Diagnosis and Molecular Characterization of Chikungunya Virus Infections

*Marta E. Álvarez-Argüelles, Susana Rojo Alba, Mercedes Rodríguez Pérez, Jose Antonio Boga Riveiro and Santiago Melón García*

## **Abstract**

In recent years, large-scale outbreaks of chikungunya arbovirus (CHIKV), which is transmitted by the *Aedes* mosquito, have enabled the rapid propagation of the virus across the world. After acute infection phase with commonly fever, joint pain, headache, or rash, chronic rheumatism (arthralgia or myalgia, anorexia, and concentration disorders) up to 40% of cases is observed. The chronic form is defined by symptoms persisting for more than 3 months, and up to years, after initial diagnosis. Chronic discomfort has been linked to one of the four genotypes described. These genotypes represent different geographic lineages (classification based on partial sequence of viral E1 glycoprotein): West African, East-Central-South-African (ECSA), ECSA-diverged or Indian Ocean Lineage (IOL), and Asian. The first marker detected in CHIK infection is the viral RNA, usually by reverse transcription-polymerase chain reaction (RT-PCR). This marker can be identified in samples within 8 days of symptom onset. The infection can also be diagnosed with serological testing to detect CHIKV-specific immunoglobulin IgG and/or IgM. Sequencing studies can determine the infecting genotype.

**Keywords:** chikungunya, genotype, chronic

#### **1. Introduction**

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that causes severe acute febrile infection, often debilitating polyarthralgias and arthritis [1], and its symptoms are similar. The name chikungunya is derived from an African language *makonde* which means "that which bends up" or "stooped walk" because of the incapacitating arthralgia caused by the disease.

Chikungunya virus, the first reported case of which was in Tanzania in 1952 [2], is endemic in certain parts of West Africa, human serosurveys having identified antibodies to chikungunya virus in 35–50% of the population in some areas [3, 4].

#### **1.1 Epidemiology, structure, and classification of chikungunya**

Since 2004 CHIKV has spread into novel locations, with outbreaks having occurred in other parts of Africa as well as Asia, Europe, and the islands of the Indian and Pacific Oceans, and more recently in the Americas, mostly during the tropical rainy season. The risk of CHIKV being imported into further new areas is ever present because of the high attack rates, which affect between one-third to three-quarters of the population in areas where the virus is circulating, associated with the recurrence of epidemics, along with the high levels of viremia in infected humans and the worldwide distribution of the vectors, *Aedes aegypti* and *Aedes albopictus* [4]. Infected travelers can import chikungunya into new areas [1, 5–7], and in areas with *A. aegypti* and/or *A. albopictus* mosquitoes, local transmission can follow.

CHIKV multiple outbreaks have been identified as a cause of dengue-like illness with arthralgia in Africa and febrile hemorrhagic disease in Asia [2]. Although outbreaks of CHIKV were limited from the 1970s onward, in 2004–2005 the virus was detected in Kenya and the islands of the Indian Ocean in 2004–2005 [4, 8–10], and in the last decade, there have been massive outbreaks in new areas as well as in those where the virus is endemic [11]. What is more, local transmission of CHIKV has also been reported for the first time in parts of Europe, Asia [12], Australia [13], and America [9, 14, 15].

In the Americas, autochthonous CHIKV transmission was first reported in the Caribbean, on the island of St. Martin, in December 2013 [16], from which it is known to have spread to 45 countries or regions in Central/South/North America, where, according to the Pan American Health Organization, there had been 2 million suspected cases by 2014 [17]. It was detected in Central and South America in 2014, where there have since been multiple widescale outbreaks [13, 17], and between June and November of that same year, cases were reported in people returning to Northwest Italy from the Caribbean and Central America, even though there had been no reported cases of CHIKF in either region for 3 years [18]. More recently, in 2016–2017, autochthonous CHIKV transmission was reported in India [19], Pakistan [20], and Italy [21]. It can be assumed that the combination of increased global travel and trade, the wide distribution of the mosquito vectors, and a lack of herd immunity have all contributed to the introduction and rapid spread of CHIKV in naïve populations.

*Dengue* and *Zika* viruses are transmitted by the same mosquito vectors as chikungunya so all three viruses can co-circulate in a geographic region, and coinfections have been documented [22, 23]. This clearly has implications for diagnosis in the laboratory.

CHIKV belongs to the *Togaviridae* family, genus *Alphavirus*, within the Semliki Forest antigenic complex. Among the other members of this antigenic complex are the Mayaro, O'Nyong-nyong, and Ross River viruses, all of which are capable of causing disease in humans [24].

Chikungunya is a positive-sense single-stranded RNA virus with approximately 12 kb. It is spherical in shape and has a diameter of about 70 nm. The viral particle comprises 240 copies of the capsid protein contained within a lipid bilayer envelope which has 80 trimer-shaped spikes formed by E1 and E2 glycoproteins protruding from it [25]. There are two open reading frames (ORFs) in the genome: the 5'ORF (genomic RNA) is responsible for encoding the nonstructural proteins nsP1, nsP2, nsP3, and nsP4, and the 3'ORF (subgenomic RNA) is responsible for encoding a polyprotein that is processed into the structural capsid and envelope proteins (E1 and E2) and two peptides (E3 and 6 K) [26].

Several CHIKV genotypes which have been described are shown below: East-Central-South-African (ECSA) isolates which comprise the East, Central and South African, and West Africa isolates (West Africa) and Asian isolates (Asian). The Indian Ocean Lineage (IOL) was identified in 2004 as a descendant of the ECSA lineage [27, 28].

**33**

**diagnosis**

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections*

CHIKV can be genetically classified into three major lineages: West African (WA); East, Central, and South African (ECSA); and Asian. Prior to 2004, these were rarely found outside the geographic limits implied by their names. The epidemics subsequent to 2004 were mainly due to isolates forming a distinct clade within the ECSA lineage [29], likely originating in eastern Kenya before spreading to cause large outbreaks affecting millions in islands of the Indian Ocean, India, and Asia [30], as well as numerous imported cases in previously nonendemic regions, including Europe and the Americas. More recently, since 2013, the Asian genotype

Additionally, the Indian Ocean (IOL) sublineage emerged within the ECSA clade, and the Asian/American sublineage emerged within the Asian clade [31, 32]. While differences in epidemiological and pathological characteristics among outbreaks involving different CHIKV lineages and sublineages have been suggested, few targeted investigations comparing lineage virulence levels have been reported. The most important clades in terms of public health impact are the Asian lineage (including Asian/American) and the IOL sublineage, as well as some other ECSA lineage strains responsible for African outbreaks. They are responsible for multiple CHIKV outbreaks over the last 15 years involving millions of people [29, 32, 33],

CHIKV phylogenetic analysis, based on previous work speculating on the existence of distinct lineages [36], has established that the three principal currently circulating genotypes emerged from a common ancestor less than 500 years ago and

West African genotype is particularly well adapted to sylvatic mosquitoes or to a

The Asian genotype that once circulated in India during the 1950s and 1960s formed a clade that was phylogenetically distinct from the Southeast Asian (SEA) strains that continue to circulate today. There is a remarkable spatial and temporal pattern in the evolution of the SEA lineage, spreading from Thailand to Indonesia

Chikungunya virus is transmitted to people, as mentioned above, primarily via mosquito bites (primarily during the day but also at night). Mosquitoes become infected when they feed on a person already infected with the virus and then spread the virus to other people via biting, after the virus reaches the mosquito salivary glands. Other less-frequent routes of transmission are through blood products,

Pregnant women infected with CHIKV are not at increased risk of atypical or severe disease, but maternal CHIKV infection has been associated with miscarriage in the first trimester [16, 37, 38]. However, when maternal infection occurs toward the end of pregnancy, only 12% of newborns are expected to be symptomatic, clinical manifestations—fever, poor feeding, tenderness, unexplained apnea, peripheral edema, thrombocytopenia, and rash—appearing 3–7 days after delivery [37, 39]. More severe symptoms have also been observed, such as encephalopathy and myocardial disease, and neurocognitive development was found to be poor in

that the ECSA and Asian genotypes separated within the past 150 years [29].

**2. Transmission, pathogenesis, clinical disease, and differential** 

has also caused significant outbreaks in the Caribbean and the Americas.

which continue to arise in Asia and Africa [34, 35].

and then to the Philippines and the South Pacific.

organ transplantation, and maternal-fetal.

children with perinatal CHIKV infection [40].

certain vector-host combination within the sylvatic cycle.

*DOI: http://dx.doi.org/10.5772/intechopen.86957*

*1.1.1 Chikungunya genotypes*

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections DOI: http://dx.doi.org/10.5772/intechopen.86957*

#### *1.1.1 Chikungunya genotypes*

*Current Topics in Neglected Tropical Diseases*

and America [9, 14, 15].

CHIKV in naïve populations.

causing disease in humans [24].

and E2) and two peptides (E3 and 6 K) [26].

the laboratory.

and Pacific Oceans, and more recently in the Americas, mostly during the tropical rainy season. The risk of CHIKV being imported into further new areas is ever present because of the high attack rates, which affect between one-third to three-quarters of the population in areas where the virus is circulating, associated with the recurrence of epidemics, along with the high levels of viremia in infected humans and the worldwide distribution of the vectors, *Aedes aegypti* and *Aedes albopictus* [4]. Infected travelers can import chikungunya into new areas [1, 5–7], and in areas with *A. aegypti*

CHIKV multiple outbreaks have been identified as a cause of dengue-like illness

with arthralgia in Africa and febrile hemorrhagic disease in Asia [2]. Although outbreaks of CHIKV were limited from the 1970s onward, in 2004–2005 the virus was detected in Kenya and the islands of the Indian Ocean in 2004–2005 [4, 8–10], and in the last decade, there have been massive outbreaks in new areas as well as in those where the virus is endemic [11]. What is more, local transmission of CHIKV has also been reported for the first time in parts of Europe, Asia [12], Australia [13],

In the Americas, autochthonous CHIKV transmission was first reported in the Caribbean, on the island of St. Martin, in December 2013 [16], from which it is known to have spread to 45 countries or regions in Central/South/North America, where, according to the Pan American Health Organization, there had been 2 million suspected cases by 2014 [17]. It was detected in Central and South America in 2014, where there have since been multiple widescale outbreaks [13, 17], and between June and November of that same year, cases were reported in people returning to Northwest Italy from the Caribbean and Central America, even though there had been no reported cases of CHIKF in either region for 3 years [18]. More recently, in 2016–2017, autochthonous CHIKV transmission was reported in India [19], Pakistan [20], and Italy [21]. It can be assumed that the combination of increased global travel and trade, the wide distribution of the mosquito vectors, and a lack of herd immunity have all contributed to the introduction and rapid spread of

