017;**6**:169-170

[35] Mandavdhare HS, Shah J, Prasad KK, Agarwala R, Suri V, Kumari S, et al. Gastrointestinal histoplasmosis: A case series from a non-endemic region in North India. Intestinal Research. 2019;**17**:149-152

[36] Muhammed H, Nampoothiri RV, Gaspar BL, Jain S. Infectious causes of Addison's disease: 1 organ-2 organisms!

BMJ Case Reports. 2018:1-3

Lung India. 2018;**35**:41-46

India. 2018;**35**:41-46

[37] Pandey M, Bajpai J, Kant S, Mishra P, Verma S. Histoplasmosis presenting as an intrathoracic mass.

[38] Pangeni R, Mittal S, Arava S, Hadda V, Ramam M, Mohan A, et al. A 44-year-old man with hemoptysis. Lung

[39] Patra S, Nimitha P, Kaul S, Valakkada J, Verma KK, Ramam M, et al. Primary cutaneous histoplasmosis in an immunocompetent patient presenting with severe pruritus. Indian Journal of Dermatology, Venereology and Leprology. 2018;**84**:465-468

[40] Philips CA, Augustine P, Kumar L, Mahadevan P. Addison's darling

[32] Khullar G, Saxena AK,

2019;**58**:423-424

*The Review of Histoplasmosis Endemicity and Current Status in Asia DOI: http://dx.doi.org/10.5772/intechopen.92448*

[32] Khullar G, Saxena AK, Ramesh V, Sharma S. Asymptomatic indurated plaque on the tongue in an immunocompetent man. International Journal of Dermatology. 2019;**58**:423-424

Histoplasma *and Histoplasmosis*

1995;**12**:235-238

2009;**20**:643-645

2007;**13**:127-129

2018;**37**:472-474

2019;**102**:352-354

[17] Yilmaz GG, Yilmaz E, Coşkun M, Karpuzoǧlu G, Gelen T, Yeǧin O. Cutaneous histoplasmosis in a child with hyper IgM. Pediatric Dermatology. immunocompromised patient: A histologic surprise. Journal of Gastroenterology and Hepatology.

[25] Gupta R, Majumdar K, Saran R, Srivastava S, Sakhuja P, Batra V. Role of endoscopic ultrasound-guided fineneedle aspiration in adrenal lesions: Analysis of 32 patients. Journal of

[26] Gupta R, Majumdar K, Srivastava S, Varakanahalli S, Saran R. Endoscopic ultrasound-guided cytodiagnosis of adrenal histoplasmosis with reversible CD4 T-lymphocytopenia and jejunal lymphangiectasia. Journal of Cytology.

[27] Gupta S, Jain SK, Kumar R, Saxena R. Idiopathic CD4 lymphocytopenia presenting with progressive disseminated

Forces India. 2018;**74**:280-283

Medical Journal. 2019;**32**:1-5

[29] Jawale PM, Gulwani HV.

2017;**27**:573-576

2019;**47**:834-836

ulcer in a post-renal transplant male: An unusual case from India. Journal de Mycologie Medicale.

Histoplasmosis presenting as laryngeal

[30] Kapatia G, Gupta P, Harshal M, Usha D, Rajwanshi A. Isolated lymph nodal histoplasmosis: A rare presentation. Diagnostic Cytopathology.

[31] Khetarpal S, Mhapsekar T, Nagar R,

Parihar A. Oral histoplasmosis masquerading as acute necrotizing ulcerative gingivitis: A rare case report. International Journal of Health Sciences

(Qassim). 2019;**13**:37-40

[28] Gupta N, Vinod KS, Mittal A, Kumar APA, Kumar A, Wig N. Histoplasmosis, heart failure, hemolysis and haemophagocytic lymphohistiocytosis. The Pan African

histoplasmosis. Medical Journal, Armed

2018;**33**:769

2018;**5**:4-8

Cytology. 2008;**5**:4-8

[18] Yehia MM, Abdulla ZA. Isolation of *Histoplasma capsulatum* and *Blastomyces dermatitidis* from Iraqi patients with lower respiratory tract infections. Journal of the Islamic Medical

Association of North America. 2011;**43**

[19] Pourfarziani V, Taheri S. Is pulmonary histoplasmosis a risk factor for acute renal failure in renal transplant recipients? Saudi Journal of Kidney Diseases and Transplantation.

[20] Lai CH, Huang CK, Chin C, Yang YT, Lin HF, Lin HH. Indigenous case of disseminated histoplasmosis, Taiwan. Emerging Infectious Diseases.

[21] Chang YT, Huang SC, Hu SY, Tsan YT, Wang LM, Wang RC.

[22] Bhut B, Kulkarni A, Rai V, Agrawal V, Verma A, Jain M, et al. A rare case of disseminated histoplasmosis

in a patient with Crohn's disease on immunosuppressive treatment. Indian Journal of Gastroenterology.

[23] Choudhury AK, Mishra AK, Gautam DK, Tilak R, Tilak V, Gambhir IS, et al. Histoplasmosis accompanying disseminated

[24] Dawra S, Mandavdhare HS, Prasad KK, Dutta U, Sharma V. Gastrointestinal: Unusual cause of ileocecal thickening in an

tuberculosis in an immunocompetent adolescent boy. The American Journal of Tropical Medicine and Hygiene.

Disseminated histoplasmosis presenting as haemolytic anaemia. Postgraduate Medical Journal. 2010;**86**:443-444

**24**

[33] Kumar V, Bhatia A, Madaan GB, Marwah S, Nigam AS. Role of bone marrow examination in the evaluation of infections: Clinico-hematological analysis in a tertiary care centre. Turk Patoloji Dergisi. 2020;**36**:17-22

[34] Mahto S, Jamal A, Gupta P, Majumdar P, Grewal V, Agarwal N. A rare cause of nodular skin lesions with fever in an immunocompetent individual. Journal of Family Medicine and Primary Care. 2017;**6**:169-170

[35] Mandavdhare HS, Shah J, Prasad KK, Agarwala R, Suri V, Kumari S, et al. Gastrointestinal histoplasmosis: A case series from a non-endemic region in North India. Intestinal Research. 2019;**17**:149-152

[36] Muhammed H, Nampoothiri RV, Gaspar BL, Jain S. Infectious causes of Addison's disease: 1 organ-2 organisms! BMJ Case Reports. 2018:1-3

[37] Pandey M, Bajpai J, Kant S, Mishra P, Verma S. Histoplasmosis presenting as an intrathoracic mass. Lung India. 2018;**35**:41-46

[38] Pangeni R, Mittal S, Arava S, Hadda V, Ramam M, Mohan A, et al. A 44-year-old man with hemoptysis. Lung India. 2018;**35**:41-46

[39] Patra S, Nimitha P, Kaul S, Valakkada J, Verma KK, Ramam M, et al. Primary cutaneous histoplasmosis in an immunocompetent patient presenting with severe pruritus. Indian Journal of Dermatology, Venereology and Leprology. 2018;**84**:465-468

[40] Philips CA, Augustine P, Kumar L, Mahadevan P. Addison's darling

crisis. Indian Journal of Pathology & Microbiology. 2017;**60**:612-613

[41] Pujari A, Rakheja V, Bajaj M, Sen S, Yadav B. Isolated conjunctival histoplasmosis in an elderly patient: A rare but important scenario. Canadian Journal of Ophthalmology. 2019;**54**:e15-e16

[42] Rajeshwari M, Xess I, Sharma MC, Jain D. Acid-fastness of histoplasma in surgical pathology practice. Journal of Pathology and Translational Medicine. 2017;**51**:482-487

[43] Ramesh V, Narreddy S, Gowrishankar S, Barigala R, Nanda S. A challenging case of pyrexia of unknown origin: Adrenal histoplasmosis mimicking tuberculosis in a patient with chronic hepatitis C. Tropical Doctor. 2018:3-5

[44] Samaddar A, Sarma A, Kumar PHA, Srivastava S, Shrimali T, Gopalakrishnan M, et al. Disseminated histoplasmosis in immunocompetent patients from an arid zone in Western India: A case series. Medical Mycology Case Reports. 2019;**25**:49-52

[45] Santosh T, Kothari K, Singhal SS, Shah VV, Patil R. Disseminated histoplasmosis in an immunocompetent patient—Utility of skin scrape cytology in diagnosis: A case report. Journal of Medical Case Reports. 2018;**12**:10-13

[46] Sethi J, Gupta KL, Mohanty T, Gupta S, Ahluwalia J, Kohli HS. Fever of unknown origin in a renal transplant recipient: Lactate dehydrogenase as an important clue to diagnosis. Experimental and Clinical Transplantation. 2019:2018-2019

[47] Sondhi P, Singh S, Khandpur S, Agarwal S. A case of disseminated histoplasmosis presenting with facial and laryngeal involvement. Indian Journal of Dermatology, Venereology and Leprology. 2018;**84**:6-15

[48] Shastri P, Gupta P. Fulminant histoplasmosis presenting as pyrexia of unknown origin in immunocompetent adult diabetic patient. Indian Journal of Critical Care Medicine. 2019;**23**:193-195

[49] Singh A, Gauri M, Gautam P, Gautam D, Haq M, Handa AC, et al. Head and neck involvement with histoplasmosis; the great masquerader. American Journal of Otolaryngology and Head and Neck Surgery. 2019;**40**:678-683

[50] Singh M, Chandy DD, Bharani T, Marak RSK, Yadav S, Dabadghao P, et al. Clinical outcomes and cortical reserve in adrenal histoplasmosis—A retrospective follow-up study of 40 patients. Clinical Endocrinology. 2019;**90**:534-541

[51] Tiwari S, Ojha S. Disseminated histoplasmosis diagnosed on bone marrow aspiration in a case of fever with thrombocytopenia. Journal of Global Infectious Diseases. 2018;**10**:112-113

[52] Abdulla M, Narayan R, Mampilly N, Kumar P. Subdural empyema in disseminated histoplasmosis. Annals of Indian Academy of Neurology. 2017;**20**:169-172

[53] Yadav A, Bansal R, Tandon A, Wadhwa N, Bhatt S. Disseminated histoplasmosis: A rare case presentation. Tropical Doctor. 2018;**48**:152-154

[54] Yadhav CSC, Elumalai K, Nishad SS, Sivannan S, Srinivasan S. Development of acrocyanosis associated with pain and increased creatinine level in histoplasmosis patient: Medication therapy. Aging Medicine. 2018;**1**:220-223

[55] Abdulla M, Mustaq S, Narayan R. Sequential surprises; non endemic mycoses revealing immunodeficiency. Indian Journal of Pathology & Microbiology. 2019;**62**:512-514

[56] Agrawal S, Goyal A, Agarwal S, Khadgawat R. Hypercalcaemia, adrenal insufficiency and bilateral adrenal histoplasmosis in a middle-aged man: A diagnostic dilemma. BML Case Reports. 2019;**12**:10-13

[57] Bansal RK, Choudhary NS, Patle SK, Agarwal A, Kaur G, Sarin H, et al. Endoscopic ultrasound-guided fine-needle aspiration of enlarged adrenals in patients with pyrexia of unknown origin: A single-center experience of 52 cases. Indian Journal of Gastroenterology. 2018;**37**:108-112

[58] Bansal N. Maltese cross birefringence in histoplasmosis. ACG Case Reports Journal. 2019;**6**:1-2

[59] Bansal N, Sethuraman N, Gopalakrishnan R, Ramasubramanian V, Kumar SD, Nambi SP, et al. Can urinary histoplasma antigen test improve the diagnosis of histoplasmosis in a tuberculosis endemic region? Mycoses. 2019;**62**:502-507

[60] Rahim MA, Zaman S, Amin MR, Nazim Uddin K, Chowdhury MJ. Adrenal histoplasmosis among immunocompetent Bangladeshi patients. Tropical Doctor. 2019;**49**: 132-133

[61] Shaikh MS, Majeed MA. Disseminated histoplasmosis in an immuno-competent young male: Role of bone marrow examination in rapid diagnosis. Diagnostic Cytopathology. 2018;**46**:273-276

[62] Sigera LSM, Gunawardane SR, Malkanthi MA, Jayasinghe RD, Sitheeque MAM, Tilakaratne WM, et al. *Histoplasma capsulatum* caused a localized tongue ulcer in a non-HIV patient—A case from nonendemic country. Ear, Nose, & Throat Journal. 2019 14556131984424

[63] Pan B, Chen M, Pan W, Liao W. Histoplasmosis: A new endemic fungal infection in China? Review and analysis of cases. Mycoses. 2013;**56**:212-221

**27**

2017;**5**:1-5

*The Review of Histoplasmosis Endemicity and Current Status in Asia*

[72] Pihua G, Zhaolong C, Xinlin M, Xiaosong D, Keqiang W, Rui'e F, et al. The clinical-radiologic-pathologic features of imported pulmonary histoplasmosis. Chinese Journal of Tuberculosis and Respiratory Diseases.

Paitoonpong L, Snabboon T. Bilateral adrenal histoplasmosis. Balkan Medical

[74] Jordan AS, Chavada R, Nagendra V, Mcneil HP, Kociuba K, Gibson KA. A budding surprise from the joint. The Medical Journal of Australia.

[75] Hung M, Sun H, Hsueh P, Hung C, Chang S. Meningitis due to *Histoplasma* 

[76] Murty O. Cystic tumor of papillary muscle of heart: A rare finding in sudden death. The American Journal of Forensic Medicine and Pathology.

[77] Sharma R, Lipi L, Gajendra S, Mohapatra I, Goel RK, Duggal R, et al. Gastrointestinal histoplasmosis: A case series. International Journal of Surgical

[78] Marsilla MM, Khairunisa AA, Azyani Y, Petrick P. Disseminated histoplasmosis mimicking an acute appendicitis. The Malaysian Journal of

[79] Van TC, Nguyen SV, Nguyen TV, Hoang HTT, Pham PTM, Do HTT, et al. An unusual presentation of disseminated histoplasmosis in a non-HIV patient from Vietnam. Revista Iberoamericana de Micología.

Pathology. 2017;**25**:592-598

Pathology. 2019;**41**:223-227

2019;**36**:147-150

*capsulatum* and *Mycobacterium tuberculosis* in a returned traveler with acquired immunodeficiency syndrome. Journal of the Formosan Medical Association. 2005;**104**:860-863

2015;**38**:23-28

[73] Porntharukchareon T, Khahakaew S, Sriprasart T,

Journal. 2019;**36**:359-360

2013;**199**:700-701

2009;**30**:201-203

*DOI: http://dx.doi.org/10.5772/intechopen.92448*

[64] Zhu L, Wang J, Wang Z, Wang Y,

Gastroenterology. 2016;**22**:4027-4033

[65] Zhao C, Zhao S, Liu G, Xu X. Risk factors on invasive fungal infections in patients admitted to non-hematological oncology department and pediatric intensive care unit. Chinese Journal of

[66] Dang Y, Jiang L, Zhang J, Pan B, Zhu G, Zhu F, et al. Disseminated histoplasmosis in an immunocompetent individual diagnosed with gastrointestinal endoscopy: A case report. BMC

[67] Huang L, Wu Y, Miao X. Localized *Histoplasma capsulatum* osteomyelitis of the fibula in an immunocompetent teenage boy: A case report. BMC Infectious Diseases. 2013;**13**:1-5

[69] Wang Y, Pan B, Wu J, Bi X, Liao W, Pan W, et al. Short report: Detection and phylogenetic characterization of a case of *Histoplasma capsulatum* infection in Mainland China. The American Journal of Tropical Medicine and Hygiene. 2014;**90**:1180-1183

[70] Xiong X, Fan L, Kang M, Wei J, Cheng D. Disseminated histoplasmosis: A rare clinical phenotype with difficult diagnosis. Respirology Case reports.

[71] Yan Z, Xiaoli SU, Yuanyuan LI,

Pinhua PAN. Clinical comparative analysis for pulmonary histoplasmosis

histoplasmosis. Journal of Central South

Ruoxi HE, Chengping HU,

and progressive disseminated

University. 2016;**41**:1345-1351

Yang J, Zhu L, et al. Intestinal histoplasmosis in immunocompetent

adults. World Journal of

Pediatrics. 2013;**51**:598-601

Infectious Diseases. 2019:1-5

[68] Liu B, Qu L, Zhu J, Yang Z, Yan S. Histoplasmosis mimicking metastatic spinal tumour. The Journal of International Medical Research.

2017;**45**:1440-1446

*The Review of Histoplasmosis Endemicity and Current Status in Asia DOI: http://dx.doi.org/10.5772/intechopen.92448*

[64] Zhu L, Wang J, Wang Z, Wang Y, Yang J, Zhu L, et al. Intestinal histoplasmosis in immunocompetent adults. World Journal of Gastroenterology. 2016;**22**:4027-4033

Histoplasma *and Histoplasmosis*

[48] Shastri P, Gupta P. Fulminant histoplasmosis presenting as pyrexia of unknown origin in immunocompetent adult diabetic patient. Indian Journal of Critical Care Medicine. 2019;**23**:193-195

insufficiency and bilateral adrenal histoplasmosis in a middle-aged man: A diagnostic dilemma. BML Case Reports.

[57] Bansal RK, Choudhary NS, Patle SK, Agarwal A, Kaur G, Sarin H, et al. Endoscopic ultrasound-guided fine-needle aspiration of enlarged adrenals in patients with pyrexia of unknown origin: A single-center experience of 52 cases. Indian Journal of Gastroenterology. 2018;**37**:108-112

[58] Bansal N. Maltese cross

[59] Bansal N, Sethuraman N,

2019;**62**:502-507

2018;**46**:273-276

2019 14556131984424

132-133

birefringence in histoplasmosis. ACG Case Reports Journal. 2019;**6**:1-2

Gopalakrishnan R, Ramasubramanian V, Kumar SD, Nambi SP, et al. Can urinary histoplasma antigen test improve the diagnosis of histoplasmosis in a tuberculosis endemic region? Mycoses.

[60] Rahim MA, Zaman S, Amin MR, Nazim Uddin K, Chowdhury MJ. Adrenal histoplasmosis among immunocompetent Bangladeshi patients. Tropical Doctor. 2019;**49**:

[61] Shaikh MS, Majeed MA. Disseminated histoplasmosis in an immuno-competent young male: Role of bone marrow examination in rapid diagnosis. Diagnostic Cytopathology.

[62] Sigera LSM, Gunawardane SR, Malkanthi MA, Jayasinghe RD, Sitheeque MAM, Tilakaratne WM, et al. *Histoplasma capsulatum* caused a localized tongue ulcer in a non-HIV patient—A case from nonendemic country. Ear, Nose, & Throat Journal.

[63] Pan B, Chen M, Pan W, Liao W. Histoplasmosis: A new endemic fungal infection in China? Review and analysis of cases. Mycoses. 2013;**56**:212-221

2019;**12**:10-13

[49] Singh A, Gauri M, Gautam P, Gautam D, Haq M, Handa AC, et al. Head and neck involvement with histoplasmosis; the great masquerader. American Journal of Otolaryngology

and Head and Neck Surgery.

[50] Singh M, Chandy DD, Bharani T, Marak RSK, Yadav S, Dabadghao P, et al. Clinical outcomes and cortical reserve in adrenal histoplasmosis—A retrospective follow-up study of 40 patients. Clinical Endocrinology. 2019;**90**:534-541

[51] Tiwari S, Ojha S. Disseminated histoplasmosis diagnosed on bone marrow aspiration in a case of fever with thrombocytopenia. Journal of Global Infectious Diseases. 2018;**10**:112-113

[52] Abdulla M, Narayan R, Mampilly N,

Kumar P. Subdural empyema in disseminated histoplasmosis. Annals of Indian Academy of Neurology.

[53] Yadav A, Bansal R, Tandon A, Wadhwa N, Bhatt S. Disseminated histoplasmosis: A rare case presentation.

Tropical Doctor. 2018;**48**:152-154

[54] Yadhav CSC, Elumalai K, Nishad SS, Sivannan S, Srinivasan S. Development of acrocyanosis associated with pain and increased creatinine level in histoplasmosis patient: Medication therapy. Aging Medicine. 2018;**1**:220-223

[55] Abdulla M, Mustaq S, Narayan R. Sequential surprises; non endemic mycoses revealing immunodeficiency.

[56] Agrawal S, Goyal A, Agarwal S, Khadgawat R. Hypercalcaemia, adrenal

Indian Journal of Pathology & Microbiology. 2019;**62**:512-514

2017;**20**:169-172

2019;**40**:678-683

**26**

[65] Zhao C, Zhao S, Liu G, Xu X. Risk factors on invasive fungal infections in patients admitted to non-hematological oncology department and pediatric intensive care unit. Chinese Journal of Pediatrics. 2013;**51**:598-601

[66] Dang Y, Jiang L, Zhang J, Pan B, Zhu G, Zhu F, et al. Disseminated histoplasmosis in an immunocompetent individual diagnosed with gastrointestinal endoscopy: A case report. BMC Infectious Diseases. 2019:1-5

[67] Huang L, Wu Y, Miao X. Localized *Histoplasma capsulatum* osteomyelitis of the fibula in an immunocompetent teenage boy: A case report. BMC Infectious Diseases. 2013;**13**:1-5

[68] Liu B, Qu L, Zhu J, Yang Z, Yan S. Histoplasmosis mimicking metastatic spinal tumour. The Journal of International Medical Research. 2017;**45**:1440-1446

[69] Wang Y, Pan B, Wu J, Bi X, Liao W, Pan W, et al. Short report: Detection and phylogenetic characterization of a case of *Histoplasma capsulatum* infection in Mainland China. The American Journal of Tropical Medicine and Hygiene. 2014;**90**:1180-1183

[70] Xiong X, Fan L, Kang M, Wei J, Cheng D. Disseminated histoplasmosis: A rare clinical phenotype with difficult diagnosis. Respirology Case reports. 2017;**5**:1-5

[71] Yan Z, Xiaoli SU, Yuanyuan LI, Ruoxi HE, Chengping HU, Pinhua PAN. Clinical comparative analysis for pulmonary histoplasmosis and progressive disseminated histoplasmosis. Journal of Central South University. 2016;**41**:1345-1351

[72] Pihua G, Zhaolong C, Xinlin M, Xiaosong D, Keqiang W, Rui'e F, et al. The clinical-radiologic-pathologic features of imported pulmonary histoplasmosis. Chinese Journal of Tuberculosis and Respiratory Diseases. 2015;**38**:23-28

[73] Porntharukchareon T, Khahakaew S, Sriprasart T, Paitoonpong L, Snabboon T. Bilateral adrenal histoplasmosis. Balkan Medical Journal. 2019;**36**:359-360

[74] Jordan AS, Chavada R, Nagendra V, Mcneil HP, Kociuba K, Gibson KA. A budding surprise from the joint. The Medical Journal of Australia. 2013;**199**:700-701

[75] Hung M, Sun H, Hsueh P, Hung C, Chang S. Meningitis due to *Histoplasma capsulatum* and *Mycobacterium tuberculosis* in a returned traveler with acquired immunodeficiency syndrome. Journal of the Formosan Medical Association. 2005;**104**:860-863

[76] Murty O. Cystic tumor of papillary muscle of heart: A rare finding in sudden death. The American Journal of Forensic Medicine and Pathology. 2009;**30**:201-203

[77] Sharma R, Lipi L, Gajendra S, Mohapatra I, Goel RK, Duggal R, et al. Gastrointestinal histoplasmosis: A case series. International Journal of Surgical Pathology. 2017;**25**:592-598

[78] Marsilla MM, Khairunisa AA, Azyani Y, Petrick P. Disseminated histoplasmosis mimicking an acute appendicitis. The Malaysian Journal of Pathology. 2019;**41**:223-227

[79] Van TC, Nguyen SV, Nguyen TV, Hoang HTT, Pham PTM, Do HTT, et al. An unusual presentation of disseminated histoplasmosis in a non-HIV patient from Vietnam. Revista Iberoamericana de Micología. 2019;**36**:147-150

[80] Story H, Tennessee F, Turhan V. Tennessee (Abd)' DenTürkı̇ye' Ye Hı̇stoplazmozun Öyküsü Histoplasmosis Story From Tennessee, USA To Turkey. Mikrobiyoloji Bülteni. 2009:339-351

[81] Aslani J, Jeyhounian M. Pulmonary histoplasmosis in Iran: A case report. Kowsar Medical Journal. 2001;**6**:21-24

[82] Kathuria S, Capoor MR, Yadav S, Singh A, Ramesh V. Disseminated histoplasmosis in an apparently immunocompetent individual from north India: A case report and review. Medical Mycology. 2013;**51**:774-778

[83] Sanyal M, Thammayya A. *Histoplasma capsulatum* in the soil of Gangetic Plain in India. The Indian Journal of Medical Research. 1975;**63**:1020-1028

[84] Malhotra S, Dhundial R, Kaur N, Kaushal M, Duggal N. Cutaneous histoplasmosis and role of direct microscopy—A case report. Clinical Microbiology Open Access. 2017;**06**:2-4

[85] Gupta N, Vyas SP, Kothari DC. Primary histoplasmosis of oral mucosa: A rare case report. International Journal of Dental and Medical Research. 2015;**1**:91-93

[86] Kriplani D, Kante K, Maniar J, Khubchandani S. Histoplasmosis in an immunocompetent host: A rare case report Dipsha. Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology. 2012;**24**:106-109

[87] Sethi SK, Wadhwani N, Jha P, Duggal R, Sharma R, Bansal S, et al. Uncommon cause of fever in a pediatric kidney transplant recipient: Answers. Pediatric Nephrology. 2017;**32**:1527-1529

[88] Choudhury TA, Baruah R, Shah N, Lahkar B, Ahmed K, Sarmah BJ. Disseminated histoplasmosis presenting as oropharyngeal mass lesion. Medical Mycology Case Reports. 2019;**24**:78-81

[89] Viswanathan R, Chakravarty SC, Randhawva HS. Pilot histoplasmosis survey in Delhi area. British Medical Journal. 1960;**1**:399-400

[90] Mochi A, Edwards PQ. Geographical distribution of histoplasmosis and histoplasmin sensitivity. Bulletin of the World Health Organization. 1952;**5**:259-291

[91] Dhariwal G, Chakravarty N. Histoplasmin, coccidioidin, blastomycin, tuberculin sensitivity in relation to tropical eosinophilia and pulmonary calcifications. Indian Medical Gazette. 1954;**89**:23-28

[92] Islam N, Islam M, Muazzam M. A histoplasmosis survey in east Pakistan. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1962;**56**:246-249

[93] Siddiqi SH, Stauffer JC. Prevalence of histoplasmin sensitivity in Pakistan. The American Journal of Tropical Medicine and Hygiene. 1980;**29**:109-111

[94] Uragoda CG, Wijenaike A, Han ES. Histoplasmin and coccidioidin sensitivity in Ceylon. Bulletin of the World Health Organization. 1971;**45**:689-691

[95] Wongprommek P, Chaakulkeeree M. Clinical characteristics of histoplasmosis in Siriraj hospital. Journal of the Medical Association of Thailand. 2016;**99**:257-261

[96] Norkaew T, Ohno H, Sriburee P. Detection of environmental sources of *Histoplasma capsulatum* in Chiang Mai, Thailand, by nested PCR. Mycopathologia. 2013;**176**:395-402

[97] Ng KH, Siar CH. Review of oral histoplasmosis in Malaysians. Oral Surgery, Oral Med Oral Pathol Oral Radiol Endodontology. 1996;**81**:303-307

**29**

*The Review of Histoplasmosis Endemicity and Current Status in Asia*

Prevalence of histoplasmin sensitivity in healthy adults and tuberculosis patients in Southwest China. Journal of Medical and Veterinary Mycology.

