Epidemiology and Immunopathology of Leishmaniases

## **Chapter 2** Epidemiology of Leishmaniasis

*Stephen Kyari*

#### **Abstract**

A zoonotic illness of importance to the public's health is leishmaniasis. *Leishmania donovani*, *Leishmania tropica*, *Leishmania major*, *Leishmania infantum*, *Leishmania chagasi*, *Leishmania mexican*, and *Leishmania braziliensis* are the most recognised and widely distributed leishmania parasite species, and they are also the ones that cause the disease. On every continent and in more than 90 countries, the disease is present, however it appears to be absent from Australia. The disease is spread by Phlebotomus sandflies, and people, rodents, and other domestic animals act as reservoirs and unintentional hosts. Cutaneous leishmaniasis, diffuse cutaneous leishmaniasis, mucocutaneous leishmaniasis, and visceral leishmaniasis are the four ways the illness can present. The spread of the disease, as well as its appearance and reemergence, are caused by risk factors include regional warfare and wars, political instability, migration of people, substandard housing, climate, vegetation cover, p7oor socioeconomic standard of life, and lack of access to quality medical care. To eradicate the disease, particularly in poor nations where it is still a threat, there is a need for increased public education, government involvement, proper surveillance, and disease reporting.

**Keywords:** epidemiology, distribution, risk factors, Phlebotomus sandfly, leishmaniasis

#### **1. Introduction**

Leishmaniasis is a disease that is caused by the parasite Leishmania; comprising of at least 20 species that affect humans. The parasite is primarily transmitted by the sand flies that belongs to the Phlebotomus in the old world and Lutzomyia in the new world; however, other *principal hosts* are rodents such as *Rhombomys opimus, Tatera indica, Meriones hurrianae, and Meriones libycus gerbils which are of the Zoonotic cutaneous leishmaniasis (ZCL)* [1–5]*. The disease is associated* with and limited by the geographical distribution of its sand fly vectors. Fifteen geographical entities have been classified worldwide, of which 13 zoonotic in nature. In recent years, there have been significant increase in the number of regions becoming *Leishmania*-endemic, accompanied with an increased number of animal and human cases. These parasites have been reported to be maintained and circulated in mammals with each species having a specific reservoir host [6]*.* The clinical spectrum of the disease in humans can be asymptomatic infections in some individual and can result in high mortality, which depends on the form of the disease and can be categorised into three (4) namely; visceral leishmaniasis *(*VL*)*, cutaneous leishmaniasis *(*CL*), diffuse cutaneous leishmaniasis (DCL)* and mucocutaneous leishmaniasis (MCL*)*. In dogs, *Leishmania infantum* and *Lieshmania chagasi are* now regarded as synonyms *and are the species known* to be

responsible for chronic visceral-cutaneous disease in this host (canine leishmaniosis). Asymptomatic infections occur in dogs and other canines thereby maintaining the presence of the parasite in endemic regions [6]. The ability of the host macrophages to effectively destroy the parasite is as a result of host- parasite interaction which is responsible for clinical appearance. Leishmaniasis is classified as a prominent neglected disease of the tropics due to the number of death per year (40,000) with a global distribution with Indian sub-continent having the highest number of cases followed by Bangladesh, Sudan, South Sudan, Ethiopia, and Brazil [7, 8].

#### **1.1 Cutaneous leishmaniasis**

The aetiological agent responsible for cutaneous leishmaniasis are as follows: *Leishmania tropica*, *Leishmania major*, and *Leishmania aethiopica*, also known as oldand new-world leishmania species, are found in southern Europe, the Middle East, Asia, and Africa, respectively. These species include *Leishmania guyanensis*, *L. viannia braziliensis*, *L. viannia amazonesis*, *L. viannia panamensis*, and the *Leishmania mexicana* complex (Latin America). Skin ulcers on the exposed body parts, such as the face, arms, and legs, as well as lesions that leave the patient permanently disfigured and prone to social judgement, are the hallmarks of cutaneous leishmaniasis. The incubation time is roughly 2 to 6 weeks [9]; cases of up to 3 years have been documented for Old World cutaneous leishmaniasis [10], whereas the incubation period for New World cutaneous leishmaniasis is typically 2 to 8 weeks [11]. Over 85% of newly discovered CL cases in 2018 that were reported to the WHO originated in Afghanistan, Algeria, Bolivia, Brazil, Colombia, Iran, Iraq, Pakistan, Syria, and Tunisia, while over 95% of newly discovered LV cases originated in Brazil, China, Ethiopia, India, Iraq, Kenya, Nepal, Somalia, and Sudan. Finally, four nations—Brazil, Bolivia, Ethiopia, and Peru reported almost 90% of all new cases of mucocutaneous leishmaniasis (MCL) [12].

#### **1.2 Mucocutaneous leishmaniasis**

*Leishmania viannia braziliensis*, *leishmania viannia amazonesis*, *leishmania viannia panamensis*, and *leishmania viannia guyanesis* are the leishmania species that cause mucocutaneous leishmaniasis. The progression of this illness is controlled by parasite virulence and host cell mediated immunity. The condition starts with erythema and ulceration of the nose, which progresses to continuous degradation of the cartilage in the upper airway and facial structures, oronaso-pharyngeal mucosa, disfigurement, and obstruction of the airway [13].

#### **1.3 Diffuse cutaneous leishmaniasis**

Even in nations where leishmaniasis is endemic, diffuse cutaneous leishmaniasis is a rare manifestation of the illness. *Leishmania mexicana* and *Leishmania aethiopica* are the parasites that cause this type of leishmaniasis. A small, painless bump at the site of the innoculation causes spreading, non-ulcerating macules, nodules, and plaques that range in colour from erythematous to violet. The face, upper and lower limbs, and buttocks are the area most commonly affected [14, 15].

#### **1.4 Visceral leishmaniasis**

*Leishmania donovani* is the cause of visceral leishmaniasis. The most serious type of the disease, which affects internal organs like the spleen and liver and can

#### **Figure 1.**

*Shows the geographical distribution pattern of leishmaniasis [20].*


#### **Table 1.**

*Shows old world and new world cutaneous leishmaniasis [21–23].*

be deadly if neglected, is Kala-azar cutaneous leishmaniasis, which ranges from hypopigmented macules to infiltrating papules [16]. The incubation period ranges from three to eight months [17]; symptoms include fever, weight loss, hepatosplenomegaly, lymphadenopathy, pancytopenia, and hypergammaglobulinaemia; skin pigmentation may also be present [18]; "kala azar," also known as black disease, may be asymptomatic and self-resolving, but typically has a chronic course and can be fatal with or without treatment [19]; death typically results due to bacterial infections. In the Indian subcontinent, Bangladesh, India, and Nepal are the most severely affected and account for 28% of cases, while Brazil contributes 20%. The three eco-epidemiological hotspots where VL has been identified are East Africa, where countries like Ethiopia, Kenya, Somalia, South Sudan, Sudan, and Uganda contribute 45% of global cases (**Figure 1** [20] and **Table 1** [21–23]) [24].

#### **2. Main text**

#### **2.1 Distribution pattern, vector and causative agents of Leishmaniasis**

Leishmaniasis is endemic in several parts of Africa. In North Africa, nations including Morocco, Algeria, Tunisia, Libya, and Egypt have recorded numerous instances of the disease, with cutaneous and visceral leishmaniasis being the most prevalent types [25]. Important species that are spread by the phlebotomus sand fly include *L. major, L. tropica, L. infantum*, and *Labrus donovani. L. major* is the main cause of cutaneous leishmaniasis, with *L. tropica* and *L. infantum* playing a less significant role. Visceral leishmaniasis (VL) has been linked to *L. donovani*, *L. infantum*, and infrequently *L. tropica*. Zoonotic cutaneous leishmaniasis (ZCL) and Anthropogenic cutaneous leishmaniasis (ACL) are the two kinds of cutaneous leishmaniasis seen in this region [6, 26–28]; with ZCL being caused by *L. major* and transmitted by mainly by *P. papatasi, P. duboscqi* and *P. salehi with P. papatasi* considered among the most widespread invasive sandflies species in these regions and the also has a worldwide distribution; *Psammomys obesus*, *Meriones libycus*, *Meriones shawi*, *Nesokia indica*, and *Rhombomys opimus* rodents also transmit the disease, while ACL is caused by *L. tropica* is transmitted by *P. sergenti* and gundi and hyraxes and *Leishmania killicki* with dogs also serving as accidental host in some cases [6, 29–33]*.* Additionally, there are two types of visceral leishmaniasis: (ZVL). Anthropogenic visceral leishmaniasis (AVL), which is caused by *L. infantum*, is primarily zoonotic, with candida serving as the principal reservoir and humans serving as unintentional hosts [34–36]. The most common form of leishmaniasis in Morocco, Algeria, and Lybia is cutaneous leishmaniasis, which is caused by the *L. tropica* species [37–40]. According to reports, *L. tropica* is a reservoir host in Ethiopia and Chad has noted an upsurge in visceral leishmaniasis in Central Africa [41, 42].

The countries of Sudan, South Sudan, Ethiopia, Kenya, Uganda, and Somalia make up one of the main geographic regions in East Africa most severely affected by VL, where the disease is primarily brought on by *Leishmania donovani* and, in certain cases, Leishmania infantum [43–45]. With the confirmation of *Phlebotomus orientalis, Phlebotomus martini, Phlebotomus celiae,* and *Phlebotomus rodhaini* as VL vectors [46–50], man and dogs serve as potential reservoir hosts. Subgenera such as Paraphlebotomus, Synphlebotomus, Larroussius, and Anaphlebotomus have also been reported [51, 52]. In East Africa, children under the age of 15 make up about 65% of VL cases, and having inadequate levels of protein, energy, iron, vitamin A, and zinc increases the risk of VL manifestation [53]. In East Africa, cutaneous and mucosal leishmaniasis have both been attributed to *Leishmania donovani* [54–56]. VL has been reported and is prevalent in Kenya, Uganda, and Somalia along their borders; *L. donovani* and *L. infantum* are the parasites responsible for the disease [57–59]. Every year, Sudan reports over two-thirds of the VL cases in East African nations; the majority of these cases are presumed to be caused by *L. donovani* [60, 61], and recent outbreaks in South Sudan have been linked to over 100,000 fatalities between 1983 and 2005 [62]. It is possible for VL to develop complications that lead to post-kalaazar cutaneous leishmaniasis, which is characterised by a macular or nodular rash that frequently starts at the mouth and is transmitted by *P. argentipes* and *P. orientalis* [63].

The disease has also been documented in Cameroon, Burkina Faso, Mauritania, Gambia, Guinea, and Nigeria in West Africa. Lieshmaniasis was originally identified in Niger, then in Nigeria, Senegal, and Mali. Mauritania, Gambia, Senegal, Nigeria, Cameroon, and Ghana are among the countries where CL is endemic and is caused by *L. major* [64, 65]. The only known case of MCL in the area is in Senegal, while the only instances of VL have been recorded in Togo and Burkina Faso with *L. donovani* as the causing agent [66]. Rodents including *Mastomys erythroleucus, Tatera gambiana, Arvicanthis niloticus*, and *Mastomys erythroleucus*, as well as dogs in Gambia, serve as the reservoir host for L. major. The parasites *L. major* and *L. donovani*, which transmit VL and CL in Ghana, Nigeria, and Senegal, respectively, are the most prevalent forms to be recorded together with *P. duboscqi* and *P. rodhaini* [66–69].

#### *Epidemiology of Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.110490*

Syria has historically been endemic to CL due to *L. tropica* for centuries and, at one point, had the highest burden of this form of leishmaniasis in the region. Recent research indicates that political unrest, wars, and population migration have contributed to an increase in cases in the Middle East [70]. Due to the influx of migrants, these circumstances have caused outbreaks to be reported in 2012 in other countries including Lebanon, which have seen a rise in the number of cases recorded [71, 72]. *L. tropica* has also been claimed to be endemic in Israel and the Philistines, while the cycle of transmission is not completely understood. With a widespread geographic distribution throughout the region, *Phlebotomus (Paraphlebotomus) sergenti* has been identified as the primary vector of *L. tropica* [73–75]. *Phlebotomus arabicus* and *Phlebotomus similis* have also been documented. Leishmania major has reportedly been linked to CL in Southern Israel; this type of the illness is self-healing but makes the patient anxious. The reservoir host is the obese sand rat *Psammomys obesus* (Cricetidae: Gerbillinae), and *P. obesus* and the sand fly *Phlebotomus papatasi* (Diptera: Psychodidae) serve as the vectors [76]. *Leishmania majori*, the parasite that causes CL and VL, is the primary carrier of both the disease and its primary vector, *P. papatasi*. VL is primarily brought on by *Leishmania infantum*, though *L. tropica* can also be to blame in some cases. CL comes in two forms: ZCL and ACL, which are caused by *L. major* and *L. tropica*, respectively. Rodents are the main reservoir hosts, and some of the more endemic species include *Rhombomys* (*R*.) *opimus*, *Meriones libycus*, *Tatera* (*T.*) *indica*, and *Meriones hurrianae*. *Phlebotomus papatasi* transmits *L. major*, whereas *Phlebotomus sergenti* transmits *L. tropica* [70, 77–79]. ACL and ZCL are present, *L. tropica* and *L. major* are endemic in Saudi Arabia, and CL is declining with fewer instances being reported [80–83]. The most common kind of leishmaniasis in Iraq is CL, which is caused by the parasites *Leishmania tropica* and *Leishmania major* [84–86]. ACL and ZCL are the two types of CL that are endemic in Pakistan [87]; *L. tropica* and *L. major* are the two known forms of the parasite that transmit CL [88–90]. The frequency of ACL is higher than ZCL in Afghanistan, where *L. tropica* is known to account for 96 to 98% of CL cases [91]. ACL is said to be endemic in Turkmenistan, with *L. major* as the causative agent [8].

Countries in Europe, including Greece, have reported occasional cases of CL caused by *L. tropica*; *Phlebotomus sergenti* has been identified as the disease's primary vector and is thought to be responsible for its spread [92]. There have been reports of *Phlebotomus tobbi* and *Phlebotomus perfiliewi* as potential *L. tropica* vectors [93]. Although CL caused by *L. tropica* has not been documented in Europe, *P. sergenti* was found in Greek camps where 21 different sand fly species had significant levels of *L. tropica* infection. The emerging and re-emerging of leishmaniasis in Europe has been attributed to the introduction of exotic *Leishmania* species or strains via worldwide travelling of humans and domestic dogs; this is also responsible for the spread of visceral and cutaneous leishmaniasis caused by *L. infantum* and *L. tropicai* due to their endemicity from the Mediterranean region of Europe to neighbouring temperate areas where there are vectors without disease [90, 94]. A high risk of leishmaniasis spreading to Europe is posed by both zoonotic cutaneous and visceral leishmaniasis caused by *L. infantum* in the Mediterranean region and anthroponotic cutaneous leishmaniasis caused by *L. tropica* in Greece. Additionally, the presence of Phlebotomus sandflies without the disease is of public health concern [95]. A retrospective study of leishmaniasis across Europe revealed that cutaneous leishmaniasis was the prevalent form of the disease that occurred in 11 countries followed by visceral and mucosal disease with *L. donovani, L. infantum* (syn. *L. chagasi* in New World), *L. major, L. amazonensis* (syn. *L. garnhami), L. Mexicana, L. aethiopica,* 

*L. tropica, L. braziliensis, L. peruviana, L. guyanensis, L. panamensis, L. lainsoni, L. naiffi, L. siamensis /Lechytia martiniquensis* been the parasites identified and responsible for the disease [96].

In North America, parasite species in the genus Leishmania are responsible for different clinical pathologies of the disease which include VL that is deadly and caused by *L. chagasi*, as well as MCL, localised Leishmaniasis, diffuse Leishmaniasis; MCL has been reported to be caused by *L. brasiliensis*, *L. panamensis* and *L. guyanensis*; DCL are related to *L. m. mexicana, L. amazonensis, L. pifanoi, L. guyanensis*, and *L. panamensis* [97]. Female blood-feeding sand flies of the genus Lutzomyia (Diptera: Psychodidae: Phlebotomidae) are the transmission vector in Mexico, the United States, and Canada; humans are incidental hosts while mammals serve as reservoir hosts; *L. mexicana* is responsible for the majority of human cases that have been reported [98]. Leishmania infantum, which causes canine visceral leishmaniasis, has been reported in dogs and cats. While cutaneous *Leishmania mexicana* is endemic to the United States, visceral leishmaniasis is not considered endemic among humans but primarily affects dogs, which are thought to be the reservoir host for humans [99, 100].

*Leishmania (Viannia) braziliensis, L. (Viannia) amazonensis, L. (Viannia) guyanensis*, and *L. Infantum*, associated with visceral leishmaniasis, were the four species of Leishmania cases reported in humans in South America. In addition, 28 species of Phlebotominae (23 species of Lutzomyia, 4 species of Brumptomyia, and 1 species of [101]. Both CL and MCL are induced by *L. Braziliensis*: *Lutzomyia neivai*, *L. Whitmani*, *L. Cortelezzii* complex (*L. Cortelezzii, L. Sallesi*), *L. Migonei*, and *L. Pessoai*, while V is brought on by *L. Infantum* was *L. Longipalpis* [102]. However, domestic and wild canids have been well established as the reservoir host of leishmania, with more than 20 species of the parasites being transmitted by the phlebotomine vectors (Diptera, Psychodidae); CL and VL are the two major forms of the disease where *Leishmania (Viannia) braziliensis*, *Leishmania (Leishmania) amazonensis, L. mexicana, L. donovani* causes VL in the old world, but *L. infantum* does so in the new world [103–107]. In Colombia several species of *Leishmania* parasites have been reported to infect humans with CL been the major form of the disease followed by MCL and VL; *L. braziliensis*, *L. panamensis* and *L. guyanensis* have been responsible for the outbreaks of CL; other parasites include *L. lainsoni*, *L. amazonensis*, *L. infantum chagasi*, *L. Mexicana*, *L. colombiensis* and *L. equatoriensis,* this country is ranked second only to Brazil in terms of cases of leishmaniasis [108–110]. In Bolivia, *Leishmania (Viannia) lainsoni, Leishmania (Viannia) guyanensis, Leishmania (Viannia) panamensis and Leishmania (Leishmania) amazonensis* have been described as a causal agent of Tegumentary leishmaniasis (TL) (CL, MCL, DCL) with transmission also reported to be zoonotic and caused by *Nyssomyia neivai,* Cortelezzii complex, *Evandromyia sallesi*, *Migonemyia migonei* and *Micropygomyia quinquefer* [111].

The parasite *L. donovani*, the vector *Phlebotomus annandalei*, Phlebotomus argentipes sensu stricto, and Phlebotomus glaucus, and the disease CL, MCL, and VL are all endemic throughout Asia [112]. VL, which is indigenous to Nepal and brought on by *L. donovani*, can occasionally lead to post-Kala-azar cutaneous leishmaniasis [113]. Similar to Kala-azar sickness, VL is chronic in Bangladesh and can lead to it. *L. donovani* is thought to be the main cause of the illness, while *Phlebotomus papatasi* is the disease's vector in Asia [114, 115].

*Leishmania donovani* is the chronic disease that causes VL on the Indian subcontinent; however, *L. tropica* or *L. infantum*, particularly when co-infected with HIV, can also cause the illness; *Phlebotomus argentipes* is the vector for VL, with humans


**Table 2.**

*The table below shows the clinical form of leishmaniasis and their geographical distribution [21–23].*

functioning as reservoir hosts. PKDL does not have an animal reservoir, and transmission is thought to be anthroponotic [116, 117]. VL is also known as kala-azar, which is the Hindi word for "black disease" (**Table 2**).

#### **3. Leishmaniasis and HIV co-infection**

There have been reports of leishmania-HIV co-infection in 45 countries in 2021, with Brazil, Ethiopia, and Bihar in India having the highest number of cases [118]. According to a report by the WHO in 2022, people who have leishmaniasis and HIV are at an increased risk of developing full clinical disease, high relapse rates, and mortality rates. Leishmaniasis and HIV co-infection have also been shown to occur in CL and VL [119, 120]. A change in immune response brought on by immunosuppression caused by a low CD4 T-lymphocyte count and the development of immunological activation with cell senescence are two examples of the clinical manifestation of VL [121].

#### **4. Risk factors in the distribution of Leishmaniasis**

Conflict, climate (temperature, rainfall, relative humidity, precipitation), physical (geographical barriers), and biotic (distribution and abundance of vertebrate hosts) factors, socioeconomic circumstances, environmental (land degradation, agricultural activities, deforestation due to urbanisation), domestic animals and living standards, immune response of hosts, and conflict have all been linked to the distribution of

leishmaniasis [6]. Although no consistent pattern has been identified, civil wars, social unrest, migration, forced population displacement, rainfall, humidity, temperature, soil types and moisture contents, and land cover type are significantly associated with the distribution pattern of these sandflies and contribute to emerging and re-emerging outbreaks [18].