*Dengue* and *Zika* viruses are transmitted by the same mosquito vectors as chikungunya so all three viruses can co-circulate in a geographic region, and coinfections have been documented [22, 23]. This clearly has implications for diagnosis in

CHIKV belongs to the *Togaviridae* family, genus *Alphavirus*, within the Semliki Forest antigenic complex. Among the other members of this antigenic complex are the Mayaro, O'Nyong-nyong, and Ross River viruses, all of which are capable of

Chikungunya is a positive-sense single-stranded RNA virus with approximately 12 kb. It is spherical in shape and has a diameter of about 70 nm. The viral particle comprises 240 copies of the capsid protein contained within a lipid bilayer envelope which has 80 trimer-shaped spikes formed by E1 and E2 glycoproteins protruding from it [25]. There are two open reading frames (ORFs) in the genome: the 5'ORF (genomic RNA) is responsible for encoding the nonstructural proteins nsP1, nsP2, nsP3, and nsP4, and the 3'ORF (subgenomic RNA) is responsible for encoding a polyprotein that is processed into the structural capsid and envelope proteins (E1

Several CHIKV genotypes which have been described are shown below: East-Central-South-African (ECSA) isolates which comprise the East, Central and South African, and West Africa isolates (West Africa) and Asian isolates (Asian). The Indian Ocean Lineage (IOL) was identified in 2004 as a descendant of the ECSA

and/or *A. albopictus* mosquitoes, local transmission can follow.

**32**

lineage [27, 28].

CHIKV can be genetically classified into three major lineages: West African (WA); East, Central, and South African (ECSA); and Asian. Prior to 2004, these were rarely found outside the geographic limits implied by their names. The epidemics subsequent to 2004 were mainly due to isolates forming a distinct clade within the ECSA lineage [29], likely originating in eastern Kenya before spreading to cause large outbreaks affecting millions in islands of the Indian Ocean, India, and Asia [30], as well as numerous imported cases in previously nonendemic regions, including Europe and the Americas. More recently, since 2013, the Asian genotype has also caused significant outbreaks in the Caribbean and the Americas.

Additionally, the Indian Ocean (IOL) sublineage emerged within the ECSA clade, and the Asian/American sublineage emerged within the Asian clade [31, 32].

While differences in epidemiological and pathological characteristics among outbreaks involving different CHIKV lineages and sublineages have been suggested, few targeted investigations comparing lineage virulence levels have been reported.

The most important clades in terms of public health impact are the Asian lineage (including Asian/American) and the IOL sublineage, as well as some other ECSA lineage strains responsible for African outbreaks. They are responsible for multiple CHIKV outbreaks over the last 15 years involving millions of people [29, 32, 33], which continue to arise in Asia and Africa [34, 35].

CHIKV phylogenetic analysis, based on previous work speculating on the existence of distinct lineages [36], has established that the three principal currently circulating genotypes emerged from a common ancestor less than 500 years ago and that the ECSA and Asian genotypes separated within the past 150 years [29].

West African genotype is particularly well adapted to sylvatic mosquitoes or to a certain vector-host combination within the sylvatic cycle.

The Asian genotype that once circulated in India during the 1950s and 1960s formed a clade that was phylogenetically distinct from the Southeast Asian (SEA) strains that continue to circulate today. There is a remarkable spatial and temporal pattern in the evolution of the SEA lineage, spreading from Thailand to Indonesia and then to the Philippines and the South Pacific.

### **2. Transmission, pathogenesis, clinical disease, and differential diagnosis**

Chikungunya virus is transmitted to people, as mentioned above, primarily via mosquito bites (primarily during the day but also at night). Mosquitoes become infected when they feed on a person already infected with the virus and then spread the virus to other people via biting, after the virus reaches the mosquito salivary glands. Other less-frequent routes of transmission are through blood products, organ transplantation, and maternal-fetal.

Pregnant women infected with CHIKV are not at increased risk of atypical or severe disease, but maternal CHIKV infection has been associated with miscarriage in the first trimester [16, 37, 38]. However, when maternal infection occurs toward the end of pregnancy, only 12% of newborns are expected to be symptomatic, clinical manifestations—fever, poor feeding, tenderness, unexplained apnea, peripheral edema, thrombocytopenia, and rash—appearing 3–7 days after delivery [37, 39]. More severe symptoms have also been observed, such as encephalopathy and myocardial disease, and neurocognitive development was found to be poor in children with perinatal CHIKV infection [40].

#### **2.1 Clinical disease**

The "natural" history of symptomatic CHIKV infection has been classified into three phases: acute, post-acute, and chronic.

*Acute phase* is considered the first 3 weeks of clinical manifestations. The incubation period is 3–7 days (range 1–14 days), and clinical manifestations begin abruptly, usually with high fever (>39°C), during 3–5 days (range 1–10 days), and malaise [41]. More than 85% of patients are symptomatic [1]. The duration of acute phase is usually 7–10 days.

Polyarthralgia, commonly bilateral and symmetric, begins 2–5 days after onset of fever and involves multiple joints: hands (50–76% of infected individuals), wrists (29–81%), ankles (41–68%), and axial skeleton (34–52%) [42, 43]. Pain may be intense and disabling, leading to immobilization.

Rash (maculopapular) has been reported in 40–75% of patients [43] and pruritus in 25–50% of patients.

Serious complications are not common, except in patients older than 65 years and patients with underlying pathologies, where the infection and symptoms can contribute to the cause of death. These severe forms usually involve the central nervous system, respiratory system, and urinary system.

Most patients recover fully, but in some cases joint pain may persist, or the patient experiences relapses of signs and symptoms for several months or even years [26].

In the *post-acute phase*, only a small proportion of patients remain completely asymptomatic 2–3 weeks after the onset of disease [44]. Generally, most patients exhibit only transitory improvements in their clinical condition, and relapses occur after a brief "healing" period. Most studies have indicated that, on average, clinical manifestations persist in 50–90% of patients after the second or third week, and the percentage of patients with persistent polyarthralgia after the acute phase of CHIKV infection is more frequent in those older than 40 years and in women [45].

Clinical manifestations observed during the post-acute phase, which indicate the persistence of the initial inflammatory process, include arthritis/arthralgia, edematous polyarthritis of fingers and toes, morning pain and stiffness, and severe tenosynovitis [42].

A set of nonspecific clinical manifestations that are not always associated with CHIKV usually occurs, such as chronic fatigue, changes in skin color, alopecia, decompensated endocrine and metabolic diseases, as well as the decompensation of other preexisting chronic diseases, depression, and anxiety [44].

*Chronic phase*: It is estimated that the percentage of patients infected with chikungunya virus who progress to chronic phase (more than 3 months) varies from 40 to 80% [45–47], and they may endure clinical manifestations for a few months or even years [44]. Although no clear evidence exists to explain the pathogenesis of persistent symptoms following infection, two hypotheses have been proposed: (a) That viral and/or antigenic debris remains in the tissues of joints and muscles. Unfortunately, to date, the virus has not been isolated from such tissue. However, CHIKV proteins have been found in macrophages and muscle cell tissue of relapsing CHIKV patients, supporting the notion that there may be low-grade replication of the virus or non-replicative viral debris present. (b) That infection triggers a persistent immune response. Studies are underway with mouse models to determine whether, and in what way, immunological mechanisms might be altered in patients with persistent symptoms [48, 49].

Chronic phase sufferers can be divided into three groups in terms of disease progression: those whose symptoms disappear either spontaneously or following treatment and who have no long-term complications, which accounts for the majority of such cases; a group who experiences prolonged and persistence generalized

**35**

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections*

clinical symptoms, with or without joint problems; and another set of patients in whom the degenerative or inflammatory process is exacerbated and their condition

Twenty-five percent of CHIKV cases have been estimated to result in chronic inflammatory rheumatism, although this study did not address the issue of differences between genotypes [50]. Other symptoms, such as chronic pain, mental health issues, and nonspecific manifestations, are also common in this phase [51–54], with arthralgia and arthritis with pain accompanied by articular edema or morning joint stiffness being the most frequent clinical manifestations [55]. Tenosynovitis is also frequent (with two or more tendons affected), and less common symptoms are fatigue and neuritis [44]. Clearly the daily life of patients with long-term chronic CHIKV symptoms is considerably impacted [54], and interestingly there is some evidence that the incidence of chronic symptoms is greater in high-income than low-income countries, albeit that the confidence intervals overlapped [54]. These same authors did consider the relationship between virus genotype and self-reported chronic discomfort and found this symptom to be less prevalent in those infected by the ECSA group, followed by those carrying the Asian strain. The highest prevalence was linked to the ECSA-diverged/IOL genotypes,

There is no commercial vaccine against CHIKV, although development is underway [56–58] and there is no specific antiviral therapy for acute infection. Treatment in this phase consists of supportive care for as long as the infection is confirmed. It is, however, known that systemic glucocorticoids and other immunosuppressive

Fever and polyarthralgia are key symptoms of CHIKV infection but are far from being reliable diagnostic markers since they have a sensitivity of only 84%, a positive predictive value (PPV) of 71%, and an 83% negative predictive value (NPV) [25]. Thus differential diagnosis of both the acute and the chronic manifestations of the disease can be complex and take time due to the nonspecific nature of the typical symptoms of arthralgia, high fever, and rash. The shared vectors, as well as symptoms, of CHIKV and the DENV, ZK, O'nyong-nyong, and Mayaro viruses, as well as the fact that the viruses all co-exist and are known to co-infect, mean that

Therefore, it is difficult to differentiate from those of other febrile illnesses or be misdiagnosed in areas where dengue occurs. Other infections that should be considered include malaria, yellow fever, leptospirosis, measles, mononucleosis, or

An additional diagnostic complication is the similarity of chronic arthralgia caused by chikungunya fever with other more common causes, and it has been demonstrated that patients with underlying joint disease prior to CHIKV infection have worse prognosis and increased morbidity [60]. Differential serological diagnosis includes the exclusion of rheumatoid and systemic arthritis, lupus erythematosus

medications should be avoided in patients during acute infection [44].