1996;**34**:171-174

1988;**19**:1063-1066

[107] Held JL, Berkowitz RK, Grossman ME. Use of touch preparation for rapid diagnosis of disseminated candidiasis. Journal of the American Academy of Dermatology.

[108] India HIV Estimations. 2017

IDF Diabetes Atlas Ninth. 2019

[110] Sicree R, Shaw J, Zimmet P. Diabetes and impaired glucose tolerance. Diabetes Atlas. 2010:1-105

[111] Hu Y, Zhang J, Li X, Yang Y, Zhang Y, Ma J, et al. *Penicillium marneffei* infection: An emerging disease in Mainland China. Mycopathologia. 2013;**175**:57-67

[112] Chan JFW, Lau SKP, Yuen KY, Woo PCY. *Talaromyces* (Penicillium) *marneffei* infection in non-HIV-infected

patients. Emerging Microbes & Infections. 2016;**5**:e19-e19

[109] International Diabetes Federation.

*DOI: http://dx.doi.org/10.5772/intechopen.92448*

[98] Yew Hu AS, Fong Hu AS, Hu CH. Histoplasmosis with addisonian crisis: Call for bird control. The Medical Journal of Malaysia. 2015;**70**:104-105

[99] Hu RCH. The Malaysian Medical Association's role in public health control for reduction of bird dropping hazards in Sarawak. Australian and New Zealand Journal of Public Health.

[100] Sirait SP, Bramono K, Hermanto N. Correlation of CD4 counts with clinical and histopathological findings in disseminated histoplasmosis: A 10-year retrospective study. International Journal of Dermatology. 2017;**56**:926-931

[101] Wahyuningsih R, Sjam R, Suriadireja A, Djauzi S. Touch biopsy: A diagnosis method for cutaneous mycoses of AIDS patients in poor resources laboratory setting. Mycoses.

[102] Tucker HA, Kvisseelgaard N. Histoplasmin and tuberculin sensitivity in Burma: Study of tests on 3,558 subjects. Bulletin of the World Health

Organization. 1952;**7**:189-200

[103] Mardianto AT. Prevalensi histoplasmosis pada mahasiswa

Kedokteran. 1997;**29**:139-144

2001;**149**:69-71

kedokteran Universitas Islam Sumatera Utara dan hubungan hewan peliharaan dengan tes histoplasmin. Berkala Ilmu

[104] Bulmer AC, Bulmer GS. Incidence of histoplasmin hypersensitivity in the Philippines. Mycopathologia.

[105] Zhao B, Xia X, Yin J, Zhang X, Wu E, Shi Y, et al. Epidemiological investigation of *Histoplasma capsulatum* infection in China. Chinese Medical

[106] Wen FQ, Sun YD, Watanabe K, Yoshida M, Wu JNS, Baum GL.

Journal. 2001;**114**:743-746

2009;**33**:194-195

2011;**54**:89

*The Review of Histoplasmosis Endemicity and Current Status in Asia DOI: http://dx.doi.org/10.5772/intechopen.92448*

[98] Yew Hu AS, Fong Hu AS, Hu CH. Histoplasmosis with addisonian crisis: Call for bird control. The Medical Journal of Malaysia. 2015;**70**:104-105

Histoplasma *and Histoplasmosis*

[80] Story H, Tennessee F, Turhan V. Tennessee (Abd)' DenTürkı̇ye' Ye Hı̇stoplazmozun Öyküsü Histoplasmosis Story From Tennessee, USA To Turkey. Mikrobiyoloji Bülteni. 2009:339-351

as oropharyngeal mass lesion. Medical Mycology Case Reports. 2019;**24**:78-81

[89] Viswanathan R, Chakravarty SC, Randhawva HS. Pilot histoplasmosis survey in Delhi area. British Medical

Journal. 1960;**1**:399-400

[90] Mochi A, Edwards PQ. Geographical distribution of histoplasmosis and histoplasmin sensitivity. Bulletin of the World Health

Organization. 1952;**5**:259-291

[91] Dhariwal G, Chakravarty N. Histoplasmin, coccidioidin, blastomycin, tuberculin sensitivity in relation to tropical eosinophilia and pulmonary calcifications. Indian Medical Gazette. 1954;**89**:23-28

[92] Islam N, Islam M, Muazzam M. A histoplasmosis survey in east Pakistan. Transactions of the Royal Society of Tropical Medicine and Hygiene.

[93] Siddiqi SH, Stauffer JC. Prevalence of histoplasmin sensitivity in Pakistan. The American Journal of Tropical Medicine and Hygiene. 1980;**29**:109-111

[94] Uragoda CG, Wijenaike A, Han ES. Histoplasmin and coccidioidin sensitivity in Ceylon. Bulletin of the World Health

[95] Wongprommek P, Chaakulkeeree M. Clinical characteristics of histoplasmosis in Siriraj hospital. Journal of the Medical Association of Thailand. 2016;**99**:257-261

[96] Norkaew T, Ohno H, Sriburee P. Detection of environmental sources of *Histoplasma capsulatum* in Chiang Mai, Thailand, by nested PCR. Mycopathologia. 2013;**176**:395-402

[97] Ng KH, Siar CH. Review of oral histoplasmosis in Malaysians. Oral Surgery, Oral Med Oral Pathol Oral Radiol Endodontology. 1996;**81**:303-307

Organization. 1971;**45**:689-691

1962;**56**:246-249

[81] Aslani J, Jeyhounian M. Pulmonary histoplasmosis in Iran: A case report. Kowsar Medical Journal. 2001;**6**:21-24

[82] Kathuria S, Capoor MR, Yadav S, Singh A, Ramesh V. Disseminated histoplasmosis in an apparently immunocompetent individual from north India: A case report and review. Medical Mycology. 2013;**51**:774-778

[83] Sanyal M, Thammayya A. *Histoplasma capsulatum* in the soil of Gangetic Plain in India. The Indian Journal of Medical Research.

[84] Malhotra S, Dhundial R, Kaur N, Kaushal M, Duggal N. Cutaneous histoplasmosis and role of direct microscopy—A case report. Clinical Microbiology Open Access. 2017;**06**:2-4

[85] Gupta N, Vyas SP, Kothari DC. Primary histoplasmosis of oral mucosa: A rare case report. International Journal

of Dental and Medical Research.

[86] Kriplani D, Kante K, Maniar J, Khubchandani S. Histoplasmosis in an immunocompetent host: A rare case report Dipsha. Journal of Oral and Maxillofacial Surgery, Medicine, and

[87] Sethi SK, Wadhwani N, Jha P, Duggal R, Sharma R, Bansal S, et al. Uncommon cause of fever in a pediatric kidney transplant recipient: Answers. Pediatric Nephrology.

[88] Choudhury TA, Baruah R, Shah N, Lahkar B, Ahmed K, Sarmah BJ.

Disseminated histoplasmosis presenting

Pathology. 2012;**24**:106-109

2017;**32**:1527-1529

1975;**63**:1020-1028

2015;**1**:91-93

**28**

[99] Hu RCH. The Malaysian Medical Association's role in public health control for reduction of bird dropping hazards in Sarawak. Australian and New Zealand Journal of Public Health. 2009;**33**:194-195

[100] Sirait SP, Bramono K, Hermanto N. Correlation of CD4 counts with clinical and histopathological findings in disseminated histoplasmosis: A 10-year retrospective study. International Journal of Dermatology. 2017;**56**:926-931

[101] Wahyuningsih R, Sjam R, Suriadireja A, Djauzi S. Touch biopsy: A diagnosis method for cutaneous mycoses of AIDS patients in poor resources laboratory setting. Mycoses. 2011;**54**:89

[102] Tucker HA, Kvisseelgaard N. Histoplasmin and tuberculin sensitivity in Burma: Study of tests on 3,558 subjects. Bulletin of the World Health Organization. 1952;**7**:189-200

[103] Mardianto AT. Prevalensi histoplasmosis pada mahasiswa kedokteran Universitas Islam Sumatera Utara dan hubungan hewan peliharaan dengan tes histoplasmin. Berkala Ilmu Kedokteran. 1997;**29**:139-144

[104] Bulmer AC, Bulmer GS. Incidence of histoplasmin hypersensitivity in the Philippines. Mycopathologia. 2001;**149**:69-71

[105] Zhao B, Xia X, Yin J, Zhang X, Wu E, Shi Y, et al. Epidemiological investigation of *Histoplasma capsulatum* infection in China. Chinese Medical Journal. 2001;**114**:743-746

[106] Wen FQ, Sun YD, Watanabe K, Yoshida M, Wu JNS, Baum GL.

Prevalence of histoplasmin sensitivity in healthy adults and tuberculosis patients in Southwest China. Journal of Medical and Veterinary Mycology. 1996;**34**:171-174

[107] Held JL, Berkowitz RK, Grossman ME. Use of touch preparation for rapid diagnosis of disseminated candidiasis. Journal of the American Academy of Dermatology. 1988;**19**:1063-1066

[108] India HIV Estimations. 2017

[109] International Diabetes Federation. IDF Diabetes Atlas Ninth. 2019

[110] Sicree R, Shaw J, Zimmet P. Diabetes and impaired glucose tolerance. Diabetes Atlas. 2010:1-105

[111] Hu Y, Zhang J, Li X, Yang Y, Zhang Y, Ma J, et al. *Penicillium marneffei* infection: An emerging disease in Mainland China. Mycopathologia. 2013;**175**:57-67

[112] Chan JFW, Lau SKP, Yuen KY, Woo PCY. *Talaromyces* (Penicillium) *marneffei* infection in non-HIV-infected patients. Emerging Microbes & Infections. 2016;**5**:e19-e19

**31**

Section 3

Diagnosis

Section 3 Diagnosis

**33**

**Chapter 3**

**Abstract**

performance

**1. Introduction**

mised hosts [1, 4].

examination of the sample [3].

*Histoplasma capsulatum* and

*Emilie Guemas, Loïc Sobanska and Magalie Demar*

for the Diagnosis

includes an integrated medical approach.

*Histoplasmosis:* Current Concept

Histoplasmosis is a global deep mycosis caused by *Histoplasma capsulatum (Hc)*, a dimorphic fungus. It exists on two main varieties *Hc capsulatum* and *Hc duboisii* that could be distinguished by their epidemiology, their clinical presentation, and the morphological aspect of the fungus at direct examination of the sample. Laboratory diagnosis of *Hc* remains a real challenge as it required experience and equipment. Through a general review of literature, the different diagnosis tools for *Histoplasma* sp. are analyzed, and strengths and weaknesses are pointed according to the context-based value. Isolation of *Hc* on culture is the gold standard for diagnosis of histoplasmosis. However, it remains less sensitive (sensitivity: up to 77%) and implies long time to result, which can be inappropriate or in adapted for an emergency diagnosis. So, nonculture methods as antigen testing, serology, and molecular biology become available and allow a rapid diagnosis. However, the optimal diagnostic method depends on many parameters as the very wide range of symptomatology, the immune status. Indeed, Ag detection is the best diagnosis tool for PHD (sensitivity: 92–95%) and SCN histoplasmosis (sensitivity: 66%) and serology for the subacute/chronic form (sensitivity: 85–93%). Thus, the clinico-biological dialog is essential, and histoplasmosis management

**Keywords:** *Histoplasma capsulatum*, diagnosis, non-culture methods, culture,

Histoplasmosis caused by *Histoplasma capsulatum* leads to a wide spectrum of symptomatology [1–3] varying from subclinical or acute to chronic form. Histoplasmosis can be localized or it causes a disseminated, life-threatening infection involving various tissues and organs of the body, notably in immunocompro-

This is a thermally dimorphic fungus presenting as a yeast at body temperature and as a hyaline mold in the natural environment. Human histoplasmosis is due to the two varieties of the pathogen: *H. capsulatum* var. capsulatum *(Hcc)* and *H. capsulatum* var. duboisii *(Hcd),* which could be distinguished by their epidemiology, their clinical presentation, and the morphological aspect of the fungus at direct

#### **Chapter 3**

## *Histoplasma capsulatum* and *Histoplasmosis:* Current Concept for the Diagnosis

*Emilie Guemas, Loïc Sobanska and Magalie Demar*

#### **Abstract**

Histoplasmosis is a global deep mycosis caused by *Histoplasma capsulatum (Hc)*, a dimorphic fungus. It exists on two main varieties *Hc capsulatum* and *Hc duboisii* that could be distinguished by their epidemiology, their clinical presentation, and the morphological aspect of the fungus at direct examination of the sample. Laboratory diagnosis of *Hc* remains a real challenge as it required experience and equipment. Through a general review of literature, the different diagnosis tools for *Histoplasma* sp. are analyzed, and strengths and weaknesses are pointed according to the context-based value. Isolation of *Hc* on culture is the gold standard for diagnosis of histoplasmosis. However, it remains less sensitive (sensitivity: up to 77%) and implies long time to result, which can be inappropriate or in adapted for an emergency diagnosis. So, nonculture methods as antigen testing, serology, and molecular biology become available and allow a rapid diagnosis. However, the optimal diagnostic method depends on many parameters as the very wide range of symptomatology, the immune status. Indeed, Ag detection is the best diagnosis tool for PHD (sensitivity: 92–95%) and SCN histoplasmosis (sensitivity: 66%) and serology for the subacute/chronic form (sensitivity: 85–93%). Thus, the clinico-biological dialog is essential, and histoplasmosis management includes an integrated medical approach.

**Keywords:** *Histoplasma capsulatum*, diagnosis, non-culture methods, culture, performance

#### **1. Introduction**

Histoplasmosis caused by *Histoplasma capsulatum* leads to a wide spectrum of symptomatology [1–3] varying from subclinical or acute to chronic form. Histoplasmosis can be localized or it causes a disseminated, life-threatening infection involving various tissues and organs of the body, notably in immunocompromised hosts [1, 4].

This is a thermally dimorphic fungus presenting as a yeast at body temperature and as a hyaline mold in the natural environment. Human histoplasmosis is due to the two varieties of the pathogen: *H. capsulatum* var. capsulatum *(Hcc)* and *H. capsulatum* var. duboisii *(Hcd),* which could be distinguished by their epidemiology, their clinical presentation, and the morphological aspect of the fungus at direct examination of the sample [3].

*Hcc*, responsible for "small cell" histoplasmosis or American histoplasmosis, is well-documented in the United States mainly in the valleys of Ohio and Mississippi, South America, and Asia. Hcd responsible for "large-form" histoplasmosis or African histoplasmosis is endemic in Central and West Africa and Madagascar [3, 5–6]. We point out a third variety, *H. capsulatum* var. farcimimosum, involved in equine pathology that will not be detailed in this chapter.

Histoplasmosis can be challenging to diagnose because it is laborious and requires special characteristics for its revealing. According to the criteria recommended by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) and those of the Council of State and Territorial Epidemiologists (CSTE) [7, 8], an integrated approach including clinical, radiographic, and laboratory evidence is required. For laboratory diagnosis, several techniques are available as microbiology, histopathology, and immune-serological assays, depending on the clinical context and laboratory capabilities. Since 1978, the introduction of the *Histoplasma* antigen assay significantly improved the diagnosis by allowing a rapid, noninvasive, and sensitive method. Then, if culture remains the reference approach in most laboratories in endemic and non-endemic areas, nonculture methods are developing and much more improving [2, 9–11].

This review describes the current diagnostics for the laboratory identification of *H. capsulatum*, with focus on their performance and context-based value.

#### **2. Clinical presentations and type of histoplasmosis**

*For Histoplasma capsulatum* var. capsulatum*,* the inhalation of conidia from the environment (bat or bird guano) leads to Histoplasmosis syndrome or asymptomatic carriage in immunocompetent individuals. It can be self-limited especially in the lung or a progressive disseminated disease. Upon immunosuppression, the fungus can reactivate and be responsible for a localized or disseminated disease. Disseminated histoplasmosis has been classified as an AIDS-defining infection. There is a significant overlap of the pathophysiology and the clinical presentation as tuberculosis or cryptococcosis, sarcoidosis, and malignancy [1–4, 6, 12].

*The* histoplasmosis syndromes can be evoked according the clinical presentation, the epidemiology, and the disabled. It can be expressed as [1–2, 6]:


For *Histoplasma capsulatum* var. duboisii*,* many features remain undiscovered as the reservoir niche, pathogenesis, and epidemiology because of the relatively low

**35**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

number of case reports. It expresses much more in skin and subcutaneous tissues

All types of clinical specimens can be processed according to symptomatology

• Bronchoalveolar lavage (BAL), sputum, and lung biopsy should be performed

• Organ biopsies (lymph node, digestive tract, liver, skin, oral mucous)

Different microbiological approaches can be performed as on one hand mycological examination (direct, culture) and molecular assays (qPCR) and, on the other hand, histopathology. The performances depend on the specimens tested and

Examination of liquid smears or fine-needle aspiration and/or tissue apposi-

Yeasts are spherical, small (2–5 μm in diameter), intensely violet colored, and surrounded by a clear halo. These yeasts are narrow-based budding yeast cells, usually intracellular (macrophage, histiocyte, etc.), and do not produce any filaments (**Figure 1**). The differential diagnosis includes *Leishmania* species, which is similar in size but has one kinetoplast [3, 6, 13, 14], *Candida* mainly *glabrata* and *Cryptococcus*

Yeasts cells are oval and large (8–15 μm by 4–6 μm), with a "hourglass" or "figure eight" budding form, a thick wall and a narrow budding. They may have inside fat

tion on slides from all types of samples is carried out by staining with May-Grünwald-Giemsa (MGG). Using 10% KOH with or without calcofluoric acid, a chitin-binding fluorescent stain for the fungal cell wall, the yeasts can be easily visualized between slide and slip cover. For some specimens as the peripherical blood, cerebrospinal fluid, bronchoalveolar fluid, and cytocentrifugation may be

Diagnosis is possible according to the morphology of the yeasts.

yeast cell which is predominantly extracellular and strongly MGG stained.

• Peripheral blood (EDTA or fungal blood culture vacutainers)

*DOI: http://dx.doi.org/10.5772/intechopen.92782*

[1–2, 4, 6–8]:

than in lungs. Association as well with HIV is rare [5].

**3. Types of specimens and context base value**

for patients with pulmonary symptoms

• Punctures of lymph node, pus, or exudates

are much more details on paragraph 5.4 (**Table 1**).

**4. Direct examination for mycology**

**4.1** *H. capsulatum* **var. capsulatum**

**4.2** *H. capsulatum* **var. duboisii**

required [3, 13, 14].

• Bone marrow aspiration

• Cerebrospinal fluid (CSF)

number of case reports. It expresses much more in skin and subcutaneous tissues than in lungs. Association as well with HIV is rare [5].

### **3. Types of specimens and context base value**

All types of clinical specimens can be processed according to symptomatology [1–2, 4, 6–8]:


Histoplasma *and Histoplasmosis*

*Hcc*, responsible for "small cell" histoplasmosis or American histoplasmosis, is well-documented in the United States mainly in the valleys of Ohio and Mississippi, South America, and Asia. Hcd responsible for "large-form" histoplasmosis or African histoplasmosis is endemic in Central and West Africa and Madagascar [3, 5–6]. We point out a third variety, *H. capsulatum* var. farcimimosum, involved in

Histoplasmosis can be challenging to diagnose because it is laborious and requires special characteristics for its revealing. According to the criteria recommended by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) and those of the Council of State and Territorial Epidemiologists (CSTE) [7, 8], an integrated approach including clinical, radiographic, and laboratory evidence is required. For laboratory diagnosis, several techniques are available as microbiology, histopathology, and immune-serological assays, depending on the clinical context and laboratory capabilities. Since 1978, the introduction of the *Histoplasma* antigen assay significantly improved the diagnosis by allowing a rapid, noninvasive, and sensitive method. Then, if culture remains the reference approach in most laboratories in endemic and non-endemic areas, non-

This review describes the current diagnostics for the laboratory identification of

*For Histoplasma capsulatum* var. capsulatum*,* the inhalation of conidia from the environment (bat or bird guano) leads to Histoplasmosis syndrome or asymptomatic carriage in immunocompetent individuals. It can be self-limited especially in the lung or a progressive disseminated disease. Upon immunosuppression, the fungus can reactivate and be responsible for a localized or disseminated disease. Disseminated histoplasmosis has been classified as an AIDS-defining infection. There is a significant overlap of the pathophysiology and the clinical presentation as

*The* histoplasmosis syndromes can be evoked according the clinical presenta-

• A pulmonary histoplasmosis presenting as acute, subacute, chronic, or pulmo-

some particular populations (extreme age groups, immunosuppressed people including AIDS/HIV, iatrogenic origin). However, immunocompetent people defined as without obviously immunodeficiency can develop such syndrome

• A progressive disseminated histoplasmosis (PDH) that mainly concerns

• Other clinical findings as mediastinal histoplasmosis (adenitis, granuloma,

For *Histoplasma capsulatum* var. duboisii*,* many features remain undiscovered as the reservoir niche, pathogenesis, and epidemiology because of the relatively low

equine pathology that will not be detailed in this chapter.

culture methods are developing and much more improving [2, 9–11].