#### **5. Climatic conditions**

Leishmaniasis is a disease that is sensitive to climate because variations in temperature, precipitation, and humidity have a significant impact on the life cycles of the parasite's vectors and reservoir hosts. Climate changes can also affect the distribution, survival, and population of the vectors and reservoir hosts [122]. Sandflies have been reported to be abundant between the months of June and September with *P. argentipes* being active between this period in India with temperature ranging between 27 and 31°C [123, 124]. Fluctuations in temperature can negatively affect the developmental life cycle of leishmania promastigote in sandflies, which can result in the transmission of the parasite into areas not previously endemic for the disease.

#### **5.1 Vegetation cover**

Leishmaniasis outbreaks have been shown to decrease with the improvement of vegetation types that limit rodent growth [125]; it has also been established that areas with thick or dense vegetation cover have a higher risk of leishmaniasis infection and transmission [126]. Forest fragmentation and deforestation have been linked to an increased risk for leishmaniasis.

#### **5.2 Age and gender**

Leishmaniasis has been found to affect adults more frequently than children; this could be because younger people are less exposed to the parasites and vectors that cause the disease, especially in rural areas where adults are more engaged in farming and other socioeconomic activities than children are. It has also been found that men are generally more susceptible to infection, though this could vary [127, 128].

#### **5.3 Housing and socio-economic conditions**

Poor housing and the lack of good sanitary conditions in terms of waste management, sewage disposal, and drainages in the majority of rural and semi-urban centre provide breeding and resting sites for sandflies, bringing this vector closer to human habitation. These sandflies feed on blood meal. Other good breeding sites for these vectors include cracks and crevices in walls, roofs that are poorly made, and thatched homes [129]. The disease spreads as a result of people moving from rural to urban areas for economic reasons [130–132].

#### **5.4 Domestic animal breeding**

It has been discovered that there may be a significant risk of infection in houses where animals are housed, bred, or live in close proximity to people. This is because some of these animals may act as reservoir hosts for the disease; leishmaniasis, particularly the VL, affects dogs and felines [133].

#### **5.5 Host immune responses and parasite factor**

In order for the infection to take hold, promastigotes must infiltrate macrophages and avoid inducing host defences. Additionally, the progression of intracellular infection by amastigotes depends on the preservation of macrophages in an inert, deactivated state. Through cell-mediated immunity and delayed-type hypersensitivity, an immunocompetent host defence system is equipped with and reacts to non-specific innate and antigen-specific acquired mechanisms; these inflammatory responses play a critical role in disease expression and may result in asymptomatic infection and self-healing or may result in clinical manifestation of disease. People with weakened immune systems, such as HIV patients, are more susceptible to infection since their immune systems are unable to defend them against the parasites. The parasite, however, can alter intracellular kinases, phosphatases, signalling pathways, the responsiveness of macrophages to the parasite and secretion of cytokines, as well as dendritic cell and macrophage mechanisms. This allows the parasites in the case of an infection to alter, temper, or hinder with intracellular kinases, phosphatases, signalling pathways, the responsiveness of macrophages to the parasite and secretion of the parasite can also disrupt dendritic cells, which are important for antigen presentation, T-cell co-stimulation, and the effective establishment of acquired Th1 responses [134].

#### **6. Life cycle of leishmania parasite**

Leishmania parasites go through two main stages in their life cycle: the extracellular stage in invertebrate hosts and the intracellular stage in vertebrate hosts. The parasite has two primary morphologies: amastigote and promastigote, with the former occurring in vertebrate hosts and the latter in invertebrate hosts. The promastigotes are injected into the host circulation by female sandflies during a blood meal on a vertebrate host; after this inoculation, the promastigotes are phagocytosed by the host macrophages; the macrophage ruptures and releases a large number of amastigotes into circulation. Monocytes and macrophages in the spleen, liver, lymph nodes, bone marrow, and other tissues of reticulo-endothelial cells are invaded by the released amastigotes. The female sandflies then consume free amastigotes from the blood as well as intracellular amastigotes from the monocytes. The promastigotes in the sand fly's midgut move over a period of 6 to 9 days to its pharynx and buccal cavity, where the sand fly can spread the parasites to a new host after a blood meal [135].

#### **7. The vector for leishmaniasis**

Only 21 of the 700 species of sandflies (family Phlebotominae) are known to be disease vectors. The majority of these species belong to the genera Phlebotomus, Sergentomyia, and Lutzomyia; the Lutzomyia group includes species from the old and new worlds. Old-world species are those that can be found in South and Central Asia, the Indian subcontinent, the Middle East, North and East Africa, and Southern Europe; new-world species are those that can be found in Brazil and

other Latin American nations, Mexico, and the United States. These fly species are absent from Australia but are indigenous to over 90 other nations and are found on every continent [136]. The following list includes the phlebotomous sandfly species that cause leishmaniasis in the old world. The transmission of *L. major, L. tropica, L. infantum,* and *L. aethiopica* is carried out by *P. longicuspis, P. papatasi, P. ariasi, P. pedifer, P. duboscqi, P. rhodaini, P. segenti, and P. sergenti.* The new world sandfly genus Lutzomyia includes species including *L. hartmanni*, *L. gomezi*, *L. longipalpis, L. Ayacuchensis, L. Sanguinaria, L. Trapidoire* responsible for the transmission of *L. infantum, L. vianna equatorensis, L. colombiensis*, and *L. vianna braziliensis*, among others. The Psychodopygus, which includes species like *P. panamensis, P. carrerai*, and *P. amazonensis*, is another type of sandfly of significant medical and veterinary significance [42, 134–144].

### **8. Conclusions**

*L. donovani*, *L. tropica*, and *L. major* are the species that cause infection in Africa, L. tropica, L. major, and Leishmania infantum are the species that cause infection in the Middle East, *L. tropica, L. infantum*, and Europe, *L. mexican*a and *L. chagasi* are the species that cause infection in North America, and *Leishmania (Viannia) braziliensis* is the species that causes infection in South America. The disease is spread by numerous different species of Phlebotomus sandflies. Risk factors for the disease include climatic factors, vegetation cover, age and gender, housing and socioeconomic circumstances, animal breeding, and host immunity. Additionally, migration of people from other continents has been to blame for the emergence and re-emergence of the disease in Europe where there is civil and regional strife, political instability, and increased disease transmission and dissemination between continents. Obstacles to the quest to eradicate the disease include limited access to health care, a lack of national policy, and the maintenance of such policy, if it exists, particularly in developing nations.

### **Acknowledgements**

Non to be disclosed.

### **Conflict of interest**

The authors declare no conflict of interest.

*Epidemiology of Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.110490*

### **Author details**

Stephen Kyari Department of Veterinary Parasitology and Entomology, Ahmadu Bello University, Zaria, Kaduna State, Nigeria

\*Address all correspondence to: kyaristephen757@gmail.com

© 2023 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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#### **Chapter 3**

## Epidemiology of Visceral Leishmaniasis in India

*Rajan R. Patil and Prabir K. Chatterjee*

#### **Abstract**

Kala-azar is a leading parasitic infection of great epidemic and mortality potential. More than 90% of Incident cases brought to notice of WHO in 2019 were mainly reported from 10 countries. Four endemic states in India namely Bihar, Jharkhand, Uttar Pradesh and Jharkhand have high disease burden of Kalazar. All 4 endemic states have to mandatorily notify cases to the National Vector Borne Disease Control Programme (NVBDCP) every month, even if there are zero cases. In recent years of Kala-azar cases, India have witnessed reduction of 97% largely due to the introduction of single-dose AmBisome –in India has been the game changer. There are three forms of leishmaniasis seen in India Kala-azar, they are Visceral Leishmaniasis, Post Kalazar Dermal Leishmaniasis (PKDL) and Cutaneous leishmaniasis. PKDL patients harbor the parasite and may be the source of new infection to the vector even 20 years later. Poverty enhances the risk for Kala-azar. Poor housing and domestic sanitary conditions are good breeding ground for sandfly which is the vector for Visceral Leishmaniasis, as well as resting sites and their ease of sandfly contact with humans. Kala-azar is a climate-sensitive disease as any change in temperature and humidity influences vector breeding.

**Keywords:** Kalazar, epidemiology, leishmaniasis, tribal population, India

#### **1. Introduction**

Kalazar is the widely used term for Visceral leishmaniasis (VL) in India. As per WHO Global Health Observatory, which is basically distribution maps of VL cases using the DHIS2 platform and has an interactive dashboard [1], VL is endemic in 78 countries. India, East Africa, and Brazil are the countries where most cases occur. Worldwide, about 50,000 to 90,000 new KA cases occur every year, however, only 25–45% are reported to WHO [2].

Kala-azar is a leading parasitic infection of great epidemic and mortality potential. More than 90% of incident cases that are brought to the notice of WHO in 2019 were mainly reported from 10 countries: India, Eiteria, Iraq, Somalia, Brazil, Ethiopia, Kenya, South Sudan, Sudan and Nepal. India's share of global burden of Kala-azar in 2020 has been 18%. Kala-azar remains a significant public health issue in India with 54 districts scattered in four states — Bihar (33 out of 38 districts), Jharkhand (4 out of 24 districts), Uttar Pradesh (6 out of 75 districts) and West Bengal (11 out of 23 districts). Stratified analysis showed that there are 633 blocks spread over four states which are endemic. Sporadic cases are also detected in states like Gujarat, Assam, Himachal Pradesh Haryana Tamil Nadu, Puducherry, Madhya Pradesh, Kerala, Sikkim, Uttaranchal and Jammu & Kashmir [3].

As per the instructions received from National Vector Borne Disease Control Programme (NVBDCP), All 4 states endemic to Kala-azar, namely Bihar, Jharkhand, West Bengal and Uttar Pradesh, have to mandatorily notify cases of the NVBDCP every month, even if there are zero cases. The spot map of Indian subcontinent (**Figure 1**) shows the distribution of various species of Leishmaniasis prevalent in India and its neighboring countries. Within India, Kalazar is clustering in eastern India as per data collected in last 2015 to 2019 [4].

*Leishmania donovani* is the pathogenic organism for Kalazar, and *Phlebotomus argentipes* is the main vector, particularly in India, commonly called as sand fly. Animal carriers are not known in India, though in a few countries, animal carriers [5] do exist in Europe e.g., foxes, and in China dogs are known to carry the infection. Disease spread of Kalazar is highly localized: over 3/4th of inferred infectors are seen to be at a mean distance <300 m to their respective secondary VL cases.

*Epidemiology of Visceral Leishmaniasis in India DOI: http://dx.doi.org/10.5772/intechopen.112444*

After many years (since 1992) of accelerated Kalazar elimination programme implementation, the cases of Kalazar cases in India have witnessed a reduction by 97%, while the reported deaths have fallen from 1419 in 1992 to 58 in 2018, and only 37 deaths were reported in 2020, formally [6].

Kalazar elimination as a public health problem is defined as achieving an annual incidence of less than 1 case per 10,000 population at the district level in Nepal and at the subdistrict level in Bangladesh and India.

National Kalaazar Elimination Programme NKEP, now integrated into National Vector Borne Disease Control Programme (NVBDCP) has set an ambitious goal of zero KA transmission by 2030. This goal will be attained through early diagnosis and prompt treatment, integrated vector management, operational research, effective disease surveillance, supervision, monitoring and evaluation and social/community mobilization and partnerships [7].

#### **2. Kalaazar (visceral leishmaniasis)**

Kalazar in its severe form is associated with high mortality in the absence of prompt diagnosis and treatment. Kalazar is prevalent in all the countries (**Figure 2**) in Indian subcontinent [4]. After Malaria, Kalazar comes second in ranking for causing maximum number of deaths by a parasitic disease. It is reported that 20,000


#### **Table 1.**

*Signs and diagnosis of Kalazar.*

to 30,000 deaths occur worldwide due to Kalazar. The term Kalazar is derived from the word 'kala' which means black referring to black discoloration of skin in kalazar affected person. Since there is great similarity between the symptomatology of Kalazar and Malaria, the propensity to misdiagnose the disease is common. Misdiagnosis is perilous to the patient as the case fatality rate in Kalazar cases is 100% in the absence of proper treatment. In severe Kalazar, the death of the patient is not due to pathogen (L D bodies) but instead, due to opportunistic infection resulting from immune-compromised status due to Kalazar disease. The death by Kalazar is often due to secondary infections such as Dysentery, Pneumonia and Tuberculosis. which are highly prevalent in the communities where Kalazar is rampant [9, 10].

Symptoms of Kalazar are fever with chills. Generally, fever occurs twice a day. If not diagnosed and treated, the fever continues, though it is not so high after the initial days. Loss of appetite and severe weakness are other symptoms (**Table 1**).

#### **3. Post-kala-azar dermal leishmaniasis**

Post Kalazar dermal Leishmaniasis is a complication of Kala-azar, and is mainly seen in India, South-East Asia, and East Africa. It is manifested by discolored (hypopigmented) flat skin (macular) rash, combined with some slightly elevated (maculopapular) or elevated (nodular) rash. It is manifested in Kalazar patients who are cured of the VL. Manifestation of Post-kalazar dermal leishmaniasis (PKDL) usually occurs in the period of six months to one or more years after treatment and cure of Kala-azar, at the same time it may occur earlier or even along with Kala-azar. PKDL heals spontaneously in most cases in Africa but rarely in patients in India [11, 12]. PKDL patients harbor the parasite and may be the source of new infection in susceptible children even 20 years later.

In 6 districts of Bihar peak PKDL was in 2018 and in 2 districts it was in 2017. Recent papers suggest that the 2 districts achieved control around 2015 and elimination is likely between 2021 and 2023. In other districts it will not happen before 2022 given current spray rates (less than 50% in Kishanganj, between 60 and 70% in Gopalganj and Patna), endemicity (>2) and delays in treatment (60 days). PKDL was increasing in Jharkhand. There were 1800 reported cases of PKDL in India in 2020: 1034 were in Bihar, 502 in Jharkhand, 108 in UP and 156 in West Bengal. (NVBDCP, Ministry of Health, Govt of India.)

#### **4. Cutaneous Leishmaniasis**

Cutaneous Leishmaniasis is Less prevalent in India- sporadic cases are found in western India. Cutaneous manifestations are characterized by skin lesions mostly ulcers especially on exposed parts such as face, neck, and extremities. Skin lesions are the major reason for disability and stigma. As per the reported cases, 95% of cutaneous leishmaniasis worldwide comes from American continent, the Mediterranean region. There is gross under reporting of cutaneous leishmaniasis, it is understood that the actual number of cases may be in the range of 600,000 to 1,000,000, however only 200,000 cases are reported to WHO.

In the Indian region, Cutaneous Leishmaniasis is a highly localized public health problem in western India, restricted to one state of Rajasthan, especially in the Thar desert of Bikaner district. It is located at the center of Thar desert. The Bikaner city is surrounded by stone walls. Bikaner being a desert city expectedly the weather will be hot and dry with very little rainfall [13, 14].

Less common in India- mostly found in western India. CL cases were reported in 4 in Jammu, 2 in Punjab, 1 in Haryana, 1 in Delhi, 16 in Rajasthan, 2 in UP, 2 in Assam/Atypical CL 13 in Himachal, 2 in W Bengal, 5 in Kerala, 1 in Andhra, 2 in Maharashtra, 2 in Gujarat. (NVBDCP, Ministry of Health, Govt of India.)

#### **5. Kala-azar-HIV co-infection**

The chance of developing visceral Leishmaniasis clinical manifestation of disease accompanied by high relapse and death rates is very high in Kalazar-HIV coinfected cases due to Immuno- compromised status. Prompt treatment with antiviral treatment decreases the chance of disease development, keeping in check the relapses and enhancing the survival rates [15].

As of 2021, leishmania-HIV co-infection has been reported in 45 countries. Brazil, Ethiopia, and the state of Bihar in India have reported high Leishmania-HIV coinfection rates [16].

Due to the low immunity in Kalazar patients, many other co-infections, such as Tuberculosis and Dysentery, are commonly seen. Malaria and TB co-exist in Jharkhand.

Bleeding from Gums occurs due to low platelet count. Also seen is Noma (a Cancrum oris like growth) a known complication VL-HIV co-infection cases were 141 in 2018 and 91 in 2019 in Bihar. They were 7 in 2018 in Jharkhand and 2 in 2019. (NVBDCP, Ministry of Health, Govt of India.)

#### **6. Treatment**

Treatment of Kalazar is not simple, the drug administration should necessarily be done by trained health personnel since most of the anti-leishmanials are injectable drugs. All the Kalazar affected cases are required to comply with the drug regimen and take complete treatment [16].

Early diagnosis and prompt treatment of Kalazar result in decline in the prevalence of the Kalazar disease and hence reduction in the resultant disability and death. Epidemiologically, it helps in interrupting the chain of transmission subsequently reduction in disease burden in the community. Although anti-leishmanial drugs are

costly, however highly subsidized procumbent by WHO has brought down the cost of medicine. Affordable medicines have helped improve access to essential medicine making in public health institutions. Good part is anti-leishmanials that are absolutely free to all the Kalazar patients [17, 18].

Liposomal Amphotericin-B (AmBisome) is the safest and effective injection. Other medicines include Sodium Stibonate (Antimony) Gluconate or SSG injections. Oral medicine Miltefosine, and Paramomycin (Injection)- which appear to be less effective.

#### **6.1 Dosing**

AmBisome is given as a Single dose in India and as 6/7 Doses in other countries (4 mg/kg/body wt.). SSG is given as 20 mg/kg body weight once a day for 30 days.

Miltefosine is given as 100 mg/day. Metclopramide – 5 mg/day is given along with Miltefosine.

Although single-dose AmBisome treatment is available in all endemic areas and satisfactory cure and relapse rates are reported (>95%). Significant delays between onset of symptoms and treatment were noted in Bihar and Jharkhand, due to the lack of diagnostic capacity at facility and case management.

#### **7. Major risk factors**

Socioeconomic conditions: Poverty enhances the risk for Kala-azar. Poor housing and domestic sanitary conditions are good breeding ground for sandfly which is the vector for Visceral Leishmaniasis, as well as resting sites and their ease of sandfly contact with humans. The mud plastered on a bamboo framework with cracks in the walls of houses is a typical breeding place. Lack of sunlight (due to there being no windows) is another feature as cool, dark spaces attract sandflies. Houses with crowding attract sandflies as these provide a good source of blood meals. Human behavior, such as sleeping outside or on the ground, may increase risk. Sandflies are zoophilic. So, keeping cattle inside the house and sleeping close by increases the chance of human beings getting Kalazar [19].

Malnutrition: Diets lacking in protein-energy, iron, vitamin A and zinc increase the risk that an infection will progress to a full-blown disease.

Population migration and movements of non-immune population in areas with active infection circulation are ideal conditions for epidemics of cutaneous as well as Kalazar.

#### **8. Environmental changes**

Kalazar is climate-sensitive as it affects the epidemiology in several ways:


*Epidemiology of Visceral Leishmaniasis in India DOI: http://dx.doi.org/10.5772/intechopen.112444*

• Disasters like flood, drought and famine lead to massive human migration of population to newer areas that may or may not have Kalazar which may initiate fresh Kalazar transmission [21].

India has significantly improved Kalazar control programme. In endemic villages that have reported cases of Kala-azar over the past 3 years, 2 rounds of indoor residual spraying are being applied. Development of resistance in vectors has been the major challenge in efforts towards Kalazar elimination especially to pesticides such as DDT has led to the introduction of synthetic parathyroid for indoor residual spray in 2015 [22].

#### **9. Public health intervention for Kalazar elimination**

A series of public health measures have sustained the Indian Kala-azar elimination drive. These include:


The KA Management Information System (KAMIS) for NKEP which is essentially a web-based online platform has been in use since 2014. KAMIS holds electronic records of all KA and PKDL cases since 2013, along with data on drugs and diagnostics, IRS monitoring and vector surveillance. Data are entered in real-time at the implementation level by trained data operators.

To ensure prompt diagnosis and effective treatment, financial incentives are provided to community health workers, and wage losses have been compensated for KA and PKDL cases, with a good referral system with free of cost ambulance service. All Kalazar and PKDL patients are beneficiaries of the national health insurance scheme called Ayushman Bharat and avail absolutely free of cost health services including admission.

#### **9.1 The reasons**

Despite wonderful progress, last-mile challenges remain to eliminate Kala-azar. The reasons are varied ranging from factors such as difficult geographical terrain,

indigenous populations, poor socio-economic conditions poor health-seeking behavior conditions, inadequate housing, and difficulties facing the implementation of the Kala-azar programme [24, 25].

With a progressive decline in cases, it is very important to maintain strong surveillance – this is now being integrated with those of other health surveillance programmes. Intensified governmental initiatives and political will has kept Kalazar elimination on high action alert.

#### **10. Challenges in Kalazar elimination**

There are several challenges that are encountered in attaining Kalazar elimination in India broadly they can be categorized as public health management, Biological factors & adverse impact pandemic on the operational aspect of Kalazar [26].