The management of persistent or relapse symptoms, particularly joint disease, depends upon the duration of the symptoms. Treatment with antiinflammatory drugs and analgesics is appropriate in the acute phase. On the other hand, for symptoms persisting for more than 3 months after infection, the use of disease-modifying antirheumatic drug (DMARD) therapy, such as methotrexate (MTX) and hydroxychloroquine [44], or, as an alternative, physiotherapy

*DOI: http://dx.doi.org/10.5772/intechopen.86957*

though there was overlap with the Asian strain.

they must all be ruled out in the differential diagnosis.

Reiter arthritis, rheumatoid arthritis, and hepatitis C.

becomes serious [44].

is indicated [59].

**2.2 Differential diagnosis**

African tick bite fever.

#### *Diagnosis and Molecular Characterization of Chikungunya Virus Infections DOI: http://dx.doi.org/10.5772/intechopen.86957*

*Current Topics in Neglected Tropical Diseases*

three phases: acute, post-acute, and chronic.

intense and disabling, leading to immobilization.

nervous system, respiratory system, and urinary system.

The "natural" history of symptomatic CHIKV infection has been classified into

Polyarthralgia, commonly bilateral and symmetric, begins 2–5 days after onset of fever and involves multiple joints: hands (50–76% of infected individuals), wrists (29–81%), ankles (41–68%), and axial skeleton (34–52%) [42, 43]. Pain may be

Rash (maculopapular) has been reported in 40–75% of patients [43] and pruri-

Serious complications are not common, except in patients older than 65 years and patients with underlying pathologies, where the infection and symptoms can contribute to the cause of death. These severe forms usually involve the central

Most patients recover fully, but in some cases joint pain may persist, or the patient experiences relapses of signs and symptoms for several months or even years [26]. In the *post-acute phase*, only a small proportion of patients remain completely asymptomatic 2–3 weeks after the onset of disease [44]. Generally, most patients exhibit only transitory improvements in their clinical condition, and relapses occur after a brief "healing" period. Most studies have indicated that, on average, clinical manifestations persist in 50–90% of patients after the second or third week, and the percentage of patients with persistent polyarthralgia after the acute phase of CHIKV infection is more frequent in those older than 40 years and in women [45]. Clinical manifestations observed during the post-acute phase, which indicate the persistence of the initial inflammatory process, include arthritis/arthralgia, edematous polyarthritis of fingers and toes, morning pain and stiffness, and severe

A set of nonspecific clinical manifestations that are not always associated with CHIKV usually occurs, such as chronic fatigue, changes in skin color, alopecia, decompensated endocrine and metabolic diseases, as well as the decompensation of

*Chronic phase*: It is estimated that the percentage of patients infected with chikungunya virus who progress to chronic phase (more than 3 months) varies from 40 to 80% [45–47], and they may endure clinical manifestations for a few months or even years [44]. Although no clear evidence exists to explain the pathogenesis of persistent symptoms following infection, two hypotheses have been proposed: (a) That viral and/or antigenic debris remains in the tissues of joints and muscles. Unfortunately, to date, the virus has not been isolated from such tissue. However, CHIKV proteins have been found in macrophages and muscle cell tissue of relapsing CHIKV patients, supporting the notion that there may be low-grade replication of the virus or non-replicative viral debris present. (b) That infection triggers a persistent immune response. Studies are underway with mouse models to determine whether, and in what way, immunological mechanisms might be altered in patients

Chronic phase sufferers can be divided into three groups in terms of disease progression: those whose symptoms disappear either spontaneously or following treatment and who have no long-term complications, which accounts for the majority of such cases; a group who experiences prolonged and persistence generalized

other preexisting chronic diseases, depression, and anxiety [44].

*Acute phase* is considered the first 3 weeks of clinical manifestations. The incubation period is 3–7 days (range 1–14 days), and clinical manifestations begin abruptly, usually with high fever (>39°C), during 3–5 days (range 1–10 days), and malaise [41]. More than 85% of patients are symptomatic [1]. The duration of acute

**2.1 Clinical disease**

phase is usually 7–10 days.

tus in 25–50% of patients.

tenosynovitis [42].

with persistent symptoms [48, 49].

**34**

clinical symptoms, with or without joint problems; and another set of patients in whom the degenerative or inflammatory process is exacerbated and their condition becomes serious [44].

Twenty-five percent of CHIKV cases have been estimated to result in chronic inflammatory rheumatism, although this study did not address the issue of differences between genotypes [50]. Other symptoms, such as chronic pain, mental health issues, and nonspecific manifestations, are also common in this phase [51–54], with arthralgia and arthritis with pain accompanied by articular edema or morning joint stiffness being the most frequent clinical manifestations [55]. Tenosynovitis is also frequent (with two or more tendons affected), and less common symptoms are fatigue and neuritis [44]. Clearly the daily life of patients with long-term chronic CHIKV symptoms is considerably impacted [54], and interestingly there is some evidence that the incidence of chronic symptoms is greater in high-income than low-income countries, albeit that the confidence intervals overlapped [54]. These same authors did consider the relationship between virus genotype and self-reported chronic discomfort and found this symptom to be less prevalent in those infected by the ECSA group, followed by those carrying the Asian strain. The highest prevalence was linked to the ECSA-diverged/IOL genotypes, though there was overlap with the Asian strain.

There is no commercial vaccine against CHIKV, although development is underway [56–58] and there is no specific antiviral therapy for acute infection. Treatment in this phase consists of supportive care for as long as the infection is confirmed. It is, however, known that systemic glucocorticoids and other immunosuppressive medications should be avoided in patients during acute infection [44].

The management of persistent or relapse symptoms, particularly joint disease, depends upon the duration of the symptoms. Treatment with antiinflammatory drugs and analgesics is appropriate in the acute phase. On the other hand, for symptoms persisting for more than 3 months after infection, the use of disease-modifying antirheumatic drug (DMARD) therapy, such as methotrexate (MTX) and hydroxychloroquine [44], or, as an alternative, physiotherapy is indicated [59].

#### **2.2 Differential diagnosis**

Fever and polyarthralgia are key symptoms of CHIKV infection but are far from being reliable diagnostic markers since they have a sensitivity of only 84%, a positive predictive value (PPV) of 71%, and an 83% negative predictive value (NPV) [25]. Thus differential diagnosis of both the acute and the chronic manifestations of the disease can be complex and take time due to the nonspecific nature of the typical symptoms of arthralgia, high fever, and rash. The shared vectors, as well as symptoms, of CHIKV and the DENV, ZK, O'nyong-nyong, and Mayaro viruses, as well as the fact that the viruses all co-exist and are known to co-infect, mean that they must all be ruled out in the differential diagnosis.

Therefore, it is difficult to differentiate from those of other febrile illnesses or be misdiagnosed in areas where dengue occurs. Other infections that should be considered include malaria, yellow fever, leptospirosis, measles, mononucleosis, or African tick bite fever.

An additional diagnostic complication is the similarity of chronic arthralgia caused by chikungunya fever with other more common causes, and it has been demonstrated that patients with underlying joint disease prior to CHIKV infection have worse prognosis and increased morbidity [60]. Differential serological diagnosis includes the exclusion of rheumatoid and systemic arthritis, lupus erythematosus Reiter arthritis, rheumatoid arthritis, and hepatitis C.

It is thus clear that differential diagnosis is essential to establish CHIKV infection and ensure the appropriate public health response as well as the optimal treatment regime for the patient. Employing a single PCR which targets all three infectious agents (CHIKV, DENV, ZV), available from US Centers for Disease Control and Prevention (CDC) and approved laboratories, would expediate diagnosis [61–64].

### **3. Diagnosis**

Chikungunya virus infection should be suspected in patients with acute onset of fever and polyarthralgia and who meet the relevant epidemiologic exposure criteria (residence in or travel to an area where mosquito-borne transmission of chikungunya virus infection has been reported).

The laboratory diagnosis of CHIKV infection can be achieved in the majority of cases by following two different strategies: the detection of viral RNA (virological diagnosis) and the identification of the specific immune response (serological diagnosis). To this end, choosing the appropriate timing of specimen collection and of the use of the most suitable diagnostic methodologies is crucial for accurate diagnosis. The algorithm developed by the US Center for Disease Control and Prevention (CDC) to diagnose CHIKV infections is based on the characteristics of CHIKV infection and the timing of specimen collection (**Figure 1**).

CHIKV replicates rapidly to high titers in the host, and viral RNA generally can be detected by real-time RT-PCR in the first week after onset of clinical illness, while immunoglobulin M (IgM) antibodies are normally detectable in serum by days 5–7 after onset of illness [65]. Consequently, the kinetic replication and pathogenesis of CHIKV infection, including the duration of viremia and the response of the host immune response, should be considered when selecting the appropriate diagnostic tests [66]. Molecular assays (TaqMan real-time PCR, RT-LAMP assay, and reverse transcription PCR) are more sensitive in the early stage of chikungunya fever (2–5 days p.o.i.) when CHIKV-specific IgM is not yet detectable. In the later stages of chikungunya fever (>5 days p.o.i.), CHIKVspecific IgM is a more reliable indicator.

**37**

is preferable.

**Figure 2.**

*infection. Source of CDC.*

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections*

In the CHIKV testing algorithm developed by the CDC arbovirus diagnostic laboratory, samples collected <6 days after onset of illness are first tested by virological methods (CHIKV real-time RT-PCR using two different set primers/probe

*The Centers for Disease Control and Prevention diagnostic testing algorithm for detection of chikungunya virus* 

The CDC guidelines indicate that samples collected on or after day 6 of illness and samples with negative real-time RT-PCR results are tested by serological methods (the CHIKV IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) or an indirect fluorescent antibody assay (IFA) and plaque reduction

Samples for chikungunya diagnosis have to be collected as soon as possible. Two different types of sample are suitable for virological and/or serological diagnosis. Viral diagnosis depends on the characteristics of CHIKV infection and the time elapsed since the viral infection, the type of patient (e.g., fetus, pregnant, etc.), and clinical manifestation of infection. Virological diagnosis can be made using samples of blood (whole blood, plasma, or serum), urine, blister fluid, cerebrospinal fluid,

Samples must be collected in strictly aseptic conditions and in the right container: sterile recipients for fluids and with transport viral medium for the others

The best type of tube for serological diagnosis is serum separator. Heparin and EDTA are unsuitable for antibodies CHIK testing [71]. Serum used for serological test can be used for virological diagnosis, but plasma or whole blood (leukocytes)

that were developed to detect different genotypes (**Figure 2**)).