**2. Clinical presentations and type of histoplasmosis**

*H. capsulatum*, with focus on their performance and context-based value.

tuberculosis or cryptococcosis, sarcoidosis, and malignancy [1–4, 6, 12].

tion, the epidemiology, and the disabled. It can be expressed as [1–2, 6]:

**34**

nary nodules.

mediastinitis).

when important inoculum.

• A cerebral nervous system (CNS) histoplasmosis.


Different microbiological approaches can be performed as on one hand mycological examination (direct, culture) and molecular assays (qPCR) and, on the other hand, histopathology. The performances depend on the specimens tested and are much more details on paragraph 5.4 (**Table 1**).

### **4. Direct examination for mycology**

Examination of liquid smears or fine-needle aspiration and/or tissue apposition on slides from all types of samples is carried out by staining with May-Grünwald-Giemsa (MGG). Using 10% KOH with or without calcofluoric acid, a chitin-binding fluorescent stain for the fungal cell wall, the yeasts can be easily visualized between slide and slip cover. For some specimens as the peripherical blood, cerebrospinal fluid, bronchoalveolar fluid, and cytocentrifugation may be required [3, 13, 14].

Diagnosis is possible according to the morphology of the yeasts.

#### **4.1** *H. capsulatum* **var. capsulatum**

Yeasts are spherical, small (2–5 μm in diameter), intensely violet colored, and surrounded by a clear halo. These yeasts are narrow-based budding yeast cells, usually intracellular (macrophage, histiocyte, etc.), and do not produce any filaments (**Figure 1**). The differential diagnosis includes *Leishmania* species, which is similar in size but has one kinetoplast [3, 6, 13, 14], *Candida* mainly *glabrata* and *Cryptococcus* yeast cell which is predominantly extracellular and strongly MGG stained.

#### **4.2** *H. capsulatum* **var. duboisii**

Yeasts cells are oval and large (8–15 μm by 4–6 μm), with a "hourglass" or "figure eight" budding form, a thick wall and a narrow budding. They may have inside fat


Histoplasma *and Histoplasmosis*

**36**

**Table 1.**

**37**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

droplets. These yeasts are intra- or extracellular, sometimes arranged in short chains

Histoplasma capsulatum *var. capsulatum on bone marrow smear, intramacrophagic yeast form, microscopic* 

Specimens as pus from abscess, draining sinuses and bone marrow lesions, colic biopsy, or BAL smears are relatively contributive to the diagnosis [4, 6, 15] in

Direct examination is inexpensive, and according to the relatively typical appearance of the levuriform elements, it allows to quickly and easily provide presumptive evidence for histoplasmosis. However, doubts may remain, and then it requires trained personnel and further investigations. Indeed confusion can con

cern *Hcc* versus *Cryptococcus sp., Candida glabrata*, and *Leishmania sp*. and/or if the clinical picture is not very classical [3, 6, 13, 14] or *Hcd* versus *Blastomyces dermatiti*

Histopathology consists in the research for elements evocative of the yeast form

*.* Different stains can be carried out such as Gomori

μm in diameter). *Hcc* is predominantly

*dis*, which looks like another yeast cell and differs with its broad base budding.

Methenamine Silver (GMS), Giemsa, or periodic acid-Schiff (PAS) stains that are the most relevant for the diagnosis. Hematoxylin and eosin (H&E) staining is

This concept of juggling between different stains and the description of mor

phologies of the microorganism (shape, size variation, cell disposition) and the tissue (cell response) permit to distinguish other pathogens as *Cryptococcus* spp., *Blastomyces dermatitis*, *Candida glabrata*, *Pneumocystis jirovecii*, *Coccidioides* spp., *Talaromyces* (for

merly *Penicillium*) *marneffei*, *Leishmania* spp., *Toxoplasma gondii*, and *Trypanosoma* 

It usually shows a granulomatous reaction with "giant cells," containing large

found phagocytosed within macrophages, histiocytes, and giant cells, often in clusters of many organisms, but sometimes they are in extracellular spaces.

**2** .

insensitive to detect the presence of *H. capsulatum* [3, 13, 14, 16].

*cruzi. T*hese differences are represented in **Table**

**5.1** *H. capsulatum* **var. capsulatum** *(Hcc)*

rounded or oval-shaped elements (2–4


*-*



*DOI: http://dx.doi.org/10.5772/intechopen.92782*

of 2 or 3 [3, 6, 13, 14].

**Figure 1.**

**5. Histopathology**

of *H. capsulatum* inside tissues

disseminated histoplasmosis.

*appearance, magnification ×1000 (photo by Emilie GUEMAS).*

*Preferred specimens for diagnosis according the histoplasmosis syndromes [1, 2].*

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.92782*

#### **Figure 1.**

Histoplasma *and Histoplasmosis*

**36**

**PHD**

**Acute** 

**Subacute** 

**Chronic** 

**Pulmonary** 

**Mediastinal** 

**CNS** 

**Other manifestations** 

**histoplasmosis**

**histoplasmosis**

**(pericarditis,** 

**rheumatological),**

**nodules**

**pulmonary** 

**pulmonary** 

**syndrom**

**syndrom**

**pulmonary** 

**syndrom**

Culture

Pulmonary tissue

x

x

x

x

(x):

differential

diagnosis

x (if associated

PHD)

or secretions

(LBA, sputum)

Bone marrow Lymph nodes

Exudates Organ biopsy Peripheral blood

Cerebrospinal

fluid

histopathology

x

x

x

(x)

Non viable

yeasts

Cytopathologic

examination of

BAL (50%)

Serology

Antigen

x serum,

x (83%):

(x) up to

(x)

40% (urine)

serum, urine

+/-LBA

serum, urine

+/-LBA or if

associated PDH

*(): not preferred but can be contributive, PDH: Progressive histoplasmosis disseminated, CNS: Central nervous system, BAL: Broncho-alveolar fluid.*

urine

+/-LBA

to repeat

**Table 1.** *Preferred specimens for diagnosis according the histoplasmosis syndromes [1, 2].*

(x)

x

x

X (low

x

X x

x

sensitivity)

x

(x)

x

x

(x)

mediastinal

(x)

(x)

Non viable

Non viable

yeasts

x

Non viable

yeasts

x (if associated

PHD)

x

yeasts

x Histoplasma capsulatum *var. capsulatum on bone marrow smear, intramacrophagic yeast form, microscopic appearance, magnification ×1000 (photo by Emilie GUEMAS).*

droplets. These yeasts are intra- or extracellular, sometimes arranged in short chains of 2 or 3 [3, 6, 13, 14].

Specimens as pus from abscess, draining sinuses and bone marrow lesions, colic biopsy, or BAL smears are relatively contributive to the diagnosis [4, 6, 15] in disseminated histoplasmosis.

Direct examination is inexpensive, and according to the relatively typical appearance of the levuriform elements, it allows to quickly and easily provide presumptive evidence for histoplasmosis. However, doubts may remain, and then it requires trained personnel and further investigations. Indeed confusion can concern *Hcc* versus *Cryptococcus sp., Candida glabrata*, and *Leishmania sp*. and/or if the clinical picture is not very classical [3, 6, 13, 14] or *Hcd* versus *Blastomyces dermatitidis*, which looks like another yeast cell and differs with its broad base budding.

#### **5. Histopathology**

Histopathology consists in the research for elements evocative of the yeast form of *H. capsulatum* inside tissues*.* Different stains can be carried out such as Gomori Methenamine Silver (GMS), Giemsa, or periodic acid-Schiff (PAS) stains that are the most relevant for the diagnosis. Hematoxylin and eosin (H&E) staining is insensitive to detect the presence of *H. capsulatum* [3, 13, 14, 16].

This concept of juggling between different stains and the description of morphologies of the microorganism (shape, size variation, cell disposition) and the tissue (cell response) permit to distinguish other pathogens as *Cryptococcus* spp., *Blastomyces dermatitis*, *Candida glabrata*, *Pneumocystis jirovecii*, *Coccidioides* spp., *Talaromyces* (formerly *Penicillium*) *marneffei*, *Leishmania* spp., *Toxoplasma gondii*, and *Trypanosoma cruzi. T*hese differences are represented in **Table 2**.

#### **5.1** *H. capsulatum* **var. capsulatum** *(Hcc)*

It usually shows a granulomatous reaction with "giant cells," containing large rounded or oval-shaped elements (2–4 μm in diameter). *Hcc* is predominantly found phagocytosed within macrophages, histiocytes, and giant cells, often in clusters of many organisms, but sometimes they are in extracellular spaces.


*IC: intracellular, EC: extra cellular, GMS: Gromori Methenamine Silver, PAS: Periodique Acid Schiff:, GE, MGG: May-Grunwald Giemsa.*

#### **Table 2.**

*Diagnosis differential of* H. capsulatum [2, 16, 13]*.*

The yeast phase of *Hcc* is very similar to other pathogens and not distinctive in tissues from several other endemic fungi. The misidentification occurs principally with *Candida glabrata, Penicillium marneffei, Pneumocystis jiroveci, Toxoplasma gondii, Leishmania donovani*, and *Cryptococcus neoformans* [9]. However, in the appropriate clinical context, the presence of *H. capsulatum* like yeast is able to

**39**

**Figure 2.**

*GUEMAS).*

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

confidently make a diagnosis of histoplasmosis and is indicative of active infection. Moreover, the immunohistochemistry reaction with *H. capsulatum* antibodies can

*Hcc* is large (6–12 μm) and the wall is thick and highly refractive, with a pseudocapsulated appearance. The yeast is easily distinguishable from the other endemic

Isolation of *H. capsulatum* remains the gold standard for the laboratory diagnosis of histoplasmosis. However, it requires both BioSafety Level 3 (BSL3) facilities to be manipulated [17] and usually invasive methods to obtain the specimens from which

Culture is performed on Sabouraud dextrose agar with antibiotics (chloramphenicol +/− gentamicin) +/− actidione that inhibits the contaminants. Other mediums can be used: brain heart infusion (BHI), yeast extract peptone agar (YEP agar) and potato dextrose agar (PDA) [3]. Mediums are incubated at 25–30°C for

As the rate of growth is slow, the growth of the mycelial forms usually takes 2–3 weeks but may take up to 8 weeks. Colony is initially white and smooth and then becomes brown, with a granular or cottony texture. The reverse is white, yel-

*Culture of* H. capsulatum *on Sabouraud dextrose agar. Colony is white with a cottony texture (photo by Emilie* 

*DOI: http://dx.doi.org/10.5772/intechopen.92782*

**5.2** *H. capsulatum* **var. duboisii** *(Hcc)*

fungi especially *Blastomyces dermatitis* [8].

**6.1 Inoculation and culture of specimens**

All specimen types can be used from superficial to deep ones.

**6. Culture**

6–8 weeks.

culture can be performed.

low, or orange (**Figure 2**).

be used to confirm diagnosis of histoplasmosis.

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.92782*

confidently make a diagnosis of histoplasmosis and is indicative of active infection. Moreover, the immunohistochemistry reaction with *H. capsulatum* antibodies can be used to confirm diagnosis of histoplasmosis.

#### **5.2** *H. capsulatum* **var. duboisii** *(Hcc)*

*Hcc* is large (6–12 μm) and the wall is thick and highly refractive, with a pseudocapsulated appearance. The yeast is easily distinguishable from the other endemic fungi especially *Blastomyces dermatitis* [8].

#### **6. Culture**

Histoplasma *and Histoplasmosis*

**Size (μm)**

*Cryptococcus* 3 - 8 Facultative

*Blastomyces dermatitis*

*Candida glabrata*

*Pneumocystis cysts*

*Talaromyces marneffeï*

*May-Grunwald Giemsa.*

**Table 2.**

**Cell disposition**

> halo Narrow-based budding Grouped in clusters into macrophage

Spherical with characteristic halo (thick capsule) Narrow-based budding

Thick retractile

No pseudohyphal production

Confusion when endospores are outside spherules or young spherules without endospores

wall Broad-based budding

5-8 EC Cysts Minimal

shaped yeast Transverse septum No bud

kinetoplast

*IC: intracellular, EC: extra cellular, GMS: Gromori Methenamine Silver, PAS: Periodique Acid Schiff:, GE, MGG:* 

*Hcd* 6 - 12 IC Oval with halo Granulamatous

*Hcc* 2 - 5 IC Spherical with

IC

*Coccidioides* 2 - 5 IC/ EC spherical

> 15 EC Round,

1 - 4 EC Oval to round

2-5 EC Small oval-

*Leishmania* 2 - 5 IC Oval to round

*Diagnosis differential of* H. capsulatum [2, 16, 13]*.*

**Characteristic Tissue response Specific** 

Granulamatous tissue response, necrosis

tissue response

Predominantly granulomatous inflammation, necrosis, +/- fibrosis

Mixed suppurative and granulomatous inflammation

Suppurative tissue response

Mixed suppurative and granulomatous inflammation, (Splendore-Hoëppli phenomenon likely)

reaction

Mixed suppurative and granulomatous inflammation

**coloration**

GMS, PAS

GMS, PAS

Mucicarmin (capsulated yeast) Fontana-Masson (uncapsulated yeast)

GMS, PAS, H&E

GMS, PAS, H&E

GMS, H&E, +/-PAS

GMS, PAS, H&E

MGG

**38**

The yeast phase of *Hcc* is very similar to other pathogens and not distinctive in tissues from several other endemic fungi. The misidentification occurs principally with *Candida glabrata, Penicillium marneffei, Pneumocystis jiroveci, Toxoplasma gondii, Leishmania donovani*, and *Cryptococcus neoformans* [9]. However, in the appropriate clinical context, the presence of *H. capsulatum* like yeast is able to

Isolation of *H. capsulatum* remains the gold standard for the laboratory diagnosis of histoplasmosis. However, it requires both BioSafety Level 3 (BSL3) facilities to be manipulated [17] and usually invasive methods to obtain the specimens from which culture can be performed.

#### **6.1 Inoculation and culture of specimens**

All specimen types can be used from superficial to deep ones.

Culture is performed on Sabouraud dextrose agar with antibiotics (chloramphenicol +/− gentamicin) +/− actidione that inhibits the contaminants. Other mediums can be used: brain heart infusion (BHI), yeast extract peptone agar (YEP agar) and potato dextrose agar (PDA) [3]. Mediums are incubated at 25–30°C for 6–8 weeks.

As the rate of growth is slow, the growth of the mycelial forms usually takes 2–3 weeks but may take up to 8 weeks. Colony is initially white and smooth and then becomes brown, with a granular or cottony texture. The reverse is white, yellow, or orange (**Figure 2**).

#### **Figure 2.**

*Culture of* H. capsulatum *on Sabouraud dextrose agar. Colony is white with a cottony texture (photo by Emilie GUEMAS).*

#### **6.2 Identification of the fungi**

Confirmation of the cultured organism as *H. capsulatum* requires complementary tests:

#### *6.2.1 The lactophenol cotton blue test (LPCB test)*

It allows determining microscopy morphology with identification characters [18]:


The differential diagnosis includes *Sepedonium sp*. (**Figure 3**).

#### *6.2.2 Identification with the MALDI-TOF*

The identification of the colony can be carried out by matrix-assisted laser desorption/ionization time off light (MALDI-TOF). Mass spectrometry by MALDI-TOF allows to confirm species accurately and quickly.

This identification technique is based upon the detection of highly abundant proteins in a mass range of 2–20 kDa by calculating their mass (m) to charge (z), m/z values. The spectrum thus obtained is compared with the reference spectra [19, 20].

#### *6.2.3 The conversion from the mold to the yeast form*

This can be achieved using enriched media such as BHI or blood agar plates incubated at 35–37°C in a CO2-enriched atmosphere [3]. However, *H. capsulatum* does not convert easily depending on many parameters [13] as nutriments and temperature conditions, which can explain that this experiment is increasingly abandoned.

#### *6.2.4 "Historic" tests*

The urease test allows the distinction between Hcc and Hcd as it reveals the expression of urease strongly for Hcc and weakly for Hcd at 48 hours [3, 5].

Others tests performed from the cultured fungi such as DNA hybridization using a highly specific commercially kit (AccProbe; Gen-Probe, Inc., San Diego, CA®) or the detection of specific precipitin by the exoantigen test was used in some laboratories [10, 13]. They were gradually replaced by less time-consuming method as MALDI-TOF. They will not be more detailed in this chapter.

#### **6.3 Blood culture**

The lysis centrifugation method as Isolator® system followed by the inoculation of the collected buffy coat [11] into an appropriate media culture has been proved to be the most efficient for detecting *Histoplasma* in the blood in comparison with other methods such as conventional or automated methods such as Bactec MYCO/F

**41**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

Lytic bottle [21]. However, many laboratories preferred those alternative systems than the manual isolator system because they permit less pre-analytic processing and continuous automated monitoring of bottles for growth when automated [9].

Histoplasma capsulatum*, microscopic appearance with LPCB test, magnification ×400 (photo by Emilie* 

The sensitivity of culture depends on the clinical manifestation, the clinical specimen, the state of immunity of the host, and the burden of disease [2, 9, 14]. Sensitivity is lower for patients with acute pulmonary histoplasmosis (40%) than

The diagnosis of disseminated histoplasmosis blood culture processed by lysis methodology and bone marrow shows higher sensitivity (60–90%). Conversely,

The limitations of the culture are the time frame for diagnosis, the mycological expertise required, and safety. Several weeks for growth of the fungus to establish the diagnosis are not consistent with the severity of the disease in immunosuppressed patients. Moreover, *H. capsulatum* poses an infectious risk and must be

Conversely, detection of *H. capsulatum* by automated methods such as the Bactec system has not shown optimal results with sensitivity of less than 50% [2, 9–11, 13, 14] in acute and disseminated histoplasmosis that need to be rapidly diagnosed for

Several protocols do exist based on *Histoplasma*-specific antibody detection with three routinely used assays: immunodiffusion test (ID), fixation complement test (FC), and indirect immunological assays (EIA) [9–11, 13, 14, 22–26]. Serology aims to confirm a previously contact with the pathogen, and it usually does not mean that the patient is making an acute infection. For histoplasmosis, in certain circumstances, serology can detect the acute phase of histoplasmosis. It is reported

for patients with disseminated histoplasmosis (75%) [2, 9, 10].

respiratory samples presented poor sensitivity (0–60%) [2, 9, 10].

*DOI: http://dx.doi.org/10.5772/intechopen.92782*

**6.4 Performances of the culture**

**Figure 3.**

*GUEMAS).*

manipulated in a laboratory with BSL3.

the prompt initiation of therapy.

**7. Antibody detection**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.92782*

#### **Figure 3.**

Histoplasma *and Histoplasmosis*

tary tests:

[19, 20].

abandoned.

*6.2.4 "Historic" tests*

**6.3 Blood culture**

**6.2 Identification of the fungi**

*6.2.1 The lactophenol cotton blue test (LPCB test)*

late, or knobby (7–15 μm in diameter).

*6.2.2 Identification with the MALDI-TOF*

• **Hyphae**: hyaline septated (2–3 μm in diameter),

short branches or directly on the sides of the hyphae

TOF allows to confirm species accurately and quickly.

*6.2.3 The conversion from the mold to the yeast form*

The differential diagnosis includes *Sepedonium sp*. (**Figure 3**).

Confirmation of the cultured organism as *H. capsulatum* requires complemen-

It allows determining microscopy morphology with identification characters [18]:

• **Characteristic macroconidia**: large, thick-walled, round, typically tubercu-

• **Microconidia**: smooth-walled spherical, pyriform (2–5 μm in diameter) on

The identification of the colony can be carried out by matrix-assisted laser desorption/ionization time off light (MALDI-TOF). Mass spectrometry by MALDI-

This identification technique is based upon the detection of highly abundant proteins in a mass range of 2–20 kDa by calculating their mass (m) to charge (z), m/z values. The spectrum thus obtained is compared with the reference spectra

This can be achieved using enriched media such as BHI or blood agar plates incubated at 35–37°C in a CO2-enriched atmosphere [3]. However, *H. capsulatum* does not convert easily depending on many parameters [13] as nutriments and temperature conditions, which can explain that this experiment is increasingly

The urease test allows the distinction between Hcc and Hcd as it reveals the expression of urease strongly for Hcc and weakly for Hcd at 48 hours [3, 5]. Others tests performed from the cultured fungi such as DNA hybridization using a highly specific commercially kit (AccProbe; Gen-Probe, Inc., San Diego, CA®) or the detection of specific precipitin by the exoantigen test was used in some laboratories [10, 13]. They were gradually replaced by less time-consuming method

The lysis centrifugation method as Isolator® system followed by the inoculation of the collected buffy coat [11] into an appropriate media culture has been proved to be the most efficient for detecting *Histoplasma* in the blood in comparison with other methods such as conventional or automated methods such as Bactec MYCO/F

as MALDI-TOF. They will not be more detailed in this chapter.

**40**

Histoplasma capsulatum*, microscopic appearance with LPCB test, magnification ×400 (photo by Emilie GUEMAS).*

Lytic bottle [21]. However, many laboratories preferred those alternative systems than the manual isolator system because they permit less pre-analytic processing and continuous automated monitoring of bottles for growth when automated [9].

#### **6.4 Performances of the culture**

The sensitivity of culture depends on the clinical manifestation, the clinical specimen, the state of immunity of the host, and the burden of disease [2, 9, 14]. Sensitivity is lower for patients with acute pulmonary histoplasmosis (40%) than for patients with disseminated histoplasmosis (75%) [2, 9, 10].

The diagnosis of disseminated histoplasmosis blood culture processed by lysis methodology and bone marrow shows higher sensitivity (60–90%). Conversely, respiratory samples presented poor sensitivity (0–60%) [2, 9, 10].

The limitations of the culture are the time frame for diagnosis, the mycological expertise required, and safety. Several weeks for growth of the fungus to establish the diagnosis are not consistent with the severity of the disease in immunosuppressed patients. Moreover, *H. capsulatum* poses an infectious risk and must be manipulated in a laboratory with BSL3.

Conversely, detection of *H. capsulatum* by automated methods such as the Bactec system has not shown optimal results with sensitivity of less than 50% [2, 9–11, 13, 14] in acute and disseminated histoplasmosis that need to be rapidly diagnosed for the prompt initiation of therapy.

#### **7. Antibody detection**

Several protocols do exist based on *Histoplasma*-specific antibody detection with three routinely used assays: immunodiffusion test (ID), fixation complement test (FC), and indirect immunological assays (EIA) [9–11, 13, 14, 22–26]. Serology aims to confirm a previously contact with the pathogen, and it usually does not mean that the patient is making an acute infection. For histoplasmosis, in certain circumstances, serology can detect the acute phase of histoplasmosis. It is reported that the antibodies detection period is between 4 and 8 weeks but can be negative in immunodeficient patients [10, 14]. The serologic targets are the specific *Histoplasma* proteins M (a catalase) and H (a β-glucosidase) that are present in the entire yeast or an antigenic extract (histoplasmin or HMIN) used as deglycosylate or not, from mycelial culture. Another protein can be targeted, the protein C (a carbohydrate, galactomannan), that is less specific and can be responsible of cross-reactions with other fungi [9]. Their characteristics are summarized in the **Table 3**.

#### **7.1 Immunodiffusion (ID)**

Immunodiffusion assay qualitatively detects the precipitating antibodies to antigens M and H on agar gel. The H band appears after the M band, and the presence of both bands is highly significant for histoplasmosis diagnosis. Thus, M band is detectable in most patients with acute infection and persists for long periods of time up to 3 years after disease resolution and is often present in chronic forms [9, 10, 14]. It does not distinct between active from latent or resolved infection. H band is less frequent, confirms acute infection only in 7% according [13], remains present 1–2 years after disease resolution, and indicates a more severe form of the disease. This method is simple, reliable, and inexpensive.

#### **7.2 Complement fixation (FC)**

Complement fixation method quantitatively measures the presence of complex antigen antibodies in a patient's serum against the entire yeast form or mycelial antigen (HMIN) from 3 to 6 weeks following infection [13]. Interlaboratory results vary as it is entirely strain-dependent for the antigen preparation. It is quite more sensitive bus less specific than the ID. Indeed, it presents numerous cross-reactions with other fungi and interference with rheumatoid factor and cold agglutinins [13]. A threshold defined as up to 1:32 or titer a 4-fold rise indicates an active infection.