#### **10.1 Challenges in surveillance and case management**

Even though KA clusters are known, early diagnosis remains a challenge. KA symptoms are non-descriptive and nonspecific, ruling out other prevalent febrile diseases is the best way of finding KA-suspected cases. The first point of contact in endemic rural areas for Kalazar with fever is over-the-counter consultation with pharmacist or rural health practitioner, which is responsible for delay in treatmentseeking by 35–39 days at public health institutions. However, treatment-seeking delay at public health institutions ranged from 35 days in Bihar, however, it was lower in Jharkhand, 30 days. In lower endemic states the delay was much higher, 45 days in up and 50 days in West Bengal.

There are great hindrances in KA Surveillance when villages that have not previously reported KA begin to report KA cases from such villages, although they are located within known endemic blocks and districts. The number of such villages is declining each year, from 1001 in 2018 to 627 in 2019 and 87 in 2020.

#### **10.2 KA elimination programme**

Two significant challenges in KA–HIV coinfection and PKDL which are being obstacle to KA elimination. The percentage of KA cases with HIV has remained stable at 2–5%, while surveillance for PKDL has been fluctuating, with no known baseline of the disease burden.

Little is known about the outcomes of treatment of KA-HIV coinfected and PKDL cases. Certainly, the quality of diagnosis has improved significantly, however, identification of relapsed cases is being done in a few tertiary care health facilities [27].

#### **10.3 Indoor residual spraying**

According to national protocol, challenges in IRS as per guidelines, 2 rounds of IRS are conducted every year for sand fly control; however, the timing of IRS and the duration of spray rounds are affected by local contexts, IRS activity is affected by crop harvesting, elections, rains, migrations for work, all of which lead to delay in spraying activities or incomplete coverage [28].

As the spray pumps used in the programme are imported, obtaining spare parts (controlled flow valves, nozzle tips, lead gaskets, filter strainer) has been a challenge.

#### **10.4 Impact of Covid-19 on Kalazar control programme**

The COVID-19 pandemic has adversely impacted KA elimination programme since early 2020; despite the pandemic essential vector control and case management activities have been continued. The impact of COVID-19 on NKEP is yet to be assessed. With the shrinking geographical distribution of KA cases, having a strong surveillance system becomes paramount, in an effort to integrate KA case searches with those of other health programmes in a few KA-endemic states. Access to health care services especially for KA-HIV co-infected cases is challenge as they have to travel 160–240 km for treatment at a specialized tertiary hospital, Institutions of excellence are being set up to improve access health care to specialized care for complex KA cases and to build the trained health workers for case management [29].

Inter-sectoral Coordination among health and non-health departments and clarity in roles and responsibilities among all sectors, community mobilization will be key to sustaining the gains in the post-elimination phase [30].

#### **11. Conclusions**

Strengthening of Kalazar surveillance is key component of Kalazar elimination programme through strategic human resource planning in the health system. In the last leg of Kalazar elimination efforts have to be in mission mode. Both types of management styles are bottom-up as well as top to bottom. On paper, India has one of the finest national guidelines for the elimination of Kalazar, however, translating it to practical ground application with required resource mobilization would be primary pre-requisite. The use of quality epidemiological data is necessary to make an evidenced-based decision. Keeping with the spirit of national health policy there is a need for avoiding temptation of health systems to run Kalazar control programme as a vertical program, instead of integration with primary health care. Vector control against sand flies needs to take the holistic approach of integrated vector control methods.

#### **Author details**

Rajan R. Patil1 \* and Prabir K. Chatterjee2

1 KLE University, Belagavi, India

2 Amader Haspatal, Bankura, India

\*Address all correspondence to: rajanpatil@yahoo.com

© 2023 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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#### **Chapter 4**

## Leishmaniasis Epidemiology and Psychosocial Aspect

*Ghulam Rahim Awab*

#### **Abstract**

Leishmaniasis is a neglected tropical and the most important vector-borne disease caused by protozoan parasites of the genus *Leishmania*, transmitted by female sandfly vector to the vertebrate host including human, highly correlated with poverty, malnutrition, climate and environmental factors such as crowded living conditions and poor sanitation that affect health, wellbeing, and livelihoods of millions of people around the world. Transmission is complex due to relationships between reservoir hosts, parasites, sand fly vectors, and socio-environmental risk factors. There are various clinical manifestations, ranging from spontaneously healing cutaneous lesions to potentially fatal visceral leishmaniasis caused by different *Leishmania* species. The psychological long-term manifestations leading to stigmatization, social exclusion, discrimination, and psychosocial impacts, advocating the importance of the One Health approach to combat these diseases effectively.

**Keywords:** leishmaniasis, neglected tropical diseases, vector-borne disease, epidemiology, sand fly, parasite

#### **1. Introduction**

Epidemiology of Leishmaniasis is the study of the characteristics of the parasite and sand-fly species, ecological characteristics of the transmission sites, current and past exposure of the human population to the parasite, human behavior, geographical distribution, and impacts on human populations [1]. Leishmaniasis is a multifactorial anthropo-zoonotic protozoal disease, responsible for various syndromes in humans, by way of anthroponotic and/or zoonotic transmission. The interactions among all of the agent, vector, climatic, environmental, and socioeconomic factors influence the risk of leishmaniasis [2]. Infection and transmission are initiated by inoculating of the causative agent (free promastigotes of *Leishmania*) by vector (female sand flies of the genus *Phlebotomus* and *Lutzomyia*) bite in vertebrate hosts [1]. The vertebrate hosts are attacked by female sand flies for blood meals necessary for eggs development cycle.

It is a global public health threat, a neglected grave tropical disease that portends possible poor prognosis and fatal consequences due to broad range of clinical manifestations observed worldwide with numerous endemic zones in various continents and cases are raising due to urbanization, deforestation [1, 3].

Leishmaniasis epidemiology has a major role in clarifying the etiology of particular *Leishmania* various species and variants as the cause of specific diseases, in improving our understanding of the overall characteristics of specific vectors, in determining factors affecting host susceptibility and immunity, in unraveling modes of transmission, in clarifying the interaction of parasite with environmental determinants of disease, in determining the safety, efficacy, and utility of preventive and curative weapons (personal protection, vaccines, drugs) especially in alerting and directing disease prevention and control actions [4]. Information on morbidity burden and biopsychosocial properties rates contributes directly to the establishment of priorities for prevention and control programs, whether this involves insecticide, drug and vaccine development and delivery, environmental and hygienic improvements, enhancement of socio-economic and nutritional status, personal or community behavior, agricultural and constructional processing enhancements, reservoir host and vector control, and international cooperation and communication.

#### **2. Problem statement**

Leishmaniasis is among the top three vector-borne diseases of parasite origin along with malaria and filariasis that impacts significantly on the health, wellbeing, and livelihoods of affected communities. It is also the third most common infectious disease in terms of morbidity, after tuberculosis and malaria with great diversity of parasite species, reservoirs, and vectors that play role in transmission [5]. The disease is considered as an endemic tropical and subtropical zoonotic health threat with outbreak and mortality potential occurs primarily in countries with (sub) tropical climates, disproportionally affecting individuals living with poverty, poor sanitation, malnutrition, restricted healthcare access, displacement, poor housing, weak immunity, and lack of financial resources. Existence in non-endemic areas linked to globalization, urbanization and environmental changes, migration, tourism and other international types of population movements [1]. Different species might cause different clinical features, and the severity differs from spontaneously healing lesions to life-threatening visceral disease that might be ended with more than 90% deaths if not promptly treated particularly in people living with HIV/AIDS [6]. The cutaneous leishmaniasis (CL) is the predominant clinical manifestation with skin lesions or ulcers, on the face, head, arms and legs and other exposed to sand-fly biting parts of the body [7, 8], leaving life-long scars and serious disability, disfiguration and stigma "horrific social discrimination" and cause significant maternal morbidity, and even fetal mortality during pregnancy [3, 9]. Although the disease is not lethal, the disfigurement and social stigmatization may cause or precipitate psychological disorders, restriction of social participation, reduction of health-related quality of life of affected individuals. Thus CL not only affects the physical well-being of the individual but also significantly alters their psychological, social, and economic well-being [10]. The overall leishmaniasis outcomes are determined by the inter-relationship between the individualities of the parasite, the bio-environmental aspects of the vector, and the immune response of host [1].

#### **3. Problem magnitude**

Globally, leishmaniasis is among the top ten neglected tropical health burden, endemically existing in people of every continent except Australia and Antarctica. *Leishmaniasis Epidemiology and Psychosocial Aspect DOI: http://dx.doi.org/10.5772/intechopen.110568*

Currently, 98 countries are endemic for leishmaniasis in the tropics, subtropics, and Southern Europe. More than 350 million individuals are at risk, 12 to 15 million infected, and 1.5 to 2 million new cases occur annually with 20,000–30,000 deaths [11, 12]. The endemic regions include Mediterranean Basin, Africa, Central and South America and Asia. As per WHO 2012 report, predictably, 59 countries are endemic for visceral lieshmaniasis, although cases fall excessively on seven of these as 90% of these cases happened in INDIA, South Sudan, Brazil, Ethiopia, Kenya, Sudan, Somalia and among 95% of CL cases occurred in the Americas, Middle East and Central Asia, approximately 70% reported in Syria, Brazil, Algeria, Colombia, Afghanistan, Iran, Ethiopia, Sudan, and Costa Rica [13].

Presently, over 85% of new CL cases occurred in 10 countries, namely Afghanistan, Algeria, Brazil, Colombia, Iraq, Libya, Pakistan, Peru, the Syrian Arab Republic, and Tunisia. Likewise, 10 countries: Brazil, China, Ethiopia, Eritrea, India, Kenya, Somalia, South Sudan, Sudan, and Yemen were accountable for more than 90% of new VL cases reported to WHO [11].

#### **4. Epidemiological determinants**

It is known that different species of *Leishmania* can cause in many mammalian hosts various clinical manifestations, and the severity varies from spontaneously healing lesions to life-threatening visceral disease. The outcome is determined by the inter-relationship between the characteristics of the parasite, the biology and habitat of the vector, and the behavior and immune response of host (**Table 1**) [1].

#### **4.1 Agent**

Leishmaniasis human disease is caused by more than 20 pathogenic species of *Leishmania* parasites transmitted from host to host by a tiny insect. *Leishmania* parasites are categorized into either old world (The Eastern Hemisphere) or new world (The Western Hemisphere) species, most notably the old world species: *L. major, L. tropica, L. aethiopica*, and the new world species: *L. amazonesis, L. braziliensis, L. mexicana, L. panamensis, and L. guyanensis* [15]. Old world species can be found in Asia, the Middle East, the Mediterranean Basin, and Africa, whereas new world species are found in the Americas [16].

#### **4.2 Vector**

Approximately, in over all 600 known species of sand flies, 10% act as disease vectors. And only 30 are important from public health point. Particular species of *Leishmania* are transmitted by particular adult female sand fly of *phlebotomine* belonging to either *Phlebotomus* spp. (Old World) or *Lutzomyia* spp. (New World) to either incriminated or suspected reservoir hosts [17]. The sand flies generally are active at night and inoculate the parasite when they bite humans ("from dusk to dawn") [18] and are less active in the hottest day-time, but still bite if they are bothered. Sand flies do not make noise, they are small, and their bites might not be noticed the parasite are spread. Adult sand flies often inhabit rock cracks, hollows, and rodent holes, and rest in cool, dark, and humid corners of animal shelters or human dwellings and their ability to acquire, maintain, and


#### **Table 1.**

*Disease types and transmission cycles of leishmaniasis worldwide [14].*

transmit the parasites depends on maturity, density, biting habit parasite-vectorhost interactions as well as on the ecological and epidemiological features of the infection [19]. Many sand fly species are opportunistic and feed on easy to access animals [20].

#### **4.3 Reservoir hosts (host preference)**

Humans, domestic animals, and wild animals are known to act as reservoir hosts such as up to 70 animal species, have been found as natural reservoir hosts of Leishmania parasites.

#### *4.3.1 Human*

Despite the human genetic susceptibility, hereditary factors, population ethnicity, genome-wide association, and immunological responses correlated to disease epidemiology [21, 22] the following demographic factors are of consideration.

Age: People of all ages are at risk for leishmaniasis if they live or travel to leishmaniasis endemic areas. Indigenous prevalence increases with age up to 15 years, after which the prevalence stabilizes or decreases, presumably reflecting the progressive buildup of immune protective status [23, 24]. Children under 10 years old displayed the highest proportion (>50%) of new infection [25] potentially due to lack of protective immunity or due to them collecting water or playing in gorges close to sand-fly habitat.

Gender: There is no real gender difference in leishmaniasis, but for decades, male patients have comprised the majority of reported cases, perhaps because of referral bias, more risk-taking behaviors by men or possibly, men are less likely to strictly adhere to recommended preventive measures and thus increase their risk of contracting the disease [26, 27]. The sex-related differences cannot be explained solely in terms of environmental exposure, socio-cultural determinant or healthcare access. Furthermore, transcriptomic evidence is revealing that biological sex is a variable impacting physiology, immune response, drug metabolism, and consequently, the progression of disease [28].

Poverty: Poverty increases the risk for leishmaniasis due to remote and rural inhabitance, low-income, malnutrition, poor waste management (such as open sewerage), poor housing and domestic sanitary conditions which increase sand-fly breeding and resting sites, as well as their access to humans. Sand flies are attracted to crowded housing as these provide a good source of blood-meals. Human behavior, such as sleeping outside or on the ground, may increase risk. Malnutrition and protein deficit, as well as a lack of iron, zinc, and vitamin A in the diet is also a risk factor for complications and for the disease progressing to kala-azar [29].

Population mobility: Migration, tourism, shipment to an area of endemic illness, and occupational exposure of non-immune people into areas with existing transmission cycles increase the risk of infection and epidemics leishmaniasis are often associated with massive migration, internal dislocation, and the population movement [30, 31].

#### *4.3.2 Domestic animals*

Dogs are the known reservoirs for the parasites of all types of leishmaniasis and natural host for *L. infantum, L. chagasi, L. tropica, and L. peruviana* as being infected by them, prevalently in Mediterranean, Asia, and Latin America [32]. Cats are considered secondary reservoirs of the infection in endemic areas as Leishmaniasis has been reported sporadically in several parts of the world [33, 34].

Cattle may possibly increase transmission burden by remaining as untreated reservoir for the parasite, their closeness to human owners, and because of increased availability of blood meals for the sand fly [35].

#### *4.3.3 Wild animals*

The detection of Leishmania infection in rodents, suggests that wild animals are also contributing to maintaining the life cycle and transmission of Leishmaniasis. Several rodents, namely, Mus musculus (domestic mouse), Microtus socialis, Rattusrattus (black rat), Cercomys cunicularius (wild rat), Mesocricetus auratus (Syrian hamsters), and marsupial e.g. Didelphis albiventris (opossum) in America, Africa, and Asia lead to spread of leishmaniasis [36, 37]. Other potential reservoirs are squirrels, wild canids, reptiles, and bats [38–40].

#### **4.4 Ecology/environment**

Leishmaniasis is climate-sensitive such as the pathogens, vectors, and hosts involved in the transmission cycle are environmentally sensitive [41] and impacted by climate change and human modification of ecosystems, therefore, changes in ecological settings (range from rain forests to deserts), rainfall, atmospheric temperature, and humidity resulting in fluctuations in sand fly numbers [42] due to variation in the developmental time and metabolism of sand flies and Leishmania development cycle within vector [43]. Humidity and moisture from rainfall or in the soil dealing with survival, development, and activity of sand flies. The matter of hygiene, (which is generally neglected in rural areas), presence of waste or stables in the vicinity of houses, use of traditional building materials for house construction, are all environmental factors that favor the development of the vector and reservoirs, therefore impact the incidence and distribution of leishmaniasis in different regions of the world. Environmental changes such as conversion of natural forest to other land uses, habitat destruction, altered landscape composition, urbanization, human incursion into forested areas, tourism into natural areas, the creation of roadways, energy networks, new farmlands, water management, and poorly planned urban development play an important role in Leishmaniasis epidemiology [44].

#### **5. Transmission of pathogens**

Understanding the transmission cycles is very important in effective prevention of leishmaniasis. There is no direct transmission from person to person and the indirect (vector-born) transmission of Leishmania species that cause VL or CL can be zoonotic or anthroponotic [45] depends on the characteristics of the parasite and sand-fly species, characteristics of the transmission sites, current and past exposure of people to the parasite, and human behavior. The zoonotic transmission from canine to humans, is found in the Mediterranean region and many other drier regions of Latin America. Leishmania species reported from dogs and occasionally from cats include *L. mexicana, Labrus donovani*, and *L. braziliensis*. Cats are at risk of infection especially in areas where these parasites are endemic [19]. In many endemic areas, infected people are not necessarily maintaining the transmission cycle, but infected animals along with sand flies, maintain the cycle. Only for anthroponotic transmission,

infected people are needed to maintain the cycle (human-sand fly-human), parasite transmission being mostly nocturnal and typically seasonal [46].

The incubation period in human is quite variable, generally 1 to 4 months; range is 10 days to 3 years. Visceral leishmaniasis is 3–8 months (range 10 days to 34 months) and of the Cutaneous leishmaniasis is 2 weeks to several months (rarely up to 3 years).

#### **6. Psycho-social impact**

Leishmaniasis due to disfigurement and social stigmatization causes or predisposes severe psychological disorders and negatively affects the quality of life varied from minor domestic restrictions to severe physical and emotional isolation that results in poor mental health [47]. The adolescents with lesions are often faced with difficulty in social relations such as finding job, friends, spouse or deemed unsuitable for marriage or social communal life. While the children disfigured by lesions or scars, due to painful treatment, because of exclusion from play with other children are also psychologically affected and emotionally damaged [48].

The social exclusion, discrimination, and psychosocial impacts negatively affect socioeconomic opportunities which showed that depression and anxiety symptoms were higher in patients. Body satisfaction was also impaired in the groups with active CL and healed scars. Culturally, older people were more accustomed to the CL scar, but younger generations had less acceptance of any lifelong stigma and disfigurement on the face [49].

Understanding the psychosocial consequences, economic cost, health importance and the interaction of pathogenic protozoan, sand flies vectors, environmental impact is encouraging prevention, control, and surveillance of Leishmaniasis under a holistic view and integrated approach of human and veterinary medicine, environmental science, and wildlife conservation under the "One Health" approach.

#### **Author details**

Ghulam Rahim Awab1,2

1 Medical Faculty, Nangarhar University, Jalalabad, Afghanistan

2 Oxford Tropical Diseases Research Unit (MORU Tropical Network), Mahidol, Bangkok, Thailand

\*Address all correspondence to: awabgr@yahoo.com

© 2023 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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[47] Hofstraat K, van Brakel WH. Social stigma towards neglected tropical diseases: A systematic review. International Health. 2016;**8**(Suppl. 1): i53-i70. DOI: 10.1093/inthealth/ihv071

[48] Pal B, Murti K, Siddiqui NA, Das P, Lal CS, Babu R, et al. Assessment of quality of life in patients with post kalaazar dermal leishmaniasis. Health and Quality of Life Outcomes. 2017;**15**(1):1-7

[49] Chahed MK, Bellali H, Ben Jemaa S, Bellaj T. Psychological and psychosocial consequences of zoonotic cutaneous leishmaniasis among women in Tunisia: Preliminary findings from an exploratory study. PLoS Neglected Tropical Diseases. 2016;**10**(10):e0005090

## **Chapter 5** Cutaneous Leishmaniasis

*Azhar Rafique, Sayydah Surrya Sani, Salma Sultana, Tayyaba Sultana, Asma Ashraf and Muhammad Shahid Mahmood*

#### **Abstract**

Cutaneous leishmaniasis (CL) is a widespread parasitic infection caused by the Leishmania, which is carried by female sandflies. The symptoms include basic ulcer to lethal systemic disease *i.e*., formation of widely dispersed skin lesions of diverse types. Almost 350 million individuals are at danger and the disease is endemic in more than 98 countries. There are globally 12 million cases, with 2–2.5 million new cases annually. Cutaneous leishmaniasis is considered as critically neglected disease by WHO. Earlier it was difficult to identify the infecting parasite, but modern DNA techniques make it quite simple to identify the Leishmania species, allowing quick treatment decisions. The quick identification of Leishmania is made possible using the PCR method. There is currently no vaccination to prevent leishmaniasis, and pharmacological treatment is frequently ineffectual. There is a need for broad and well-conducted investigations to help its control. Amphotericin B, pentamidine isethionate, paromomycin, and antifungals are some of the drugs recommended for treatment. By organising direct, in-person training, which is a crucial step in improving attitudes and preventative actions toward CL and its control in endemic areas, it is necessary to underline the significance and necessity of teaching this at-risk population.