**3.1 Types of samples for chikungunya infection diagnosis**

Samples need to be sent to the laboratory quickly.

amniotic fluid, or tissues such as the placenta and brain, among others.

hemagglutination test as confirmation) [67–70].

(blisters, biopsies, swabs, etc.).

*DOI: http://dx.doi.org/10.5772/intechopen.86957*

**Figure 1.** *Time course of chikungunya virus viremia and immune response. Source of CDC.*

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections DOI: http://dx.doi.org/10.5772/intechopen.86957*

**Figure 2.**

*Current Topics in Neglected Tropical Diseases*

nya virus infection has been reported).

specific IgM is a more reliable indicator.

**3. Diagnosis**

It is thus clear that differential diagnosis is essential to establish CHIKV infection and ensure the appropriate public health response as well as the optimal treatment regime for the patient. Employing a single PCR which targets all three infectious agents (CHIKV, DENV, ZV), available from US Centers for Disease Control and Prevention (CDC) and approved laboratories, would expediate diagnosis [61–64].

Chikungunya virus infection should be suspected in patients with acute onset of fever and polyarthralgia and who meet the relevant epidemiologic exposure criteria (residence in or travel to an area where mosquito-borne transmission of chikungu-

The laboratory diagnosis of CHIKV infection can be achieved in the majority of cases by following two different strategies: the detection of viral RNA (virological diagnosis) and the identification of the specific immune response (serological diagnosis). To this end, choosing the appropriate timing of specimen collection and of the use of the most suitable diagnostic methodologies is crucial for accurate diagnosis. The algorithm developed by the US Center for Disease Control and Prevention (CDC) to diagnose CHIKV infections is based on the characteristics of

CHIKV replicates rapidly to high titers in the host, and viral RNA generally can be detected by real-time RT-PCR in the first week after onset of clinical illness, while immunoglobulin M (IgM) antibodies are normally detectable in serum by days 5–7 after onset of illness [65]. Consequently, the kinetic replication and pathogenesis of CHIKV infection, including the duration of viremia and the response of the host immune response, should be considered when selecting the appropriate diagnostic tests [66]. Molecular assays (TaqMan real-time PCR, RT-LAMP assay, and reverse transcription PCR) are more sensitive in the early stage of chikungunya fever (2–5 days p.o.i.) when CHIKV-specific IgM is not yet detectable. In the later stages of chikungunya fever (>5 days p.o.i.), CHIKV-

CHIKV infection and the timing of specimen collection (**Figure 1**).

*Time course of chikungunya virus viremia and immune response. Source of CDC.*

**36**

**Figure 1.**

*The Centers for Disease Control and Prevention diagnostic testing algorithm for detection of chikungunya virus infection. Source of CDC.*

In the CHIKV testing algorithm developed by the CDC arbovirus diagnostic laboratory, samples collected <6 days after onset of illness are first tested by virological methods (CHIKV real-time RT-PCR using two different set primers/probe that were developed to detect different genotypes (**Figure 2**)).

The CDC guidelines indicate that samples collected on or after day 6 of illness and samples with negative real-time RT-PCR results are tested by serological methods (the CHIKV IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) or an indirect fluorescent antibody assay (IFA) and plaque reduction hemagglutination test as confirmation) [67–70].

#### **3.1 Types of samples for chikungunya infection diagnosis**

Samples for chikungunya diagnosis have to be collected as soon as possible. Two different types of sample are suitable for virological and/or serological diagnosis.

Viral diagnosis depends on the characteristics of CHIKV infection and the time elapsed since the viral infection, the type of patient (e.g., fetus, pregnant, etc.), and clinical manifestation of infection. Virological diagnosis can be made using samples of blood (whole blood, plasma, or serum), urine, blister fluid, cerebrospinal fluid, amniotic fluid, or tissues such as the placenta and brain, among others.

Samples must be collected in strictly aseptic conditions and in the right container: sterile recipients for fluids and with transport viral medium for the others (blisters, biopsies, swabs, etc.).

Samples need to be sent to the laboratory quickly.

The best type of tube for serological diagnosis is serum separator. Heparin and EDTA are unsuitable for antibodies CHIK testing [71]. Serum used for serological test can be used for virological diagnosis, but plasma or whole blood (leukocytes) is preferable.

#### **3.2 Virological diagnosis**

As was described above, for virological diagnosis it is important to know the pathogenesis and the infection kinetics. The RNA viruses are found in plasma or serum specimens between 2 and 6 days following onset of fever, which corresponds to the period of incubation [41]. Viral load can subsequently rapidly reach up to 108 /109 genome copies/ml of blood [1, 26, 37].

Complete virions as well as viral fragments (antigen or genome) are frequent in this phase, although replication later declines. In line with this kinetic replication, three methods can be used in the laboratory: viral isolation and antigen or genomic detection.

#### *3.2.1 Viral isolation*

Viral isolation is generally a research tool [71–73]. For viral culture it is essential to preserve the complete virion. Culture sensitivity for chikungunya virus is high only in early infection (during the viremic phase) but drops 5 days after onset of illness. As a result, virus isolation is rarely used in the diagnosis of CHIKV infection because low sensitivity occurs after only a few days post-infection, and it is timeconsuming and laborious procedure. However, it does allow for the identification of the viral strain and can be important for epidemiologic and research purposes. Samples where chikungunya virus is suspected should be handled under Biosafety Level (BSL) 3 conditions.

A wide variety of cells are used for in vitro CHIKV cultures in order to assess the full scope of the disease, including primary human skeletal muscle myoblasts [74], human blood monocytes [75], African green monkey kidney (Vero-E6) cells [76], and C6/36 (*Aedes albopictus* clone cells). Of particular interest, given the known tropism of chronic CHIKV for bone and synovial tissues, is the use of primary human fibroblast-like synoviocytes [77] and human osteoblasts.

Culture on HEL cells is more sensitive, and the presence of a cytopathic effect can be observed earlier than in Vero cells [78].

These cultures all need to be maintained under standard conditions.

#### *3.2.2 Antigen detection*

There are commercial immunochromatography antigen detection kits which detect CHIKV with high sensitivity in the early phase (up to 4–5 days after the onset of fever, when blood-enveloping proteins are still present). These tests also have high specificity since no cross-reactions with dengue virus are known to exist, and the method is straightforward and simple and does not require specially trained laboratory personnel [79].

However, a very important issue to consider is what genotype/s the tests are capable of detecting since not all detect all the known genotypes. Thus the geographical area from which the patient comes, meaning they are more likely to have one genotype or another, needs to be taken into account for accurate diagnosis (e.g., Okabayashi's immunochromatography only detects the Asian genotype) [80].

These methods are therefore, generally, sensitivity compromised.

#### *3.2.3 Genomic detection (RT-PCR)*

The diagnosis of CHIKV infection in the acute phase of infection is typically performed by the detection of viral RNA in plasma or serum (or other sample

**39**

**Table 1.**

*strains.*

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections*

types) by RT-PCR. The viral RNA can be detected by various molecular methods,

Conventional and real-time PCRs have been used to amplify nsP1, nsP2, or even

Molecular diagnostic tests used for the detection of CHIKV include reverse transcription (RT) and amplification (PCR) assay of fragments in the nsP1, nsP2, nsP3, nsP4, or E1 regions of the CHIKV genome, in general by real-time [65, 82–86]. Labeled probes or SYBR Green is used for real-time quantification of the amplified PCR products [87, 88]. In these assays, the limits of detection range between 0.5 and 1.5 log10 RNA copies/reaction. Although the detection of positive-strand RNA is commonly used for diagnostic purposes, assays to detect negative-strand CHIKV RNA have also been developed. These include strand-specific quantitative RT-PCRs for nsP1 [89] and nsP3 [90], which use a tagged-primer system to improve PCR specificity and accuracy. In comparison to the detection of positive-strand RNA, the detection limit for negative-strand RNA assays is slightly decreased, 3 log10 RNA

While there is an ever-present possibility of CHIKV spreading further in developed countries in the Americas and Europe, CHIKV still predominantly occurs in resource-limited countries. *Loop-mediated isothermal amplification* (LAMP) amplifies nucleic acid under isothermal conditions without the use of a thermal cycler and is a fast, specific, and cost-effective technique [92]; LAMP represents a cheaper alternative amplification method, albeit with lower sensitivity than real-time RT-PCR. Monitoring by turbidity as well as observation of color change after adding SYBR Green has also been described for the detection of CHIKV [85, 92]. Visualization is made with the naked eye, meaning no sophisticated equipment is required, and thus LAMP is especially useful in developing

Another method, *microfluidic lab-on-chip* integrating multiplex molecular amplification and DNA microarray hybridization, has been developed for the simultaneous detection of 26 globally important tropical pathogens such as CHIKV, *Dengue virus* (DENV), and other arboviruses [93]. Such diagnostic capacity provides an effective and rapid means to establish the presence of specific

The CDC protocol for chikungunya infection diagnosis uses two RT-PCRs. The nucleotide sequences of the two sets of primers and probes used are listed in **Table 1** [94, 95]. The 3855 primer/probe set is specific to the ECSA genotypes although it is

CHIKV3957C TACTGGTGATACATGGTGGTTTC + TGCTGGTGACACATGGTGGTTTC

ACGAGTAATCTGCGTACTGGGACGTA + ACGAGTCATCTGCGTATTGGGACGCA

*Chikungunya virus oligonucleotide primers and probes used in the Centers for Disease Control and Prevention real-time reverse transcription-polymerase chain reaction assays designed to detect CHIKV Asian genotype* 

**Nucleotide sequence**

CHIKV3855F GAGCATACGGTTACGCAGATAG

CHIK856F ACCATCGGTGTTCCATCTAAAG CHIK962C GCCTGGGCTCATCGTTATT

CHIK908FAM (probe) ACAGTGGTTTCGTGTGAGGGCTAC

*DOI: http://dx.doi.org/10.5772/intechopen.86957*

such as nested and real-time PCRs [37].

copies [91].

countries or in field studies.

potential pathogens.

**Primer/genome 5′ position**

CHIKV3886FAM (probe)

envelope protein genes (E3, E2, or E1) [81, 82].