#### **7.3 Enzyme-linked immunosorbent assay (ELISA)**

This method does not allow differentiation between an active infection and an old infection. Indeed, antibodies require 4–8 weeks to become detectable in peripheral blood. Serology is unreliable in patients with a reduced ability to produce antibodies, and then it is often negative in immunocompromised patients [13, 14]. Antibody testing is most useful for subacute and chronic forms of histoplasmosis [2].

#### **7.4 Western blot**

Western blot immunoassay uses Ag proteins of different kDa that will complex with the specific *Histoplasma* antibodies on a nitrocellulose band. It showed good results but does not exist as a commercial kit, so it can be fastidious to set up. The sensibility is improved by the treatment of the Ag (Histoplasmin) [22].

#### **7.5 Other tests**

Interferon gamma release assay (IGRA): this test is based on the quantification of lymphocyts-released interferon (IFN) -γ after restimulation in vitro of the cellmediated immunity with the same specific antigens. Recently, Rubio-Carrasquilla et al. [23] considered it as a promising screening method to detect individuals with latent Hc infection, even decades after the primary infection.

**43**

Mechanisms

Type of Ag

delay Duration of positive antibodies after resolution

Sensitivity Specificity Histoplasmosis

meningitidis

Acute infection Chronic infection

Special remarks

Kits/tests *Histoplasmine : HMIN, deglycosylated HMIN: tpHMIN.*

**Table 3.** *Summary of serological assays [2, 9, 10, 13, 14, 22, 26].*

+ Interference with

Rheumatoid factor and

cold agglutinins

House-made tests

House-made tests

++

Band M (80%)

Band H (7-20%)

Band M Positive after skin test

Histoplasmine (M +++/H+)

3 -6 weeks Months to years

Months to years

Persistent in recurrences

72-95% 70-80% When positive (culture

usually negative (63%)

4-6 weeks IgG anti-M → IgG M+H

Prot M: up to 3 years

Prot H: 1-2 years

70-95%

100% When positive (culture

usually negative (44%)

75-100% 91-100% Specificity: 93%

Sensibility: 82%

(26)

66-100% (9-10, 13)

90% (9-10, 13)

Ribosome antigen and ptHMIN induces better

response than glycosylate HMIN antigene. Best

results with ELISA using ferrous metal

House-made tests

Commercialized tests with IgG, IgM, IgA

45-94% (13, 22)

94-100% (13, 22)

Better results when using ptHMIN

Can detect early in the infection

House-made tests

Months to years

Entire yeast or HMIN

HMIN Prot M, Prot H

Yeast cell extract, ribosome, HMIN (glycosylayte/

deglycosylate)

2 -4 weeks

Histoplasma antigens of 115, 91,88, 83, 70 and 38 kDa from HMIN (glycosylayte/deglycosylate)

**Complement fixation (FC)** A supplemented specific Ag induces Ag-Ab complexes

Precipitins Ac-Ag (H/M) on gel agar

**Immunodiffusion (ID)**

**ELISA** Indirect sandwich ELISA

Visualisation of band profiles on nitrocellulose membranes

**Western Blott**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

45-100%

94-100%

No data

*DOI: http://dx.doi.org/10.5772/intechopen.92782*

2-4 weeks Months to years


#### Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.92782*

**Table 3.** *Summary of serological assays [2, 9, 10, 13, 14, 22, 26].*

Histoplasma *and Histoplasmosis*

**7.1 Immunodiffusion (ID)**

**7.2 Complement fixation (FC)**

histoplasmosis [2].

**7.4 Western blot**

**7.5 Other tests**

that the antibodies detection period is between 4 and 8 weeks but can be negative in immunodeficient patients [10, 14]. The serologic targets are the specific *Histoplasma* proteins M (a catalase) and H (a β-glucosidase) that are present in the entire yeast or an antigenic extract (histoplasmin or HMIN) used as deglycosylate or not, from mycelial culture. Another protein can be targeted, the protein C (a carbohydrate, galactomannan), that is less specific and can be responsible of cross-reactions with

Immunodiffusion assay qualitatively detects the precipitating antibodies to antigens M and H on agar gel. The H band appears after the M band, and the presence of both bands is highly significant for histoplasmosis diagnosis. Thus, M band is detectable in most patients with acute infection and persists for long periods of time up to 3 years after disease resolution and is often present in chronic forms [9, 10, 14]. It does not distinct between active from latent or resolved infection. H band is less frequent, confirms acute infection only in 7% according [13], remains present 1–2 years after disease resolution, and indicates a more severe form of the

Complement fixation method quantitatively measures the presence of complex antigen antibodies in a patient's serum against the entire yeast form or mycelial antigen (HMIN) from 3 to 6 weeks following infection [13]. Interlaboratory results vary as it is entirely strain-dependent for the antigen preparation. It is quite more sensitive bus less specific than the ID. Indeed, it presents numerous cross-reactions with other fungi and interference with rheumatoid factor and cold agglutinins [13]. A threshold defined as up to 1:32 or titer a 4-fold rise indicates an active infection.

This method does not allow differentiation between an active infection and an old infection. Indeed, antibodies require 4–8 weeks to become detectable in peripheral blood. Serology is unreliable in patients with a reduced ability to produce antibodies, and then it is often negative in immunocompromised patients

Western blot immunoassay uses Ag proteins of different kDa that will complex with the specific *Histoplasma* antibodies on a nitrocellulose band. It showed good results but does not exist as a commercial kit, so it can be fastidious to set up. The

Interferon gamma release assay (IGRA): this test is based on the quantification of lymphocyts-released interferon (IFN) -γ after restimulation in vitro of the cellmediated immunity with the same specific antigens. Recently, Rubio-Carrasquilla et al. [23] considered it as a promising screening method to detect individuals with

[13, 14]. Antibody testing is most useful for subacute and chronic forms of

sensibility is improved by the treatment of the Ag (Histoplasmin) [22].

latent Hc infection, even decades after the primary infection.

other fungi [9]. Their characteristics are summarized in the **Table 3**.

disease. This method is simple, reliable, and inexpensive.

**7.3 Enzyme-linked immunosorbent assay (ELISA)**

**42**

Latex agglutination tests were developed as a commercial kit, but false positives results were found in patients with tuberculosis. It was compared as more sensitive than CF [13].

A hemagglutination test was available but failed to differentiate *B. dermatitidis* [13].

A recent meta-analysis [9] interested in the global sensitivity of antibody detection for disseminated histoplasmosis and showed a low sensitivity of 58% in contrast with high specificity (100%). But there is a real distinction between the different assays. WB and ELISA methods have the highest analytical performance, with sensitivity of up to 90% when used in ptHMIN. This can explain that it is relevant to associate different methods in order to improve the diagnosis.

Cross-reactivity with granulomatous disease, tuberculosis, and sarcoidosis can occur with immunodiffusion and complement fixation tests. Moreover, serologic cross-reaction can occur with other common fungal pathogens like *Blastomyces dermatitidis*, *Coccidioides immitis*, *Aspergillus fumigatus*, and *Paracoccidioides brasiliensis* [24, 25].

The presence of antibodies in the CSF allows making the diagnosis of Histoplasma meningitis [2, 26].

#### **8. Antigen detection**

#### **8.1 Target antigens**

#### *8.1.1 Specific-*Histoplasma *antigen detection*

Circulating specific-*Histoplasma* polysaccharide antigen (HPA) detection is a useful option for diagnosis [9, 13–14, 27–28]. *Histoplasma* galactomannan is the target of the antigenic detection. Since its introduction in 1986 [13–14], as a solidphase radioimmunoassay, it was developed into a sandwich enzyme immunoassay (EIA) [2, 13, 27–29] with different improved generations and recently into a lateral flow assay (LFA) [30]. Their performance is related to the choice of the antibodies used monoclonal versus polyclonal, and the conjugate (Biotine or horseradish peroxidase) [13].

#### *8.1.2 Galactomannan antigen detection*

This polysaccharide mostly found in the cell wall of *Aspergillus* sp. is commonly used for invasive aspergillosis diagnosis in high-risk immunosuppressed patients suffering of solid organ transplantation or hematological malignancies. Crossreactivity with the histoplasma antigen detection is reported suggesting that this test could be a potentially helpful diagnostic test for histoplasmosis in HIV-infected patient because of the low incidence of invasive aspergillosis in this population. The sensibility of this test for diagnosis of disseminated histoplasmosis in AIDS patients is 77% and specificity is 100% [31].

#### **8.2 Technical methods**

#### *8.2.1 Enzyme immunoassay (EIA)*

This noninvasive method can be performed in urine, serum, and other body fluids as LBA [2, 9, 10, 13–14] and is the simplest diagnostic method, easily implemented in low- and middle-income countries.

**45**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

diagnosis of subacute and chronic pulmonary histoplasmosis [2, 9].

It allows a rapid diagnosis especially for immunosuppressed patients with severe acute or disseminated histoplasmosis. It is less sensitive than serology for the

This method has also been applied to other body fluids, including BAL for patient with pulmonary symptomatology and CSF for *Histoplasma* meningitis. Antigen was detected in the CSF of 66% of immunosuppressed-infected patient

in serum and thus appears as an useful marker for treatment response [32]. Moreover, it has proven its applicability to infected animals that may act as

histoplasmosis and blastomycosis because the geographic areas overlap.

If useful for the diagnosis, it can also monitor the antigen clearance, particularly

Cross-reactivity of antigen testing occurs in patients who have other fungal infections, including infections by *Blastomyces dermatitidis, Paracoccidioides brasiliensis,* and *Penicillium marneffei* [33]*.* The most problematic issue is differentiating

Recently, a lateral flow assay (LFA) has been developed and evaluated for rapid diagnosis of histoplasmosis. This point of care antigen detection in serum allows rapid results with high analytical performance. It is based on the using of rabbit polyclonal antibody that recognizes galactomannan antigen of *H. capsulatum*. Indeed, sensitivity is under 95% and specificity is 90%. Cross-reactivity occurs

Molecular methods can improve and accelerate the histoplasmosis diagnosis with a high analytical sensibility and specificity [9, 10, 34–36]. This combined with turnaround times shorter than those of other diagnostics. It is much more safety and reduces the risk of laboratory acquired histoplasmosis when handle its positive culture. Several applications can be linked to the use of PCR based techniques as (i) human diagnosis from different samples, with an increasingly use for the imported cases diagnosis in non-endemic areas [9–11, 13], (ii) environmental exploration for revealing the histoplasmosis reservoir [35], (iii) and prevention policies and strate-

There are no currently official approved molecular assays for H. capsulatum that are directly applicable to clinical specimens and no commercially kits are available [9, 10, 34]. However, there are numerous reports of the laboratory-developed PCR assays (in house PCR) using the different type of PCR (conventional, nested and quantitative) and a variety of molecular targets. The most relevant are the Internal Transcribed Spacer (ITS) multicopy region of the ribosomal DNA, genes encoding the M antigen or the 100-kDa-like protein [9, 34]. Most studies in the literature evaluated the performance of nested PCR in the diagnosis of PDH, this assay is associated with the increased risk of amplicon contamination, because of the

Antigen detection in urine is more sensitive than in serum for the diagnosis of disseminated histoplasmosis (95% versus 86% for HIV-infected patient) [9, 10–11]. However, antigen detection in urine is less useful for pulmonary forms or chronic

*DOI: http://dx.doi.org/10.5772/intechopen.92782*

patients who had *Histoplasma* meningitis [26].

principally with paracoccidioidomycosis [30].

**9.1 Polymerase chain reaction (PCR)**

potential reservoirs for humans [28, 29].

*8.2.2 Lateral flow assay (LFA)*

**9. Molecular tests**

gies for exposure risk [2].

manipulation of amplified products.

histoplasmosis.

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.92782*

It allows a rapid diagnosis especially for immunosuppressed patients with severe acute or disseminated histoplasmosis. It is less sensitive than serology for the diagnosis of subacute and chronic pulmonary histoplasmosis [2, 9].

Antigen detection in urine is more sensitive than in serum for the diagnosis of disseminated histoplasmosis (95% versus 86% for HIV-infected patient) [9, 10–11]. However, antigen detection in urine is less useful for pulmonary forms or chronic histoplasmosis.

This method has also been applied to other body fluids, including BAL for patient with pulmonary symptomatology and CSF for *Histoplasma* meningitis. Antigen was detected in the CSF of 66% of immunosuppressed-infected patient patients who had *Histoplasma* meningitis [26].

If useful for the diagnosis, it can also monitor the antigen clearance, particularly in serum and thus appears as an useful marker for treatment response [32].

Moreover, it has proven its applicability to infected animals that may act as potential reservoirs for humans [28, 29].

Cross-reactivity of antigen testing occurs in patients who have other fungal infections, including infections by *Blastomyces dermatitidis, Paracoccidioides brasiliensis,* and *Penicillium marneffei* [33]*.* The most problematic issue is differentiating histoplasmosis and blastomycosis because the geographic areas overlap.

#### *8.2.2 Lateral flow assay (LFA)*

Histoplasma *and Histoplasmosis*

than CF [13].

*iensis* [24, 25].

Histoplasma meningitis [2, 26].

*8.1.1 Specific-*Histoplasma *antigen detection*

*8.1.2 Galactomannan antigen detection*

is 77% and specificity is 100% [31].

*8.2.1 Enzyme immunoassay (EIA)*

mented in low- and middle-income countries.

**8.2 Technical methods**

**8. Antigen detection**

**8.1 Target antigens**

peroxidase) [13].

*dis* [13].

Latex agglutination tests were developed as a commercial kit, but false positives results were found in patients with tuberculosis. It was compared as more sensitive

A hemagglutination test was available but failed to differentiate *B. dermatiti-*

Cross-reactivity with granulomatous disease, tuberculosis, and sarcoidosis can occur with immunodiffusion and complement fixation tests. Moreover, serologic cross-reaction can occur with other common fungal pathogens like *Blastomyces dermatitidis*, *Coccidioides immitis*, *Aspergillus fumigatus*, and *Paracoccidioides brasil-*

Circulating specific-*Histoplasma* polysaccharide antigen (HPA) detection is a useful option for diagnosis [9, 13–14, 27–28]. *Histoplasma* galactomannan is the target of the antigenic detection. Since its introduction in 1986 [13–14], as a solidphase radioimmunoassay, it was developed into a sandwich enzyme immunoassay (EIA) [2, 13, 27–29] with different improved generations and recently into a lateral flow assay (LFA) [30]. Their performance is related to the choice of the antibodies used monoclonal versus polyclonal, and the conjugate (Biotine or horseradish

This polysaccharide mostly found in the cell wall of *Aspergillus* sp. is commonly used for invasive aspergillosis diagnosis in high-risk immunosuppressed patients suffering of solid organ transplantation or hematological malignancies. Crossreactivity with the histoplasma antigen detection is reported suggesting that this test could be a potentially helpful diagnostic test for histoplasmosis in HIV-infected patient because of the low incidence of invasive aspergillosis in this population. The sensibility of this test for diagnosis of disseminated histoplasmosis in AIDS patients

This noninvasive method can be performed in urine, serum, and other body fluids as LBA [2, 9, 10, 13–14] and is the simplest diagnostic method, easily imple-

The presence of antibodies in the CSF allows making the diagnosis of

A recent meta-analysis [9] interested in the global sensitivity of antibody detection for disseminated histoplasmosis and showed a low sensitivity of 58% in contrast with high specificity (100%). But there is a real distinction between the different assays. WB and ELISA methods have the highest analytical performance, with sensitivity of up to 90% when used in ptHMIN. This can explain that it is relevant to associate different methods in order to improve the diagnosis.

**44**

Recently, a lateral flow assay (LFA) has been developed and evaluated for rapid diagnosis of histoplasmosis. This point of care antigen detection in serum allows rapid results with high analytical performance. It is based on the using of rabbit polyclonal antibody that recognizes galactomannan antigen of *H. capsulatum*. Indeed, sensitivity is under 95% and specificity is 90%. Cross-reactivity occurs principally with paracoccidioidomycosis [30].

#### **9. Molecular tests**

#### **9.1 Polymerase chain reaction (PCR)**

Molecular methods can improve and accelerate the histoplasmosis diagnosis with a high analytical sensibility and specificity [9, 10, 34–36]. This combined with turnaround times shorter than those of other diagnostics. It is much more safety and reduces the risk of laboratory acquired histoplasmosis when handle its positive culture. Several applications can be linked to the use of PCR based techniques as (i) human diagnosis from different samples, with an increasingly use for the imported cases diagnosis in non-endemic areas [9–11, 13], (ii) environmental exploration for revealing the histoplasmosis reservoir [35], (iii) and prevention policies and strategies for exposure risk [2].

There are no currently official approved molecular assays for H. capsulatum that are directly applicable to clinical specimens and no commercially kits are available [9, 10, 34]. However, there are numerous reports of the laboratory-developed PCR assays (in house PCR) using the different type of PCR (conventional, nested and quantitative) and a variety of molecular targets. The most relevant are the Internal Transcribed Spacer (ITS) multicopy region of the ribosomal DNA, genes encoding the M antigen or the 100-kDa-like protein [9, 34]. Most studies in the literature evaluated the performance of nested PCR in the diagnosis of PDH, this assay is associated with the increased risk of amplicon contamination, because of the manipulation of amplified products.

A multiplex approach can also be performed. For example, in a reference laboratory in Spain they developed a multiplex qPCR for *H. capsulatum, Pneumocystis jirovecii* and *Cryptococcus neoformans* [11].

As a full tool to Histoplasmosis diagnosis, molecular assays can be performed on different types of specimens including respiratory secretions, biopsies, bone marrow, blood, or sera.

For diagnosis of disseminated histoplasmosis (PDH), sensitivity and specificity are 95% and 99%, respectively [2, 9]. Sample of blood and bone marrow have an excellent sensitivity (100%) in immunocompromised patients with disseminated histoplasmosis, whereas it is weaker in immunocompetent patients [2, 11].

Moreover, sensitivity increases by testing more than one sample per patient in cases with extra-pulmonary histoplasmosis. So, in case of high suspicion, clinicians should repeat and diversify samples [2, 9, 11, 34].

Recent findings in molecular biology permit to evaluate genetic diversity using multilocus sequence typing (MLST) OR RAPD-PCR [36]. This could be Interesting to associate genotypes and clinical presentation.

#### **9.2 Loop-mediated isothermal amplification (LAMP)**

#### *9.2.1 Principe*

Loop-mediated isothermal amplification (LAMP) is a highly efficient, sensitive, specific and cost-effective isothermal amplification method that uses at least four primers, recognizing six different regions in the target sequence (**Figure 4**) and results in a self-primed DNA [37].

In LAMP, the target sequence is amplified at a constant temperature of 60–65°C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. The amount of DNA produced in LAMP is considerably higher than PCR-based amplification.

To increase the reaction sensitivity, the ITS region was used as target, since it is a multicopy sequence and also because it is considered an important barcoding sequence for fungal identification, being conserved among *H. capsulatum* strains and divergent from other fungi, ensuring high specificity.

Forward inner primer (FIP) and backward inner primer (BIP) have inverted sequences attached at the 5′ end, named F1c and B1c, which are complementary to an internal sequence from the amplified strand, forming a loop at each extremity of a single strand DNA.

The outer primers (F3 and B3) anneal upstream to the FIP and BIP, acting as a binding site for DNA polymerase, which, in the LAMP reaction, also contains the strand displacement activity.

LAMP results can be observed using several strategies with minimal ambiguity with real-time turbidimetry (magnesium pyrophosphate formation), fluorescent compounds (Sybr Green, Eva Green, SYTO, calcein), magnesium colorimetric titration, fluorescent-labeled probes, quencher-labeled primers, dye-labeled primers, and PH-sensitive dyes.

#### *9.2.2 Indications*

LAMP can be used on samples of whole blood or bone marrow for patients suspected of progressive disseminated histoplasmosis (PDH).

A new assay, the so-called ITS LAMP, showed no cross-reactivity when assayed with DNA from other pathogenic or environmental fungi. The assay is able to detect isolates from all geographical clades of *H. capsulatum*, including Hcd. In comparison with Hcp100 nPCR, it reached sensitivity of 83% and specificity of 92% [35].

**47**

to be routinely used.

*Principe of LAMP [38].*

**Figure 4.**

**11. Conclusion**

ent protocols (antigen preparation, methods, ...).

the reference method remains less sensitive.

antifungal therapy is not prescribed.

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

This method remains cost-effective with or without the extraction DNA step and did not require a thermocycler or an electrophoresis apparatus. It saves time with test performed in less than 200 minutes [35]. However, this little-known and recent method is currently underused and much more reserved for resource-limited laboratories. It should evolve in the next years to come and need further evaluation

**10. Summary of the different categories of tests and their sensitivity (%)**

The performance for each test according the clinical context is summarized in **Table 4**. It considers global sensitivity without any distinction between the differ-

It highlights that Ag detection is the best diagnosis tool for PHD and SCN histoplasmosis, serology for the subacute/chronic form, and the culture although this is

Histoplasmosis remains a severe and neglected disease for which early diagnosis of invasive fungal infections is critical to allow a prompt patient care. Indeed the mortality rate among HIV/AIDS patients diagnosed with histoplasmosis is high: 42% mortality for disseminated histoplasmosis if treatment is delayed and 100% if

*DOI: http://dx.doi.org/10.5772/intechopen.92782*

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.92782*

**Figure 4.** *Principe of LAMP [38].*

Histoplasma *and Histoplasmosis*

marrow, blood, or sera.

*9.2.1 Principe*

a single strand DNA.

strand displacement activity.

ers, and PH-sensitive dyes.

*9.2.2 Indications*

*jirovecii* and *Cryptococcus neoformans* [11].

should repeat and diversify samples [2, 9, 11, 34].

to associate genotypes and clinical presentation.

results in a self-primed DNA [37].

**9.2 Loop-mediated isothermal amplification (LAMP)**

A multiplex approach can also be performed. For example, in a reference labora-

As a full tool to Histoplasmosis diagnosis, molecular assays can be performed on different types of specimens including respiratory secretions, biopsies, bone

For diagnosis of disseminated histoplasmosis (PDH), sensitivity and specificity are 95% and 99%, respectively [2, 9]. Sample of blood and bone marrow have an excellent sensitivity (100%) in immunocompromised patients with disseminated histoplasmosis, whereas it is weaker in immunocompetent patients [2, 11].

Moreover, sensitivity increases by testing more than one sample per patient in cases with extra-pulmonary histoplasmosis. So, in case of high suspicion, clinicians

Recent findings in molecular biology permit to evaluate genetic diversity using multilocus sequence typing (MLST) OR RAPD-PCR [36]. This could be Interesting

Loop-mediated isothermal amplification (LAMP) is a highly efficient, sensitive, specific and cost-effective isothermal amplification method that uses at least four primers, recognizing six different regions in the target sequence (**Figure 4**) and

In LAMP, the target sequence is amplified at a constant temperature of 60–65°C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. The amount of DNA pro-

To increase the reaction sensitivity, the ITS region was used as target, since it is a multicopy sequence and also because it is considered an important barcoding sequence for fungal identification, being conserved among *H. capsulatum* strains

Forward inner primer (FIP) and backward inner primer (BIP) have inverted sequences attached at the 5′ end, named F1c and B1c, which are complementary to an internal sequence from the amplified strand, forming a loop at each extremity of

The outer primers (F3 and B3) anneal upstream to the FIP and BIP, acting as a binding site for DNA polymerase, which, in the LAMP reaction, also contains the

LAMP results can be observed using several strategies with minimal ambiguity with real-time turbidimetry (magnesium pyrophosphate formation), fluorescent compounds (Sybr Green, Eva Green, SYTO, calcein), magnesium colorimetric titration, fluorescent-labeled probes, quencher-labeled primers, dye-labeled prim-

LAMP can be used on samples of whole blood or bone marrow for patients

A new assay, the so-called ITS LAMP, showed no cross-reactivity when assayed with DNA from other pathogenic or environmental fungi. The assay is able to detect isolates from all geographical clades of *H. capsulatum*, including Hcd. In comparison with Hcp100 nPCR, it reached sensitivity of 83% and specificity of 92% [35].

suspected of progressive disseminated histoplasmosis (PDH).

duced in LAMP is considerably higher than PCR-based amplification.

and divergent from other fungi, ensuring high specificity.

tory in Spain they developed a multiplex qPCR for *H. capsulatum, Pneumocystis* 

**46**

This method remains cost-effective with or without the extraction DNA step and did not require a thermocycler or an electrophoresis apparatus. It saves time with test performed in less than 200 minutes [35]. However, this little-known and recent method is currently underused and much more reserved for resource-limited laboratories. It should evolve in the next years to come and need further evaluation to be routinely used.