**Keywords:** cutaneous leishmaniasis, protozoa, diagnosis, PCR, identification

### **1. Introduction**

In 190 developing nations, leishmaniasis, an infectious illness brought on by protozoa of the genus Leishmania, continues to be a significant public health issue [1]. Leishmania parasite infection can result in one of three main clinical forms, depending on the species that caused the infection. *Leishmania infantum* and *L. chagasi* have been found to be identical by genotyping [2] *Leishmania chagasi* is considerate a subpopulation of *L. infantum* that arose from imported European strains [3–5], therefore they should be regarded as synonyms and are presents in both NW and OW. The first is localised cutaneous leishmaniasis (CL), which can cause a single or numerous skin ulcers as well as satellite lesions or nodular lymphangitis. CL with mucosal involvement is the third kind, systemic visceral leishmaniasis (VL), which affects internal organs such the liver, spleen, and bone marrow and is lethal if untreated. CL without mucosal involvement (CL) is the second kind [6].

Males are more prone than females to get this sickness [7]. The leishmaniasis disease is spread by phlebotomine flies, of the genera Phlebotomus in the ancient world and Lutzomyia in the new world. The vector of this disease belongs to the order Diptera, class insecta, Family: Psychodidae [8]. The sandfly has a 3 mm length and is known for its "hopping" flight. They feature fragile, long legs, dagger-shaped mouthparts, huge, dark eyes, long antennae, and downward-facing mouthparts [9].

By being bitten by female sandflies, 20 different Leishmania species cause his illness. The disease is spread by 30 different species of sandflies. Humans and nonwild or domesticated animals serve as their reservoir hosts. Female sandflies consume reservoir hosts and contract the disease [10]. Use of contaminated syringes from sick people is how this parasite is spread. *Leishmania chagasi, Leishmania infantum,* and *Leishmania donovani* are the common species [11]. Due to the shift of individuals toward urban areas during the past 20 years, there has been a potential increase in leishmaniasis cases [12]. Leishmaniasis spread among people living in non-endemic areas through travel.

Just seven nations—Algeria, Afghanistan, Brazil, Iran, Peru, Syria and Saudi Arabia, —represent 90% of all CL cases. CL is the most common clinical type of leishmaniasis worldwide [13]. The Old World species of Leishmania parasites, such as *L. infantum*, *L. tropica* and *L. major* (common in, the Middle East, the horn of Africa, the Mediterranean basin and the Indian subcontinent), and the New World species, such as *L. chagasi*, *L. mexicana*, *L. amazonensis, L. naiffi*, (endemic in Middle and South America). Self-limiting ulcers are frequently caused by Old World species, but American tegumentary leishmaniasis, which also causes MCL and disseminated cutaneous leishmaniasis (DCL), is typically caused by New World species [14].

Leishmaniasis patients have a wide immunological spectrum, from those with a robust T cell response, as shown by delayed-type hypersensitivity (DTH) and high levels of interferon γ (IFNγ), to those who lack a DTH response but still have high antibody levels [11]. Others with a robust DTH have few parasites in their lesions because Leishmania spp. are killed by IFNγ -activated macrophages instead of being neutralised by antibodies, but people with merely a humoral response are unable to manage the parasite count [15, 16].

In addition to varying clinical symptoms based on species, Leishmania species also differ in their sensitivity to treatment options [17]. Because of this, determining the species is crucial to how leishmaniasis will manifest clinically. The identification of the Leishmania parasite that causes the disease used to be difficult, in disparity to many other infectious diseases. With the development of new DNA techniques, Leishmania parasites can now be recognised quite easily. With the motto "Small bite, big threat," the World Health Organisation (WHO) highlighted the serious and growing risk of vector-borne diseases, particularly leishmaniasis, on World Health Day 2014 [18]. In order to improve vector management, diagnosis, and the treatment toolset to prevent additional incidence and morbidity, leishmaniasis is considered a category 1 emergent and uncontrolled sickness and requires more intensive study. The major subjects of this review are the diagnosis, management, prevention, and strategies for the management and control of CL caused by both Old World and New World species.

#### **2. Sandfly and Leshmania life cycle**

In the blood meal of several hematophagus arthropods, the eaten amastigotes (protist cell, non-motile) in the infected host change into promastigotes (external

#### *Cutaneous Leishmaniasis*

*DOI: http://dx.doi.org/10.5772/intechopen.110569*

#### **Figure 1.**

*Leishmaniasis' life cycle (http://www.niaid.nih.gov/topics/leishmaniasis/pages/lifecycle.aspx).*

flagellum). When a host is inappropriate, parasites are discharged with the excrement [19]. When the blood meal is digested, trypanosomatids such as Leshmania species and others bind to the parasites on the midgut epithelium. The parasite is then kept in the gut and begins a stage differentiation [20]. Promastigotes then proceed to Sandfly's foregut, which is covered in cuticles, of the sandfly. In the foregut some attach and some remain free for next transmission by bite [21] into the vertebrate host.

Promastigotes reproduce by binary fusion in the sandfly's digestive system. After seven days, promastigotes undergo metacyclogenesis and become contagious (metacyclic promastigotes). The sandfly will puncture the host's skin while feeding in order to inject saliva and metacyclic promastigotes into the host (**Figure 1**).

Metamorphose into the amastigote form within the host macrophage. They multiply inside the phagolysosome by binary fission until the cell ruptures and spreads the infection to additional phagocytic cells. This keeps the cycle going.

#### **3. Early defence against Leishmania**

The several species of Leishmania that cause cutaneous leishmaniasis each have unique characteristics. However, the parasites do share a life cycle in which a sand fly transmits a promastigote, a flagellated form of the parasite, to mammalian hosts such as humans, dogs, and rodents [22]. The promastigotes enter various phagocytic cells after being introduced into the skin by a sand fly's bite. Promastigotes change into an amastigote, a circular, non-flagellated replicative form, inside the phagolysosome of macrophages. When sand flies consume amastigotes while feeding on a host, the amastigotes then change into promastigotes and reproduce within the sand fly, the life cycle is complete. However, during a natural infection, other elements found in the sand fly saliva are delivered in the skin that affects early immune responses. Most experimental infections involve injecting promastigotes into the skin with a needle [23]. The biological significance of studies looking at the early response to infection without taking into account the conditions present

during a natural infection, such as the inoculation site, the number of parasites, and the components present during the sand fly bite, must therefore be carefully interpreted [24, 25].

While neutrophils, dendritic cells (DCs), and monocytes that are recruited to the infection site also have a chance of contracting the parasite and play critical and distinctive roles in the establishment of the immune response to infection.

#### **4. Causes of leishmaniasis**

The most prevalent type of leishmaniasis that affects people is cutaneous leishmaniasis. It is a cutaneous condition spread by the bite of a phlebotomine sand fly and brought on by a single-celled parasite. Leishmania species that can lead to cutaneous leishmaniasis number around thirty.

#### **5. Signs and symptoms (diagnosis)**

#### **5.1 Cutaneous leishmaniasis**

Skin scrapings samples—often collected from the advantage of lesions—that have been microscopically examined are typically used to make the diagnosis. Although quick and inexpensive, this has a low sensitivity, especially in persistent lesions [26]. Although, bacteria and fungi present in the biopsy samples may contaminate the cultures of the lesions. Additionally, the needs for growth vary among species. Isoenzyme electrophoresis can be used to identify specific Leishmania species, although the procedure is time-consuming, expensive, and requires extensive parasite cultivation. Although PCR is preferable for a direct investigation of clinical material, monoclonal antibodies can also be employed to identify species in cultured strains. High specificity and sensitivity PCR is quick. Leishmania detection and genetic characterisation are also possible at the same time [27]. The PCR sensitivity for one investigation on American cutaneous leishmaniasis was 100% [28]. Due to the absence of considerable antibody generation in cutaneous leishmaniasis, antibody detection is not very sensitive.

Additionally, in cases of American cutaneous leishmaniasis, reports of crossreactivity between leishmanial antigens and antibodies made by other kinetoplastids, such as *Trypanosoma cruzi*, have been made [29]. The Montenegro (leishmanin) skin test, which looks for a particular type of delayed-type hypersensitivity on the skin, is another method for diagnosing cutaneous leishmaniasis that is currently available. *L mexicana* antigen is injected intradermally while being watched for a native reaction [30]. This test's limitations include the difficulty to distinguish between an infection that is currently present and one that has already occurred, as well as instances of false positive results in cases of other skin conditions [31].

#### **5.2 Mucocutaneous Leishmaniasis**

Mucocutaneous leishmaniasis develops after the commencement of cutaneous leishmeniasis and is defined by the destruction of the pharyngeal, oral, and nasal canals. The occurrence of this disease is also significantly influenced by genetic factors. Although nasal inflammation and stuffiness are the main early signs of

mucocutaneous leishmaniasis, the septum may slowly perforate and ulcerate. The larynx, mouth, throat, soft palate, and face are all affected by the lesion [32]. Bacterial infections may not harm the bones, but untreated illness can cause diarrhoea, pneumonia, and tuberculosis [33]. Suffocation, lung diseases, and starvation are other factors that might lead to death (due to closure of laryngeal aperture) [34].

#### **5.3 Post kala-azar dermal leishmaniasis**

A recurrence of kala-azar known as post-kala-azar dermal leishmaniasis (PKDL) may show up on a person's skin months or even up to 20 years after being partially treated, left untreated, or even in those who were thought to have had adequate treatment [35, 36]. In up to 60% of instances that are being treated in Sudan, they can be proven. They appear as facial redness or hypopigmented skin lesions (such macules, papules, and nodules). Even though PKDL can be caused by any organism that causes kala-azar, it is frequently linked to *Leishmania donovani*, which causes various disease patterns in Sudan and India. Nodules from the African type frequently ulcerate as they advance, whereas nodules from the Indian variant frequently expand with time and create plaques but seldom do so. Involvement of the nerves is more common in the African variation than in the Indian subcontinent [37]. Histology shows a variety of chronic inflammatory cells; macrophage or epitheloid granuloma may be present [38]. The variation in parasite concentration throughout investigations may be due to the less sensitive diagnostic techniques utilised in previous entries.

#### **6. Clinical features**

#### **6.1 Scratches**

An infection brought on by a sandfly bite can either remain asymptomatic or show up as an expanding, ulcerating papule after an incubation period of 1 to 12 weeks. An average scratch or lesions resembles an ulcer that is not painful and has a raised, indurated edge and a necrotic base that is frequently coated in an adherent crust of dried exudate. Most patients have 1 or 2 lesions, typically on exposed areas that range in diameter from 0.5 to 3 cm [39]. However, there is a great deal of variation: whereas some lesions develop sporotrichoid nodular lymphangitis, others do not ulcerate. Pain can be brought on by typical secondary bacterial infections. Atrophic scars are left behind after the majority of lesions heal over several months to years. *L. tropica* lesions typically take longer to heal—about 10 months—than those caused by *L. major* or *L. mexicana*, and *L. brazilinesis* lesions typically last considerably longer [40, 41]. Partial resistance to reinfection results from natural resolution [42].

In a study of 475 cases of *L. major* in Saudi Arabia, the parasite was found in 50–80% of smears (depending on the researcher and the technique employed), 70% of skin biopsies, and only 50% of cultures. In 10–20% of cases, even after combining all three procedures, the parasite remained undetectable. In New World illness, the issue is frequently worse, especially in lesions older than six months [43].

#### **6.2 Alternative methods**

Alternative methods for obtaining tissue for diagnosis include needle aspirates and slit skin smears. A needle aspirate is obtained using a 2 ml leur lock syringe, a 20 gauge needle, and 0.3 ml 0.9% saline. The needle is inserted through healthy skin and 0.1 ml is injected into the margin of the lesion. The tiny tissue fragments are aspirated after being severed from the needle track's edge while the needle is being rotated back and forth and suctioned. To create smears and inoculate cultures, use the aspirate. Slit-skin smears are made by pinching the edge of the lesion between the thumb and fingers, making a 1 mm deep slit with a scalpel, and then scraping the cut edge [44].

Because antibodies frequently go undetected or are present in low, serology is ineffective for treating cutaneous diseases [45]. Similar to the tuberculin test, the Leishmanin skin test identifies cell-mediated immunity; it turns positive after the scratches start to crust and stays that way for ever. It is unable to differentiate between current and previous infections [46].

#### **7. Morphology**

A papule that resembles an insect bite marks the beginning of the lesion. The papule persists and gradually grows in size rather than regressing. The nodular, noduloulcerative, and ulcerative forms are three common morphological presentations in the following stage. The latter features a flat foundation with marginal rolls that are either small or significant. If it is a secondary infection, the base is made up of granulation tissue and may be covered in pus.

Satellite papules, which are tiny (2–4 mm) papules along the perimeter of the lesion, are another crucial aspect of morphology. Skin crease orientation is visible in the lesions. Multiple lesions frequently cluster together; the sand fly bites the same spot repeatedly, delivering parasites with each bite.

Special clinical features of *L. major* infection are: [47].

1.Diffuse thickening without ulceration (erysipeloid form).


Leishmaniasis recidivans, also known as lupoid leishmaniasis, is a unique clinical form of *L. tropica* infection; in this situation, the sore appears to heal but recurs along the edge of the lesion, a process that may last for many years and may be disfiguring. Like cutaneous tuberculosis, it (lupus vulgaris). Typically, there are few parasites, and the LST is quite positive.

#### **8. Infections in animals**

#### **8.1 Clinical signs**

#### *8.1.1 Dog*

Wild dogs and domestic dogs are the main reservoirs of zoonotic visceral leishmaniasis caused by *L. infantum* in the Mediterranean area, Middle East, Asian countries, and Latin America. Dogs serving as the main reservoir of visceral leishmaniasis have made research into the immune response and searching for Leishmania antigens linked to protective cellular immunity in canine visceral leishmaniasis more attractive. Recent studies have shed fresh light on the genetic underpinnings, pathophysiology, immunology, and epidemiology of canine leishmaniasis. These new discoveries have improved understanding of the condition and aided in the creation of novel diagnostic techniques and infection-control strategies, such as dog collars impregnated with insecticide, new medications, and second-generation vaccines [48, 49].

Other infected dogs may remain asymptomatic or display one or more minor illness, which is known as oligosymptomatic infection [50]. Some infected dogs may experience symptoms that result in death. The distinctive histological feature in the skin, liver, and spleen is a granulomatous inflammatory response connected to Leishmania amastigotes within macrophages [51].

The clinical indications of canine visceral leishmaniasis were used to categorise a group of mixed-breed dogs with spontaneous Leishmania infections as symptomatic or asymptomatic [52].

Domestic dogs in Latin America have been observed to naturally contract *L. braziliensis, L. peruviana, L. panamensis, Leptodactylus colombiensis, and L. mexicana* [48]. There is currently no conclusive proof that dogs serve as reservoir hosts for the domestic spread of CL [53, 54]. The majority of research focuses on determining the prevalence of CL in dogs, but little is known about the parasitic and immunological aspects of the infection.

#### *8.1.2 Cats*

Leishmaniasis can occur in cats, but most infected cats are believed to remain asymptomatic. Most frequently, lesions of the skin or mucosa are described, either with or without visceral symptoms. Visceral symptoms, however, might appear without cutaneous involvement. Skin lesions can be found anywhere on a cat, although most frequently appear on the lips, nose, ears, eyelids, and paws. The most frequent lesions encountered are localised papules, nodules and chronic crusted or ulcerated lesions; regional lymphadenopathy may also be present. Rare reports of alopecia, scales, and hemorrhagic pustules or nodules have been made. Initial lesions are frequently single, although they can sometimes be numerous and occasionally spread.

There have been reports of oral, nasal, and in certain cases other mucous membranes (such the anal mucosa) being involved. Some cats can have ocular symptoms, including unilateral or bilateral uveitis, conjunctivitis, and blepharitis (which can

develop into panophthalmitis). Fever, hepatomegaly, jaundice, vomiting, diarrhoea, lymphadenopathy, dyspnea, nasal discharge, anaemia, and leukopenia are just a few of the visceral abnormalities and symptoms that have been observed in cats [55].

#### *8.1.3 Equidae*

Skin lesions can occasionally appear on horses, mules, and donkeys, especially on the head, neck, legs, and inguinal or axillary regions. Solitary or numerous papules or nodules, which frequently have ulcers, are the most typical lesions. Additionally, widespread skin illness has been documented. Although visceral leishmaniasis in horses has not been documented, parasites and *L. braziliensis* nucleic acids have been found in the blood and bone marrow of other animals in South America [55].

#### **8.2 Other domestic animals**

Rarely have medical instances in cattle or other small ruminants been reported. Only skin lesions, occasionally accompanied by lymphadenopathy, were noted in goat, sheep and cattle. In Germany, a pregnant cow with *Lechytia martiniquensis* infection had several ulcerative or plaque-like skin lesions on various body parts. After giving birth, it fully recovered. The only clinical symptom in experimentally infected sheep was a fever. Pigs with an experimental infection exhibited no symptoms.

Rarely, *L. enriettii* skin lesions, frequently on the ear, have been found in naturally infected guinea pigs. The earliest lesions in experimentally infected animals start out as redness and swelling but quickly progress into sizable, ulcerated lumps that resemble tumours. While some investigations discovered secondary lesions at other locations, including as the skin, lip, and genitalia, others claimed the lesions did not spread. Additionally, parasites were found in various internal organs. Spontaneous healing has been documented in certain studies but not others. Hamsters formed non-ulcerated nodules that went away on their own after contracting *L. enriettii* infection in an experiment.

#### **8.3 Captive wild species and wild animals**

The few known cases of leishmaniasis in wild or captive canids have resembled leishmaniasis in canines. Some nonhuman primates have been found to have visceral involvement with nonspecific symptoms (such pale mucous membranes and weight loss). A lion displayed clinical symptoms of colitis, including bloody diarrhoea, epistaxis, weight loss, and footpad sores.

*L. martiniquensis*-infected captive Australian marsupials have experienced skin lesions that include elevated, crusty or ulcerative pale nodules as well as localised to converging patches of thickened skin. Some rats infected with the *L. mexicana* complex in the wild have been reported to have skin lesions. These lesions are described as ulcers or swollen bumps with thinning hair. The tail base was said to be where they appeared most frequently, but they might also appear on the ears or toes. Numerous species have been known to have subclinical infections [55].

#### **9. Treatment**

Luckily, there are some rules to follow. A wait-and-see strategy for spontaneous cure may be acceptable because most lesions heal quickly without therapy, especially for patients who live in endemic areas because spontaneous healing is linked to the

#### *Cutaneous Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.110569*

development of protective immunity. It is recommended to actively treat lesions that are multiple or persistent, have associated lymphangitis, are on cosmetically or functionally significant places like the hands or face, have several lesions. Patients with early, non-inflamed lesions should have local therapy; patients with many lesions or more complex lesions should receive systemic therapy.

#### **9.1 Sodium stibogluconate and Meglumine antimoniate**

Patients with cutaneous leishmaniasis can receive an intralesional infiltration of 1–5 ml (100 mg/ml) sodium stibogluconate (SSG) on alternate days for three days once a month, and in the majority of instances, this causes full healing by the end of the second month [56]. For numerous and larger lesions, a higher dose and more than three regimens were required. In addition to intralesional SSG in several lesions, Sharma et al. employed intramuscular SSG (800 mg/day) [56]. Meglumine antimoniate, a substitute medication that is the top drug of choice in Ecuador [57]. In Nepal, sodium antimony gluconate (20 mg/Kg/day) is administered intramuscularly to PKDL patients for duration of 30 to 72 days [58].

Drug resistance is a problem, and cases of visceral leishmaniasis with SSG resistance were reported in Nepal [59, 60]. So far, this issue was not reported from Nepal in cutaneous leishmaniasis. It is unknown that what is the most effective treatment for American utaneous and mucocutaneous leishmaniasis (ACML)? Since the 1940s, pentavalent antimonial medications have been utilised, such as sodium stibogluconate (SSG) and meglumine antimonate (Glucantime, MA), but they are costly, poisonous, and uncomfortable [61]. It is advised to identify the precise species of Leishmania before beginning treatment because medications that are effective for one species of Leishmania may not be effective for another. Sadly, leishmaniasis is an orphan disease in affluent countries, and nearly all of the available treatments are poisonous and have serious adverse effects [61].

Compared to parenteral therapy, this results in higher local concentrations and fewer systemic side effects. 5–10 1–5 ml antimony infiltrations are performed two– three times per week. Make sure the injection is in the lesion and not in the tissues beneath the skin. It might be really painful. There is growing evidence that it is useful in treating *L. tropica* infections, as well as CL brought on by *L. major* [62].

#### **10. Physical methods**

Patients of all ages have received treatment for cutaneous leishmaniasis using a variety of physical techniques, such as surgical excision, cauterization, cryotherapy, and the use of local heat.

#### **10.1 Cryotherapy**

Cryotherapy involves applying liquid nitrogen repeatedly to a lesion up to 2 mm outside the lesion margin using a cotton-tipped applicator or a cotton swab with moderate pressure. Each application's freezing time is 15–20 seconds. The process is carried out two or three times with brief breaks, taking between 30 and 120 seconds in total. The whitening of the skin at 2–3 mm outside the lesion's edges indicates proper treatment [63–66]. The typical post-freeze pattern includes blistering of the lesion for two to three days, crusting, mild oedema and the development of an eschar [63–66].