#### *Diagnosis and Molecular Characterization of Chikungunya Virus Infections DOI: http://dx.doi.org/10.5772/intechopen.86957*

*Current Topics in Neglected Tropical Diseases*

genome copies/ml of blood [1, 26, 37].

As was described above, for virological diagnosis it is important to know the pathogenesis and the infection kinetics. The RNA viruses are found in plasma or serum specimens between 2 and 6 days following onset of fever, which corresponds to the period of incubation [41]. Viral load can subsequently rapidly reach up to

Complete virions as well as viral fragments (antigen or genome) are frequent in this phase, although replication later declines. In line with this kinetic replication, three methods can be used in the laboratory: viral isolation and antigen or genomic

Viral isolation is generally a research tool [71–73]. For viral culture it is essential to preserve the complete virion. Culture sensitivity for chikungunya virus is high only in early infection (during the viremic phase) but drops 5 days after onset of illness. As a result, virus isolation is rarely used in the diagnosis of CHIKV infection because low sensitivity occurs after only a few days post-infection, and it is timeconsuming and laborious procedure. However, it does allow for the identification of the viral strain and can be important for epidemiologic and research purposes. Samples where chikungunya virus is suspected should be handled under Biosafety

A wide variety of cells are used for in vitro CHIKV cultures in order to assess the full scope of the disease, including primary human skeletal muscle myoblasts [74], human blood monocytes [75], African green monkey kidney (Vero-E6) cells [76], and C6/36 (*Aedes albopictus* clone cells). Of particular interest, given the known tropism of chronic CHIKV for bone and synovial tissues, is the use of primary

Culture on HEL cells is more sensitive, and the presence of a cytopathic effect

There are commercial immunochromatography antigen detection kits which detect CHIKV with high sensitivity in the early phase (up to 4–5 days after the onset of fever, when blood-enveloping proteins are still present). These tests also have high specificity since no cross-reactions with dengue virus are known to exist, and the method is straightforward and simple and does not require specially trained

However, a very important issue to consider is what genotype/s the tests are capable of detecting since not all detect all the known genotypes. Thus the geographical area from which the patient comes, meaning they are more likely to have one genotype or another, needs to be taken into account for accurate diagnosis (e.g., Okabayashi's immunochromatography only detects the Asian

The diagnosis of CHIKV infection in the acute phase of infection is typically performed by the detection of viral RNA in plasma or serum (or other sample

These methods are therefore, generally, sensitivity compromised.

These cultures all need to be maintained under standard conditions.

human fibroblast-like synoviocytes [77] and human osteoblasts.

can be observed earlier than in Vero cells [78].

**3.2 Virological diagnosis**

108 /109

detection.

*3.2.1 Viral isolation*

Level (BSL) 3 conditions.

*3.2.2 Antigen detection*

laboratory personnel [79].

*3.2.3 Genomic detection (RT-PCR)*

genotype) [80].

**38**

types) by RT-PCR. The viral RNA can be detected by various molecular methods, such as nested and real-time PCRs [37].

Conventional and real-time PCRs have been used to amplify nsP1, nsP2, or even envelope protein genes (E3, E2, or E1) [81, 82].

Molecular diagnostic tests used for the detection of CHIKV include reverse transcription (RT) and amplification (PCR) assay of fragments in the nsP1, nsP2, nsP3, nsP4, or E1 regions of the CHIKV genome, in general by real-time [65, 82–86]. Labeled probes or SYBR Green is used for real-time quantification of the amplified PCR products [87, 88]. In these assays, the limits of detection range between 0.5 and 1.5 log10 RNA copies/reaction. Although the detection of positive-strand RNA is commonly used for diagnostic purposes, assays to detect negative-strand CHIKV RNA have also been developed. These include strand-specific quantitative RT-PCRs for nsP1 [89] and nsP3 [90], which use a tagged-primer system to improve PCR specificity and accuracy. In comparison to the detection of positive-strand RNA, the detection limit for negative-strand RNA assays is slightly decreased, 3 log10 RNA copies [91].

While there is an ever-present possibility of CHIKV spreading further in developed countries in the Americas and Europe, CHIKV still predominantly occurs in resource-limited countries. *Loop-mediated isothermal amplification* (LAMP) amplifies nucleic acid under isothermal conditions without the use of a thermal cycler and is a fast, specific, and cost-effective technique [92]; LAMP represents a cheaper alternative amplification method, albeit with lower sensitivity than real-time RT-PCR. Monitoring by turbidity as well as observation of color change after adding SYBR Green has also been described for the detection of CHIKV [85, 92]. Visualization is made with the naked eye, meaning no sophisticated equipment is required, and thus LAMP is especially useful in developing countries or in field studies.

Another method, *microfluidic lab-on-chip* integrating multiplex molecular amplification and DNA microarray hybridization, has been developed for the simultaneous detection of 26 globally important tropical pathogens such as CHIKV, *Dengue virus* (DENV), and other arboviruses [93]. Such diagnostic capacity provides an effective and rapid means to establish the presence of specific potential pathogens.

The CDC protocol for chikungunya infection diagnosis uses two RT-PCRs. The nucleotide sequences of the two sets of primers and probes used are listed in **Table 1** [94, 95]. The 3855 primer/probe set is specific to the ECSA genotypes although it is


#### **Table 1.**

*Chikungunya virus oligonucleotide primers and probes used in the Centers for Disease Control and Prevention real-time reverse transcription-polymerase chain reaction assays designed to detect CHIKV Asian genotype strains.*


**Table 2.**

*Primers and probes used to detect DENV, CHIKV, ZIKV, YFV, and WNV.*

capable of detecting both Asian and ECSA genotypes [95]. The 856 primer/probe set targets the Asian genotype, which has a slightly higher sensitivity than the 3855 set, which is prevalent in the Caribbean. This elevated sensitivity makes it a valuable tool for confirming diagnosis in samples where CHIKV RNA levels are reduced. These characteristics should be taken into account for testing protocols (i.e., which sets to use and in what order).

In our experience "in-house" real-time PCR is a useful tool in the chikungunya diagnosis. As such, proprietary primers and probe have been designed (**Table 2**) for CHIKV. In addition, for the syndromic diagnosis of imported viral diseases, our in-house RT-PCR can also detect other arboviruses such as Zika, dengue, yellow fever, and West Nile (**Table 2**) with sensitivities/specificities comparable to, or even superior to, commercially available techniques [63].

Genomic analysis is also used to classify and characterize viruses usually by Sanger sequencing method.

The sequencing of envelope E1 (viral structural glycoprotein) is often used for phylogenetic analysis. The three principal strains of CHIKV (ECSA, West African, and Asian) have been typed by nucleotide sequencing of a portion of the E1 region using RT-PCR [96].

Using sequencing studies to examine genomic evolution, it has been determined that global chikungunya outbreaks since 2005 have occurred as a result of a mutation of the ECSA lineage: an alanine-to-valine substitution in the E1 envelope glycoprotein at position 226 (A226V), which enabled the virus to acquire a new mosquito vector, *A. albopictus,* commonly known as the Asian tiger mosquito, conferring increased virus adaptation and replication ability in *Aedes albopictus*.

There are also specific regions that showed nucleotide variability and novel mutations making them suitable for phylogenetic analysis in nsP2 and E2 CHIKV regions [97].

**41**

in **Table 3**.

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections*

A variety of serological methods (hemagglutination inhibition, ELISA, complement fixation, and neutralization of viral infectivity using reference serum samples) can be used to characterize the alphavirus species [15, 98]. A fourfold increase in levels of CHIK virus IgG antibody in serum samples taken during the acute and the recovery phase is required to serodiagnosis. However, it is often problematic to collect paired samples, and so the existence of CHIKV-specific IgM antibodies in acute-phase samples is used instead. Also, patient serum coupled with neutralization by reference serum can be taken as definitive proof of the

Several serological assays have been developed, the large majority of which demonstrate high reliability and specificity. The most common first-line serological techniques used for CHIKV diagnosis are the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFA) assays, and the most suitable sample type is serum, generally using 1/100 dilutions. Commercially available ELISA tests, for example, have been shown to demonstrate high sensitivity (ranging from 82 to 88%) and specificity in samples from recovering

The presence of IgM antibodies indicates recent CHIKV infection [41], generally occurring in samples taken 2 weeks prior to patients becoming symptomatic. Detection of specific IgG antibodies, on the other hand, indicates previous CHIKV infection, which can be recent or as long ago as several months or years, given the

CHIKV-specific IgM and IgG can be detected in serum using enzyme-linked immunosorbent assay immunocapture. The first antibodies detected in patients with chikungunya fever is CHIKV-specific IgM, which commonly appears 3–8 days after the onset of infection and may persist for several months to 2 years. After 4–10 days since onset of infection, the IgG antibodies become detectable in serum and may persist for years (**Figure 1**) [1] and be potentially lifelong. The chances of cross-reactivity between IgG and other viruses of the Semliki antigenic serocomplex are less now than in the past due to improvements in test design and procedure, but IgG seroconversion (greater than fourfold increase) is still recommended for reliable diagnosis. Furthermore, the existence of closely related alphaviruses in the area where the patient was infected necessitates the use of plaque reduction neutral-

ELISA is a rapid and sensitive method largely used for the detection of anti-CHIKV antibodies. The most common tests used for the diagnosis of CHIKV infection are IgM antibody-capture ELISA (MAC- ELISA) and indirect ELISA (i-ELISA) for the detection of type M (IgM) and type G (IgG) immunoglobulin, respectively [102]. A list of different commercially available ELISA tests is shown

Enzyme-linked immunosorbent assays can be used to confirm the presence of anti-CHIKV antibodies, with IgM antibody levels highest 3–5 weeks post-infection

ELISA is not, however, without its disadvantages: false-positives because of cross-reactivity with other alphaviruses (Ross River virus, Barmah Forest virus, and Sindbis virus [103]) and its sensitivity being much reduced (4–20%) in serum

*DOI: http://dx.doi.org/10.5772/intechopen.86957*

**3.3 Serological diagnosis**

presence of CHIKV.

patients [99, 100].

persistence of anti-CHIKV IgG antibodies [1].

ization testing to confirm CHIKV infection [101].

and persisting for up to 2 months.

samples taken during the acute phase [104].

*3.3.1 Enzyme-linked immunosorbent assay*

## **3.3 Serological diagnosis**

*Current Topics in Neglected Tropical Diseases*

sets to use and in what order).