#### **10. Summary of the different categories of tests and their sensitivity (%)**

The performance for each test according the clinical context is summarized in **Table 4**. It considers global sensitivity without any distinction between the different protocols (antigen preparation, methods, ...).

It highlights that Ag detection is the best diagnosis tool for PHD and SCN histoplasmosis, serology for the subacute/chronic form, and the culture although this is the reference method remains less sensitive.

#### **11. Conclusion**

Histoplasmosis remains a severe and neglected disease for which early diagnosis of invasive fungal infections is critical to allow a prompt patient care. Indeed the mortality rate among HIV/AIDS patients diagnosed with histoplasmosis is high: 42% mortality for disseminated histoplasmosis if treatment is delayed and 100% if antifungal therapy is not prescribed.


#### **Table 4.**

*The different performances (sensibilité %)of the methods are summarized in this table adapted to azar et al. (2), Caceres et.al (9) and Wheat et.al (26).*

The gold standard for histoplasma diagnosis remains culture. It's a timeconsuming process and has limitations in sensitivity. Moreover, it requires invasive procedure and mycological expertise.

Nonculture methods have been developed to improve and accelerate diagnosis of histoplasmosis, such as histoplasma antigen detection, antibody detection, and molecular biology. It should have conjunction between the different tools of diagnosis to be reliable in the histoplasmosis management regarding the wide range of clinical features.

#### **Author details**

Emilie Guemas1 , Loïc Sobanska1 and Magalie Demar1,2\*

1 Academic Laboratory of Parasitology and Mycology, Cayenne Hospital, French Guiana

2 Amazonian Ecosystem and Tropical Diseases (EA 3593, EPat), University of French Guiana, Cayenne, French Guiana

\*Address all correspondence to: magalie.demar@ch-cayenne.fr

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**49**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

Infectious Diseases mycoses study group (EORTC/MSG) consensus group. Clinical Infectious Diseases.

[8] CDC. Histoplasmosis 2017 case definition [Internet]. Available from: https://wwwn.cdc.gov/ nndss/conditions/histoplasmosis/

[9] Caceres DH, Knuth M, Derado G, Lindsley MD. Diagnosis of progressived

advanced HIV: A meta-analysis of assay analytical performance. Journal of

[10] Scheel CM, Gómez BL. Diagnostic methods for Histoplasmosis: Focus on endemic countries with variable infrastructure levels. Current Tropical Medicine Reports. 2014;**1**(2):129-137. DOI: 10.1007/s40475-014-0020-0

[11] Buitrago MJ, Martín-Gómez MT. Timely diagnosis of Histoplasmosis in non-endemic countries: A laboratory challenge. Frontiers in Microbiology.

[12] Caceres DH, Valdes A. Histoplasmosis and tuberculosis co-occurrence in people with advanced HIV. Journal of Fungi. 2019;**5**(3):73. DOI: 10.3390/

[13] Guimarães AJ, Nosanchuk JD, Zancopé-Oliveira RM. Diagnosis of histoplasmosis. Brazilian Journal of Microbiology. 2006;**37**(1):1-13. DOI: 10.1590/S1517-83822006000100001

[14] Azar MM, Hage CA. Laboratory diagnostics for histoplasmosis. Journal of Clinical Microbiology.

[15] Adenis AA, Aznar C, Couppié P. Histoplasmosis in HIV-infected patients:

2017;**55**(6):1612-1620

2020;**11**:467. DOI: 10.3389/

fmicb.2020.00467

jof5030073

disseminated histoplasmosis in

2008;**46**(12):1813-1821

case-definition/2017

Fungi. 2019;**5**(3):76

*DOI: http://dx.doi.org/10.5772/intechopen.92782*

[1] Wheat LJ, Freifeld AG, Kleiman MB, et al. Clinical practice guidelines for the management of patients with histoplasmosis: 2007 update by the Infectious Diseases Society of America. Clinical Infectious Diseases. 2007;**45**(7):807-825. DOI:

[2] Azar MM, Loyd JL, Relich RF, Wheat LJ, Hage CA. Current concepts in the epidemiology, diagnosis, and Management of Histoplasmosis Syndromes. Seminars in Respiratory and Critical Care Medicine. 2020;**41**(1): 13-30. DOI: 10.1055/s-0039-1698429

[3] Société Française de Microbiologie. Chromoblastomycose et Histoplasmose. In: Rémic. Paris: Société française de microbiologie; 2018. pp. 911-926

[4] Couppié P, Herceg K, Bourne-Watrin M, et al. The broad clinical Spectrum of disseminated Histoplasmosis in HIV-infected patients:

A 30 Years' experience in French

[5] Valero C, Gago S, Monteiro MC, Alastruey-Izquierdo A, Buitrago MJ. African histoplasmosis: New clinical and microbiological insights. Medical Mycology. 2018;**56**(1):51-59. DOI:

DOI: 10.3390/jof5040115

10.1093/mmy/myx020

[6] Oladele RO, Ayanlowo OO, Richardson MD, Denning DW.

Histoplasmosis in Africa: An emerging or a neglected disease? PLoS Neglected Tropical Diseases. 2018;**12**(1):e0006046. DOI: 10.1371/journal.pntd.0006046

[7] De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et al. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of cancer/invasive fungal infections cooperative group and the National Institute of Allergy and

Guiana. Journal of Fungi. 2019;**5**(4):115.

10.1086/521259

**References**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.92782*

### **References**

Histoplasma *and Histoplasmosis*

Ag detection

Molecular Biology

**Table 4.**

**(2) (9)**

**PHD Acute** 

**pulmonary syndrom**

**48**

**Author details**

of clinical features.

Emilie Guemas1

French Guiana

, Loïc Sobanska1

French Guiana, Cayenne, French Guiana

provided the original work is properly cited.

procedure and mycological expertise.

*(2), Caceres et.al (9) and Wheat et.al (26).*

95

and Magalie Demar1,2\*

1 Academic Laboratory of Parasitology and Mycology, Cayenne Hospital,

\*Address all correspondence to: magalie.demar@ch-cayenne.fr

2 Amazonian Ecosystem and Tropical Diseases (EA 3593, EPat), University of

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

The gold standard for histoplasma diagnosis remains culture. It's a timeconsuming process and has limitations in sensitivity. Moreover, it requires invasive

*The different performances (sensibilité %)of the methods are summarized in this table adapted to azar et al.* 

**Subacute pulmonary syndrom**

Serology 75 58 64 95 83 83 59

culture 74 77 42 54 67 67 38

Pathology 76 NE 20 42 75 75

*PDH: Progressive histoplasmosis disseminated, CNS: Central nervous system, NE: Non evaluated.*

**Chronic pulmonary syndrom**

92 95 83 30 88 5 66

**Mediastinal histoplasmosis**

**CNS histoplasmosis**

81 31

63 54

40 33

Nonculture methods have been developed to improve and accelerate diagnosis of histoplasmosis, such as histoplasma antigen detection, antibody detection, and molecular biology. It should have conjunction between the different tools of diagnosis to be reliable in the histoplasmosis management regarding the wide range [1] Wheat LJ, Freifeld AG, Kleiman MB, et al. Clinical practice guidelines for the management of patients with histoplasmosis: 2007 update by the Infectious Diseases Society of America. Clinical Infectious Diseases. 2007;**45**(7):807-825. DOI: 10.1086/521259

[2] Azar MM, Loyd JL, Relich RF, Wheat LJ, Hage CA. Current concepts in the epidemiology, diagnosis, and Management of Histoplasmosis Syndromes. Seminars in Respiratory and Critical Care Medicine. 2020;**41**(1): 13-30. DOI: 10.1055/s-0039-1698429

[3] Société Française de Microbiologie. Chromoblastomycose et Histoplasmose. In: Rémic. Paris: Société française de microbiologie; 2018. pp. 911-926

[4] Couppié P, Herceg K, Bourne-Watrin M, et al. The broad clinical Spectrum of disseminated Histoplasmosis in HIV-infected patients: A 30 Years' experience in French Guiana. Journal of Fungi. 2019;**5**(4):115. DOI: 10.3390/jof5040115

[5] Valero C, Gago S, Monteiro MC, Alastruey-Izquierdo A, Buitrago MJ. African histoplasmosis: New clinical and microbiological insights. Medical Mycology. 2018;**56**(1):51-59. DOI: 10.1093/mmy/myx020

[6] Oladele RO, Ayanlowo OO, Richardson MD, Denning DW. Histoplasmosis in Africa: An emerging or a neglected disease? PLoS Neglected Tropical Diseases. 2018;**12**(1):e0006046. DOI: 10.1371/journal.pntd.0006046

[7] De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et al. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of cancer/invasive fungal infections cooperative group and the National Institute of Allergy and

Infectious Diseases mycoses study group (EORTC/MSG) consensus group. Clinical Infectious Diseases. 2008;**46**(12):1813-1821

[8] CDC. Histoplasmosis 2017 case definition [Internet]. Available from: https://wwwn.cdc.gov/ nndss/conditions/histoplasmosis/ case-definition/2017

[9] Caceres DH, Knuth M, Derado G, Lindsley MD. Diagnosis of progressived disseminated histoplasmosis in advanced HIV: A meta-analysis of assay analytical performance. Journal of Fungi. 2019;**5**(3):76

[10] Scheel CM, Gómez BL. Diagnostic methods for Histoplasmosis: Focus on endemic countries with variable infrastructure levels. Current Tropical Medicine Reports. 2014;**1**(2):129-137. DOI: 10.1007/s40475-014-0020-0

[11] Buitrago MJ, Martín-Gómez MT. Timely diagnosis of Histoplasmosis in non-endemic countries: A laboratory challenge. Frontiers in Microbiology. 2020;**11**:467. DOI: 10.3389/ fmicb.2020.00467

[12] Caceres DH, Valdes A. Histoplasmosis and tuberculosis co-occurrence in people with advanced HIV. Journal of Fungi. 2019;**5**(3):73. DOI: 10.3390/ jof5030073

[13] Guimarães AJ, Nosanchuk JD, Zancopé-Oliveira RM. Diagnosis of histoplasmosis. Brazilian Journal of Microbiology. 2006;**37**(1):1-13. DOI: 10.1590/S1517-83822006000100001

[14] Azar MM, Hage CA. Laboratory diagnostics for histoplasmosis. Journal of Clinical Microbiology. 2017;**55**(6):1612-1620

[15] Adenis AA, Aznar C, Couppié P. Histoplasmosis in HIV-infected patients: A review of new developments and remaining gaps. Current Tropical Medicine Reports. 2014;**1**(2):119-128

[16] Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st century. Clinical Microbiology Reviews. 2011;**24**(2): 247-280. DOI: 10.1128/CMR.00053-10

[17] Kauffman CA. Histoplasmosis: A clinical and laboratory update. Clinical Microbiology Reviews. 2007;**20**(1):115-132

[18] Guimarães AJ, de Cerqueira MD, Nosanchuk JD. Surface architecture of *Histoplasma capsulatum*. Frontiers in Microbiology. 18 November 2011;**2**:225. DOI: 10.3389/fmicb.2011.00225

[19] Valero C, Buitrago MJ, Gago S, Quiles-Melero I, García-Rodríguez J. A matrix-assisted laser desorption/ ionization time of flight mass spectrometry reference database for the identification of *Histoplasma capsulatum*. Medical Mycology. 2018;**56**(3):307-314. DOI: 10.1093/mmy/myx047

[20] Rychert J, Slechta ES, Barker AP, et al. Multicenter evaluation of the Vitek MS v3.0 system for the identification of filamentous fungi. Journal of Clinical Microbiology. 24 January 2018;**56**(2):e01353-17. DOI: 10.1128/ JCM.01353-17

[21] Vetter E, Torgerson C, Feuker A, et al. Comparison of the BACTEC MYCO/F lytic bottle to the isolator tube, BACTEC plus aerobic F/bottle, and BACTEC anaerobic lytic/10 bottle and comparison of the BACTEC plus aerobic F/bottle to the isolator tube for recovery of bacteria, mycobacteria, and fungi from blood. Journal of Clinical Microbiology. 2001;**39**(12):4380-4386. DOI: 10.1128/JCM.39.12.4380-4386.2001

[22] de Almeida MA, Pizzini CV, Damasceno LS, de Muniz M, Almeida-Paes R, RHS P, et al. Validation of

western blot for *Histoplasma capsulatum* antibody detection assay. BMC Infectious Diseases. 2016;**16**(1):87

[23] Rubio-Carrasquilla M, Santa CD, Rendón JP, et al. An interferon gamma release assay specific for Histoplasma capsulatum to detect asymptomatic infected individuals: A proof of concept study. Medical Mycology. 2019;**57**(6):724-732. DOI: 10.1093/mmy/ myy131

[24] Wheat J, French ML, Kamel S, Tewari RP. Evaluation of cross-reactions in Histoplasma capsulatum serologic tests. Journal of Clinical Microbiology. 1986;**23**(3):493-499

[25] Negroni R, De Flores CI, Robles AM. Study of serologic cross reactions between the antigens of Paracoccidioides brasiliensis and Histoplasma capsulatum. Revista de la Asociación Argentina de Microbiología. 1976;**8**(2):68-73

[26] Wheat J, Myint T, Guo Y, et al. Central nervous system histoplasmosis: Multicenter retrospective study on clinical features, diagnostic approach and outcome of treatment. Medicine (Baltimore). 2018;**97**(13):e0245. DOI: 10.1097/MD.0000000000010245

[27] Zhang X, Gibson B, Daly TM. Evaluation of commercially available reagents for diagnosis of histoplasmosis infection in immunocompromised patients. Journal of Clinical Microbiology. 2013;**51**(12):4095-4101

[28] Cunningham L, Cook A, Hanzlicek A, Harkin K, Wheat J, Goad C, et al. Sensitivity and specificity of Histoplasma antigen detection by enzyme immunoassay. Journal of the American Animal Hospital Association. 2015;**51**(5):306-310

[29] Rothenburg L, Hanzlicek AS, Payton ME. A monoclonal antibodybased urine Histoplasma antigen

**51**

Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis*

Reyes-Montes MR, Taylor ML. Analyses

population structures of Histoplasma capsulatum clinical isolates from Mexico, Guatemala, Colombia and Argentina, using a randomly amplified polymorphic DNA-PCR assay. Epidemiology and Infection. 2019;**147**:e204. DOI: 10.1017/

of the genetic diversity and

S0950268819000931

[37] da Zatti MS, Arantes TD, Fernandes JAL, Bay MB, Milan EP, Naliato GFS, et al. Loop-mediated isothermal amplification and nested PCR of the internal transcribed spacer (ITS) for Histoplasma capsulatum detection. PLoS Neglected Tropical Diseases. 2019;**13**(8):e0007692

[38] Alhassan A, Li Z, Poole CB, Carlow CKS. Expanding the MDx toolbox for filarial diagnosis and surveillance. Trends in Parasitology.

2015;**31**(8):391-400

*DOI: http://dx.doi.org/10.5772/intechopen.92782*

[30] Cáceres DH, Gómez BL, Tobón AM, Chiller TM, Lindsley MD. Evaluation of a Histoplasma antigen lateral flow assay for the rapid diagnosis of progressive disseminated histoplasmosis

enzyme immunoassay (IMMY®) for the diagnosis of histoplasmosis in cats. Journal of Veterinary Internal Medicine.

2019;**33**(2):603-610

in Colombian patients with

AIDS. Mycoses. 2020;**63**(2):139-144

[31] Iriart X, Blanchet D, Menard S, Lavergne R-A, Chauvin P, Adenis A, et al. A complementary tool for management of disseminated Histoplasma capsulatum var.

capsulatum infections in AIDS patients.

[32] Myint T, Anderson AM, Sanchez A, Farabi A, Hage C, Baddley JW, et al. Histoplasmosis in patients with human immunodeficiency virus/ acquired immunodeficiency syndrome (HIV/AIDS): Multicenter study of outcomes and factors associated with relapse. Medicine (Baltimore).

International Journal of Medical Microbiology. 2014;**304**(8):1062-1065

[33] Wheat LJ. Improvements in diagnosis of histoplasmosis. Expert Opinion on Biological Therapy.

[34] Vasconcellos ICDS, Dalla Lana DF, Pasqualotto AC. The role of molecular tests in the diagnosis of disseminated Histoplasmosis. Journal of Fungi. 2019;**6**(1):1. DOI: 10.3390/jof6010001

2014;**93**(1):11-18

2006;**6**(11):1207-1221

[35] Gómez LF, Arango M, McEwen JG, et al. Molecular epidemiology of Colombian Histoplasma capsulatum isolates obtained from human and

heliyon.2019.e02084

chicken manure samples. Heliyon. 2019;**5**(7):e02084. DOI: 10.1016/j.

[36] Sahaza JH, Duarte-Escalante E, Canteros C, Rodríguez-Arellanes G, Histoplasma capsulatum *and* Histoplasmosis: *Current Concept for the Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.92782*

enzyme immunoassay (IMMY®) for the diagnosis of histoplasmosis in cats. Journal of Veterinary Internal Medicine. 2019;**33**(2):603-610

Histoplasma *and Histoplasmosis*

[16] Guarner J, Brandt ME.

2007;**20**(1):115-132

A review of new developments and remaining gaps. Current Tropical Medicine Reports. 2014;**1**(2):119-128 western blot for *Histoplasma capsulatum*

[23] Rubio-Carrasquilla M, Santa CD, Rendón JP, et al. An interferon gamma release assay specific for Histoplasma capsulatum to detect asymptomatic infected individuals: A proof of concept study. Medical Mycology. 2019;**57**(6):724-732. DOI: 10.1093/mmy/

[24] Wheat J, French ML, Kamel S, Tewari RP. Evaluation of cross-reactions in Histoplasma capsulatum serologic tests. Journal of Clinical Microbiology.

antibody detection assay. BMC Infectious Diseases. 2016;**16**(1):87

myy131

1986;**23**(3):493-499

1976;**8**(2):68-73

[25] Negroni R, De Flores CI, Robles AM. Study of serologic cross reactions between the antigens of Paracoccidioides brasiliensis and Histoplasma capsulatum. Revista de la Asociación Argentina de Microbiología.

[26] Wheat J, Myint T, Guo Y, et al. Central nervous system histoplasmosis: Multicenter retrospective study on clinical features, diagnostic approach and outcome of treatment. Medicine (Baltimore). 2018;**97**(13):e0245. DOI: 10.1097/MD.0000000000010245

[27] Zhang X, Gibson B, Daly TM. Evaluation of commercially available reagents for diagnosis of histoplasmosis infection in immunocompromised patients. Journal of Clinical

Microbiology. 2013;**51**(12):4095-4101

Goad C, et al. Sensitivity and specificity of Histoplasma antigen detection by enzyme immunoassay. Journal of the American Animal Hospital Association.

[28] Cunningham L, Cook A, Hanzlicek A, Harkin K, Wheat J,

[29] Rothenburg L, Hanzlicek AS, Payton ME. A monoclonal antibodybased urine Histoplasma antigen

2015;**51**(5):306-310

Histopathologic diagnosis of fungal infections in the 21st century. Clinical Microbiology Reviews. 2011;**24**(2): 247-280. DOI: 10.1128/CMR.00053-10

[17] Kauffman CA. Histoplasmosis: A clinical and laboratory update. Clinical Microbiology Reviews.

[18] Guimarães AJ, de Cerqueira MD, Nosanchuk JD. Surface architecture of *Histoplasma capsulatum*. Frontiers in Microbiology. 18 November 2011;**2**:225.

DOI: 10.3389/fmicb.2011.00225

DOI: 10.1093/mmy/myx047

JCM.01353-17

[19] Valero C, Buitrago MJ, Gago S, Quiles-Melero I, García-Rodríguez J. A matrix-assisted laser desorption/ ionization time of flight mass

spectrometry reference database for the identification of *Histoplasma capsulatum*. Medical Mycology. 2018;**56**(3):307-314.

[20] Rychert J, Slechta ES, Barker AP, et al. Multicenter evaluation of the Vitek MS v3.0 system for the identification of filamentous fungi. Journal of Clinical Microbiology. 24 January 2018;**56**(2):e01353-17. DOI: 10.1128/

[21] Vetter E, Torgerson C, Feuker A, et al. Comparison of the BACTEC MYCO/F lytic bottle to the isolator tube, BACTEC plus aerobic F/bottle, and BACTEC anaerobic lytic/10 bottle and comparison of the BACTEC plus aerobic F/bottle to the isolator tube for recovery of bacteria, mycobacteria, and fungi from blood. Journal of Clinical Microbiology. 2001;**39**(12):4380-4386. DOI: 10.1128/JCM.39.12.4380-4386.2001

[22] de Almeida MA, Pizzini CV, Damasceno LS, de Muniz M, Almeida-Paes R, RHS P, et al. Validation of

**50**

[30] Cáceres DH, Gómez BL, Tobón AM, Chiller TM, Lindsley MD. Evaluation of a Histoplasma antigen lateral flow assay for the rapid diagnosis of progressive disseminated histoplasmosis in Colombian patients with AIDS. Mycoses. 2020;**63**(2):139-144

[31] Iriart X, Blanchet D, Menard S, Lavergne R-A, Chauvin P, Adenis A, et al. A complementary tool for management of disseminated Histoplasma capsulatum var. capsulatum infections in AIDS patients. International Journal of Medical Microbiology. 2014;**304**(8):1062-1065

[32] Myint T, Anderson AM, Sanchez A, Farabi A, Hage C, Baddley JW, et al. Histoplasmosis in patients with human immunodeficiency virus/ acquired immunodeficiency syndrome (HIV/AIDS): Multicenter study of outcomes and factors associated with relapse. Medicine (Baltimore). 2014;**93**(1):11-18

[33] Wheat LJ. Improvements in diagnosis of histoplasmosis. Expert Opinion on Biological Therapy. 2006;**6**(11):1207-1221

[34] Vasconcellos ICDS, Dalla Lana DF, Pasqualotto AC. The role of molecular tests in the diagnosis of disseminated Histoplasmosis. Journal of Fungi. 2019;**6**(1):1. DOI: 10.3390/jof6010001

[35] Gómez LF, Arango M, McEwen JG, et al. Molecular epidemiology of Colombian Histoplasma capsulatum isolates obtained from human and chicken manure samples. Heliyon. 2019;**5**(7):e02084. DOI: 10.1016/j. heliyon.2019.e02084

[36] Sahaza JH, Duarte-Escalante E, Canteros C, Rodríguez-Arellanes G, Reyes-Montes MR, Taylor ML. Analyses of the genetic diversity and population structures of Histoplasma capsulatum clinical isolates from Mexico, Guatemala, Colombia and Argentina, using a randomly amplified polymorphic DNA-PCR assay. Epidemiology and Infection. 2019;**147**:e204. DOI: 10.1017/ S0950268819000931

[37] da Zatti MS, Arantes TD, Fernandes JAL, Bay MB, Milan EP, Naliato GFS, et al. Loop-mediated isothermal amplification and nested PCR of the internal transcribed spacer (ITS) for Histoplasma capsulatum detection. PLoS Neglected Tropical Diseases. 2019;**13**(8):e0007692

[38] Alhassan A, Li Z, Poole CB, Carlow CKS. Expanding the MDx toolbox for filarial diagnosis and surveillance. Trends in Parasitology. 2015;**31**(8):391-400

**53**

**Chapter 4**

**Abstract**

tives are discussed.