#### **11. Treatment in animals**

Animals rarely receive topical treatments, but radio-frequency-induced heat therapy was effective in treating two dogs with several localised mucocutaneous lesions on the snout. In certain animals, such as some cats and some horses, cutaneous lesions did not recur following surgical resection; but, in other instances, surgical resection alone was ineffective.

#### **11.1 Vector control**

Theoretically, infection might be controlled by preventing transmission by the sandfly vector. Depending on the species, some sandflies are endophagic and eat indoors, while others are exophagic and eat outdoors. Examples of tactics include deterrents, particularly pyrethroids, and insecticides like DDT. The majority of the times, sandflies are still quite sensitive to insecticides, despite reports of DDT resistance. Spraying has been shown effective at the local level, but it is unclear what effect blanket spraying would have on the sandfly population, and these programmes are challenging to maintain. Bednets offer defence against species that consume living things [67] with pyrethroid-impregnated nets providing extra protection dropping biting rates by up to 64–100% [68]. The success of bednets as a long-term control method depends on routine, replacing damaged nets, re-impregnation and dissemination to rural areas. The ability of bednets to stop the spread of malaria to children has raised incentive to improve this method of malaria control, including research into bednets treated with long-lasting insecticides [68].

#### **11.2 Animal reservoirs**

Leishmaniasis is a zoonotic disease that is widespread throughout the world with significant reservoirs of infection in sylvatic and domestic animals. The dog population has been targeted throughout the Mediterranean basin and Brazil. The use of dog collars impregnated with delta methrin has proven to be the most successful tactic, offering dogs up to 86% protection during high transmission seasons [69]. Dog killing has also been used, and modelling from a Brazilian study suggests that in areas with low endemicity, both dog killing and dog collars should have a greater proportional impact. However, as transmission rates rise, the relative benefit of dog collars decreases [70]. However, the effectiveness of vector and animal reservoir control programmes has been diminished due to decreased pesticide use and rising prevalence among urban populations. Given the wide range of epidemiological situations, the multiplicity of factors that affect disease transmission, and the continuous understanding regarding the biology of the parasite, its vector, and its reservoir hosts, disease management has thus proven to be extremely difficult to achieve. To avoid these problems, a vaccine for humans or dogs is a preferable option.

#### **11.3 Vaccines**

A plausible basis for vaccine development can be found in the discovery that strong immunity against re-infection accompanies spontaneous or medicationinduced recovery from CL or VL. This finding gave rise to the long-standing technique of "leishmanization," or the use of live parasites retrieved from skin lesions to cause lesions in desired body regions in order to prevent sickness on re-infection.

#### *Cutaneous Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.110569*

Such a custom has been practised for at least 2000 years. Between 1982 and 1986, Iran had about 1.2 million patients receive this live vaccination [71]. 93% of people who had skin lesions after receiving the vaccination had a positive leishman-delayed hypersensitivity skin test, which is a good field predictor of population immunity. Skin lesions occurred in almost 50% of vaccination recipients. Additionally, a considerable decrease in disease incidence was seen, going from 14% in the group that had not gotten the immunisation to 2.5% in the group that had. Although it has not always produced cross-species protection, the use of heterologous organisms with lower pathogenicity as vaccines against a more virulent species is justified by the greater extent of immunological cross-reactivity between species at the humoral and cellular levels. Leishmanization is no longer used due to the risk of localised sickness and HIV-related spread, as well as the impossibility of delivering new cultures of a live vaccination in the field. A different approach with attenuated organisms enables exposure to a considerably wider spectrum of antigens than is possible with more complex subunit vaccines, resulting in the establishment of an immune response that is most similar to that of a natural infection. However, even using naturally pathogenic organisms in such a way to treat human or experimental murine leishmaniasis [72], exposed organisms [73] or genetically manipulated organisms [74], there has been little achievement. Similar to this, dead vaccinations have low immunogenicity and effectiveness even when supplemented with adjuvants, such as bacille Calmette-Guérin BCG or alum [75]. Interestingly, BCG alone caused a positive leishmanin skin test in some people, most likely because Mycobacteria and Leishmania have antigenic cross-reactivity. While the effectiveness of the single-dose *L. major* vaccination combined with BCG in dogs was around 70% [76]. It has showed promise as a possible vaccination policy against a natural *L. infantum* infection to administer to dogs naturally excreted secretory antigens extracted from culture supernatant of *L. infantum* promastigotes [77]. TSA, LeIF, and LmSTI1 are three recombinant leishmanial antigens that have shown immunogenicity in canine [78]. Recent developments in the creation of a vaccination that prevents transmission are hopeful given the significance of dogs as virus reservoirs. 92–97% protection against zoonotic visceral leishmaniasis was produced by the *Labrus donovani* fucose-mannose ligand (FML) antigen in combination with saponin (FML vaccination and Leishmune), with considerable protection shown out to 12 months [79]. Investigating the use of specific compounds as human vaccinations is an alternative strategy. Animal models for a gp63 peptide vaccination were successful [80]. The failure to adequately elicit cellular immunity, a necessary component for the management of intracellular infections, has, nevertheless, contributed to the generally poor effectiveness of these vaccines in humans.

#### **11.4 DNA vaccines**

The creation of DNA vaccines has provided an innovative solution to this issue. According to Wolff et al., intramuscular injection of plasmid DNA expressing a variety of reporter genes could cause muscle cells to produce proteins [81]. This study provides a solid basis for the concept that pure recombinant nucleic acids can be delivered in vivo to regulate protein production. Later studies found that DNA vaccines could defend mice from CL [82]. The ability to retain information over the long term is one of the main determinants of a vaccine's effectiveness. DNA encoding leishmanial antigen LACK is better to leishmanial protein and IL-12 protein immunisation for maintaining antigen-specific Th1 responses that can restrict *L. major* infection [83]. Antigen persistence and IL-12 activation by CpG motifs are two factors that explain

why DNA vaccination is more effective than protein and adjuvant. In an experiment with leishmaniasis, a study finding that anti-IL-10R antibody resulted to sterile cure but loss of immunity against re-infection showed that parasite persistence was essential for the preservation of immunity [84]. However, DNA vaccines can nonetheless induce long-lasting immunity in the absence of noticeable antigen. This is done either by allowing undetectable antigen to remain, potentially in follicular DCs, or by inducing antigen-independent immune responses [85]. According to studies, memory T cells are diverse, with one fraction (central memory T cells) migrating through lymph nodes and another migrating to tissues and producing effector cytokines [86]. Recent research shows that even in the absence of parasites, central memory T cells can mediate long-term memory [87]. Finding the pathways that cause central memory T cells to be induced would therefore be a significant problem for DNA vaccines. The potential for improving DNA vaccine immunogenicity has been investigated in a number of ways. By acting as TLR ligands, novel adjuvants including CpG motifs and monophosphoryl lipid A release IL-12 and encourage a Th1 response. Heterologous primeboost immunisation has been used as another method to improve human responses to vaccination. Various combinations have been studied, but priming with DNA and boosting with MVA have received the most attention [88]. This work demonstrates that IL-10 plays a crucial role in the immune response by showing that the efficacy of the vaccine against these antigens in the BALB/c model was dictated by IL-10 from regulatory T cells. The most frequently sampled gene among expressed sequence tags from cDNA libraries of *L. major* was TryP, according to early investigations [89], in vulnerable BALB/c mice as a reproducibly protective antigen against infection. LACK, *L. major* stress-inducible protein 1, Leishmania elongation factor, and HASPB1, a stage-specific hydrophilic acylated surface protein in mice, are other antigens that are effective in mice and non-human primates. In order to immunise against the saliva-containing *L. major* challenge, the use of salivary antigens in plasmid DNA has also been examined [90]. The 8500 discovered genes now that *L. major* Friedlin's genome sequence is complete serve as a source of potential vaccine candidates [91]. There is no presumed requirement that the target antigen be a surface molecule because the parasite is inside, which significantly expands the pool of potential vaccine candidates. One such thorough vaccine screening of 100 different amastigote-expressed Leishmania genes was done in a BALB/c mouse model challenged with the *L. major* LV39 substrain using DNA vaccination [90]. A heterologous prime-boost strategy for immunising against experimental visceral leishmaniasis in dogs was successful using DNA/recombinant cysteine proteinases type I and type II [92]. We are eagerly awaiting the outcomes of additional dog experiments as well as a human study of a DNA vaccination.

#### **12. Conclusion**

Renewing hope that control is possible is the international acknowledgement of this disease's significance, coordinated by WHO programmes, particularly in India, where 70% of the world's VL burden is found. A better coordination of control programmes should be possible thanks to new techniques for early case detection. Only when the local infrastructure is developed to support the provision of healthcare will more extensive control be possible in the world's less developed areas. This attempt is hampered by the introduction of HIV, drug-resistant strains, and changes in the epidemiology of the vector. The development of new techniques and, eventually, a protective vaccine will require a sustained worldwide effort and the requisite funding.

### **Acknowledgements**

Dr. Azhar Rafique, Doctor of Philosophy (PhD) in Zoology from University of Agriculture Faisalabad (UAF), Assistant Professor Department of Zoology at Government College University Faisalabad, Pakistan, provided help in language, edited and proofread the chapter.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Azhar Rafique1 \*, Sayydah Surrya Sani1 , Salma Sultana1 , Tayyaba Sultana1 , Asma Ashraf1 and Muhammad Shahid Mahmood2

1 Department of Zoology, Government College University Faisalabad, Punjab, Pakistan

2 Institute of Microbiology, University of Agriculture, Faisalabad, Punjab, Pakistan

\*Address all correspondence to: azharrafique96@gmail.com

© 2023 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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#### **Chapter 6**

## Visceral Leishmaniasis: An Overview and Integrated Analysis of the Current Status, Geographical Distribution and Its Transmission

*Kaushal Kumar Mahto, Pooja Prasad, Mohan Kumar, Intzar Ali, Vikram Vohra and Deepak Kumar Arya*

#### **Abstract**

Visceral leishmaniasis (VL) is a vector–borne disease transmitted by Phlebotomine sandflies, with up to 350 million people are at risk of developing infection globally. VL has a severe influence on the impoverished and undeveloped populations among several subcontinents. Early and accurate diagnosis and treatment remain crucial to the management of VL, which still depends on vector control. The present chapter objectives are to provide an overview of visceral leishmaniasis and to raise knowledge of the most recent progress in this condition's management, treatment, and prevention. Additionally, this chapter could be helpful for comprehending the difficulties and knowledge gaps in eliminating this protozoan disease as well as for learning the planning lessons from the global management of diseases like malaria and tuberculosis.

**Keywords:** visceral leishmania, endemicity, surveillance, strategy, transmission control, treatment

#### **1. Introduction**

Visceral leishmaniasis (VL), also referred to as kala–azar in the Hindu vernacular, is caused by an obligate intracellular protozoon species of the genus *Leishmania* (Trypanosomatida: Trypanosomatidae) [1]. VL is transmitted by the bite of female sandfly's (Diptera, Psychodidae) [2]. VL is usually transmitted by the two species i.e. *Labrus donovani* and *L. infantum* (*L. chagasi infantum*) depending on the geographical area and generally affects internal organs such as spleen, liver, and bone marrow. It is one among the three leishmaniasis where it is life threatening, if untreated. It is mainly distributed throughout in tropical and subtropical areas and can range from asymptomatic to severe however; other forms such as cutaneous and mucosal leishmaniasis can cause substantial morbidity. VL caused by *L. infantum* mainly affects children under the age of 5 years whereas *L. donovani* infects all age groups and this may be due to malnutrition and other conditions of immunosuppression [3].

#### **Figure 1.**

The spatial distribution and the burden of VL is up surging year after year and it is now became a growing health concern worldwide. VL is thought to be an anthroponotic disease and is prevalent in several foci across Africa and the Indian subcontinent [4]. Most VL infections occur in the least developed countries and the most underdeveloped areas of middle–income countries. More than 90% of new cases reported to WHO in 2020 took place in ten countries: South Sudan, Sudan, Somalia,Yemen, Kenya, Ethiopia, Eritrea, India, Brazil, China respectively [5]. It should be possible to eradicate it with rapid case detection, treatment, and local vector control. Insecticide–impregnated materials, active case detection, and treatment are indeed the cornerstones of the current control techniques for VL.

**Figure 1** represents the number of VL cases across 83 countries. In which 28 countries data are not available for the year 2020. Maximum number of VL cases were found from Sudan (2563) and most of the countries had very smaller number of cases which indicate endemicity of VL. Country like Sudan, Brazil, and India were reported maximum number of cases of VL.

#### **2. VL and India**

In India, it is estimated that 165.4 million population are at risk in 4 states viz., Bihar (33 districts, 458 blocks), Jharkhand (4 districts, 33 blocks), West Bengal (11 districts, 120 blocks), and Uttar Pradesh where kala–azar is endemic (6 districts, 22 blocks) [6] (**Table 1**). India reported 810 cases and 39 deaths in the year 2022. In order to overcome remaining obstacles in eradication of visceral leishmaniasis, India is stepping up its efforts. India has made strengthening a widely spread network of comprehensive primary health care facilities, backed by health education and social mobilization, a top priority in its national health strategy in order to attain universal

*Visceral Leishmaniasis: An Overview and Integrated Analysis of the Current Status… DOI: http://dx.doi.org/10.5772/intechopen.110567*


**Table 1.**

*VL cases and deaths in India since 2014 [7].*

health coverage. For instance, there is one community health volunteer, or certified social health activist (ASHA), for every 1000 residents of a village. India now uses a 5% wettable powder formulation of alpha–cypermethrin for indoor residual spraying. Sandflies that settle on surfaces of sprayed walls are killed by it.

#### **3. VL and suspected countries**

There are 53 *Leishmania* species found worldwide in five genera (*Leishmania, Viannia, Sauroleishmania, L. enrietti complex, and Paraleishmaia*) and 31 species are known to be mammalian parasite and out of which approximately 20 different species of *Leishmania* infect animals, reservoir host includes domestic dogs and cats, wild canids and many of them can contribute to leishmaniasis in humans [8] (**Table 2**).

Major risk factors associated with VL are socioeconomic conditions, malnutrition, population mobility, environmental changes, and climate changes.

#### **4. Growth and development of sandfly**

Sandflies are tiny holometabolous insects, completes their development in four stages i.e. egg, larva, pupa and adult (**Figure 2**). Female sandflies usually lay 30–70 eggs during a single gonotrophic cycle, which are deposited in cracks and holes in the ground or in building, animal burrows and among tree roots [15]. Eggs are elongated, oval–shaped and pale at first and darkening on exposure to air. Larvae feed on dead organic material. Larvae are mainly scavengers, feeding on organic matters such as dead and decaying leaves, decomposing insets etc. The pupal stage lasts 6–13 days before the adult sandflies emerge.


#### **Table 2.**

*Suspected countries with reservoir hosts of visceral leishmaniasis worldwide.*

*Visceral Leishmaniasis: An Overview and Integrated Analysis of the Current Status… DOI: http://dx.doi.org/10.5772/intechopen.110567*

**Figure 2.** *A complete life cycle of sandfly.*

#### **4.1 Life cycle of** *Leishmania* **parasite in sandfly and host**

The life cycle often involves two hosts: a phalebotomine sandfly and a vertebrate host (such as a dog or human).

According to Dawit [16], there are two evolutive forms: promastigotes for invertebrate hosts and amastigotes for vertebrate hosts.

The Following steps are involved to complete the life cycle of visceral leishmaniasis vector sandfly.


The life cycle of the *Leishmania* parasite is indicated in below in **Figure 3**.

#### **4.2 Source of infection**

In both urban and rural environments, the domestic dog (*Canis familiaris*) is the primary reservoir. Foxes (*Cerdocyon thous* and *Lycalopex vetulus*) and other marsupials are among the identified reservoirs in the wild (*Didelphis spp.)* [17]. In a study

**Figure 3.** *Life cycle of Leishmania parasite in host and sandfly.*

conducted in Iran, Gavgani *et al.* [18], the use of collars medicated with deltamethrin decreased the incidence of infection in dogs (by 54%) and children (by 43%). If a successful vaccine could be devised, vaccination of dogs would be the best approach.

#### **4.3 Mode of transmission of VL**

Leishmaniasis is caused the bite of infected female phlebotomine sand flies. The infected sand flies regurgitates the parasites infective stage (i.e., promastigotes) during blood meals According to some research studies the use of needles also contributes to parenteral and congenital transmission. No direct transfer occurs from one person to another [17]. The kind of transmission where (human—sandfly—human) involved is known as anthroponotic transmission because it occurs in some areas of the world where infected individuals are needed to keep the cycle going. For instance, the spread of *L. donovani* is anthroponotic in the Indian subcontinent (South Asia). Early diagnosis and appropriate treatment of those who are infected in these areas can act as a control strategy, whereas insufficient care can result in the development and spread of drug resistance. Spraying with residual–action pesticides and using bed nets sprayed with long–lasting insecticides may offer protection since the spread is intra– and peridomiciliary (rather than sylvatic).

#### **4.4 Signs and symptoms**

A wide spectrum of clinical manifestations, from mild to moderate to severe clinical symptoms, are present in the infection. Although it can be as long as 24 months, the incubation phase usually lasts between 2 and 6 months.

Clinical symptoms visceral infection has the following stereotypical symptoms [3, 19].

• Fever

*Visceral Leishmaniasis: An Overview and Integrated Analysis of the Current Status… DOI: http://dx.doi.org/10.5772/intechopen.110567*


Lymphadenopathy has been reported in a few locations, most significantly Sudan and South Sudan. Other signs and symptoms include jaundice, bleeding from the mouth or nose, abdominal fluid buildup, respiratory issues, gastrointestinal issues including vomiting and diarrhea, and in extreme instances, malnutrition and lower limb edoema. In these persons deaths are eventually brought on by bacterial infection or hemorrhage.

Despite the fact that the terms kala–azar and visceral leishmaniasis are occasionally used interchangeably, kala–azar—which in Hindi is described as "black fever"—is frequently used to refer to severe (advanced) cases of visceral leishmaniasis. If untreated, visceral leishmaniasis in its most severe forms typically causes mortality, either directly from the disease or indirectly through its side effects, such as secondary Mycobacterium infection or hemorrhage.

#### **5. Post–kala–azar dermal leishmaniasis**

The dermal leishmaniasis known as post–kala–azar dermal leishmaniasis (PKDL), which typically follows visceral leishmaniasis, manifests as a macular, papular, or nodular rash on the face, upper arms, trunks, and other areas of the body [20]. The infection is said to develop in 5–10% of kala–azar patients, and it mostly affects people in East Africa and the Indian subcontinent [21] (**Table 3**). India reported 613 cases in the year 2022 where state Bihar has the heighted number of cases reported (311) followed by Jharkhand (164), West Bengal (109), and Uttar Pradesh (29). Although it can develop earlier, it often manifests 6 months to a year or more after kala–azar has purportedly been treated [6]. The Kala-azar Elimination Program (KAEP) is built around five pillars: surveillance, vector management, societal mobilization, early case detection for rapid diagnosis, treatment, and operational research. *Leishmaniasis* is thought to be transmitted by people who have PKDL.

#### **6. VL and HIV co–infection**

Due to the vulnerability of HIV–infected patients to the disease, *leishmania* and HIV confections have been a challenge to the treatment and eradication of visceral leishmaniasis. HIV and *leishmania* reinforce one another, creating serious clinical and public health challenges. Both conditions induce immune system suppression, which leads to increased death rates, exposure to drugs with higher toxicity, and more severe morbidity with fewer available treatments and higher rates of relapse. The coinfection was first identified in southern Europe in the middle of the 1980s, but is now known to exist in as many as 45 countries [6]. Brazil, Ethiopia, and the Indian state of Bihar have all recorded increased incidence. In India the subsequent cases of VL + HIV has been decreased from 9241 in 2014 to 881 cases in 2022 (**Table 4**).


#### **Table 3.**

*PKDL situation in India since 2014 [7].*


**Table 4.**

*VL and HIV coinfection status and situation in India from 2014 onwards [7].*

Liposomal amphotericin B injections are the current treatment standard for HIV/ visceral leishmaniasis co–infection (LAmB). The new treatment regimen combines LAmB and miltefosine, an oral medication. Patients who are co–infected are at risk of developing a variety of forms of stigma and human rights issues in addition to other comorbid conditions including TB and cryptococcal meningitis. The co–infection of pulmonary TB with visceral leishmaniasis is a problem for public health in many countries. *Leishmania* infection can change the immune system's protective response to the BCG TB vaccine [22].

#### **7. Prevention and control**

No vaccination exists to protect against VL disease, however; the following are some preventive care practices for the human population to prevent vector contact [3].