**Table 2.**

Sanger sequencing method.

using RT-PCR [96].

superior to, commercially available techniques [63].

*Primers and probes used to detect DENV, CHIKV, ZIKV, YFV, and WNV.*

capable of detecting both Asian and ECSA genotypes [95]. The 856 primer/probe set targets the Asian genotype, which has a slightly higher sensitivity than the 3855 set, which is prevalent in the Caribbean. This elevated sensitivity makes it a valuable tool for confirming diagnosis in samples where CHIKV RNA levels are reduced. These characteristics should be taken into account for testing protocols (i.e., which

**Virus Name Sequence (5′- 3′) Amplicon size** DEN.ZIK-TR-S AAGGACTAGAGGTTAGAGGAGACCC Dengue DEN-TR-A GAGACAGCAGGATCTCTGGTCT 83 bp DEN-FAM AACAGCATATTGACGCTGG DEN.ZIK-TR-S AAGGACTAGAGGTTAGAGGAGACCC Zika ZIK-TR-A3 GGCCAGCGTGGTGGAA 96 bp ZIK-FAM AAACAGCATATTGACG CHK-TR-S CGGCGACCATTTGTGATCA Chikungunya CHK-TR-A TTCGTATTCCGTTGCGTTCTG 127 bp CHK-FAM CCGGCATCCTTGCTA WN.FA-TR-S GGCTGGGGCAAYGGCT Yellow fever FA-TR-A CCTCAAACAAACTCATGGATTTG 91 bp WN-FA-FAM CATGCGCCAAATT WN.FA-TR-S GGCTGGGGCAAYGGCT West Nile WN-TR-A CAAGATGGTTCTTCCTATTGCCT 96 bp WN-FA-FAM CATGCGCCAAATT

In our experience "in-house" real-time PCR is a useful tool in the chikungunya diagnosis. As such, proprietary primers and probe have been designed (**Table 2**) for CHIKV. In addition, for the syndromic diagnosis of imported viral diseases, our in-house RT-PCR can also detect other arboviruses such as Zika, dengue, yellow fever, and West Nile (**Table 2**) with sensitivities/specificities comparable to, or even

Genomic analysis is also used to classify and characterize viruses usually by

The sequencing of envelope E1 (viral structural glycoprotein) is often used for phylogenetic analysis. The three principal strains of CHIKV (ECSA, West African, and Asian) have been typed by nucleotide sequencing of a portion of the E1 region

Using sequencing studies to examine genomic evolution, it has been determined that global chikungunya outbreaks since 2005 have occurred as a result of a mutation of the ECSA lineage: an alanine-to-valine substitution in the E1 envelope glycoprotein at position 226 (A226V), which enabled the virus to acquire a new mosquito vector, *A. albopictus,* commonly known as the Asian tiger mosquito, conferring increased virus adaptation and replication ability in *Aedes* 

There are also specific regions that showed nucleotide variability and novel mutations making them suitable for phylogenetic analysis in nsP2 and E2 CHIKV

**40**

*albopictus*.

regions [97].

A variety of serological methods (hemagglutination inhibition, ELISA, complement fixation, and neutralization of viral infectivity using reference serum samples) can be used to characterize the alphavirus species [15, 98]. A fourfold increase in levels of CHIK virus IgG antibody in serum samples taken during the acute and the recovery phase is required to serodiagnosis. However, it is often problematic to collect paired samples, and so the existence of CHIKV-specific IgM antibodies in acute-phase samples is used instead. Also, patient serum coupled with neutralization by reference serum can be taken as definitive proof of the presence of CHIKV.

Several serological assays have been developed, the large majority of which demonstrate high reliability and specificity. The most common first-line serological techniques used for CHIKV diagnosis are the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFA) assays, and the most suitable sample type is serum, generally using 1/100 dilutions. Commercially available ELISA tests, for example, have been shown to demonstrate high sensitivity (ranging from 82 to 88%) and specificity in samples from recovering patients [99, 100].

The presence of IgM antibodies indicates recent CHIKV infection [41], generally occurring in samples taken 2 weeks prior to patients becoming symptomatic. Detection of specific IgG antibodies, on the other hand, indicates previous CHIKV infection, which can be recent or as long ago as several months or years, given the persistence of anti-CHIKV IgG antibodies [1].

#### *3.3.1 Enzyme-linked immunosorbent assay*

CHIKV-specific IgM and IgG can be detected in serum using enzyme-linked immunosorbent assay immunocapture. The first antibodies detected in patients with chikungunya fever is CHIKV-specific IgM, which commonly appears 3–8 days after the onset of infection and may persist for several months to 2 years. After 4–10 days since onset of infection, the IgG antibodies become detectable in serum and may persist for years (**Figure 1**) [1] and be potentially lifelong. The chances of cross-reactivity between IgG and other viruses of the Semliki antigenic serocomplex are less now than in the past due to improvements in test design and procedure, but IgG seroconversion (greater than fourfold increase) is still recommended for reliable diagnosis. Furthermore, the existence of closely related alphaviruses in the area where the patient was infected necessitates the use of plaque reduction neutralization testing to confirm CHIKV infection [101].

ELISA is a rapid and sensitive method largely used for the detection of anti-CHIKV antibodies. The most common tests used for the diagnosis of CHIKV infection are IgM antibody-capture ELISA (MAC- ELISA) and indirect ELISA (i-ELISA) for the detection of type M (IgM) and type G (IgG) immunoglobulin, respectively [102]. A list of different commercially available ELISA tests is shown in **Table 3**.

Enzyme-linked immunosorbent assays can be used to confirm the presence of anti-CHIKV antibodies, with IgM antibody levels highest 3–5 weeks post-infection and persisting for up to 2 months.

ELISA is not, however, without its disadvantages: false-positives because of cross-reactivity with other alphaviruses (Ross River virus, Barmah Forest virus, and Sindbis virus [103]) and its sensitivity being much reduced (4–20%) in serum samples taken during the acute phase [104].


#### **Table 3.**

*Commercial diagnostic test available for serological diagnosis of CHIKV infection.*

For these reasons, commercially available rapid diagnostic kits often do not provide as reliable results as RT-PCR because they all tend to detect host-derived anti-CHIKV IgM antibodies. Furthermore, since IgM antibodies are produced later in the course of infection than the antigen, this is a less sensitive test and as such can delay diagnosis and reduce the effectiveness of disease management [105].

#### *3.3.2 Indirect immunofluorescence*

IFA is an accurate and reliable technique widely used for the detection of specific anti-CHIK antibodies. IFA reveals the presence of type-specific antibodies against CHIKV by detecting the presence of virus antigens in infected cells.

The specificity of commercial IFA tests has been found to range from 75 to 100% in serum collected 5–6 days after infection [106]. That said IFA has some shortcomings in that it is laborious and the personnel carrying out the procedure need to be specially trained. In addition interpretation of the microscope examination results can be rather subjective, and there is no standardization between labs.

#### *3.3.3 Antibody neutralization assay*

The neutralization assay is generally used following ELISA or IFA results to confirm CHIKV. Neutralization involves the interaction of viral antigens and specific antibodies to block infection. The neutralization test requires the mixing of virus and serum, and the resulting mixture is then inoculated into cell culture. Inhibition of the virus can then be tested, using a variety of methods, after a number of days [107]. The microneutralization assay (MNA) evaluates the neutralizing antibodies. Although both methods have high specificity and sensitivity, there are disadvantages, namely, they are labor-intensive since only a small number of samples can be processed in each run and a Biosafety Level 3 laboratory (BSL-3) is required because live virus is being used.

The drawbacks to antibody testing include cross-reactivity with other alpha viruses and the problem of it not being able to distinguish between recent past and acute infection, as well as the fact that its sensitivity varies between clinical settings.

**43**

death [117].

*3.4.3 Children*

the A226V variant strain [122].

be investigated in the brain or other fetal tissues [40].

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections*

Diagnosis is clearly always important for appropriate patient management, but it

RNA copies/ml plasma) is likely prior to

CHIKV, like other arboviruses, can be transmitted by blood transfusion or through organ transplant from infected donors who were asymptomatic at the time of donation. Transmission via organ transplantation could also occur since chi-

In a case series of Rosso et al., 80% of the patients presented high viral load

In these patients, blood and/or tissues are essential in the diagnosis.

congenital and perinatal infection having been demonstrated.

copies/ml), and none of the solid organ transplant patients with CHIKV

Vertical transmission of CHIKV is a possibility and can affect the fetus, both

Congenital and perinatal CHIKV infections were reported following delivery to mothers with documented viremia accompanied by symptoms of CHIKF [16, 111, 112]. Vertical transmission rate of CHIKV has been estimated to range from 27.7 to 48.29%, and the same paper found transmission to be increased in mothers experiencing viremia at the time of birth. Neonatal symptoms were found to begin within 3–9 days postpartum, although there is no evidence of any link between birth

No link between CHIKV exposure in the first trimester of gestation and higher miscarriage risk or congenital malformation has been found to date [114, 115]. However, infection in the second or third trimester has been linked with increased fetal mortality, although the mechanism of this is unclear [111, 116]. Furthermore, a change in the evolution of neonates vertically infected with CHIKV has been seen following the spread of the virus to Indian Oceanic regions and Latin America in terms of the previously minor, self-limiting symptoms having been replaced by multiple severe manifestations and sometimes

Of particular note is the serious outbreak of CHIKV in 2006 in the Reunion Islands where complications were reported, including seizures and abnormal MRI scans [118, 119] as well as cardiac defects in almost 50% of patients [119]. In infants, these conditions can lead to death in the first year of life if swift and targeted medical intervention is not carried out [120, 121]. All patients in this outbreak who exhibited symptoms of neuroinvasive disease were infected with

Virus infection in fetuses can be documented by positive RT-PCR in the amniotic fluid, and placenta, and in the cases of miscarriage, the involvement of CHIKV can

The first descriptions of CHIKF in children were made in the 1960s during outbreaks in India and South Asia [123–127]. In these younger patients, the infection

*DOI: http://dx.doi.org/10.5772/intechopen.86957*

becomes crucial in several specific circumstances.

**3.4 Diagnosis in other patients**

*3.4.1 Transplantation or transfusion*

onset of symptoms [86, 108, 109].