**1. Introduction**

antibody

Laboratory Diagnosis of

*María J. Buitrago and Clara Valero*

Histoplasmosis: An Update

Early diagnosis of histoplasmosis is essential to establish a suitable antifungal therapy and reduce morbidity and mortality rates. However, laboratory diagnosis remains challenging due to the low availability of proper methods and the lack of clinical suspicion. Conventional diagnosis is still largely used even though limitations are well known. Isolating the fungus is time consuming and requires manipulation in BSL3 facilities, while direct visualization and histopathology techniques show low sensitivity and need skilled personnel. New approaches based on the detection of antibodies and antigens have been developed and commercialized last years. Although sensitivity and specificity of these methods is variable, antigen detection has been recently listed as an essential diagnostic test for AIDS patients due to its excellent performance. DNA detection methods are recognized as promising tools but there is still a lack of consensus among laboratories and there are not commercial tests available. Not all methods are widely available, thus most laboratories combine classical and other tests in order to overcome aforementioned limitations. In this chapter, we review the diagnostic pipeline currently available for the diagnosis of histoplasmosis in microbiological laboratories, from conventional to new developed tests. Most recent approaches are introduced and future perspec-

**Keywords:** histoplasmosis, diagnosis, culture, histopathology, PCR, antigen,

Histoplasmosis is caused by the thermally dimorphic fungi *Histoplasma capsulatum* and encompasses a broad variety of clinical presentations ranging from asymptomatic infections to subacute, acute and chronic pulmonary infections [1]. In immunosuppressed patients, especially in HIV positive population, it causes a progressive disseminated disease [2]. Mortality rates in AIDS population have been reported as 30–50% when treated, and 100% in absence of treatment [3]. Although this fungus has a cosmopolitan distribution, there are some areas of high endemicity as Ohio and Mississippi basins in North America, several countries from Central and South America and different regions in the African continent [4–6]. Areas of medium-high endemicity have been reported in China and some sporadic cases have been described in other regions of Asia as North of India, Thailand and Philippines [7–10]. The rest of the world is considered as non-endemic regions and reported cases are described as imported by travelers or

#### **Chapter 4**

## Laboratory Diagnosis of Histoplasmosis: An Update

*María J. Buitrago and Clara Valero*

#### **Abstract**

Early diagnosis of histoplasmosis is essential to establish a suitable antifungal therapy and reduce morbidity and mortality rates. However, laboratory diagnosis remains challenging due to the low availability of proper methods and the lack of clinical suspicion. Conventional diagnosis is still largely used even though limitations are well known. Isolating the fungus is time consuming and requires manipulation in BSL3 facilities, while direct visualization and histopathology techniques show low sensitivity and need skilled personnel. New approaches based on the detection of antibodies and antigens have been developed and commercialized last years. Although sensitivity and specificity of these methods is variable, antigen detection has been recently listed as an essential diagnostic test for AIDS patients due to its excellent performance. DNA detection methods are recognized as promising tools but there is still a lack of consensus among laboratories and there are not commercial tests available. Not all methods are widely available, thus most laboratories combine classical and other tests in order to overcome aforementioned limitations. In this chapter, we review the diagnostic pipeline currently available for the diagnosis of histoplasmosis in microbiological laboratories, from conventional to new developed tests. Most recent approaches are introduced and future perspectives are discussed.

**Keywords:** histoplasmosis, diagnosis, culture, histopathology, PCR, antigen, antibody

#### **1. Introduction**

Histoplasmosis is caused by the thermally dimorphic fungi *Histoplasma capsulatum* and encompasses a broad variety of clinical presentations ranging from asymptomatic infections to subacute, acute and chronic pulmonary infections [1]. In immunosuppressed patients, especially in HIV positive population, it causes a progressive disseminated disease [2]. Mortality rates in AIDS population have been reported as 30–50% when treated, and 100% in absence of treatment [3]. Although this fungus has a cosmopolitan distribution, there are some areas of high endemicity as Ohio and Mississippi basins in North America, several countries from Central and South America and different regions in the African continent [4–6]. Areas of medium-high endemicity have been reported in China and some sporadic cases have been described in other regions of Asia as North of India, Thailand and Philippines [7–10]. The rest of the world is considered as non-endemic regions and reported cases are described as imported by travelers or immigrants [11]. It has been estimated that about a half million of people acquire the infection per year and around 100,000 develop a disseminated disease [3]. Certain regions of Brazil and Venezuela have been considered hotspots of the disseminated form of the disease [12]. However, in other regions as the African continent the incidence is probably underestimated due to lack of studies [6]. Diagnosis of histoplasmosis is challenging in both endemic and non-endemic regions due to its similarity to other diseases as tuberculosis, usually the first clinical suspicion, and the limitations and low availability of diagnostic tools. Consequently, many efforts have been recently made with the aim of improving diagnosis and combat histoplasmosis [3]. In this chapter, we describe the conventional methods used to diagnose histoplasmosis coupled with an update about the new tests available. The advantages and limitations of each method are detailed as well as their usefulness in the diagnosis regarding the diverse clinical presentations of the disease and their availability in the different regions. The need of continuous work on the development of new methods to achieve an early diagnosis and reduce histoplasmosis morbidity and mortality is also underlined.

#### **2. Conventional diagnostic methods**

Conventional diagnostic methods are still widely used for the diagnosis of histoplasmosis. The definite diagnosis is based on the isolation of the fungus in culture or the visualization of intracellular yeasts in tissues or other clinical samples. Thus, despite its known limitations, these methods are very useful and continue to be used in many laboratories, especially in low-resources settings.

#### **2.1 Culture**

The isolation of *H. capsulatum* in culture is the gold standard for the diagnosis of histoplasmosis. The recovery of the fungus by culture from clinical samples (blood culture, bronchoalveolar lavage fluid, etc.) is recognized as the criteria for the definition of proven histoplasmosis according to the consensus definitions of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC-MSGERC) [13].

*H. capsulatum* is a so-called fastidious fungus since it can take up to 4 weeks to grow and requires Biosafety level 3 (BSL-3) containment measures [2]. At 25–30°C, it grows as a white and cottony mycelium that evolves to brown, representing the invasive form of the disease. This mycelial form is the most dangerous, as spores could be inhaled causing infection. Some laboratory-acquired infections have been reported by culture manipulation [14]. Microscopically the mycelial form produces the typical tuberculate macroconidia, which are easily identified (**Figure 1A**). However, other species as *Sepedonium* spp., an environmental organism that is usually considered as a laboratory contaminant, produce similar structures. At 37°C (human body temperature), there is a conversion from the mycelial phase to the yeast form, which is the pathogenic form of the disease. These yeasts are easily recognized by their thick wall and ovoid form with a narrow base at the smaller end (**Figure 1B**).

The sensitivity of cultures depends on the clinical status of the patient and the origin of the sample. Cultures are negative in most cases of asymptomatic and mild disease, however they are useful in disseminated and chronic pulmonary histoplasmosis, although their sensitivity varies from 50 to 85% [2, 15].

**55**

culture manipulation [19].

**Figure 1.**

*Carlos III, Madrid, Spain).*

*Laboratory Diagnosis of Histoplasmosis: An Update DOI: http://dx.doi.org/10.5772/intechopen.93305*

The main limitation of cultures is the long turnaround time needed to reach

Since aforementioned limitations make culture useless for an early diagnosis of histoplasmosis, a great effort has been made for developing alternative diagnostic

Direct examination of clinical samples or performing histopathological studies on tissues to detect yeast after staining with Gomori methenamine silver (GMS) or periodic acid-Schiff (PAS), are techniques largely used for the diagnosis of histoplasmosis. *H. capsulatum* could appear as a 2- to 4-μm (var. *capsulatum*) or 8- to 15-μm (var. *duboisii*) oval yeast. As yeasts are phagocytosed by macrophages, they could be found forming clusters, which can help the diagnosis. However, several fungi can be misled with *H. capsulatum* such as, among others, the small variant of *Blastomyces dermatitidis*, endospores of *Coccidioides* spp., *Candida glabrata* as well as the causal agents of leishmaniasis, toxoplasmosis, and Chagas' disease [20]. The higher sensitivity could be obtained in respiratory specimens or bone marrow biopsy in patients with disseminated histoplasmosis [21]. However, the low specificity and the need of skilled personnel to achieve a presumptive diagnosis are the

methods that could be used alone or in combination with cultures.

**2.2 Histopathology and direct visualization**

main limitations of this technique.

a definite diagnosis. Moreover, the microscopical observation of the grown fungus requires further confirmation by other methods. Molecular identification by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA is a powerful tool to confirm the presence of *H. capsulatum*, but extends the time response for the diagnosis even more. Alternatively, single-stranded DNA probes could also be used to detect the fungus in cultures (AccuProbe, Hologic, CA, USA), however the procedure is technically complex and time consuming [16]. In last years, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF MS) has started to be developed for the identification of *H. capsulatum* strains [17–19]. Although reports are still scarce, results in this area are promising since the system is able for recognizing different varieties (var. *capsulatum*, var. *duboisii*) and forms of the pathogen (yeast and mold), thus reducing response time and decreasing the risk of

*Photomicrograph of lactophenol cotton blue stains of both filamentous and yeast forms of* H. capsulatum*. (A) Culture at 30°C on potato dextrose agar (PDA) showing tuberculate macroconidia (63× magnification). (B) Culture at 37°C on ML-GEMA medium showing yeasts (20× magnification). Both images belong to the image library of the mycology reference laboratory (National Centre of Microbiology, Instituto de Salud* 

#### **Figure 1.**

Histoplasma *and Histoplasmosis*

morbidity and mortality is also underlined.

**2. Conventional diagnostic methods**

**2.1 Culture**

MSGERC) [13].

base at the smaller end (**Figure 1B**).

immigrants [11]. It has been estimated that about a half million of people acquire the infection per year and around 100,000 develop a disseminated disease [3]. Certain regions of Brazil and Venezuela have been considered hotspots of the disseminated form of the disease [12]. However, in other regions as the African continent the incidence is probably underestimated due to lack of studies [6]. Diagnosis of histoplasmosis is challenging in both endemic and non-endemic regions due to its similarity to other diseases as tuberculosis, usually the first clinical suspicion, and the limitations and low availability of diagnostic tools. Consequently, many efforts have been recently made with the aim of improving diagnosis and combat histoplasmosis [3]. In this chapter, we describe the conventional methods used to diagnose histoplasmosis coupled with an update about the new tests available. The advantages and limitations of each method are detailed as well as their usefulness in the diagnosis regarding the diverse clinical presentations of the disease and their availability in the different regions. The need of continuous work on the development of new methods to achieve an early diagnosis and reduce histoplasmosis

Conventional diagnostic methods are still widely used for the diagnosis of histoplasmosis. The definite diagnosis is based on the isolation of the fungus in culture or the visualization of intracellular yeasts in tissues or other clinical samples. Thus, despite its known limitations, these methods are very useful and continue to be

The isolation of *H. capsulatum* in culture is the gold standard for the diagnosis of histoplasmosis. The recovery of the fungus by culture from clinical samples (blood culture, bronchoalveolar lavage fluid, etc.) is recognized as the criteria for the definition of proven histoplasmosis according to the consensus definitions of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC-

*H. capsulatum* is a so-called fastidious fungus since it can take up to 4 weeks to grow and requires Biosafety level 3 (BSL-3) containment measures [2]. At 25–30°C, it grows as a white and cottony mycelium that evolves to brown, representing the invasive form of the disease. This mycelial form is the most dangerous, as spores could be inhaled causing infection. Some laboratory-acquired infections have been reported by culture manipulation [14]. Microscopically the mycelial form produces the typical tuberculate macroconidia, which are easily identified (**Figure 1A**). However, other species as *Sepedonium* spp., an environmental organism that is usually considered as a laboratory contaminant, produce similar structures. At 37°C (human body temperature), there is a conversion from the mycelial phase to the yeast form, which is the pathogenic form of the disease. These yeasts are easily recognized by their thick wall and ovoid form with a narrow

The sensitivity of cultures depends on the clinical status of the patient and the origin of the sample. Cultures are negative in most cases of asymptomatic and mild disease, however they are useful in disseminated and chronic pulmonary histoplas-

mosis, although their sensitivity varies from 50 to 85% [2, 15].

used in many laboratories, especially in low-resources settings.

**54**

*Photomicrograph of lactophenol cotton blue stains of both filamentous and yeast forms of* H. capsulatum*. (A) Culture at 30°C on potato dextrose agar (PDA) showing tuberculate macroconidia (63× magnification). (B) Culture at 37°C on ML-GEMA medium showing yeasts (20× magnification). Both images belong to the image library of the mycology reference laboratory (National Centre of Microbiology, Instituto de Salud Carlos III, Madrid, Spain).*

The main limitation of cultures is the long turnaround time needed to reach a definite diagnosis. Moreover, the microscopical observation of the grown fungus requires further confirmation by other methods. Molecular identification by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA is a powerful tool to confirm the presence of *H. capsulatum*, but extends the time response for the diagnosis even more. Alternatively, single-stranded DNA probes could also be used to detect the fungus in cultures (AccuProbe, Hologic, CA, USA), however the procedure is technically complex and time consuming [16]. In last years, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF MS) has started to be developed for the identification of *H. capsulatum* strains [17–19]. Although reports are still scarce, results in this area are promising since the system is able for recognizing different varieties (var. *capsulatum*, var. *duboisii*) and forms of the pathogen (yeast and mold), thus reducing response time and decreasing the risk of culture manipulation [19].

Since aforementioned limitations make culture useless for an early diagnosis of histoplasmosis, a great effort has been made for developing alternative diagnostic methods that could be used alone or in combination with cultures.

#### **2.2 Histopathology and direct visualization**

Direct examination of clinical samples or performing histopathological studies on tissues to detect yeast after staining with Gomori methenamine silver (GMS) or periodic acid-Schiff (PAS), are techniques largely used for the diagnosis of histoplasmosis. *H. capsulatum* could appear as a 2- to 4-μm (var. *capsulatum*) or 8- to 15-μm (var. *duboisii*) oval yeast. As yeasts are phagocytosed by macrophages, they could be found forming clusters, which can help the diagnosis. However, several fungi can be misled with *H. capsulatum* such as, among others, the small variant of *Blastomyces dermatitidis*, endospores of *Coccidioides* spp., *Candida glabrata* as well as the causal agents of leishmaniasis, toxoplasmosis, and Chagas' disease [20]. The higher sensitivity could be obtained in respiratory specimens or bone marrow biopsy in patients with disseminated histoplasmosis [21]. However, the low specificity and the need of skilled personnel to achieve a presumptive diagnosis are the main limitations of this technique.

#### **3. Antigen detection**

The detection of *H. capsulatum* antigens in clinical samples represented a breakthrough in the early diagnosis of histoplasmosis and has been included in the EORTC/MSG criteria for diagnosis of IFIs for almost 20 years [13, 22, 23]. The *H. capsulatum* polysaccharide antigen can be detected in both serum and urine samples with similar diagnostic value and has been recently listed as an essential diagnostic test for advanced AIDS patients [24, 25]. Two assays based on enzyme linked immunosorbent assay (ELISA) have been commercialized with good diagnostic performances (**Table 1**) although their availability is limited.

MiraVista's test presents great efficiency in serum and urine samples from patients with disseminated infection, but it is reduced in patients with pulmonary forms of the disease [26], in which BALF samples are more suitable for diagnosis [28]. This test is only performed in MVista's facilities then is not accessible out of USA limiting their use. On the other hand, IMMY has recently released an *Histoplasma* GM ELISA test which shows great performance and reproducibility, but has only be tested in urine samples [27]. Despite the high degree of crossreaction with other fungi, antigen detection assays are widely used to diagnose histoplasmosis and some reports indicate its capacity to monitor treatment response [29, 30]. Nevertheless, their use is restricted mainly to developed endemic areas but it is rarely used in non-endemic regions, probably due to its low cost-effectiveness out of endemic regions [24, 31].

In last years, point-of-care (POC) testing has been emerged as a new diagnostic methodology and immuno-chromatographic assays performed in lateral-flow devices (LFD) are good examples. These "pregnancy tests-like" assays are easy to use, have low turnaround time (less than an hour) and require minimal laboratory equipment which facilitate its implementation in low- and middle-income countries [32, 33]. This technology was first developed for *Aspergillus* GM detection with great results and MVista has recently released a similar test consisting of a dipstick sandwich immunochromatographic assay based on the recognition of *H. capsulatum* GM antigen. Although it has only been tested in serum samples from AIDS patients, its diagnostic performance was very promising (sensitivity of 96%, specificity


*a MV:* Histoplasma *Quantitative EIA test (MiraVista Diagnostics, Indianapolis, IN, USA); IMMY: CLARUS*  Histoplasma *GM EIA kit (IMMY, Norman, OK, USA).*

*b DH [26].*

*c In 95-98% [27].*

*ELISA: enzyme-linked immunosorbent assay; CSF: cerebrospinal fluid; BALF: bronchoalveolar lavage fluid; DH: disseminated histoplasmosis; APH: acute pulmonary histoplasmosis; SAPH: sub-acute pulmonary histoplasmosis; CPH: chronic pulmonary histoplamosis; GM: galactomannan.*

#### **Table 1.**

*Main characteristics and diagnostic performance of commercial assays for detecting* H. capsulatum *antigens.*

**57**

*Laboratory Diagnosis of Histoplasmosis: An Update DOI: http://dx.doi.org/10.5772/intechopen.93305*

when combined with other diagnostic methods [41, 42].

losis or candidiasis but not for endemic mycoses [13].

**5. DNA based detection methods**

**4. Antibody detection**

of 90%) [34]. However, false positive and negative results still occurred due to cross-reactivity with other endemic fungi and antibiotic treatment, respectively, and a pre-treatment step was required extending turnaround times. Currently, a new LFD test is being developed and validated by using urine samples [35].

The main advantages of antibody detection tests are the requirement of minimally invasive samples and the achievement of results when culture is still negative reducing the need of handling potentially infectious fungi [36]. Serological techniques such as complement fixation or immunodiffusion are useful when testing samples from travelers coming from endemic regions for the first time. Both techniques are commercially available (Immuno Mycologics, Norman, OK, USA) and the sensitivity of these tests in acute and subacute pulmonary histoplasmosis has been reported as 95% [37]. However, sensitivity is very limited in immunosuppressed patients due to low or absent antibody titers. A recent meta-analysis described sensitivity and specificity values of 58% and 100%, respectively, in samples from HIV patients [38]. Finally, since seropositivity remains long time after disease, interpretation of serological results could be challenging [39, 40]. Despite the limitations previously described, antibody detection tests are still considered as valuable diagnostic tools and have been demonstrated to improve diagnostic yield

PCR methods based on the detection of fungal DNA directly from clinical samples are currently implemented in the routine of several laboratories for the diagnosis of main fungal infections, but there are considerably fewer PCR tests for the diagnosis of histoplasmosis. Their advantages rely on their simplicity, high specificity and short turnaround time with the bonus that real-time PCR (qPCR) formats allow for determining the fungal burden in patients by using non-specific DNA-binding dyes or fluorescently labeled probes [36, 43]. However, this technique also has some limitations as the moderate amount of DNA in low invasive samples, the lack of standardization and the low availability of widely validated commercial systems [44, 45]. Recently, PCR based methods have been included in the EORTC/ MSG criteria for the diagnosis of some fungal infections such as invasive aspergil-

The majority of PCR tests for the diagnosis of histoplasmosis have been developed in house and none of them has been commercialized. They have been recently reviewed in several reports with different purposes [31, 38, 46]. Most developed methods targeted specific multicopy regions of the ribosomal DNA or the single-copy Hcp100 gene and were performed by using conventional, nested or qPCR formats. The sensitivity and specificity of these assays depends on the type of sample analyzed, the clinical characteristics of patients and the PCR format used for DNA detection. These tests showed an excellent analytical performance (overall sensitivity of 95% and specificity of 99%) when testing samples of HIV patients [38], but sensitivity decreased when testing blood and serum samples from immunocompetent patients [31, 46]. Panfungal or broad-range PCRs are used when there is not a clear suspicion of the fungus involved in the infection, since universal primers are used to detect any fungal DNA in the clinical sample. Although studies are scarce, several reports achieved the detection of

of 90%) [34]. However, false positive and negative results still occurred due to cross-reactivity with other endemic fungi and antibiotic treatment, respectively, and a pre-treatment step was required extending turnaround times. Currently, a new LFD test is being developed and validated by using urine samples [35].

### **4. Antibody detection**

Histoplasma *and Histoplasmosis*

out of endemic regions [24, 31].

MV Serum,

plasma, urine, CSF, BALF, other body fluids

**Testa Samples Methodology Turnaround** 

IMMY Urine ELISA <2:15H <sup>c</sup>

Histoplasma *GM EIA kit (IMMY, Norman, OK, USA).*

*CPH: chronic pulmonary histoplamosis; GM: galactomannan.*

The detection of *H. capsulatum* antigens in clinical samples represented a breakthrough in the early diagnosis of histoplasmosis and has been included in the EORTC/MSG criteria for diagnosis of IFIs for almost 20 years [13, 22, 23]. The *H. capsulatum* polysaccharide antigen can be detected in both serum and urine samples with similar diagnostic value and has been recently listed as an essential diagnostic test for advanced AIDS patients [24, 25]. Two assays based on enzyme linked immunosorbent assay (ELISA) have been commercialized with good diag-

MiraVista's test presents great efficiency in serum and urine samples from patients with disseminated infection, but it is reduced in patients with pulmonary forms of the disease [26], in which BALF samples are more suitable for diagnosis [28]. This test is only performed in MVista's facilities then is not accessible out of USA limiting their use. On the other hand, IMMY has recently released an *Histoplasma* GM ELISA test which shows great performance and reproducibility, but has only be tested in urine samples [27]. Despite the high degree of crossreaction with other fungi, antigen detection assays are widely used to diagnose histoplasmosis and some reports indicate its capacity to monitor treatment response [29, 30]. Nevertheless, their use is restricted mainly to developed endemic areas but it is rarely used in non-endemic regions, probably due to its low cost-effectiveness

In last years, point-of-care (POC) testing has been emerged as a new diagnostic

methodology and immuno-chromatographic assays performed in lateral-flow devices (LFD) are good examples. These "pregnancy tests-like" assays are easy to use, have low turnaround time (less than an hour) and require minimal laboratory equipment which facilitate its implementation in low- and middle-income countries [32, 33]. This technology was first developed for *Aspergillus* GM detection with great results and MVista has recently released a similar test consisting of a dipstick sandwich immunochromatographic assay based on the recognition of *H. capsulatum* GM antigen. Although it has only been tested in serum samples from AIDS patients, its diagnostic performance was very promising (sensitivity of 96%, specificity

**time**

<24 h Serum, plasma, CSF: 24 h

*MV:* Histoplasma *Quantitative EIA test (MiraVista Diagnostics, Indianapolis, IN, USA); IMMY: CLARUS* 

*ELISA: enzyme-linked immunosorbent assay; CSF: cerebrospinal fluid; BALF: bronchoalveolar lavage fluid; DH: disseminated histoplasmosis; APH: acute pulmonary histoplasmosis; SAPH: sub-acute pulmonary histoplasmosis;* 

*Main characteristics and diagnostic performance of commercial assays for detecting* H. capsulatum *antigens.*

ELISA Urine, BAL:

**Sensitivity/ specificity**

b DH: 92% APH: 83% SAPH: 30% CPH: 88%

**Limitations**

95-98%/97-98% • Cross-reactivity

• Cross-reactivity with other fungi • Samples are required to be shipped to the company's facilities

with other fungi

nostic performances (**Table 1**) although their availability is limited.

**3. Antigen detection**

**56**

**Table 1.**

*a*

*b DH [26]. c*

*In 95-98% [27].*

The main advantages of antibody detection tests are the requirement of minimally invasive samples and the achievement of results when culture is still negative reducing the need of handling potentially infectious fungi [36]. Serological techniques such as complement fixation or immunodiffusion are useful when testing samples from travelers coming from endemic regions for the first time. Both techniques are commercially available (Immuno Mycologics, Norman, OK, USA) and the sensitivity of these tests in acute and subacute pulmonary histoplasmosis has been reported as 95% [37]. However, sensitivity is very limited in immunosuppressed patients due to low or absent antibody titers. A recent meta-analysis described sensitivity and specificity values of 58% and 100%, respectively, in samples from HIV patients [38]. Finally, since seropositivity remains long time after disease, interpretation of serological results could be challenging [39, 40]. Despite the limitations previously described, antibody detection tests are still considered as valuable diagnostic tools and have been demonstrated to improve diagnostic yield when combined with other diagnostic methods [41, 42].