1.For the human population: Avoid outdoor activities from dusk to dawn, use mosquito nets, wear protective clothes, and use insect repellents are some of the recommended personal protection measures to prevent contact with vectors.

*Visceral Leishmaniasis: An Overview and Integrated Analysis of the Current Status… DOI: http://dx.doi.org/10.5772/intechopen.110567*

People with clinical symptoms of the disease should receive treatment as soon as possible.


Drugs that are already available in the market have significant limitations in terms of cost, stability, resistance, and safety. When administered alone, they have a poor tolerability, a prolonged course of treatment, and are challenging to deliver. Most often, pentavalent antimonials are used to treat visceral leishmaniasis. However, while choosing a medicine, it is important to take the patients' clinical circumstances, the existence of any co–infections, and pregnancy in to the consideration (**Table 5**).

#### **8. Diagnosis**

**Clinical**: People from endemic areas are considered a high clinical suspicion of disease if they have a chronic condition, an unexplained fever, and suggestive signs and symptoms [3].

**Laboratory**: Following immunological and parasitological tests are performed to treat VL condition. The rapid immunochromatographic test based on recombinant rK39 antigen and the alternative Direct Agglutination Test (DAT) to confirm infection are the only immunological tests currently available at the primary level. Other levels of care also use indirect immunofluorescence (IIF) and enzyme immunoassay (ELISA) [24, 25].


#### **Table 5.**

*Anti–leishmanial drugs in current use and issues [23].*

**Figure 4.** *Methods used for the diagnosis of Visceral leishmaniasis.*

By conducting direct examinations or isolating parasites in culture, parasitological procedures can identify parasites in affected organs, primarily the bone marrow (in vitro). PCR could also be used to find out *Leishmania* infection (**Figure 4**).

#### **9. Planning lessons from the global management of diseases like malaria and tuberculosis**


*Visceral Leishmaniasis: An Overview and Integrated Analysis of the Current Status… DOI: http://dx.doi.org/10.5772/intechopen.110567*

• To decide whether to use alternative therapies, it is necessary to rigorously and continuously assess resistance. Insecticides and next-generation drugs should both be the subject of concurrent study.

#### **10. Conclusion**

In high–burden countries, early detection, management, and treatment results should be improved by good community knowledge of the condition, the availability of resources, the development of clinical and health professionals' capacities, and reliable surveillance data. In summary, there is a strong need for continuous investment in VL diagnostic, treatment, and prevention, even though much can be accomplished with the already available tools and techniques. All of the existing drugs have one or more limitations, thus further funding for drug development is still desperately necessary to fill the pipeline with novel drugs. The investigation of combination therapy using already available medicines continues to be a top goal in the meantime. In areas where *L. infantum* is prevalent, novel approaches to reduce the animal reservoir (such as dog collars) or minimize human infection are needed (such as insecticide impregnated bed nets or blankets).

#### **Acknowledgements**

Authors are gratefully acknowledging National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India for providing the suitable facilities that help us to draft the proposed chapter.

#### **Conflict of interest**

The authors declare that they have no known competing financial interests or personal ties that would seem to have influenced the work presented in this study.

### **Author details**

Kaushal Kumar Mahto1 \*, Pooja Prasad2,3, Mohan Kumar4 , Intzar Ali5 , Vikram Vohra1 and Deepak Kumar Arya3

1 National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India

2 National Institute of Malaria Research, New Delhi, India

3 Department of Zoology, Kumaun University, Nainital, Uttarakhand, India


\*Address all correspondence to: kausha63\_sit@jnu.ac.in

© 2023 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.

*Visceral Leishmaniasis: An Overview and Integrated Analysis of the Current Status… DOI: http://dx.doi.org/10.5772/intechopen.110567*

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[4] Ready PD. Epidemiology of visceral leishmaniasis. Clinical Epidemiology. 2014;**6**:147-154. DOI: 10.2147/CLEP. S44267

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[8] Akhoundi M et al. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and Sandflies. PLoS Neglected Tropical Diseases. 2016;**10**:e0004349. DOI: 10.1371/journal.pntd.0004349

[9] Thakur L et al. Atypical leishmaniasis: A global perspective with emphasis on the Indian subcontinent. PLoS Neglected Tropical Diseases. 2018;**12**(9):e0006659. DOI: 10.1371/journal.pntd.0006659

[10] Zhao-Rong L et al. Visceral leishmaniasis in China: An endemic disease under control. Clinical Microbiology Reviews. 2015;**28**(4):987- 1004. DOI: 10.1128/CMR.00080-14

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[12] Gadisa E et al. Eco–epidemiology of visceral leishmaniasis in Ethiopia. Parasites & Vectors. 2015. Available from: https://bit.ly/3zckvBd

[13] Akhoundi M et al. Geographical distribution of *Leishmania* species of human cutaneous Leishmaniasis in Fars Province, Southern Iran. Iranian Journal of Parasitology. 2013;**8**:85-91

[14] Desbois N et al. *Leishmania* (*Leishmania*) *martiniquensis* n. sp. (Kinetoplastida: Trypanosomatidae), description of the parasite responsible for cutaneous leishmaniasis in Martinique Island (French West Indies). Parasite. 2014;**21**:12. DOI: 10.1051/ parasite/2014011

[15] Service M. Phlebotomine sand– flies. In: Service M, editor. Medical Entomology for Students. 4th ed. Vol. 18. Cambridge: Cambridge University Press; 2008

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### **Chapter 7**

From Biology to Disease: Importance of Species-Specific *Leishmania* Antigens from the Subgenera *Viannia* (*L. braziliensis*) and *Leishmania* (*L. amazonensis*) in the Pathogenesis of American Cutaneous Leishmaniasis

*Fernando T. Silveira, Marliane B. Campos, Silvia F. Müller, Patrícia K. Ramos, Luciana V. Lima, Thiago V. dos Santos, Claudia Maria Gomes, Márcia D. Laurenti, Vania Lucia da Matta and Carlos Eduardo Corbett*

### **Abstract**

American cutaneous leishmaniasis (ACL) is one of the most complex parasitic diseases from a clinical-immunopathological point of view due to the great heterogeneity of *Leishmania* species responsible for the disease. Currently, fifteen *Leishmania* species of the subgenera *Leishmania*, *Viannia* and *Mundinia* may give rise to ACL in Latin America. In Brazil, seven species are associated to the disease, but *L. (V.) braziliensis* and *L. (L.) amazonensis* stand out for producing the broadest clinicalimmunopathological spectrum: localized cutaneous leishmaniasis [LCL: DTH+/++], borderline disseminated cutaneous leishmaniasis [BDCL: DTH+/−], mucocutaneous or mucosal leishmaniasis [MCL/ML: DTH++++], and anergic diffuse cutaneous leishmaniasis [ADCL: DTH− ]. Although human genetic profile plays important factor in the immunopathogenesis of ACL, it deserves to be highlighted the crucial role of speciesspecific antigens of *L. (V.) braziliensis* and *L. (L.) amazonensis* [lipophosphoglycans, phosphatidylserine, proteophosphoglycans, glycoprotein-63 and CD200 – a macrophage activation inhibitor molecule] in the modulation of T-cell immune response (CD4+ /CD8+ ) that will define the infection evolution.

**Keywords:** American cutaneous leishmaniasis, *Leishmania (V.) braziliensis*, *Leishmania (L.) amazonensis*, species-specific antigens, pathogenesis

#### **1. Introduction**

American cutaneous leishmaniasis (ACL) is an infectious, non-contagious, chronic disease caused by fifteen well recognized species of protozoan parasites of the genus *Leishmania* Ross 1903, widely distributed throughout Latin America. The disease is characterized by involvement of the cutaneous and/or mucosal tissue, where leishmaniotic ulcer represents the most usual clinical manifestation of ACL. However, other types of cutaneous lesions can be seen, such as: papule, tubercle, nodule, infiltrated plaque and verrucous or keloid-like vegetating lesion, among others. The mucosal lesion can be installed in the nose, mouth or pharynx alone, or even simultaneously, however, it is more frequent in the nasal mucosa, giving it a granulomatous ulcerative aspect, which, depending on its depth, can reach the cartilaginous tissue causing perforation of the nasal septum [1–10].

In Brazil, the leishmaniotic nature of the skin and/or mucosal lesions of ACL were only confirmed, for the first time, in 1909, by Lindenberg, who found amastigote forms of *Leishmania* in smears made from the skin and/or mucosal lesions of patients coming from the interior of the State of São Paulo [southeastern Brazil]. Nevertheless, it was Vianna who described the new parasite, *Leishmania braziliensis* Vianna 1911, the first *Leishmania* species recognized in the New World. From then on, the etiology of ACL referred to as "Bauru's ulcer", "brave wound" or "tapir nose" was finally characterized [11, 12].

Until the early 1970s, all clinical forms of ACL in Brazil were attributed to a single *Leishmania* species, *Leishmania braziliensis* [13]. However, research carried out in the Brazilian Amazon pointed to the presence of a new *Leishmania* species responsible for some cases of the "anergic diffuse cutaneous form" named *Leishmania mexicana amazonensis* Lainson & Shaw 1972 [=*Leishmania (Leishmania) amazonensis* Lainson & Shaw 1987] [14]. Later, as new *Leishmania* species were identified in association with ACL [15, 16], great heterogeneity was noticed among these parasites which motivated a new classification of these protozoan parasites of the genus *Leishmania*, whose taxonomic character of greater expression was the biological behavior of the parasite within the gut of the sandfly vectors, represented by the terms: "hypopylaria, peripylaria and suprapilaria" [14]. Thus, these authors proposed the taxonomic classification of all *Leishmania* species into two subgenera: *Leishmania* Saf 'janova 1982, and *Viannia* Lainson & Shaw 1987, in which the species that were previously allocated within the "*mexicana*" complex [suprapylaria = with development from the midgut to the foregut] were then allocated within the subgenus *Leishmania* [the same name of the genus], while those that were previously allocated within the "*braziliensis*" complex [peripylaria = with development from the hindgut (pylorus) to the foregut] were allocated within the subgenus *Viannia*.

Currently, these two subgenera [*Leishmania* and *Viannia*] include all seven *Leishmania* species and a hybrid parasite responsible for ACL in Brazil, mainly in the Brazilian Amazon, such as: *L. (V.) braziliensis*, *L. (V.) guyanensis*, *L. (V.) shawi*, *L. (V.) lainsoni*, *L. (V.) lindenbergi*, *L. (V.) naiffi*, *L. (L.) amazonensis* and *L. (V.) guyanensis*/*L. (V.) shawi* [9–12, 17, 18]. Nevertheless, it is important to note that, more recently, a new molecular approach of the V7V8 SSU rRNA, Hsp70, and gGAPDH genes enabled the description of a new subgenus named *Mundinia* Shaw, Camargo & Teixeira 2016 [19], where *L. (M.) martiniquensis* Desbois et al. 2014 is classified, an ACL agent in the Caribbean region (**Table 1**).

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*


#### **Table 1.**

*Taxonomic classification of Leishmania parasites responsible for the etiology of American cutaneous leishmaniasis [ACL].*

In Brazil, from an eco-epidemiological point of view, ACL is regarded as a primary zoonotic infection of wild mammals, such as rodents, marsupials, edentates and primates, whose transmission of the parasites between their mammalian hosts is carried out by the infected females of different species of sandfly vectors [Diptera: Psychodidae: Phlebotominae] [11, 12, 20–24]. In this country, the disease represents an important public health problem, mainly in the North, Northeast and Midwest regions, where ecological, climatic and socio-economic factors have contributed to the formation and maintenance of large foci of the disease [9, 10, 25, 26]. In the North region, that is, in the Brazilian Amazon, it deserves special attention not only due to its high casuistry, but also due to its great heterogeneity of *Leishmania* species, mainly in the State of Pará, where *L. (V.) braziliensis* and *L. (L.) amazonensis* have high pathogenic potential to give rise to one of the most complex parasitic diseases.

In this sense, it is worth mentioning that, together, *L. (V.) braziliensis* and *L. (L.) amazonensis* are responsible for the broadest clinical-immunopathological spectrum of ACL already known, consisting of four clinical forms: localized cutaneous leishmaniasis [LCL], the most common form placed at the center of this spectrum and supported by a well-balanced T-cell immune response, including the delayed-type hypersensitivity reaction (DTH), mainly in cases by *L. (V.) braziliensis* [DTH+/++/ CD4<sup>+</sup> < CD8+ /Th1 > Th2], but with a high prevalence rate [≥80%] suppression of the T-cell hypersensitivity response in cases by *L. (L.) amazonensis* [DTH±/−/ CD4<sup>+</sup> < CD8+ /Th1 > Th2]; mucocutaneous or mucosal leishmanaisis [MCL/ML], the extreme expression of the T-cell hypersensitivity response linked to a strong species-specific CD4+ /Th1-type immune response against to infection [DTH++++/ CD4<sup>+</sup> > CD8+ /Th1 > Th2]; anergic diffuse cutaneous leishmaniasis [ADCL], the extreme expression of the T-cell hyposensitivity response linked to a strong speciesspecific CD4+ /Th2-type immune response against to infection [DTH<sup>−</sup> /CD4+ < CD8+ / Th1 < Th2]; and borderline disseminated cutaneous leishmaniasis [BDCL], an

#### **Figure 1.**

*Some clinical and immunopathological features of the ACL spectrum caused by* L. (V.) braziliensis *and* L. (L.) amazonensis *in Brazil. Anergic DCL = diffuse cutaneous leishmaniasis; BDCL = borderline disseminated cutaneous leishmaniasis; LCL = localized cutaneous leishmanaisis; ML = mucosal leishmaniasis; DTH = delayedtype hypersensitivity reaction; CD4 = lymphocyte TCD4; CD8 = lymphocyte TCD8; Th1 = Th1-type immune response; Th2 = Th2-type immune response; TLR = tool-like receptors 2, 4 and 9; TNF-α = tumor necrosis factor-alpha; IFN-у = interferon-gamma; IL-10 = interleukin-10; TGF-β = tumor growth factor-beta. (−) no reaction; (±) borderline reaction; (+) weak reaction; (++) moderate reaction; (+++) strong reaction; and (++++) exacerbated reaction.*

intermediary form between the central LCL and the two polar MCL/ML and ADCL forms, which is distinguished by a partial or incomplete suppression of the T-cell immune response more evident in those cases due to *L. (L.) amazonensis* [DTH− / CD4<sup>+</sup> < CD8+ /Th1 ≥ Th2] than those due to *L. (V.) braziliensis* [DTH± /CD4+ > CD8+ / Th1 ≥ Th2] (**Figure 1**) [10, 18, 27–30].

That said, one could question the real significance of the taxonomic classification of the genus *Leishmania* proposed by Lainson & Shaw [14] based on a merely biological taxonomic character in relation to the pathogenesis of ACL. In fact, perhaps not even the authors themselves at that time realized how significant that taxonomic classification would have in relation to the pathogenesis of ACL. However, the immunopathogenic competence of those *Leishmania* species of the subgenera *Leishmania* and *Viannia* play in the pathogenesis of ACL is now better understood. In this way, it has been shown a clear dichotomy on the interaction between *L. (L.) amazonensis* and *L. (V.) braziliensis* with human T-cell immune response; while *L. (L.) amazonensis* shows a clear tendency to lead infection from the LCL, a persistent suppressed T-cell hypersensitivity response [DTH±/−] clinical form in the center of the spectrum, towards the ADCL at the T-cell hyposensitivity pole [DTH<sup>−</sup> ] and with a marked CD4<sup>+</sup> /Th2-type immune response, *L. (V.) braziliensis* shows an opposite trend from the well-balanced form of the T-cell hypersensitivity [DTH+/++] in the center of the spectrum, leading infection towards the MCL/ML in the T-cell hypersensitivity pole [DTH++++] and with a prominent CD4+ /Th1-type immune response [18, 27, 29–33]. More than that, there is now some revealing evidence regarding the importance of some species-specific *Leishmania* antigens of the subgenera *Leishmania* and *Viannia*, represented by a dense layer of glycoconjugate molecules also known as virulence

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

factors, such as, lipophosphoglycans [LPG], glucoinositolphospholipids [GIPLs], proteophosphoglycans [PPGs], glycoprotein-63 [GP-63], phosphatidylserine [PS], and CD200 [macrophage activation inhibitor molecule], playing a crucial role in modulating T-cell immune response against infection, which will be the reason for a more complete approach later in this chapter [18, 34–36].

#### **2. Clinical and immunopathological spectrum of ACL due to** *L. (V.) braziliensis* **and** *L. (L.) amazonensis*

Firstly, what reasons would there be today to highlight the importance of *L. (V.) braziliensis* and *L. (L.) amazonensis* as the species of greatest medical interest regarding the clinical-immunopathological spectrum of ACL in Brazil, mainly in the Brazilian Amazon. Without a doubt, based on what has already been said above and also taking in account that *L. (V.) braziliensis* is historically the first *Leishmania* species described in the New World, where it shows the widest geographic distribution in Latin America, from Central America [where it has been identified as an agent of ACL in Honduras, Belize, Costa Rica, Ecuador, Guatemala, Mexico, Nicaragua, and Panama] to South America [where it is strongly associated to ACL, mainly in Brazil, but also in Colombia, Venezuela, Peru, Bolivia, Paraguay, and Argentina], it seems fair enough to regard *L. (V.) braziliensis* as the main *Leishmania* species in the context of ACL [10–12, 37].

On the other hand, alongside *L. (V.) braziliensis* in Brazil, there seems to be no doubt that *L. (L.) amazonensis* occupies the second position in the ranking of the most medically important *Leishmania* species in reason of its capacity to develop the ADCL, the extreme expression of T-cell hyposensitivity pole [DTH− ], which although it occurs with low frequency is capable of reproducing severe [nodules and infiltrated plaques] and disseminated [sparing only the scalp] chronic and incurable skin lesions in patients with strong suppression of the T-cell immune response [CD4<sup>+</sup> < CD8+ / Th1 < Th2/IL-10 and TGF-β > TNF-α and IFN-γ] [18, 27, 29, 30, 38].

Based on what was said above, it does not seem inappropriate to assume that *L. (V.) braziliensis* and *L. (L.) amazonensis* are recognized as the two *Leishmania* species with the greatest pathogenic potential for reproducing the clinical-immunopathological spectrum of ACL in Brazil: LCL and BDCL by both *Leishmania* species, while MCL/ ML by *L. (V.) braziliensis* alone, as well as ADCL by *L. (L.) amazonensis* alone, which, from now on, reinforces the crucial role of the species-specific antigens also known as virulence factors [LPGs, GIPLs, PPGs, GP-63, PS and CD200] of these *Leishmania* species in the orchestration of the clinical-immunopathological spectrum of ACL. In other words, while species-specific *L. (V.) braziliensis*-antigens modulate a strong pro-inflammatory response in MCL/ML with production of high levels of major proinflammatory cytokines [IFN-γ/TNF-α], species-specific *L. (L.) amazonensis*-antigens modulate a strong anti-inflammatory response in ADCL with production of high levels of major regulatory cytokines [IL-10/TGF-β], even though higher (*P* < 0.05) antigenpresenting cells densities [dendritic Langerhans cell (LC): CD1a– MHCII+ , Langerin+ , and dermal dendritic cell (dDC): CD11c–MHCII+ , Langerin− and MHCI+ , Langerin+ ] have been shown in ADCL clinical form, in the T cell hyposensitivity pole [DTH− ], compared to those in LCL clinical form, a well-balanced T-cell hypersensitivity form [DTH+/++] in the ACL spectrum, evidencing a crucial role of these species-specific antigens of *L. (L.) amazonensis* and *L. (V.) braziliensis* in modulating T-cell immune response (CD4+ /CD8+ ) against infection [18, 27, 29, 30, 39–41].

Nevertheless, one could question the non-inclusion of *L. (V.) guyanensis*, alongside *L. (V.) braziliensis*, among the species with pathogenic potential to cause MCL/ ML in Brazil. In fact, recent evidence has shown *L. (V.) guyanensis* as a causative agent of MCL/ML in the Brazilian Amazon [42], principally in the north bank of the Amazon River in the State of Amazonas, which undoubtedly limits its importance as a causative agent of MCL/ML not only in the Brazilian territory, but in the Brazilian Amazon as well. In the State of Pará, for example, where MCL/ML patients are regularly present in the outpatient clinic of the "Ralph Lainson leishmaniasis laboratory" at the Evandro Chagas Institute [Science, Technology and Innovation Secretariat, Ministry of Health, Brazil] in the municipality of Ananindeua, whose demand for patients comes from all regions of the State of Pará and to a lesser extent from the States of Amapá, Maranhão, Tocantins and Mato Grosso, the only leishmanine parasite that has been identified from MCL/ML in the last forty years is *L. (V.) braziliensis*, which leaves no doubt as to its importance as the main agent of MCL/ML in Brazil [9–12, 14, 18, 27, 43, 44].