*3.4.2 Maternal-fetal transmission*

(>106

kungunya viremia (which may exceed 109

infection developed graft rejection or died [110].

method and incidence of congenital disease [113].

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections DOI: http://dx.doi.org/10.5772/intechopen.86957*

#### **3.4 Diagnosis in other patients**

*Current Topics in Neglected Tropical Diseases*

*3.3.2 Indirect immunofluorescence*

**Table 3.**

*3.3.3 Antibody neutralization assay*

because live virus is being used.

For these reasons, commercially available rapid diagnostic kits often do not provide as reliable results as RT-PCR because they all tend to detect host-derived anti-CHIKV IgM antibodies. Furthermore, since IgM antibodies are produced later in the course of infection than the antigen, this is a less sensitive test and as such can

IFA is an accurate and reliable technique widely used for the detection of specific anti-CHIK antibodies. IFA reveals the presence of type-specific antibodies against

The specificity of commercial IFA tests has been found to range from 75 to 100% in serum collected 5–6 days after infection [106]. That said IFA has some shortcomings in that it is laborious and the personnel carrying out the procedure need to be specially trained. In addition interpretation of the microscope examination results

The neutralization assay is generally used following ELISA or IFA results to confirm CHIKV. Neutralization involves the interaction of viral antigens and specific antibodies to block infection. The neutralization test requires the mixing of virus and serum, and the resulting mixture is then inoculated into cell culture. Inhibition of the virus can then be tested, using a variety of methods, after a number of days [107]. The microneutralization assay (MNA) evaluates the neutralizing antibodies. Although both methods have high specificity and sensitivity, there are disadvantages, namely, they are labor-intensive since only a small number of samples can be processed in each run and a Biosafety Level 3 laboratory (BSL-3) is required

The drawbacks to antibody testing include cross-reactivity with other alpha viruses and the problem of it not being able to distinguish between recent past and acute infection, as well as the fact that its sensitivity varies between clinical

delay diagnosis and reduce the effectiveness of disease management [105].

**Company IgM IgG Test** Euroimmun + + ELISA

Novatec / + ELISA

Abcam + + ELISA

DRG / + ELISA

GenWay / + ELISA Standard diagnostic + + ELISA Euroimmun + + IFA

*Commercial diagnostic test available for serological diagnosis of CHIKV infection.*

Novatec + / IgM-capture ELISA

IBL + / IgM-capture ELISA IBL / + IgG-capture ELISA

DRG + / IgM-capture ELISA

GenWay + / IgM-capture ELISA

CHIKV by detecting the presence of virus antigens in infected cells.

can be rather subjective, and there is no standardization between labs.

**42**

settings.

Diagnosis is clearly always important for appropriate patient management, but it becomes crucial in several specific circumstances.

#### *3.4.1 Transplantation or transfusion*

CHIKV, like other arboviruses, can be transmitted by blood transfusion or through organ transplant from infected donors who were asymptomatic at the time of donation. Transmission via organ transplantation could also occur since chikungunya viremia (which may exceed 109 RNA copies/ml plasma) is likely prior to onset of symptoms [86, 108, 109].

In a case series of Rosso et al., 80% of the patients presented high viral load (>106 copies/ml), and none of the solid organ transplant patients with CHIKV infection developed graft rejection or died [110].

In these patients, blood and/or tissues are essential in the diagnosis.

#### *3.4.2 Maternal-fetal transmission*

Vertical transmission of CHIKV is a possibility and can affect the fetus, both congenital and perinatal infection having been demonstrated.

Congenital and perinatal CHIKV infections were reported following delivery to mothers with documented viremia accompanied by symptoms of CHIKF [16, 111, 112].

Vertical transmission rate of CHIKV has been estimated to range from 27.7 to 48.29%, and the same paper found transmission to be increased in mothers experiencing viremia at the time of birth. Neonatal symptoms were found to begin within 3–9 days postpartum, although there is no evidence of any link between birth method and incidence of congenital disease [113].

No link between CHIKV exposure in the first trimester of gestation and higher miscarriage risk or congenital malformation has been found to date [114, 115]. However, infection in the second or third trimester has been linked with increased fetal mortality, although the mechanism of this is unclear [111, 116]. Furthermore, a change in the evolution of neonates vertically infected with CHIKV has been seen following the spread of the virus to Indian Oceanic regions and Latin America in terms of the previously minor, self-limiting symptoms having been replaced by multiple severe manifestations and sometimes death [117].

Of particular note is the serious outbreak of CHIKV in 2006 in the Reunion Islands where complications were reported, including seizures and abnormal MRI scans [118, 119] as well as cardiac defects in almost 50% of patients [119]. In infants, these conditions can lead to death in the first year of life if swift and targeted medical intervention is not carried out [120, 121]. All patients in this outbreak who exhibited symptoms of neuroinvasive disease were infected with the A226V variant strain [122].

Virus infection in fetuses can be documented by positive RT-PCR in the amniotic fluid, and placenta, and in the cases of miscarriage, the involvement of CHIKV can be investigated in the brain or other fetal tissues [40].

#### *3.4.3 Children*

The first descriptions of CHIKF in children were made in the 1960s during outbreaks in India and South Asia [123–127]. In these younger patients, the infection

presented as a febrile illness, with rash and joint pain or dengue-like illness, hemorrhagic fever, or CSF infection. Asian lineage infection of children resulted in a lower frequency of arthralgia than in those infected by the African lineages, although hemorrhagic fever was more frequent in children testing positive for the former [117].

Lately, in the regions surrounding the Indian Ocean and the expansion of CHIKV into the Western Hemisphere, CHIKV-WH infections in children and neonates are reported more frequently and with more severe syndromes and sequelae [117].

## **4. Summary**

In recent years, large-scale outbreaks of the chikungunya arbovirus (CHIKV) have permitted rapid propagation of the virus across the globe. CHIKV is transmitted by the *Aedes* mosquito, and the acute infection phase typically results in fever, joint pain, headache or rash, and chronic rheumatism (arthralgia or myalgia, anorexia, and concentration disorders) in up to 40% of cases.

The chronic form is defined by symptoms which persist for more than 3 months, and up to years, after initial diagnosis. The negative impact of CHIKV infection on the patient's health-related quality of life is quite severe and is not limited to the acute disease, and the repercussions may last for several months after clinical recovery. The highest prevalence of chronic discomfort was linked to one of the four genotypes described. These genotypes represent different geographic lineages (classification based on partial sequence of viral E1 glycoprotein): West African, East-Central-South-African (ECSA), ECSA-diverged or Indian Ocean Lineage, and Asian lineage.

The first marker detected in CHIK infection is the viral RNA, usually by RT-PCR. This marker can be present in samples taken within 8 days of onset of the illness. After this initial acute phase, the infection can also be diagnosed by serological detection of CHIKV-IgG and/or IgM-specific immunoglobulin, and sequencing studies can determine the infecting genotype.

The correct diagnosis of chikungunya infection enables the disease progression to be better predicted and patients' treatment to be more effectively managed.

**45**

Spain

**Author details**

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections*

Marta E. Álvarez-Argüelles\*, Susana Rojo Alba, Mercedes Rodríguez Pérez,

Microbiology Department, Hospital Universitario Central de Asturias, Oviedo,

© 2019 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,

Jose Antonio Boga Riveiro and Santiago Melón García

provided the original work is properly cited.

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

*DOI: http://dx.doi.org/10.5772/intechopen.86957*

*Diagnosis and Molecular Characterization of Chikungunya Virus Infections DOI: http://dx.doi.org/10.5772/intechopen.86957*

## **Author details**

*Current Topics in Neglected Tropical Diseases*

former [117].

sequelae [117].

**4. Summary**

cases.

Asian lineage.

managed.

studies can determine the infecting genotype.

presented as a febrile illness, with rash and joint pain or dengue-like illness, hemorrhagic fever, or CSF infection. Asian lineage infection of children resulted in a lower frequency of arthralgia than in those infected by the African lineages, although hemorrhagic fever was more frequent in children testing positive for the

Lately, in the regions surrounding the Indian Ocean and the expansion of CHIKV into the Western Hemisphere, CHIKV-WH infections in children and neonates are reported more frequently and with more severe syndromes and

In recent years, large-scale outbreaks of the chikungunya arbovirus (CHIKV) have permitted rapid propagation of the virus across the globe. CHIKV is transmitted by the *Aedes* mosquito, and the acute infection phase typically results in fever, joint pain, headache or rash, and chronic rheumatism (arthralgia or myalgia, anorexia, and concentration disorders) in up to 40% of

The chronic form is defined by symptoms which persist for more than 3 months,

and up to years, after initial diagnosis. The negative impact of CHIKV infection on the patient's health-related quality of life is quite severe and is not limited to the acute disease, and the repercussions may last for several months after clinical recovery. The highest prevalence of chronic discomfort was linked to one of the four genotypes described. These genotypes represent different geographic lineages (classification based on partial sequence of viral E1 glycoprotein): West African, East-Central-South-African (ECSA), ECSA-diverged or Indian Ocean Lineage, and

The first marker detected in CHIK infection is the viral RNA, usually by RT-PCR. This marker can be present in samples taken within 8 days of onset of the illness. After this initial acute phase, the infection can also be diagnosed by serological detection of CHIKV-IgG and/or IgM-specific immunoglobulin, and sequencing

The correct diagnosis of chikungunya infection enables the disease progression to be better predicted and patients' treatment to be more effectively

**44**

Marta E. Álvarez-Argüelles\*, Susana Rojo Alba, Mercedes Rodríguez Pérez, Jose Antonio Boga Riveiro and Santiago Melón García Microbiology Department, Hospital Universitario Central de Asturias, Oviedo, Spain

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

© 2019 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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**46**

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Fourmaintraux A, Schuffenecker I, et al. Vertical maternal fetal transmission of the chikungunya virus: Ten cases among 84 pregnant women. Presse Médicale.

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Real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus. Diagnostic Microbiology and Infectious Disease.

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[92] Reddy V, Ravi V, Desai A, Parida M, Powers AM, Johnson BW. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of chikungunya fever. Journal of Medical Virology.

[93] Tan JL, Capozzoli M, Mitsuharu S, Watthanaworawit W, Ling CL, Mauduit M, et al. An integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens. PLoS Neglected Tropical Diseases.

[94] Lanciotti RS, Valadere AM. Transcontinental movement of Asian genotype chikungunya virus. Emerging Infectious Diseases.