#### **5. DNA based detection methods**

PCR methods based on the detection of fungal DNA directly from clinical samples are currently implemented in the routine of several laboratories for the diagnosis of main fungal infections, but there are considerably fewer PCR tests for the diagnosis of histoplasmosis. Their advantages rely on their simplicity, high specificity and short turnaround time with the bonus that real-time PCR (qPCR) formats allow for determining the fungal burden in patients by using non-specific DNA-binding dyes or fluorescently labeled probes [36, 43]. However, this technique also has some limitations as the moderate amount of DNA in low invasive samples, the lack of standardization and the low availability of widely validated commercial systems [44, 45]. Recently, PCR based methods have been included in the EORTC/ MSG criteria for the diagnosis of some fungal infections such as invasive aspergillosis or candidiasis but not for endemic mycoses [13].

The majority of PCR tests for the diagnosis of histoplasmosis have been developed in house and none of them has been commercialized. They have been recently reviewed in several reports with different purposes [31, 38, 46]. Most developed methods targeted specific multicopy regions of the ribosomal DNA or the single-copy Hcp100 gene and were performed by using conventional, nested or qPCR formats. The sensitivity and specificity of these assays depends on the type of sample analyzed, the clinical characteristics of patients and the PCR format used for DNA detection. These tests showed an excellent analytical performance (overall sensitivity of 95% and specificity of 99%) when testing samples of HIV patients [38], but sensitivity decreased when testing blood and serum samples from immunocompetent patients [31, 46]. Panfungal or broad-range PCRs are used when there is not a clear suspicion of the fungus involved in the infection, since universal primers are used to detect any fungal DNA in the clinical sample. Although studies are scarce, several reports achieved the detection of

*H. capsulatum* DNA in clinical samples by using this technique, which could be especially useful in non-endemic regions [47–51].

Non-PCR based methods are also able to amplify and detect *H. capsulatum* DNA from clinical samples. Loop-mediated isothermal amplification (LAMP) rests on the use of a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA [52]. Some reports described the implementation of this technique for the molecular diagnosis of histoplasmosis showing great results [53, 54]. The high efficiency, sensitivity, specificity and costeffectiveness of these assays make them good candidates to be implemented in the diagnostic routine of resource-limited laboratories.

#### **6. Conclusions and future perspectives**

Early diagnosis of histoplasmosis is essential to establish a suitable antifungal therapy, which results in the reduction of mortality rates [55, 56]. This becomes especially important in certain hyper-endemic regions since they usually are disfavored areas where patients develop the disease in its disseminated form. While culture and histopathological examination are considered the gold standards methods for histoplasmosis diagnosis, these techniques show moderate sensitivity. In addition, culture is time consuming, requiring handling fungi in BSL-3 facilities. New approaches as MALDI-ToF MS technology allow for a rapid identification, but studies are still scarce. Antibody and antigen detection are useful tools for an early detection of the pathogen in low invasive clinical samples such as serum and urine. Despite limitations concerning sensitivity in certain populations and specificity have been widely reported, *H. capsulatum* antigen detection has been recently included in the second edition of the list of essential in vitro diagnostics (https:// www.who.int/medical\_devices/diagnostics/selection\_in-vitro/selection\_in-vitromeetings/sage-ivd2nd-meeting/190318-2ndEditionofEDL-open-session.pdf?ua=1). Regarding molecular methods, in the last decade a great effort has been made on the development of PCR tests for the detection of *H. capsulatum* DNA in a broad spectrum of clinical samples showing excellent diagnostic performance. However, a consensus among laboratories about the best samples, targets and procedures is mandatory. Consequently, no commercial tests are available to date limiting the use of this technique. To date, only an inter-laboratory study focused on molecular techniques for the diagnosis of histoplasmosis has been published [57]. In this work, authors concluded that qPCR targeting a multicopy genomic region was the best option for a suitable sensitivity. A European initiative is being launched to carry out multicenter studies in this regard.

All these problems have gained attention thanks to different initiatives coming from researchers from hyper-endemic regions [58] or international foundations as the Global Action Fund for Fungal Infections (GAFFI). As a result, proposals such as the Manaus declaration have been launched, specifically to get access to rapid testing for histoplasmosis in the Americas and Caribe until 2025 (https://www. gaffi.org/the-manaus-declaration-on-histoplasmosis-in-the-americas-and-caribbean-100-by-2025/). However, in addition to these excellent initiatives further work is required to improve diagnosis. In this sense, novel techniques as next generation sequencing (NGS) have been found to be useful in the diagnosis of several infections. Sequences obtained from clinical samples through NGS can be compared against reference databases enabling their identification to the genus and species level [59]. This technique has been used recently to identify *H. capsulatum* as the causal agent of a case of chronic meningitis [60] and for the differential diagnosis among histoplasmosis, leishmaniosis and talaromycosis [61]. Another approach

**59**

**Conflict of interest**

**Figure 2.**

*Laboratory Diagnosis of Histoplasmosis: An Update DOI: http://dx.doi.org/10.5772/intechopen.93305*

in constant development is based on determining host and fungal biomarkers that could indicate the presence of *H. capsulatum* in human body fluids. Recent advances in antibody detection are related to the refinement of antibody detection platforms (*Histoplasma* antibody IgG IgM EIA, MiraVista Diagnostics), while most efforts in antigen detection are focused on improving diagnostic yields of POC methods [35]. In summary, the aim of this chapter was to summarize the diagnostic pipeline currently available for the diagnosis of histoplasmosis in microbiological laboratories (**Figure 2**). Although so much progress has been made in the area, much certainly remains to be done to improve the early diagnosis of histoplasmosis, allowing the establishment of a prompt antifungal therapy and consequently

*cytology showing intra- and extracellular yeasts of* H. capsulatum *[63]. MALDI-ToF MS: matrix-assisted laser desorption/ionization-fime of flight mass spectrometry; ID: identification; EIA: enzyme immunoassay; LFD: lateral flow device; POC: point of care; qPCR: quantitative PCR; NGS: next generation sequencing.*

*Histopathological* 

*Direct visualization of fine needle aspiration* 

*Diagnostic pipeline currently available for the microbiological diagnosis of histoplasmosis. a*

*preparation showing a* H. capsulatum *yeast budding [62]. <sup>b</sup>*

reducing morbidity and mortality rates of this infection.

The authors declare no conflict of interest.

*Laboratory Diagnosis of Histoplasmosis: An Update DOI: http://dx.doi.org/10.5772/intechopen.93305*

#### **Figure 2.**

Histoplasma *and Histoplasmosis*

especially useful in non-endemic regions [47–51].

diagnostic routine of resource-limited laboratories.

**6. Conclusions and future perspectives**

multicenter studies in this regard.

*H. capsulatum* DNA in clinical samples by using this technique, which could be

Non-PCR based methods are also able to amplify and detect *H. capsulatum* DNA from clinical samples. Loop-mediated isothermal amplification (LAMP) rests on the use of a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA [52]. Some reports described the implementation of this technique for the molecular diagnosis of histoplasmosis showing great results [53, 54]. The high efficiency, sensitivity, specificity and costeffectiveness of these assays make them good candidates to be implemented in the

Early diagnosis of histoplasmosis is essential to establish a suitable antifungal therapy, which results in the reduction of mortality rates [55, 56]. This becomes especially important in certain hyper-endemic regions since they usually are disfavored areas where patients develop the disease in its disseminated form. While culture and histopathological examination are considered the gold standards methods for histoplasmosis diagnosis, these techniques show moderate sensitivity. In addition, culture is time consuming, requiring handling fungi in BSL-3 facilities. New approaches as MALDI-ToF MS technology allow for a rapid identification, but studies are still scarce. Antibody and antigen detection are useful tools for an early detection of the pathogen in low invasive clinical samples such as serum and urine. Despite limitations concerning sensitivity in certain populations and specificity have been widely reported, *H. capsulatum* antigen detection has been recently included in the second edition of the list of essential in vitro diagnostics (https:// www.who.int/medical\_devices/diagnostics/selection\_in-vitro/selection\_in-vitromeetings/sage-ivd2nd-meeting/190318-2ndEditionofEDL-open-session.pdf?ua=1). Regarding molecular methods, in the last decade a great effort has been made on the development of PCR tests for the detection of *H. capsulatum* DNA in a broad spectrum of clinical samples showing excellent diagnostic performance. However, a consensus among laboratories about the best samples, targets and procedures is mandatory. Consequently, no commercial tests are available to date limiting the use of this technique. To date, only an inter-laboratory study focused on molecular techniques for the diagnosis of histoplasmosis has been published [57]. In this work, authors concluded that qPCR targeting a multicopy genomic region was the best option for a suitable sensitivity. A European initiative is being launched to carry out

All these problems have gained attention thanks to different initiatives coming from researchers from hyper-endemic regions [58] or international foundations as the Global Action Fund for Fungal Infections (GAFFI). As a result, proposals such as the Manaus declaration have been launched, specifically to get access to rapid testing for histoplasmosis in the Americas and Caribe until 2025 (https://www. gaffi.org/the-manaus-declaration-on-histoplasmosis-in-the-americas-and-caribbean-100-by-2025/). However, in addition to these excellent initiatives further work is required to improve diagnosis. In this sense, novel techniques as next generation sequencing (NGS) have been found to be useful in the diagnosis of several infections. Sequences obtained from clinical samples through NGS can be compared against reference databases enabling their identification to the genus and species level [59]. This technique has been used recently to identify *H. capsulatum* as the causal agent of a case of chronic meningitis [60] and for the differential diagnosis among histoplasmosis, leishmaniosis and talaromycosis [61]. Another approach

**58**

*Diagnostic pipeline currently available for the microbiological diagnosis of histoplasmosis. a Histopathological preparation showing a* H. capsulatum *yeast budding [62]. <sup>b</sup> Direct visualization of fine needle aspiration cytology showing intra- and extracellular yeasts of* H. capsulatum *[63]. MALDI-ToF MS: matrix-assisted laser desorption/ionization-fime of flight mass spectrometry; ID: identification; EIA: enzyme immunoassay; LFD: lateral flow device; POC: point of care; qPCR: quantitative PCR; NGS: next generation sequencing.*

in constant development is based on determining host and fungal biomarkers that could indicate the presence of *H. capsulatum* in human body fluids. Recent advances in antibody detection are related to the refinement of antibody detection platforms (*Histoplasma* antibody IgG IgM EIA, MiraVista Diagnostics), while most efforts in antigen detection are focused on improving diagnostic yields of POC methods [35].

In summary, the aim of this chapter was to summarize the diagnostic pipeline currently available for the diagnosis of histoplasmosis in microbiological laboratories (**Figure 2**). Although so much progress has been made in the area, much certainly remains to be done to improve the early diagnosis of histoplasmosis, allowing the establishment of a prompt antifungal therapy and consequently reducing morbidity and mortality rates of this infection.

#### **Conflict of interest**

The authors declare no conflict of interest.

Histoplasma *and Histoplasmosis*

### **Author details**

María J. Buitrago1 \* and Clara Valero1,2

1 Reference Laboratory in Mycology, National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain

2 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil

\*Address all correspondence to: buitrago@isciii.es

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**61**

ajtmh.14-0175

a:1007277602576

*Laboratory Diagnosis of Histoplasmosis: An Update DOI: http://dx.doi.org/10.5772/intechopen.93305*

> [9] Kathuria S, Capoor MR, Yadav S, Singh A, Ramesh V. Disseminated histoplasmosis in an apparently immunocompetent individual

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**Author details**

María J. Buitrago1

Ribeirão Preto, Brazil

Salud Carlos III, Madrid, Spain

\* and Clara Valero1,2

\*Address all correspondence to: buitrago@isciii.es

provided the original work is properly cited.

1 Reference Laboratory in Mycology, National Center for Microbiology, Instituto de

2 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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[54] Zatti MDS, Arantes TD, Fernandes JAL, Bay MB, Milan EP, Naliato GFS, et al. Loop-mediated isothermal amplification and nested PCR of the internal transcribed spacer (ITS) for *Histoplasma capsulatum* detection. PLoS Neglected Tropical Diseases. 2019;**13**(8):e0007692. DOI: 10.1371/journal.pntd.0007692

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[47] Ala-Houhala M, Koukila-Kahkola P, Antikainen J, Valve J, Kirveskari J, Anttila VJ. Clinical use of fungal PCR from deep tissue samples in the diagnosis of invasive fungal diseases: A retrospective observational study. Clinical Microbiology and Infection.

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[55] Adenis AA, Aznar C, Couppie P. Histoplasmosis in HIV-infected patients: A review of new developments and remaining gaps. Current Tropical Medicine Reports. 2014;**1**:119-128. DOI: 10.1007/s40475-014-0017-8

[56] Scheel CM, Gómez BL. Diagnostic methods for histoplasmosis: Focus on endemic countries with variable infrastructure levels. Current Tropical Medicine Reports. 2014;**1**(2):129-137. DOI: 10.1007/s40475-014-0020-0

[57] Buitrago MJ, Canteros CE, Frías De León G, González Á, Marques-Evangelista De Oliveira M, Muñoz CO, et al. Comparison of PCR protocols for detecting *Histoplasma capsulatum* DNA through a multicenter study. Revista Iberoamericana de Micología. 2013;**30**(4):256-260. DOI: 10.1016/j. riam.2013.03.004

[58] Nacher M, Adenis A, Mc Donald S, Do Socorro Mendonca Gomes M, Singh S, Lopes Lima I, et al. Disseminated histoplasmosis in HIVinfected patients in South America: A neglected killer continues on its rampage. PLoS Neglected Tropical Disease. 2013;**7, 11**:e2319. DOI: 10.1371/ journal.pntd.0002319

[59] Kidd SE, Chen SC, Meyer W, Halliday CL. A new age in molecular diagnostics for invasive fungal disease: Are we ready? Frontiers in Microbiology. 2019;**10**:2903. DOI: 10.3389/fmicb.2019.02903

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**67**

Section 4

Treatment

Section 4

Treatment

**69**

**Chapter 5**

**Abstract**

amphotericin B, HIV

**1. Introduction**

Treatment of Histoplasmosis

**Keywords:** *Histoplasma capsulatum*, histoplasmosis, itraconazole,

Histoplasmosis is an uncommon endemic mycosis caused by the fungus *Histoplasma capsulatum* that usually causes an asymptomatic infection but occasionally results in severe multisystem disease [1, 2]. Two main varieties of the saprophytic, thermally dimorphic fungus of the genus *Histoplasma* affect humans; *Histoplasma capsulatum var. capsulatum* and *Histoplasma capsulatum var. duboisii* [3–5]. Histoplasmosis affects over 10,000 people globally; it is neglected, worryingly under-diagnosed, and often misdiagnosed as cancer or tuberculosis with fatal consequences [6–8]. The spectrum of the clinical manifestation of histoplasmosis is very broad, ranging from an asymptomatic or minimally symptomatic acute pulmonary disease following inhalation of a large inoculum of *Histoplasma* microconidia to chronic pulmonary disease in patients with underlying structural lung disease, to acute progressive disseminated disease in patients with severe immunodeficiency [9–13]. In immunocompetent patients, acute histoplasmosis is

*Lucy Grace Asio and Akaninyene A. Otu*

*Felix Bongomin, Richard Kwizera, Joseph Baruch Baluku,* 

Histoplasmosis, caused by the thermally dimorphic fungus *Histoplasma capsulatum,* is an uncommon multisystem disease with a global distribution. The spectrum of clinical manifestations ranges from an asymptomatic or minimally symptomatic acute pulmonary disease following inhalation of a large inoculum of *Histoplasma* microconidia to chronic pulmonary disease in patients with underlying structural lung disease. It also extends to acute progressive disseminated disease in patients with severe immunodeficiency. Generally, antifungal therapy is indicated for patients with progressive acute pulmonary histoplasmosis, chronic pulmonary histoplasmosis and acute progressive disseminated histoplasmosis. In immunocompetent patients, acute pulmonary histoplasmosis may be a self-limiting disease without the need for systemic antifungal therapy. Oral triazole antifungal drugs alone are recommended for less severe disease. However, moderate-to-severe acute pulmonary histoplasmosis requires intravenous amphotericin B therapy for at least 1–2 weeks followed by oral itraconazole for at least 12 weeks. For acute progressive disseminated histoplasmosis, intravenous amphotericin B therapy is given for at least 2 weeks (4–6 weeks if meningeal involvement) or until a patient can tolerate oral therapy, followed by oral itraconazole (or an alternative triazole) for at least 12 months. Chronic cavitary pulmonary histoplasmosis is treated with oral itraconazole for 1–2 years. There is insufficient evidence to support the use of isavuconazole or the echinocandins for the treatment of histoplasmosis.

#### **Chapter 5**

## Treatment of Histoplasmosis

*Felix Bongomin, Richard Kwizera, Joseph Baruch Baluku, Lucy Grace Asio and Akaninyene A. Otu*

#### **Abstract**

Histoplasmosis, caused by the thermally dimorphic fungus *Histoplasma capsulatum,* is an uncommon multisystem disease with a global distribution. The spectrum of clinical manifestations ranges from an asymptomatic or minimally symptomatic acute pulmonary disease following inhalation of a large inoculum of *Histoplasma* microconidia to chronic pulmonary disease in patients with underlying structural lung disease. It also extends to acute progressive disseminated disease in patients with severe immunodeficiency. Generally, antifungal therapy is indicated for patients with progressive acute pulmonary histoplasmosis, chronic pulmonary histoplasmosis and acute progressive disseminated histoplasmosis. In immunocompetent patients, acute pulmonary histoplasmosis may be a self-limiting disease without the need for systemic antifungal therapy. Oral triazole antifungal drugs alone are recommended for less severe disease. However, moderate-to-severe acute pulmonary histoplasmosis requires intravenous amphotericin B therapy for at least 1–2 weeks followed by oral itraconazole for at least 12 weeks. For acute progressive disseminated histoplasmosis, intravenous amphotericin B therapy is given for at least 2 weeks (4–6 weeks if meningeal involvement) or until a patient can tolerate oral therapy, followed by oral itraconazole (or an alternative triazole) for at least 12 months. Chronic cavitary pulmonary histoplasmosis is treated with oral itraconazole for 1–2 years. There is insufficient evidence to support the use of isavuconazole or the echinocandins for the treatment of histoplasmosis.

**Keywords:** *Histoplasma capsulatum*, histoplasmosis, itraconazole, amphotericin B, HIV

#### **1. Introduction**

Histoplasmosis is an uncommon endemic mycosis caused by the fungus *Histoplasma capsulatum* that usually causes an asymptomatic infection but occasionally results in severe multisystem disease [1, 2]. Two main varieties of the saprophytic, thermally dimorphic fungus of the genus *Histoplasma* affect humans; *Histoplasma capsulatum var. capsulatum* and *Histoplasma capsulatum var. duboisii* [3–5]. Histoplasmosis affects over 10,000 people globally; it is neglected, worryingly under-diagnosed, and often misdiagnosed as cancer or tuberculosis with fatal consequences [6–8]. The spectrum of the clinical manifestation of histoplasmosis is very broad, ranging from an asymptomatic or minimally symptomatic acute pulmonary disease following inhalation of a large inoculum of *Histoplasma* microconidia to chronic pulmonary disease in patients with underlying structural lung disease, to acute progressive disseminated disease in patients with severe immunodeficiency [9–13]. In immunocompetent patients, acute histoplasmosis is

typically a self-limiting disease with no need for antifungal therapy [14]. Pneumonia remains the most common disease presentation but extrapulmonary dissemination can occur, especially in immunocompromised patients [12]. A definite diagnosis of histoplasmosis is based on the isolation of the organisms in fungal culture [15]. Rapid detection of the *H. capsulatum* polysaccharide antigen using enzyme immunoassay (EIA) in urine, blood or bronchoalveolar lavage fluid is also available and very useful especially among immunocompromised patients with disseminated or acute pulmonary disease [13, 16]. Serologic testing for antibodies can be achieved by EIA, immunodiffusion, and complement fixation [13, 16, 17]. These antibody tests may be falsely negative in immunosuppressed patients and are most valuable when combined with a compatible clinical presentation and epidemiologic risk factors.

This chapter provides an overview of the currently available treatment options for the different manifestations of histoplasmosis.

#### **2. Antifungal agents used in the treatment of histoplasmosis**

Histoplasmosis is primarily treated with 2 distinct classes of antifungal agents: The triazoles and the polyenes. Data is lacking on the use of the echinocandins for the treatment of histoplasmosis.

#### **2.1 Triazole**

The triazole class of antifungals examples of which includes drug like fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole [18, 19]. Of these, itraconazole is the drug of choice for the treatment of the various forms of histoplasmosis as a sole therapy or as a step-down therapy following amphotericin B infusion [13, 14, 20].

Triazole antifungals inhibit the cytochrome P450-dependent enzyme 14-alphalanosterol demethylase (CYP51) encoded by the *ERG11* gene that converts lanosterol to ergosterol in the cell membrane, inhibiting fungal growth and replication (fungistatic) [18, 19]. Drug-drug and drug-food interactions, as well as side effects are major issues associated with itraconazole therapy. Common side effects of itraconazole include drug-induced hepatitis, gastrointestinal discomfort, heart failure, ankle edema, alopecia, erectile dysfunction, gynaecomastia, peripheral neuropathy, visual disturbance and headaches [21]. Itraconazole interacts with agents used in the treatment of HIV (especially, nevirapine) and tuberculosis (especially, rifampicin) leading to decreased or increased exposures to itraconazole and therefore increased risk of hepatotoxicity. With the exception of fluconazole, voriconazole and posaconazole are used as alternative agents for the treatment of histoplasmosis if there is a contraindication to the use of itraconazole [13, 14, 20]. However, to the best of our knowledge, there is no data to support the use of isavuconazole for the treatment of histoplasmosis.

#### **2.2 Polyene**

The polyene antibiotic, amphotericin B derived from the fermentation product of the filamentous bacteria, *Streptomyces nodosus* is a broad-spectrum antifungal agent administered as deoxycholate (conventional amphotericin B) or liposomal formulation [18, 19].

In its mechanism of action, amphotericin B preferentially binds ergosterol in the fungal cell membrane (and to a lesser extent, cholesterol in cell membrane of humans) to form channels through which small molecules leak from the inside of the fungal cell to the outside, resulting in death of the fungal cell (fungicidal) [18, 19].

**71**

12–18 months [27].

*Treatment of Histoplasmosis*

*DOI: http://dx.doi.org/10.5772/intechopen.92984*

syndrome being managed (**Table 1**).

rate and survival benefit [14, 20].

**3.1 Central nervous system histoplasmosis**

infusion site phlebitis, and acute kidney injury [21].

**3. Overview of antifungal treatment of histoplasmosis**

Common side effects of amphotericin B include, acute infusion-related reactions characterised by nausea, vomiting, rigors, fever, hyper or hypotension, and hypoxia mainly driven by the effects of amphotericin B on pro-inflammatory cytokine production. Nephrotoxicity is another very important side effect of amphotericin B occurring in about 34–60% of patients. Blood disorders, especially anaemia and electrolyte disturbances, especially hypomagnesaemia and hypokalemia are common but can be part of the rare Fanconi syndrome. Other side effects include

It is important to understand the indications and the choices of antifungal agents in the treatment of histoplasmosis. Manifestations of histoplasmosis that are a typically treated include moderate to severe acute pulmonary histoplasmosis, symptomatic chronic cavitary pulmonary histoplasmosis, acute progressive disseminated disease, and histoplasmosis in immunocompromised individuals [12, 13], (**Table 1**). Treatment is also indicated for complications of histoplasmosis such as mediastinal granulomas and adenitis [14], (**Table 1**). Itraconazole can be used for the treatment of symptomatic immunocompetent patients with indolent non-meningeal infection, including chronic cavitary pulmonary histoplasmosis [12, 13]. Itraconazole is also used as a step-down oral agent following initial treatment of severe disease with amphotericin B [12, 13]. Itraconazole is administered at a dose 200 mg 3 times daily for 3 days, as loading dose and then 200 mg once or twice daily, for maintenance therapy [12, 13]. Duration of itraconazole therapy depends on the histoplasmosis

Amphotericin by intravenous infusion is the drug of choice for the initial treatment of fulminant or moderate to severe infections, followed by a course of oral. Following successful treatment, itraconazole can be used for secondary prophylaxis against relapse until immune reconstitution is realised [12, 13]. The deoxycholate formulation of amphotericin B is administered at a dose of 0.7–1.0 mg/kg daily by intravenous infusion, meanwhile the lipid formulation of amphotericin B can be administered at a higher dose of 3.0–5.0 mg/kg daily. Liposomal formulation is preferred to the deoxycholate due to its superior side effect profile, better response

There are no known prospective studies that have evaluated treatment of central nervous system (CNS) histoplasmosis. Treatment recommendations are guided by

several case reports, retrospective case series and expert opinion [12, 13]. Liposomal amphotericin B achieves higher CNS concentrations than deoxycholate formulations and together with triazoles, is the recommended therapy for CNS histoplasmosis [22]. While the choice of the optimal triazole is in doubt, there is evidence from an animal model to suggest that fluconazole may be antagonistic when combined with amphotericin B [23]. Voriconazole could have a role among patients with good performance status as monotherapy or in combination with amphotericin B, but the evidence is limited and the occurrence of hepatotoxicity and hypersensitivity may limit its use [24–26]. One case series of 11 cases reported a morbidity free survival of 54.5% when patients were treated with intravenous amphotericin B deoxycholate for 8 weeks followed by maintenance therapy with fluconazole or itraconazole for

#### *Treatment of Histoplasmosis DOI: http://dx.doi.org/10.5772/intechopen.92984*

Histoplasma *and Histoplasmosis*

for the different manifestations of histoplasmosis.

the treatment of histoplasmosis.