#### **2.1 Localized cutaneous leishmaniasis (LCL) due to** *L. (V.) braziliensis* **and** *L. (L.) amazonensis*

In Brazil, LCL is, without a doubt, the most common clinical manifestation of ACL, having as causal agent any of the seven *Leishmania* species and the hybrid parasite [*L. (V.) guyanensis/L. (V.) shawi*] mentioned above, that is, six species of the subgenus *Viannia*, including the species of greatest medical interest like *L. (V.) braziliensis*, and one of the subgenus *Leishmania*, with *L. (L.) amazonensis* being the only species associated with ACL in Brazil. However, it is necessary to record that, although the medical importance of these two species is well recognized, the association of *L. (V.) braziliensis* with the disease is much greater than that of *L. (L.) amazonensis*, which is explained due to the higher anthropophilic potential of the main sandfly vectors of *L. (V.) braziliensis*, such as, *Lutzomyia intermedia*, *Lut. whitmani*, *Lut. migonei*, *Psychodopygus wellcomei*, *Psy. complexus* and *Psy. davisi* than that of *Lut. flaviscutellata*, the principal sandfly vector of *L. (L.) amazonensis* in Brazil [9, 10, 45–48].

From a clinical point of view, however, it is important to say that, although the typical ulcerated skin lesion, with a raised and infiltrated edge and a granulomatous background, represents the major clinical sign most frequently observed in patients with LCL caused by *L. (V.) braziliensis* or any other species of the subgenus *Viannia*, especially after one to two months of disease progression (**Figure 2A**), the same cannot be said in relation to the skin lesion in patients with LCL caused by *L. (L.) amazonensis* as, in most cases, it is represented by a nodule or plaque with a highly infiltrated base and edge, with a thickened appearance, evidencing discrete ulceration or even an exulceration on the surface (**Figure 2E,F**), without that typical ulcerative character seen in the skin lesion caused by *L. (V.) braziliensis*. This clinical difference in the skin lesion of LCL between *L. (V.) braziliensis* and *L. (L.) amazonensis* clearly reflects distinct physiopathogenic mechanisms on the interaction of species-specific antigens of these *Leishmania* species with human T-cell immune response [CD4<sup>+</sup> /CD8<sup>+</sup> ]. On the one hand, while *L. (V.) braziliensis* is able to induce a moderate to overt DTH(+/++) in LCL patients, on the other, *L. (L.) amazonensis* interacts in contrary to this by suppressing the DTH(±/−) in more than 80% of LCL patients, which might explain the greater severity of dermal necrosis and subsequent ulceration seen in the skin lesion induced by *L. (V.) braziliensis*, since dermal

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

necrosis represents a defense mechanism in the elimination of the parasite strongly related to DTH response [49, 50]. On the contrary, in the skin lesion induced by *L. (L.) amazonensis* dermal necrosis is a more discrete phenomenon due to the suppression of DTH(±/−), resulting in a heavy parasite load on the vacuolated macrophages and lesser intense ulcerative process compared to that seen in the skin lesion of LCL by *L. (V.) braziliensis* [6, 18, 27, 29, 51–53].

Another clinical feature that deserves to be highlighted here refers to the polymorphism of the skin lesions that can be seen in patients with LCL caused by *L. (V.) braziliensis*, where not only the typical ulcerated skin lesion can be seen but also papule-like lesions, tubercle, nodule, infiltrated plaque and verrucous or keloid vegetating lesion (**Figure 2B**–**D**), while in patients with LCL caused by *L. (L.) amazonensis* the skin lesions do not go beyond the nodule or infiltrated plaque with a highly infiltrated base and edge, with a thickened appearance, evidencing discrete ulceration or even an exulceration on the surface (**Figure 2E,F**). However, in general, these clinical features are not optimized by many authors in Brazil, perhaps due to the low frequency of patients with LCL caused by *L. (L.) amazonensis* outside the Brazilian Amazon or the lack of a specific diagnosis of the etiologic agent involved. The fact is that, in the State of Pará, in the Brazilian Amazon, a regular number (194) of patients with LCL caused by *L. (L.) amazonensis* have been observed in the last forty years in the outpatient clinic of the "Ralph Lainson leishmaniasis laboratory" at the Evandro Chagas Institute [Science, Technology and Innovation Secretariat, Ministry of Health, Brazil] (Silveira, personal observation), more precisely from the northeast of the State of Pará, which has allowed a significant gain regarding the clinical, histopathological and immunopathological understanding of LCL caused by *L. (L.) amazonensis* [18, 27, 29, 38, 51–55].

However, before the establishment of *Leishmania*-infection, either by *L. (V.) braziliensis* or *L. (L.) amazonensis* and the subsequent development of the skin lesion, that is, of the LCL itself, one cannot fail to remember the crucial role of those species-specific antigens of *L. (V.) braziliensis* and *L. (L.) amazonensis* play during the two major events in the development of infection: first, the event that determines *Leishmania*-infection, represented by the interaction parasite-macrophage or parasite-neutrophil-macrophage; and second, the event that determines the evolution of *Leishmania*-infection, represented by the interaction parasitedermal dendritic cell and/or parasite-Langerhans dendritic cell with human T-cell immune response [CD4<sup>+</sup> /CD8<sup>+</sup> ] [9, 10, 18, 29, 32, 33, 40, 56, 57]. The recognition of these two events markedly influenced by the species-specific antigens of *L. (V.) braziliensis* and *L. (L.) amazonensis* is of great interest for a better understanding of the clinical-immunopathological spectrum of ACL caused by these *Leishmania* species.

Thus, after the establishment of the infection in the macrophage, some individuals who show effective activation of the innate immune response via IFN-γ and TNF-α are able to neutralize the infection [asymptomatic], while the majority develop different degrees of susceptibility [symptomatic], which may give rise to the central LCL form of the clinical-immunopathological spectrum of ACL that, depending on the species-specific *Leishmania*-antigens involved, such as, *L. (V.) braziliensis* or *L. (L.) amazonensis*, may present different clinical, histopathological and immunopathological profiles of infection [29, 32, 58].

In LCL caused by *L. (V.) braziliensis*, it has already been seen above that the predominant skin lesion is the typical leishmaniotic ulcer, which can generally be recognized after one to two months of disease progression, revealing, in most cases,

#### **Figure 2.**

*Localized cutaneous leishmaniasis caused by* L. (V.) braziliensis *[A: Typical ulcerated skin lesion; B: Keloid skin lesion; C: Verrucous skin lesion; D: Ulcer-infiltrated skin lesion on the hand, with bone deformity (osteomyelitis)] and* L. (L.) amazonensis *[E and F: Skin lesions in the form of a highly infiltrated plaque, with a thick base and exulcerated surface]; G and H: Histological sections of skin lesions caused by* L. (L.) amazonensis *and*  L. (V.) braziliensis*, respectively, stained for eosin & hematoxylin (HE), showing in (G) dermal cellular infiltrate predominantly of heavily parasitized vacuolated macrophages (arrow), in addition to lymphocytes and plasma cells; and in (H) epithelioid granulomatous reaction, with giant cells (arrow), and lymphoplasmacytic infiltrate, with few macrophages. Bars = 20 μm.*

the epithelioid granuloma into the dermis, with the presence of giant cells, some necrosis areas, typical lymphoplasmocytic cellular infiltrate and scanty parasitized macrophages (**Figure 2H**) [59, 60]. From the immunopathological point of view,

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

some parameters previously studied deserve to be highlighted here, such as: most patients [≥95%] present moderate to overt DTH(+/++), accompanied by high levels of lymphocyte proliferation assay, while into the dermis of the skin lesion the cell immunohistochemical labeling with specific monoclonal antibodies has revealed higher (*P* < 0.05) CD8<sup>+</sup> T-cells density compared to that of their CD4+ T-cells counterpart, although the CD4+ /Th1-type immune response has been shown to outperform the CD4+ /Th2-type one, in addition to higher (*P* < 0.05) cell densities expressing proinflammatory cytokines [TNF-α, IFN-γ] compared to those of regulatory ones [IL-10, TGF-β], as well as greater CD68+ macrophage densities expressing *Toll-like* receptors 2 and 4 (*TLR*2, 4) compared to that of *TLR*9 were evidenced in the double immunohistochemical cell labeling (**Figure 1**) [9, 10, 18, 27, 29, 30, 53, 61–63].

On the other hand, in LCL caused by *L. (L.) amazonensis* it has also been seen that the main clinical feature of the skin lesion observed in this form of the disease is the nodule or plaque with a highly infiltrated base and edge, with a thickened appearance, evidencing discrete ulceration or even an exulceration on the surface, in which the monocyte cell infiltrate into the dermis stands out at the expense, mainly, of the non-activated vacuolated macrophage of the M2 phenotype, intensely parasitized, with few plasma cells and lymphocytes in the interstitium, characterizing a true vacuolated macrophage reaction into the dermis of patients with LCL caused by *L. (L.) amazonensis* (**Figure 2G**). It is interesting to note the harmony between these histopathological findings with those of immunopathological nature, so that, in most patients [≥80%] the delayed-type hypersensitivity reaction is absent [DTH<sup>−</sup> ] not only after challenge with axenic promastigote antigens of *L. (L.) amazonensis* or *L. (V.) braziliensis*, but also with axenic amastigote antigen of *L. (V.) lainsoni*, as well as low lymphocyte proliferation rates and few areas with epithelioid granuloma and/or necrosis into the dermis of the skin lesion in these patients are found [10, 18, 27, 51–53, 64, 65].

In addition, regarding the immunopathological findings searched by immunohistochemical cell labeling with specific monoclonal antibodies, it seems notorious certain similarity with those in LCL caused by *L. (V.) braziliensis*, such as, a higher (*P* < 0.05) CD8<sup>+</sup> T-cells density compared to that of their CD4<sup>+</sup> T-cells counterpart, as well as a CD4<sup>+</sup> /Th1-type immune response over than that of CD4<sup>+</sup> /Th2-type one, although not as evident as that seen in LCL caused by *L. (V.) braziliensis*. In this way, it is worth mentioning the higher (*P* < 0.05) cell densities expressing some regulatory cytokines [IL-10, TGF-β] than that of pro-inflammatory one [TNF-α], but not than that of IFN-γ. It is quite possible that the significant expression of IFN-γ together with that of *TLR*2 [evidenced by double immunohistochemical cell labeling] has ensured the superiority of the CD4+/Th1-type immune response over the CD4+/Th2-type in LCL caused by *L. (L.) amazonensis*, although some findings seem to signal a persistent suppression of the T-cell immune response as evidenced by high prevalence [≥80%] of negative DTH(−), as well as low rates of lymphocyte proliferation assay in these patients (**Figure 1**) [10, 18, 27, 29, 30, 32, 51, 53].

In summary, taking into account these clinical, histopathological and immunopathological features of LCL caused by *L. (V.) braziliensis* and *L. (L.) amazonensis*, it would be appropriate to say that: i) the ulcerated character of the skin lesion is more associated with *L. (V.) braziliensis*-infection [in *L. (L.) amazonensis*-infection, the skin lesion is less ulcerated, with a more infiltrated and thickened border], ii) where the presence of parasitized macrophages into the dermis is more scanty, iii) frequently [≥95% of cases] associated to positive DTH(+/++) [unlike *L. (L.) amazonensis*-infection with negative DTH(−) in ≥80% of cases] and high rates of lymphocyte proliferation

assay, iv) with inflammatory response mainly represented by the epithelioid granuloma, giant cells, necrosis areas and consistent lymphoplasmacytic cell infiltrate [in *L. (L.) amazonensis*-infection, the inflammatory response is basically formed by non-activated vacuolated macrophages of the M2 phenotype, intensely parasitized, with few plasma cells and lymphocytes, characterizing a true vacuolated macrophage reaction], v) while the immunopathological scenario shows a significant protector role of CD8+ T-cells stands out, in greater density than their CD4<sup>+</sup> T-cells counterpart, both in LCL by *L. (V.) braziliensis* and *L. (L.) amazonensis*, vi) the CD4+ /Th1-type immune response over than that of its CD4<sup>+</sup> /Th2-type one, more evident in *L. (V.) braziliensis*-infection, vii) with higher cell densities of pro-inflammatory cytokines [IFN-γ, TNF-α] compared to those of regulatory ones [IL-10, TGF-β], in agreement with greater expressions of *TLR*2, 4 than that of *TLR*9 [which has shown higher interaction with *L. (L.) amazonensis*-infection, in which there has been demonstrated high prevalence [80%] of the T-cell immune response suppression].

#### **2.2 Borderline disseminated cutaneous leishmaniasis (BDCL) due to** *L. (V.) braziliensis* **and** *L. (L.) amazonensis*

The first point that must be clarified is why the term "borderline" is being used here to designate this form of the disease that is referred by most authors as "Disseminated cutaneous leishmaniasis" [66–73]. In fact, this term was first used by Moriearty et al. [74] to designate a clinical case of ACL in a patient with long-lasting disseminated skin lesions, and atypical intradermal reaction to *Leishmania*-antigen [DTH**−**] from northeast Brazil [Três Braços municipality, Bahia State], where the species *L. (V.) braziliensis* predominates. Considering that this patient also did not respond to other intradermally tested antigens, his ACL clinical form was labeled by these authors as "Borderline cutaneous leishmaniasis" [74]. However, considering that this was a typical clinical case with long-lasting disseminated skin lesions, the first step would be to designate the case as "Borderline disseminated cutaneous leishmaniasis". Anyway, considering that the term "borderline" was used by these authors in an attempt to characterize a probable patient's T-cell immune response dysfunction against infection, a similar interpretation was made by the authors of this chapter to also characterize a partial or incomplete suppression of the T-cell immune response against *Leishmania*-infection [mainly evidenced by suppression of the delayed-type hypersensitivity reaction (DTH) and lymphocyte proliferation assay] in some individuals with long-lasting disseminated skin lesions caused by *L. (V.) braziliensis* or *L. (L.) amazonensis*, favoring the spread of infection and the consequent establishment of the clinical form that has been designated by the authors of this chapter as "borderline disseminated cutaneous leishmaniasis" [BDCL] [10, 18, 27–29].

The second point that deserves to be clarified refers to the physiopathogenic mechanisms involved in the process of the parasite dissemination, which, once again, seems to be strongly influenced by the species-specific antigens of *L. (V.) braziliensis* and *L. (L.) amazonensis*. In this way, what stands out the most is the dynamics of the parasite dissemination and its relationship with the partial or incomplete suppression of the patient's T-cell immune response. In terms of BDCL caused by *L. (V.) braziliensis*, it has been observed that the parasite dissemination is relatively fast, so that in around three months, tens or even hundreds papular, acneiform or ulcerative skin lesions mainly can be counted, distributed in the vicinity of the primary skin lesion [spread by contiguity] or in different regions of the patient's body [lymphohematogenous systemic spread] (**Figure 3A**–**D**). Furthermore, recent evidence has shown

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

that *L. (V.) braziliensis* isolates from patients with BDCL from an endemic area in the municipality of Três Braços, Bahia State, in northeastern Brazil, were able to induce, *in vitro*, higher infectivity rates and greater expressions of *TLR*2, 4 than isolates from LCL patients, which reinforces the central idea of this chapter on the crucial role of species-specific antigens of *L. (V.) braziliensis* and *L. (L.) amazonensis*, or even among intra-specific isolates of these *Leishmania* species in the pathogenesis of ACL. Another alternative would be that these *L. (V.) braziliensis* polymorphic variants would be producing lower systemic levels of TNF-α and IFN-γ in BDCL patients than those LCL ones, which may account for parasite dissemination due to the decreased ability to control parasite growth [18, 27, 29, 66, 68, 69, 71, 75–77].

Regarding BDCL caused by *L. (L.) amazonensis*, however, the parasite dissemination is clearly slower, taking around a year for the first metastatic lesions to appear, mainly papules that progress to non-ulcerated nodules and/or infiltrated plaques [more than ten were never observed], also close to the primary skin lesion [spread by contiguity] or at greater distances [lymphohematogenous systemic spread] (**Figure 3E**–**H**). Therefore, the clinical-biological behavior of *L. (L.) amazonensis*infection, mainly with regard to the parasite dissemination and its relationship with T-cell immune response, is quite different from that by *L. (V.) braziliensis*; whereas *L. (V.) braziliensis*-infection disseminates faster and exerts a more discrete suppression of the T-cell immune response [DTH<sup>±</sup> ], *L. (L.) amazonensis*-infection disseminates more slowly, but with a more intense suppression of the T-cell immune response [DTH<sup>−</sup> ], which is reflected in the response to antimony therapy as patients with BDCL caused by *L. (L.) amazonensis* require double [four series] the dosage of antimony therapy used in the treatment of patients with BDCL caused by *L. (V.) braziliensis* [see that dosage below]. Additionally, it is interesting to note that, similarly to what has been shown in *L. (V.) braziliensis* isolates from BDCL patients, it has already been shown, also *in vitro*, greater infectivity rates of *L. (L.) amazonensis* isolates from BDCL than those from LCL, confirming our previous suspicious that intra-specific antigenic differences within *L. (L.) amazonensis* [or even within *L. (V.) braziliensis*] strains/races/populations from distinct geographical regions in Brazil may be influencing the pathogenesis of ACL [10, 18, 27–29, 38, 78].

As for the histopathological repercussion of these processes, the findings into the dermis of the skin lesions in BDCL patients do not differ much in qualitative terms from those already mentioned in LCL caused by *L. (V.) braziliensis* and *L. (L.) amazonensis*, however, it is necessary to emphasize a greater intensity of these findings that take part in these processes. For example, in BDCL caused by *L. (V.) braziliensis* what stands out is the presence of a more consistent lymphoplasmacytic cell infiltrate, accompanied by few to moderate parasitized macrophages, with few necrosis areas and epithelioid granuloma (**Figure 4B**). In BDCL caused by *L. (L.) amazonensis*, what also stands out is the exuberance of the vacuolated macrophage reaction [mainly from the M2 phenotype], intensely parasitized, accompanied by moderate lymphoplasmacytic cellular infiltrate, without necrosis areas, but in some cases with long-lasting evolution [more than a year] there may be found some epithelioid granulomas with giant cells (**Figure 4A**), which seem to represent a T-cell immune response reserve, since, as in LCL caused by this *Leishmania* species or by *L. (V.) braziliensis*, antimony therapy [12 mg/Sbv /kg weight/22 days each series] has been used successfully at twice [four series] the dosage of that used in LCL [two series] [9, 10, 18, 27–29, 64].

In terms of the immunopathology of BDCL, it should be emphasized that during the parasite dissemination, which means a critical stage of the infection, both the DTH reaction and lymphocyte proliferation assay remain negative, clearly reflecting

#### **Figure 3.**

*Borderline disseminated cutaneous leishmaniasis (BDCL) caused by* L. (V.) braziliensis *and* L. (L.) amazonensis*, respectively, with disseminated acneiform ulcerative skin lesions in A, B, C and D [L.(V.)b]; and infiltrated plaque or nodular (non-ulcerated) skin lesions in E, F, G, and H [L.(L.)a].*

#### **Figure 4.**

*Histological sections of skin lesions stained for eosin & hematoxylin (HE) in borderline disseminated cutaneous leishmaniasis (BDCL) caused by* L. (L.) amazonensis *(A) and* L. (V.) braziliensis *(B), where can be seen in a typical vacuolated macrophage reaction, intensely parasitized (arrows), with few lymphocytes and plasma cells and, in B intense lymphoplasmacytic infiltrate with few macrophages. Bars = 20 μm.*

some degree of T-cell immune response suppression, mainly in those BDCL patients due to *L. (L.) amazonensis* whom present a more intense T-cell immune response suppression [DTH− ] than those due to *L. (V.) braziliensis* whom show a more discrete T-cell immune suppression [DTH± ], which, recently, was also noted by higher (*P* < 0.05) cell densities expressing regulatory cytokines [IL-10, TGF-β] than those pro-inflammatory one [TNF-α] in BDCL caused by both *L. (V.) braziliensis* and *L. (L.) amazonensis*. Moreover, it is interesting to point out that regarding the CD4+ / CD8<sup>+</sup> T-cells expressions in BDCL, it was also recently recorded that in *L. (V.) braziliensis*-infection there was a higher (*P* < 0.05) CD4+ T-cells expression than that of their CD8<sup>+</sup> T-cells counterpart, while in *L. (L.) amazonensis*-infection there was a lower (*P* < 0.05) CD4+ T-cells expression than that of their CD8+ T-cells counterpart, *From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

which suggests a crucial role of CD4+ T-cell immune response against infection, since in BDCL caused by *L. (L.) amazonensis*, where there was a more intense T-cell immune response suppression [DTH− ], the CD4<sup>+</sup> T-cells expression was lower (*P* < 0.05) than that of their CD8+ T-cells one, although the CD4+ Th1-type immune response is still slightly over than the CD4+ Th2-type immune response. Finally, looking at the expressions of *TLR*2, 4 and 9, it was seen once again that *TLR*2,4 had greater (*P* < 0.05) expressions in BDCL by *L. (V.) braziliensis*, where the T-cell immune response suppression had less impact [DTH± ], while *TLR*9 had greater (*P* < 0.05) expression in BDCL by *L. (L.) amazonensis*, where T-cell immune response suppression was more impactful [DTH− ] (**Figure 1**) [9, 10, 18, 27–30].