**2.1 Triazole**

infusion [13, 14, 20].

**2. Antifungal agents used in the treatment of histoplasmosis**

Histoplasmosis is primarily treated with 2 distinct classes of antifungal agents: The triazoles and the polyenes. Data is lacking on the use of the echinocandins for

The triazole class of antifungals examples of which includes drug like fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole [18, 19]. Of these, itraconazole is the drug of choice for the treatment of the various forms of histoplasmosis as a sole therapy or as a step-down therapy following amphotericin B

Triazole antifungals inhibit the cytochrome P450-dependent enzyme 14-alphalanosterol demethylase (CYP51) encoded by the *ERG11* gene that converts lanosterol to ergosterol in the cell membrane, inhibiting fungal growth and replication (fungistatic) [18, 19]. Drug-drug and drug-food interactions, as well as side effects are major issues associated with itraconazole therapy. Common side effects of itraconazole include drug-induced hepatitis, gastrointestinal discomfort, heart failure, ankle edema, alopecia, erectile dysfunction, gynaecomastia, peripheral neuropathy, visual disturbance and headaches [21]. Itraconazole interacts with agents used in the treatment of HIV (especially, nevirapine) and tuberculosis (especially, rifampicin) leading to decreased or increased exposures to itraconazole and therefore increased risk of hepatotoxicity. With the exception of fluconazole, voriconazole and posaconazole are used as alternative agents for the treatment of histoplasmosis if there is a contraindication to the use of itraconazole [13, 14, 20]. However, to the best of our knowledge, there is no data to

The polyene antibiotic, amphotericin B derived from the fermentation product of the filamentous bacteria, *Streptomyces nodosus* is a broad-spectrum antifungal agent administered as deoxycholate (conventional amphotericin B) or liposomal

In its mechanism of action, amphotericin B preferentially binds ergosterol in the fungal cell membrane (and to a lesser extent, cholesterol in cell membrane of humans) to form channels through which small molecules leak from the inside of the fungal cell to the outside, resulting in death of the fungal cell (fungicidal) [18, 19].

support the use of isavuconazole for the treatment of histoplasmosis.

typically a self-limiting disease with no need for antifungal therapy [14]. Pneumonia remains the most common disease presentation but extrapulmonary dissemination can occur, especially in immunocompromised patients [12]. A definite diagnosis of histoplasmosis is based on the isolation of the organisms in fungal culture [15]. Rapid detection of the *H. capsulatum* polysaccharide antigen using enzyme immunoassay (EIA) in urine, blood or bronchoalveolar lavage fluid is also available and very useful especially among immunocompromised patients with disseminated or acute pulmonary disease [13, 16]. Serologic testing for antibodies can be achieved by EIA, immunodiffusion, and complement fixation [13, 16, 17]. These antibody tests may be falsely negative in immunosuppressed patients and are most valuable when combined with a compatible clinical presentation and epidemiologic risk factors. This chapter provides an overview of the currently available treatment options

**70**

**2.2 Polyene**

formulation [18, 19].

Common side effects of amphotericin B include, acute infusion-related reactions characterised by nausea, vomiting, rigors, fever, hyper or hypotension, and hypoxia mainly driven by the effects of amphotericin B on pro-inflammatory cytokine production. Nephrotoxicity is another very important side effect of amphotericin B occurring in about 34–60% of patients. Blood disorders, especially anaemia and electrolyte disturbances, especially hypomagnesaemia and hypokalemia are common but can be part of the rare Fanconi syndrome. Other side effects include infusion site phlebitis, and acute kidney injury [21].

#### **3. Overview of antifungal treatment of histoplasmosis**

It is important to understand the indications and the choices of antifungal agents in the treatment of histoplasmosis. Manifestations of histoplasmosis that are a typically treated include moderate to severe acute pulmonary histoplasmosis, symptomatic chronic cavitary pulmonary histoplasmosis, acute progressive disseminated disease, and histoplasmosis in immunocompromised individuals [12, 13], (**Table 1**). Treatment is also indicated for complications of histoplasmosis such as mediastinal granulomas and adenitis [14], (**Table 1**). Itraconazole can be used for the treatment of symptomatic immunocompetent patients with indolent non-meningeal infection, including chronic cavitary pulmonary histoplasmosis [12, 13]. Itraconazole is also used as a step-down oral agent following initial treatment of severe disease with amphotericin B [12, 13]. Itraconazole is administered at a dose 200 mg 3 times daily for 3 days, as loading dose and then 200 mg once or twice daily, for maintenance therapy [12, 13]. Duration of itraconazole therapy depends on the histoplasmosis syndrome being managed (**Table 1**).

Amphotericin by intravenous infusion is the drug of choice for the initial treatment of fulminant or moderate to severe infections, followed by a course of oral. Following successful treatment, itraconazole can be used for secondary prophylaxis against relapse until immune reconstitution is realised [12, 13]. The deoxycholate formulation of amphotericin B is administered at a dose of 0.7–1.0 mg/kg daily by intravenous infusion, meanwhile the lipid formulation of amphotericin B can be administered at a higher dose of 3.0–5.0 mg/kg daily. Liposomal formulation is preferred to the deoxycholate due to its superior side effect profile, better response rate and survival benefit [14, 20].

#### **3.1 Central nervous system histoplasmosis**

There are no known prospective studies that have evaluated treatment of central nervous system (CNS) histoplasmosis. Treatment recommendations are guided by several case reports, retrospective case series and expert opinion [12, 13].

Liposomal amphotericin B achieves higher CNS concentrations than deoxycholate formulations and together with triazoles, is the recommended therapy for CNS histoplasmosis [22]. While the choice of the optimal triazole is in doubt, there is evidence from an animal model to suggest that fluconazole may be antagonistic when combined with amphotericin B [23]. Voriconazole could have a role among patients with good performance status as monotherapy or in combination with amphotericin B, but the evidence is limited and the occurrence of hepatotoxicity and hypersensitivity may limit its use [24–26]. One case series of 11 cases reported a morbidity free survival of 54.5% when patients were treated with intravenous amphotericin B deoxycholate for 8 weeks followed by maintenance therapy with fluconazole or itraconazole for 12–18 months [27].


**Table 1.**

**73**

*Treatment of Histoplasmosis*

*DOI: http://dx.doi.org/10.5772/intechopen.92984*

system is reconstituted [13, 14, 28].

**3.2 Histoplasmosis in pregnancy**

The Infectious Diseases Society of America (IDSA) recommends treating CNS disease with liposomal amphotericin B (3.0–5.0 mg/kg daily for a total of 175 mg/kg given over 4–6 weeks), followed by a maintenance phase with itraconazole for at least a year at a dose of 200 mg, given 2–3 times a day [14]. Resolution of CSF abnormalities, including a negative antigen test is the recommended treatment target. Specific recommendations for CSF monitoring are: (a) worsening disease in the initial 2 weeks of therapy or lack of improvement by 1 month of therapy, in order to re-evaluate the diagnosis, (b) when amphotericin B is being replaced by a triazole, (c) if relapse is suspected and (d) after 1 year of therapy, to make the decision of treatment continuation or stoppage. Therapeutic drug monitoring for itraconazole is recommended during treatment to ensure adequate drug exposure. Treatment recommendations for histoplasmosis meningitis or CNS masses are the same. Also, for patients with concurrent pulmonary disease, chronic suppressive therapy with 200 mg of itraconazole, given once a day, is indicated until the patient's immune

The role of steroids is not well described, although case reports indicate they can be successfully used [14] . Similarly, routine brain or spinal cord surgery is not recommended by IDSA but is chronicled in case reports [14, 28]. Hydrocephalus complicating CNS histoplasmosis may be managed with shunt placement when the patient has received at least 2 weeks of amphotericin B therapy [22]. The manage

ment of increased intracranial pressure and the long-term sequelae that require rehabilitation is similar to what is done for stroke and brain tumours [14, 28]. In a sizable cohort of patients, the one-year survival of patients with CNS histoplasmosis was reported to be 75% among patients who were initially treated

All presenting clinical syndromes of histoplasmosis in pregnancy require antifungal therapy due to the increased risk of trans-placental transmission of the infection to the developing fetus [29]. Preferably, pregnant women can be treated with amphotericin B preparations for 4–6 weeks. Lipid formulation of amphoteri

cin B is given at a dose of 3–5 mg/kg/day. For pregnant women with low risk for nephrotoxicity, amphotericin B deoxycholate (0.7–1.0 mg/kg/day) may be offered as a substitute [14, 30]. Coupled with this, post-partum monitoring of the child for any evidence of the infection is vital. In any case of the newborn showing signs of histoplasmosis infection, it is recommended that treatment of amphotericin B

Itraconazole is generally considered teratogenic to the growing fetus and is therefore best avoided in pregnancy. However, itraconazole may be considered for treatment in pregnant women with systemic histoplasmosis, but only after the first trimester [31, 32]. Amphotericin B therefore remains the drug of choice in manag

ing histoplasmosis in pregnancy, as azole antifungals should generally be avoided.

**3.3 Histoplasmosis-associated immune reconstitution inflammatory syndrome** 

Immune reconstitution inflammatory syndrome (IRIS) is a spectrum of inflam

matory disorders linked with paradoxical worsening of pre-existing infectious diseases (previously diagnosed or subclinical) in HIV-infected patients. It typically follows, the initiation of antiretroviral therapy (ART): whereby the ART improves the patient's immune system enough to mount an inflammatory response tends to unmask the underlying infectious processes, such as histoplasmosis [33–36].

**in the context of human immunodeficiency virus disease**

with liposomal or deoxycholate formulations of amphotericin [28]

deoxycholate (1 mg/kg/day) is given for 4 weeks [14, 30].





**.**

*Treatment of histoplasmosis syndromes and their complications [13, 14].*

Histoplasma *and Histoplasmosis*

#### *Treatment of Histoplasmosis DOI: http://dx.doi.org/10.5772/intechopen.92984*

Histoplasma *and Histoplasmosis*

**72**

**Histoplasmosis** 

**Risk factor**

**Clinical** 

**Indication for treatment**

**Treatment**

**Antifungal therapy** 

**duration**

**time-course**

**syndrome or** 

**complication**

Acute progressive

Immunosuppression such as

1–2 weeks

Always

Mild disease

Moderate

Amphotericin B for 1–2 weeks in

non-meningeal and 4–6 weeks

in meningeal disease followed

by itraconazole as step-down

therapy

to severe

disease

Itraconazole

12 months followed by

maintenance antifungal

suppression until

immune recovery

advanced HIV disease

disseminated

histoplasmosis

Acute pulmonary

High inoculum exposure to

1–2 weeks

Moderate or severe disease

Mild disease

Severe

Amphotericin B for 1–2 weeks

followed by itraconazole as step-

down therapy

disease

Itraconazole

12 weeks

or immunosuppressed

patient

*Histoplasma* conidia

histoplasmosis

Subacute pulmonary

Low inoculum exposure to

Weeks to

If symptoms last

Itraconazole

6–12 weeks

>1 months or

immunosuppressed

patient

months

*Histoplasma* conidia

histoplasmosis

Chronic cavitary

Chronic obstructive

Months to

Always

Itraconazole

1–2 years and until

radiologic resolution or

stabilisation

6–12 weeks

years

pulmonary disease and other

lung diseases Smoking

pulmonary

histoplasmosis

Mediastinal adenitis

Mediastinal

Coalesced necrotic

Early or late

If compressive symptoms

Surgery and itraconazole

Stenting, arterial embolization or surgery

Not applicable

6–12 weeks

complication

present

If compressive symptoms

mediastinal lymph nodes

Fibrosis of mediastinal

Late

complication

present

structures

granuloma

Mediastinal fibrosis

**Table 1.** *Treatment of histoplasmosis syndromes and their complications [13, 14].*

Reactive and enlarged

Early

If compressive symptoms

Itraconazole and steroids

present or adenitis last

>1 months

complication

mediastinal lymph nodes

The Infectious Diseases Society of America (IDSA) recommends treating CNS disease with liposomal amphotericin B (3.0–5.0 mg/kg daily for a total of 175 mg/kg given over 4–6 weeks), followed by a maintenance phase with itraconazole for at least a year at a dose of 200 mg, given 2–3 times a day [14]. Resolution of CSF abnormalities, including a negative antigen test is the recommended treatment target. Specific recommendations for CSF monitoring are: (a) worsening disease in the initial 2 weeks of therapy or lack of improvement by 1 month of therapy, in order to re-evaluate the diagnosis, (b) when amphotericin B is being replaced by a triazole, (c) if relapse is suspected and (d) after 1 year of therapy, to make the decision of treatment continuation or stoppage. Therapeutic drug monitoring for itraconazole is recommended during treatment to ensure adequate drug exposure. Treatment recommendations for histoplasmosis meningitis or CNS masses are the same. Also, for patients with concurrent pulmonary disease, chronic suppressive therapy with 200 mg of itraconazole, given once a day, is indicated until the patient's immune system is reconstituted [13, 14, 28].

The role of steroids is not well described, although case reports indicate they can be successfully used [14] . Similarly, routine brain or spinal cord surgery is not recommended by IDSA but is chronicled in case reports [14, 28]. Hydrocephalus complicating CNS histoplasmosis may be managed with shunt placement when the patient has received at least 2 weeks of amphotericin B therapy [22]. The management of increased intracranial pressure and the long-term sequelae that require rehabilitation is similar to what is done for stroke and brain tumours [14, 28].

In a sizable cohort of patients, the one-year survival of patients with CNS histoplasmosis was reported to be 75% among patients who were initially treated with liposomal or deoxycholate formulations of amphotericin [28]**.**

#### **3.2 Histoplasmosis in pregnancy**

All presenting clinical syndromes of histoplasmosis in pregnancy require antifungal therapy due to the increased risk of trans-placental transmission of the infection to the developing fetus [29]. Preferably, pregnant women can be treated with amphotericin B preparations for 4–6 weeks. Lipid formulation of amphotericin B is given at a dose of 3–5 mg/kg/day. For pregnant women with low risk for nephrotoxicity, amphotericin B deoxycholate (0.7–1.0 mg/kg/day) may be offered as a substitute [14, 30]. Coupled with this, post-partum monitoring of the child for any evidence of the infection is vital. In any case of the newborn showing signs of histoplasmosis infection, it is recommended that treatment of amphotericin B deoxycholate (1 mg/kg/day) is given for 4 weeks [14, 30].

Itraconazole is generally considered teratogenic to the growing fetus and is therefore best avoided in pregnancy. However, itraconazole may be considered for treatment in pregnant women with systemic histoplasmosis, but only after the first trimester [31, 32]. Amphotericin B therefore remains the drug of choice in managing histoplasmosis in pregnancy, as azole antifungals should generally be avoided.

#### **3.3 Histoplasmosis-associated immune reconstitution inflammatory syndrome in the context of human immunodeficiency virus disease**

Immune reconstitution inflammatory syndrome (IRIS) is a spectrum of inflammatory disorders linked with paradoxical worsening of pre-existing infectious diseases (previously diagnosed or subclinical) in HIV-infected patients. It typically follows, the initiation of antiretroviral therapy (ART): whereby the ART improves the patient's immune system enough to mount an inflammatory response tends to unmask the underlying infectious processes, such as histoplasmosis [33–36].

In studies done among HIV patients, histoplasmosis-associated - IRIS was associated with histoplasmosis; IRIS was reported to be uncommon with incidence rates of 0.74 cases per 1000 HIV-infected person-years 0.5% [37–39]. However according to another study among 271 patients, the emergence of IRIS tended to be quite common in people with HIV and disseminated histoplasmosis; whether the IRIS is triggered by the Histoplasma or other co-infections, is still unclear [40].

For HIV patients with histoplasmosis to be considered to have histoplasmosis associated IRIS, they should fulfil most or all of the following criteria: AIDS with low pretreatment CD4 count (≤100 cells/μL), a positive immunological and virological response to ART, clinical manifestations of an inflammatory condition, association between ART initiation and appearance of clinical features of the inflammatory condition and; absence of evidence of ART resistance, patient non-compliance, drug allergy or adverse reactions, a concomitant non- fungal infection, or decreased drug levels due to malabsorption or drug-drug interactions [36, 38, 41–43].

The clinical manifestations vary from case to case, and they commonly include fever, lymphadenopathy, mucocutaneous lesions, and disseminated disease. The timeline of occurrence from initiation of ART is also varied from a few days to months, and symptoms can set in when the patient is already on the ART course or when the ART is just introduced [39, 44, 45]. Histoplasmosis-associated IRIS in HIV patients may have a predilection for females over males, with one study showing that it was four times more frequent in females than males [39].

If the symptoms of IRIS are mild, the patient is managed symptomatically. The role of steroids in treating histoplasmosis-IRIS is yet unclear [39].

However if the presentation is severe, corticosteroids may be used as they have been seen to be of benefit in TB-associated IRIS, though they should be used with caution [46].

Once a patient exhibits features of IRIS while already on effective ART, the ART should be continued and the patient should be initiated on histoplasmosis treatment immediately. However if the patient is not receiving ART, a two-week delay is encouraged before starting ART while the patient is on antifungal therapy (amphotericin B or itraconazole) as per the guidelines [14].

#### **4. Conclusions**

Itraconazole alone is used for the treatment of mild forms of histoplasmosis and as a step-down therapy in severe disease and for secondary prophylaxis to prevent relapse in the immunocompromised after induction therapy with amphotericin B. Moderate-to-severe acute pulmonary histoplasmosis as well as acute progressive disseminated histoplasmosis require intravenous amphotericin B therapy for at least 2 weeks (4–6 weeks if meningeal involvement) or until a patient can tolerate oral therapy, then oral itraconazole (or an alternative triazole) for at least 12 weeks (for acute pulmonary) or 12 months (for acute progressive disseminated histoplasmosis). Chronic cavitary pulmonary histoplasmosis is treated with itraconazole for 12–24 months. There is insufficient evidence to support the use of isavuconazole and the echinocandins for the treatment of histoplasmosis.

**75**

*Treatment of Histoplasmosis*

**Author details**

Felix Bongomin1

and Akaninyene A. Otu5

Gulu University, Gulu, Uganda

\*, Richard Kwizera<sup>2</sup>

Sciences, Makerere University, Kampala, Uganda

4 Department of Programs, Mildmay Uganda, Uganda

\*Address all correspondence to: drbongomin@gmail.com

provided the original work is properly cited.

1 Department of Medical Microbiology and Immunology, Faculty of Medicine,

2 Translational Research Laboratory, Infectious Diseases Institute, College of Health

3 Division of Pulmonology, Mulago National Referral Hospital, Kampala, Uganda

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

5 Department of Internal Medicine, University of Calabar, Calabar, Nigeria

, Joseph Baruch Baluku3,4, Lucy Grace Asio1

*DOI: http://dx.doi.org/10.5772/intechopen.92984*

*Treatment of Histoplasmosis DOI: http://dx.doi.org/10.5772/intechopen.92984*

Histoplasma *and Histoplasmosis*

[36, 38, 41–43].

caution [46].

**4. Conclusions**

In studies done among HIV patients, histoplasmosis-associated - IRIS was associated with histoplasmosis; IRIS was reported to be uncommon with incidence rates of 0.74 cases per 1000 HIV-infected person-years 0.5% [37–39]. However according to another study among 271 patients, the emergence of IRIS tended to be quite common in people with HIV and disseminated histoplasmosis; whether the IRIS is triggered by the Histoplasma or other co-infections, is still unclear [40]. For HIV patients with histoplasmosis to be considered to have histoplasmosis associated IRIS, they should fulfil most or all of the following criteria: AIDS with low pretreatment CD4 count (≤100 cells/μL), a positive immunological and virological response to ART, clinical manifestations of an inflammatory condition, association between ART initiation and appearance of clinical features of the inflammatory condition and; absence of evidence of ART resistance, patient non-compliance, drug allergy or adverse reactions, a concomitant non- fungal infection, or decreased drug levels due to malabsorption or drug-drug interactions

The clinical manifestations vary from case to case, and they commonly include fever, lymphadenopathy, mucocutaneous lesions, and disseminated disease. The timeline of occurrence from initiation of ART is also varied from a few days to months, and symptoms can set in when the patient is already on the ART course or when the ART is just introduced [39, 44, 45]. Histoplasmosis-associated IRIS in HIV patients may have a predilection for females over males, with one study showing

If the symptoms of IRIS are mild, the patient is managed symptomatically. The

However if the presentation is severe, corticosteroids may be used as they have been seen to be of benefit in TB-associated IRIS, though they should be used with

Once a patient exhibits features of IRIS while already on effective ART, the ART should be continued and the patient should be initiated on histoplasmosis treatment immediately. However if the patient is not receiving ART, a two-week delay is encouraged before starting ART while the patient is on antifungal therapy (ampho-

Itraconazole alone is used for the treatment of mild forms of histoplasmosis and as a step-down therapy in severe disease and for secondary prophylaxis to prevent relapse in the immunocompromised after induction therapy with amphotericin B. Moderate-to-severe acute pulmonary histoplasmosis as well as acute progressive disseminated histoplasmosis require intravenous amphotericin B therapy for at least 2 weeks (4–6 weeks if meningeal involvement) or until a patient can tolerate oral therapy, then oral itraconazole (or an alternative triazole) for at least 12 weeks (for acute pulmonary) or 12 months (for acute progressive disseminated histoplasmosis). Chronic cavitary pulmonary histoplasmosis is treated with itraconazole for 12–24 months. There is insufficient evidence to support the use of isavuconazole

that it was four times more frequent in females than males [39].

role of steroids in treating histoplasmosis-IRIS is yet unclear [39].

tericin B or itraconazole) as per the guidelines [14].

and the echinocandins for the treatment of histoplasmosis.

**74**

### **Author details**

Felix Bongomin1 \*, Richard Kwizera<sup>2</sup> , Joseph Baruch Baluku3,4, Lucy Grace Asio1 and Akaninyene A. Otu5

1 Department of Medical Microbiology and Immunology, Faculty of Medicine, Gulu University, Gulu, Uganda

2 Translational Research Laboratory, Infectious Diseases Institute, College of Health Sciences, Makerere University, Kampala, Uganda

3 Division of Pulmonology, Mulago National Referral Hospital, Kampala, Uganda

4 Department of Programs, Mildmay Uganda, Uganda

5 Department of Internal Medicine, University of Calabar, Calabar, Nigeria

\*Address all correspondence to: drbongomin@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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nih.gov/pubmed/31404979

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Disease Clinics of North America.

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### *Edited by Felix Bongomin*

*Histoplasma and Histoplasmosis* is a book on epidemiology, diagnosis, and treatment of histoplasmosis. It is an all-inclusive source of knowledge for both specialist and non-specialist mycologists at various levels of training or work experience.Through collaborative efforts of the authors and the editor, this book provides up-to-date information on the global distribution of histoplasmosis, an updated estimation of the burden of histoplasmosis in Asia, and recent advances in laboratory diagnosis and treatment of histoplasma and histoplasmosis. This volume is an invaluable source of knowledge for trainees and practitioners alike.

Published in London, UK © 2020 IntechOpen © utah778 / iStock

Histoplasma and Histoplasmosis

Histoplasma and

Histoplasmosis

*Edited by Felix Bongomin*