In summary, BDCL represents an intermediary form of ACL that can occupy the two places between the center (LCL) and the two polar forms (MCL/ML and ADCL) in the clinical-immunopathological spectrum of the disease, having *L. (V.) braziliensis* and *L. (L.) amazonensis* as the main etiological agents in Brazil, as well as there are some clinical and immunopathological features that differ BDCL between these two *Leishmania* species. The term "borderline" was introduced to characterize the clinical evidence of partial or incomplete T-cell immune response suppression against *Leishmania*-infection in BDCL, mainly in those patients infected by *L. (L.) amazonensis* who present greater suppression of T-cell immune response [DTH− ] than those infected by *L. (V.) braziliensis* [DTH<sup>±</sup> ], which seems to be one of the main reasons for the spread of the parasite. However, in BDCL by *L. (V.) braziliensis*, the parasite spread is relatively rapid, requiring only three months to give rise to a hundred or more ulcerative skin lesions; in contrast, in BDCL by *L. (L.) amazonensis*, the parasite spread is slower, requiring around one year to produce some 5 to 10 infiltrated skin lesions. As for the histopathology, attention is drawn to the exuberance of the vacuolated macrophage reaction, mainly at the expense of the intensely parasitized M2 phenotype macrophage in the cellular infiltrate of BDCL by *L. (L.) amazonensis*, while in BDCL by *L. (V.) braziliensis* what stands out is the presence of the characteristic lymphoplasmacytic cell infiltrate with few parasitized macrophages. These histopathological findings seem to be in agreement with those immunopathological ones that revealed higher [*P* < 0.05] expression of regulatory [IL-10 and TGF-β] than pro-inflammatory [TNF-α] cytokines in BDCL by both *L. (V.) braziliensis* and *L. (L.) amazonensis*, but with higher [*P* < 0.05] expression of CD4<sup>+</sup> T-cells than their CD8<sup>+</sup> T-cells counterpart in BDCL caused by *L. (V.) braziliensis*, although the opposite was observed in BDCL caused by *L. (L.) amazonensis* [lower expression of CD4+ T-cells than their CD8<sup>+</sup> T-cells counterpart], which reinforces a higher T-cell immune response suppression in BDCL by *L. (L.) amazonensis*. However, in spite of these observations, the partial or incomplete T-cell immune response suppression found in BDCL by *L. (L.) amazonensis* has been restored following successful treatment with four LCL-therapeutic antimony regimens [12 mg/Sbv /kg weight/22 days each series with a 10-day interval between series], which suggest an evident supremacy of Th1 over Th2 immune response (Th1 ≥ Th2).

#### **2.3 Mucocutaneous or mucosal leishmaniasis (MCL/ML) due to** *L. (V.) braziliensis*

The first point that needs to be clarified concerns the possible similarities or differences between the both terms mucocutaneous and mucosal leishmaniasis. What is common to both is the involvement of nasobuccopharyngeal mucosal tissue, or just nasal [which is more frequent] or just buccopharyngeal, however, what distinguishes them is the dynamics of infection, since in some situations the infection may present simultaneous involvement of the skin and nasobuccopharyngeal mucosa tissue [mucocutaneous leishmaniasis - MCL] or only the nasobuccopharyngeal mucosa tissue [mucosal leishmaniasis - ML]. Thus, it seems clear that in the case of MCL, the involvement of the mucosal tissue is earlier (a few months after the appearance of the skin lesion) than in ML, in which the appearance of the mucosal lesion may be after years (average two to three years) of apparent healing (spontaneous or after irregular therapy) of the skin lesion. Nevertheless, it is necessary to say that in the Brazilian Amazon, especially in the State of Pará, the form more frequently observed is the ML, while the MCL form is more prevalent in the Brazilian northeast, especially in the State of Bahia, which suggests, once again, that intra-specific antigenic differences within *L. (V.) braziliensis* strains/races/ populations from distinct geographical regions in Brazil may be influencing the pathogenesis of ACL [MCL/ML] [1, 2, 10, 18, 27, 43, 79–83].

Anyway, what needs to be emphasized is the extraordinary capacity of *L. (V.) braziliensis* to migrate, via lymphohematogenous, from a skin lesion to the nasobuccopharyngeal mucosa tissue, where it will develop simultaneously to the skin lesion(s) [in the case of MCL], or years, in general, after apparent spontaneous remission or the inadequate treatment-based healing of skin lesion [in the case of ML], an inflammatory process that begins with erythema and infiltration of the mucosal tissue, followed by granulation and ulceration of the cartilaginous tissue, giving an ulcerogranulomatous aspect to the mucosal lesion (**Figure 5A**–**C**). In advanced cases, usually with more than five years of evolution, the inflammatory process can promote uncomfortable conditions such as nasal obstruction, formation of crusts, epistaxis, rhinorrhea, pruritus and even perforation of the cartilaginous tissue of the nasal septum or palate. In order of frequency, mucosal lesions primarily occur in the nasal septum, palate, pharynx, and larynx, or may be simultaneous in some cases resulting in metastatic lesions of the nasobuccopharyngeal mucosal tissue [1, 2, 4, 6, 10, 18, 27, 43, 83, 84].

Regarding the histopathology of MCL/ML, the most striking finding of this form of the disease is represented by the presence of the tuberculoid granuloma in the nasobuccopharyngeal mucosal tissue, which corroborates not only the remarkable DTH(++++) and lymphocyte proliferation assay, but also the up-regulation of CD4+ / Th1-type immune response observed in MCL/ML. In addition, other histopathological findings include abundant infiltration of lymphocytes and plasma cells, with scanty parasitized macrophages, followed by necrosis of the cartilaginous structures (**Figure 5D**) [31, 53, 59, 60].

In parallel with the histopathology of nasobuccopharyngeal mucosal lesions, it is important to note that some immunohistochemical studies have shown that MCL/ML is supported not only by the increased expression of CD4<sup>+</sup> T-cells infiltrating the mucosal lesions of patients (as compared to corresponding CD8<sup>+</sup> T-cells), as well as the increased expression of pro-inflammatory TNF-α and IFN-γ as compared to regulatory TGF-β and IL-10 cytokines and lysozyme stained activated macrophages (epithelioid cells), characterizing a typical CD4<sup>+</sup> / Th1-type immune response, which reinforces a prior hypothesis regarding the pathogenesis of MCL/ML caused by *L. (V.) braziliensis* that implies the activation of a persistent antigen-specific CD4<sup>+</sup> /Th1-type immune response at the lymph nodes, followed by the recruitment of CD4<sup>+</sup> /T-cells from the peripheral blood to the inflammatory infiltrate in the mucosal lesions. As such, those cells are preferentially primed to operate as cytokine Th1-producing cells (mainly IFN-γ and TNF-α). The strong T-cell hypersensitive response recorded in these patients can

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

#### **Figure 5.**

*Clinical aspects of mucocutaneous (MCL) or mucosal leishmaniasis (ML) caused by* L. (V.) braziliensis *in A, B and C, and in D a histological section of a mucosal lesion stained for eosin & hematoxylin [EH] showing a garnulomatous reaction with intense lymphoplasmacytic infiltrate. Bar = 20 μm.*

be attributed to a long-lasting antigen-specific CD4<sup>+</sup> /Th1 activation by speciesspecific *L. (V.) braziliensis-*antigens, resulting in the high production of IFN-γ and TNF-α cytokines. In addition, the moderate expression of *TLR*2, 4 should also be highlighted, however, higher than that of *TLR*9 in MCL, which reinforces the greater interaction of *TLR*2,4 with *L. (V.) braziliensis*-infection than *TLR*9 (**Figure 1**) [10, 18, 27, 29–31, 85]. TNF-α seems to be the major cytokine responsible for damaging the mucosal tissue [86–89]. Corroborating these findings, it should also be noted recent findings on the gene expression of immune response in ACL by *L. (V.) braziliensis*, showing high levels of IFN-γ and TNF-α cytokines in older skin lesions containing detectable levels of parasite transcripts [90].

#### **2.4 Anergic diffuse cutaneous leishmaniasis (ADCL) due to** *L. (L.) amazonensis*

Anergic diffuse cutaneous leishmaniasis (ADCL) is regarded a rare clinical form of ACL caused by leishmanine parasites belonging to the subgenera *Leishmania* and *Mundinia*. In this way, although *L. (L.) mexicana* and *L. (L.) amazonensis* have been regarded as the major agents of ADCL in Latin America, *L. (M.) martiniquensis* and *L. (L.) waltoni* have also been incriminated, more recently, as etiologic agents of the disease in that region [12, 14, 44, 91–93].

In Brazil, principally in the Brazilian Amazon, *L. (L.) amazonensis* is regarded, so far, the only species with immunopathogenic competence to determine ADCL; that is, with the ability to deviate the T-cell immune response of the infected individual to the T-cell hyposensitivity pole [DTH**−**] of the clinical-immunopathological ACL spectrum, characterized by strong expression of the T-cell immune response of CD4<sup>+</sup> /Th2-type, with high production of well-known regulatory cytokines [IL-10 and TGF-β]. It is in this subverted or down-regulated immunopathological scenario, strongly influenced by the species-specific action of *L. (L.) amazonensis*-antigens, that the infection progresses to establish a clinical picture characterized by a diffuse infiltration of the skin, as well as by papular, tuberous, infiltrating and nodular skin lesions, which rarely ulcerate, distributed throughout the integument only sparing the scalp and the palmar (hand) and plantar (foot) regions (**Figure 6A**–**C**). Due to this persistent immunosuppressive character of CD4<sup>+</sup> /Th2-type immune response, most patients (mainly children under five years old) carry, for many years (sometimes four or more decades), a severe clinical picture, sometimes highly mutilating, leading to the chronification of the process since the drugs available (pentavalent antimony, amphotericin B, pentamidine and miltefosine) for the treatment are not able to change the immunogenetic status of the patients. In cases with advanced evolution (over thirty years), bone lesions may occur in the extremities (osteomyelitis) or involvement of the nasobuccopharyngeal mucosa due to the parasite dissemination by contiguity of lesions on the face [10, 18, 27, 29, 30, 43, 94–99].

Based on the immunosuppressive character of the disease, it would not be difficult to expect that the major histopathological finding of the skin lesions would reveal a monocytic cellular infiltrate into the dermis predominantly composed by vacuolated macrophages with a M2 phenotype, intensely parasitized, in addition to a moderate presence of plasma cells and few lymphocytes (**Figure 6D**) [10, 18, 27, 28, 43, 52, 100, 101]. In general, this is the quasi-stigmatic histopathological picture of ADCL that together with the persistent suppressive CD4+ /Th2-type immune response explain why the authors of this chapter use the term "anergic" to name this type of ACL with a prognosis that is still quite reserved.

From an immunopathological point of view, there is no doubt that the specific suppression of the T-cell immune response [CD4<sup>+</sup> /CD8<sup>+</sup> ] provided by the speciesspecific *L. (L.) amazonensis*-antigens has wide repercussions on different fronts of the immune defense mechanisms against infection, evidenced by the strong suppression of the delayed-type hypersensitivity reaction (DTH<sup>−</sup> ) and lymphocyte proliferation responses, as well as by the low production, *in situ*, of the main inflammatory cytokines [IFN-γ and TNF-α] and, in contrast, the high production of regulatory ones [IL-4, IL-10 and TGF-β], revealing the inability of the non-activated dermal macrophage, mainly the M2 phenotype, to contain the parasite multiplication and leading to very weak responses to conventional therapy regimens [antimony, pentamidine and amphotericin B]. Corroborating these findings, it is also worth mentioning that it is precisely in ADCL that the lowest expression of CD4<sup>+</sup> /CD8<sup>+</sup> T-cells is shown among all ACL clinical forms, but, on the contrary, it is also in ADCL that the highest expression of Treg Foxp3<sup>+</sup> regulatory cells is recorded, confirming the extremely suppressed profile of CD4<sup>+</sup> /Th2-type immune response in ADCL, which seems to be strongly associated to low transcripts of CD4<sup>+</sup> /Th1-type and cytotoxic CD8+/T-cells and the highest expression of *TLR*9 in the clinical-immunopatholgical ACL spectrum [18, 27, 29, 30, 38, 51, 53, 64, 102, 103].

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

**Figure 6.**

*Clinical aspects of anergic diffuse cutaneous leishmaniasis (ADCL) caused by* L. (L.) amazonensis *in A, B and C, and in D a histological section of a skin lesion stained for eosin & hematoxylin (HE) exhibiting diffuse vacuolated macrophage reaction, intensely parasitized (arrows), with few lymphocytes and plasma cells. Bar = 20 μm.*

#### **3. Importance of species-specific antigens of** *L. (V.) braziliensis* **and** *L. (L.) amazonensis* **in the pathogenesis of ACL**

*Leishmania* parasites are well-known as obligate intracellular protozoan organisms because they carry out an essential phase of their reproduction within the mononuclear phagocytic cells, mainly the macrophage, in which the amastigote form of the parasite multiplies by binary division [14]. However, in order for the amastigote form of the parasite to resist the lethal action of a highly hostile environment inside the macrophage's phagolysosome [highly acidic, oxidative, containing antimicrobial peptides, proteases and restrict nutrient access], the parasite makes use of a series of molecules on the surface of its plasma membrane, called glycoconjugates, in order to neutralize the lethal action of these hostile elements into the macrophage's phagolysosome [104, 105]. This is a critical phase for the parasite that will result in its survival or death, so that, if overcome by the parasite, the infection will be definitively established in the vertebrate host [106, 107]. However, in addition to the protective effect that these glycoconjugate molecules exert on the survival of the parasite, it is also known that their action can play an important role in the virulence of the parasite, reason why these glycoconjugate molecules are recognized here as species-specific *Leishmania*-antigens of both subgenera *Leishmania* and *Viannia*, with pivotal role in the pathogenesis of the disease caused by these *Leishmania* parasites, represented here by *L. (V.) braziliensis* and *L. (L.) amazonensis*.

Among the previously mentioned glycoconjugate molecules [LPG, GIPLs, PPGs, GP-63, PS and CD200], LPG is undoubtedly one of most important species-specific *Leishmania*-antigens or, in other words, the major cell surface glycoconjugate of *Leishmania* parasites, which has the ability to protect the parasite against complement-mediated lysis, facilitating its attachment and entry into the macrophage, and inhibiting the phagolysosomal fusion and protein kinase C [108–110]. Furthermore, it seems to exert one of the broadest antigenic activity spectrum being able to induce not only a pro-inflammatory innate immune response, but also a suppressive one, depending on the subgenus of *Leishmania* species involved in the process, i.e., *Leishmania* or *Viannia*. In this sense, one cannot neglect the possibility that the species-specific antigens of these subgenera (*Leishmania* and *Viannia*) may be true messengers of glycoconjugate molecules formed from the contents of the sandfly's digestive tract segment where the parasite reproduced; that is, that of the subgenus *Leishmania* in the midgut [suprapylaria], while that of the sugenus *Viannia* in the posterior intestine [peripylaria] [14]. It is possible that this may be influencing the immunobiological role of the glycoconjugate molecules that act as species-specific antigens for *Leishmania* species of the *Leishmania* (*L. amazonensis*) and *Viannia* (*L. braziliensis*) subgenera.

In this way, it has been shown that LPG exerts a pivotal role in modulating a cascade of activated inflammatory T-cell responses in experimental *L. (V.) braziliensis*infections, including high expression of TNF-α, IL-1β, IL-6 and NO (such as those seen in ML). In addition, it should also been highlighted that *L. (V.) braziliensis*-LPG not only stimulates messenger RNA transcription, but also induces NF-kB translocation, resulting in NO expression as well as IFN-γ and TNF-α cytokines induction in ACL. That induction is mediated by linking *L. (V.) braziliensis*-LPG to *TLR*2 or *TLR*4, indicating the crucial role of those receptors in innate defenses, which was also evidenced in clinical samples of LCL caused by *L. (V.) braziliensis* [30, 34, 36]. On the other hand, LPG has also been shown to act as an important immunomodulatory factor activating *TLR*4 and promoting the preferential CD4+ /Th2-type immune response in experimental *L. (L.) amazonensis-*infections, which is strongly related to ADCL [35]. Based on these results, the most emphatic impression left is that the PLG molecule seems to act in a bidirectional manner from an immunobiological point of view, depending on the subgenus, *Leishmania* [*L. amazonensis*] or *Viannia* [*L. braziliensis*], involved in the process, although, more recently, it has been shown that LPG of *L. (L.) amazonensis* isolates from different origins, that is, from an ADCL case (State of Bahia, Brazil), from a domestic dog with visceral leishmaniasis (State of Minas Gerais, Brazil) and from a wild rodent (*Proechmys* sp.) in the Brazilian Amazon, showed a significant pro-inflammatory response with expression of TNF-α and IL-6 cytokines [111].

Another interesting species-specific *Leishmania*-antigen is the PS molecule, a phospholipid commonly located on the inner face of the parasite plasma membrane with capacity for inhibiting the inflammatory response of *Leishmania*-infected macrophages (as a strategy to evade the host T-cell immune response). In that sense, higher PS exposure on the surface of late-stationary-phase promastigotes of *L. (V.) braziliensis* has been observed in patients with LCL as compared to patients with ML [112]. However, higher PS exposure has been noted in *L. (L.) amazonensis* isolates obtained from ADCL patients as compared to those from LCL patients [113]. Those findings represent consistent evidence that the variability of PS across different strains of *Leishmania* [*L. amazonensis*] and *Viannia* [*L. braziliensis*] subgenera can influence the development of different clinical forms of ACL.

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

Finally, it is worth mentioning the important role that the CD200 molecule plays in the pathogenesis of *L. (L.) amazonensis*-infection, which is capable of inhibiting the activation of the infected macrophage via interaction with *TLR*9; that is, the CD200-dependent iNOS inhibition allows parasite growth within the macrophage, increasing the virulence of *L. (L.) amazonensis* [114]. Those findings were recently confirmed clinically, with evident interactions of *TLR*2 with clinical forms associated with *L. (V.) braziliensis*, including LCL, BDCL, and ML, while *TLR*9 showed strong interactions with clinical forms associated with *L. (L.) amazonensis* (principally the down regulated ADCL form) [30].

#### **4. Concluding remarks**

Based on what was mentioned above, there seems to be no doubt about how important it is to know the biology of the parasites of the genus *Leishmania* in order to better understand their relationships not only with their primitive hosts, invertebrates [phlebotomine sand flies] and vertebrates [wild mammals], but also with man, who is merely an accidental host [14]. This knowledge today has gained a great advance, mainly helped by the development of molecular biology, which has allowed to deepen the knowledge of the biology of these parasites at the level of the molecules of their plasma membrane' (glycoconjugates), allowing a better understanding of their more specific relationships with the human immune response, mainly the T-cell immune responses, CD4/Th1-type and CD4/Th2-type, whose modulation by these glycoconjugate molecules, referred to herein as species-specific antigens of *Leishmania* species of the *Leishmania* [*L. amazonensis*] and *Viannia* [*L. braziliensis*] subgenera, will be of the same greater importance regarding the modulation of the clinical-immunopathological spectrum of ACL in Brazil [10, 18, 27, 29, 30].

#### **Acknowledgements**

This work was supported by Evandro Chagas Institute (Secretary of Health and Environment Surveillance of Ministry of Health, Brazil); Tropical Medicine Nucleus (Federal University of Pará State, Brazil); and "Laboratório de Patologia de Moléstias Infecciosas (LIM-50), Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, SP, BR, and Grant #2014/50315-0, São Paulo Research Foundation (FAPESP)".

#### **Conflict of interests**

The author declares no conflict of interest.

### **Author details**

Fernando T. Silveira1,2\*, Marliane B. Campos1 , Silvia F. Müller3 , Patrícia K. Ramos1 , Luciana V. Lima1 , Thiago V. dos Santos1 , Claudia Maria Gomes4 , Márcia D. Laurenti4 , Vania Lucia da Matta4 and Carlos Eduardo Corbett4

1 Parasitology Department, Evandro Chagas Institute (Secretary of Science, Technology and Innovation, Ministry of Health), Ananindeua, Pará State, Brazil

2 Tropical Medicine Institute, Federal University of Pará, Belém, Pará State, Brazil

3 Dermatology Service, Federal University of Pará, Belém, Pará State, Brazil

4 Pathology Laboratory of Infectious Diseases (LIM50), Pathology Department, Medical School of São Paulo University, São Paulo, São Paulo State, Brazil

\*Address all correspondence to: fernandotobias@iec.gov.br

© 2023 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.

*From Biology to Disease: Importance of Species-Specific* Leishmania *Antigens… DOI: http://dx.doi.org/10.5772/intechopen.108967*

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## Section 3