[95] Lanciotti RS, Lambert AJ.

Phylogenetic analysis of chikungunya virus strains circulating in the Western

2009;**4**:e7468

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2008;**3**:179-192

2012;**84**:1771-1778

2014;**8**:e3043

2014;**20**:1400-1402

**52**

Tanganuchitcharnchai A, Jarman RG, Gibbons RV, Paris DH, Bailey MS, et al. Poor diagnostic accuracy of commercial antibody-based assays for the diagnosis of acute chikungunya infection. Clinical and Vaccine Immunology. 2011;**18**:1773-1775

[105] Rianthavorn P, Wuttirattanakowit N, Prianantathavorn K, Limpaphayom N, Theamboonlers A, Poovorawan Y. Evaluation of a rapid assay for detection of IgM antibodies to chikungunya. The Southeast Asian Journal of Tropical Medicine and Public Health. 2010;**41**(1):92-96

[106] Yap G, Pok KY, Lai YL, Hapuarachchi HC, Chow A, Leo YS, et al. Evaluation of chikungunya diagnostic assays: Differences in sensitivity of serology assays in two independent outbreaks. PLoS Neglected Tropical Diseases. 2010;**4**:e753

[107] Sambri V, Capobianchi MR, Cavrini F, Charrel R, Donoso-Mantke O, Escadafal C, et al. Diagnosis of West Nile virus human infections: Overview and proposal of diagnostic protocols considering the results of external quality assessment studies. Viruses. 2013;**5**:2329-2348

[108] Brouard C, Bernillon P, Quatresous I, Pillonel J, Assal A, De Valk H, et al. Estimated risk of chikungunya viremic blood donation during an epidemic on Reunion Island in the Indian Ocean, 2005 to 2007. Transfusion. 2008;**48**:1333

[109] Simmons G, Brès V, Lu K, Liss NM, Brambilla DJ, Ryff KR, et al. High

incidence of chikungunya virus and frequency of viremic blood donations during epidemic, Puerto Rico, USA, 2014. Emerging Infectious Diseases. 2016;**22**:1221

[110] Rosso F, Rodríguez S, Cedano JA, Mora BL, Moncada PA, Velez JD. Chikungunya in solid organ transplant recipients, a case series and literature review. Transplant Infectious Disease. 2018;**20**(6):e1297

[111] Touret Y, Randrianaivo H, Michault A, Schuffenecker I, Kauffmann E, Lenglet Y, et al. Early maternal-fetal transmission of the chikungunya virus. Presse Médicale. 2006;**35**:1656-1658

[112] Robillard PY, Boumahni B, Gérardin P, Michault A, Fourmaintraux A, Schuffenecker I, et al. Vertical maternal fetal transmission of the chikungunya virus: Ten cases among 84 pregnant women. Presse Médicale. 2006;**35**:785-788

[113] Torres JR, Falleiros-Arlant LH, Dueñas L, Pleitez-Navarrete J, Salgado DM, Castillo JB. Congenital and perinatal complications of chikungunya fever: A Latin American experience. International Journal of Infectious Diseases. 2016;**51**:85-88

[114] Fritel X, Catteau C, Calliez F, Brodel A, Vaillant JL, Ansquin H. Chikungunya outbreak, pregnancy outcome and perinatal mortality: Observational study about 40,000 pregnancies and deliveries on Réunion island, during 2004-2006. In: Proceedings of the 13th International Congress on Infectious Disease, 2008 June 19-22, Kuala Lumpur. Abstract published in: Int J Infect Dis. Vol. 12, No. Suppl 1. 2008. p. e328

[115] Fritel X, Rollot O, Gérardin P, Gauzere BA, Bideault J, Lagarde L, et al. Chikungunya virus infection

during pregnancy, Reunion, France, 2006. Emerging Infectious Diseases. 2010;**16**:418-425

[116] Gérardin P, LaBeaud AD, Ritz N. Chikungunya fever during pregnancy and in children: and overview on clinical and research perspectives. Intech Open Access. 2016. p. 19-41. DOI: 10.5772/64424

[117] Barr KL. Vaidhyanathan V, Chikungunya in infants and children: Is pathogenesis increasing? Viruses. 2019;**11**(3):23

[118] Lewthwaite P, Vasanthapuram R, Osborne JC, Begum A, Plank JL, Shankar MV, et al. Chikungunya virus and central nervous system infections in children, India. Emerging Infectious Diseases. 2009;**15**:329-331

[119] Ramful D, Carbonnier M, Pasquet M, Bouhmani B, Ghazouani J, Noormahomed T, et al. Mother-to-child transmission of chikungunya virus infection. The Pediatric Infectious Disease Journal. 2007;**26**:811-815

[120] Maron BJ, Tajik AJ, Ruttenberg HD, Graham TP, Atwood GF, Victorica BE, et al. Hypertrophic cardiomyopathy in infants: Mother-to-child transmission of chikungunya. Circulation. 1982;**65**:7-17

[121] Sewell EK, Keene S. Perinatal care of infants with congenital birth defects. Clinics in Perinatology. 2018;**45**:213-230

[122] Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L, Vaney MC, et al. Genome microevolution of chikungunya viruses causing the Indian ocean outbreak. PLoS Medicine. 2006;**3**:e263

[123] Jadhav M, Namboodripad M, Carman RH, Carey DE, Myers RM. Chikungunya disease in infants and children in Vellore: A report of clinical and haematological features of virologically proved cases. The Indian Journal of Medical Research. 1965;**53**:764-776

[124] Vu-Qui-Dai, Nguyen-Thi Kim-Thoa, Ly-Quoc-Bang. Study of antichikungunya antibodies in Vietnamese children in Saigon. Bulletin de la Societe de Pathologie Exotique et de Ses Filiales. 1967;**60**:353-359

[125] Carey DE, Myers RM, DeRanitz CM, Jadhav M, Reuben R. The 1964 chikungunya epidemic at Vellore, South India, including observations on concurrent dengue. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1969;**63**:434-445

[126] Halstead SB, Udomsakdi S, Singharaj P, Nisalak A. Dengue chikungunya virus infection in man in Thailand, 1962-1964. 3. Clinical, epidemiologic, and virologic observations on disease in non-indigenous white persons. The American Journal of Tropical Medicine and Hygiene. 1969;**18**:984-996

[127] Nimmannitya S, Halstead SB, Cohen SN, Margiotta M. Dengue and chikungunya virus infection in man in Thailand, 1962-1964. I. Observations on hospitalized patients with hemorrhagic fever. The American Journal of Tropical Medicine and Hygiene. 1969;**18**:954-971

**55**

**Chapter 4**

**Abstract**

**1. Introduction**

reported in Italy in 2007 [5].

cases have occurred in Florida and Texas [6].

*and Robert T. Schoen*

Clinical Features and Management

of Chronic Chikungunya Arthritis

Chikungunya virus is a single-stranded RNA alphavirus transmitted to humans by *Aedes* species mosquitos, causing an acute illness known as chikungunya fever with maculopapular rash, headache, polyarthritis/arthralgias, and gastrointestinal symptoms. Up to half of affected patients develop a chronic disabling arthritis following resolution of the acute infection, which can last for months or even years. The pathophysiology of chronic chikungunya arthritis remains controversial; it may result from a dysregulated immune response or be caused by persistent viral infection. Treatment for patients with chronic chikungunya arthritis remains investigational. Limited data suggests that immunosuppressive therapies such as methotrexate and TNF alpha inhibitors may be beneficial, though randomized clinical trials are needed.

*Joshua Britton Bilsborrow, José Kennedy Amaral* 

**Keywords:** chikungunya, alphavirus, arthritis, disability, DMARDs

Chikungunya virus (CHIKV) is a small, single-stranded RNA alphavirus transmitted to humans by *Aedes* species mosquitoes, including *Aedes aegypti* and *Aedes albopictus*. CHIKV was initially isolated in Tanzania in 1952–1953 [1]. The word "chikungunya" means "that which bends up" or "to become contorted" in the Makonde language, referring to the prostrated appearance of affected patients [2]. Prior to its isolation, chikungunya (CHIK) was often misdiagnosed as dengue [3]. During the twentieth century, chikungunya fever (CHIKF) epidemics occurred sporadically and were limited to Africa and Asia, but in the twenty-first century CHIK has become a global disease. There was a major outbreak in coastal Kenya in 2004, which subsequently spread to Réunion in 2005 and throughout the Indian Ocean region [4]. Cases among travelers returning from endemic regions were

CHIKV reached the Western Hemisphere in 2013 with an outbreak on the island of Saint Martin. Since then, the virus has spread throughout the region with more than 2 million cases documented in the Americas by the end of 2016, though the actual number is likely much higher [3]. In the United States, cases involving travelers to endemic regions have been documented in 49 states, and locally-acquired

Factors contributing to the spread of CHIK include increasing urbanization, overstrained health care infrastructures in developing countries, ease of interna-

tional travel, and climate change with expansion of mosquito vectors [7].

## **Chapter 4**

*Current Topics in Neglected Tropical Diseases*

during pregnancy, Reunion, France, 2006. Emerging Infectious Diseases. of virologically proved cases. The Indian Journal of Medical Research.

[124] Vu-Qui-Dai, Nguyen-Thi Kim-Thoa, Ly-Quoc-Bang. Study of antichikungunya antibodies in Vietnamese children in Saigon. Bulletin de la Societe de Pathologie Exotique et de Ses Filiales.

[125] Carey DE, Myers RM, DeRanitz CM,

Jadhav M, Reuben R. The 1964 chikungunya epidemic at Vellore, South India, including observations on concurrent dengue. Transactions of the Royal Society of Tropical Medicine and

Hygiene. 1969;**63**:434-445

[126] Halstead SB, Udomsakdi S, Singharaj P, Nisalak A. Dengue chikungunya virus infection in man in Thailand, 1962-1964. 3. Clinical, epidemiologic, and virologic observations on disease in non-indigenous white persons. The American Journal of Tropical Medicine

and Hygiene. 1969;**18**:984-996

[127] Nimmannitya S, Halstead SB, Cohen SN, Margiotta M. Dengue and chikungunya virus infection in man in Thailand, 1962-1964. I. Observations on hospitalized patients with hemorrhagic fever. The American Journal of Tropical Medicine and Hygiene. 1969;**18**:954-971

1965;**53**:764-776

1967;**60**:353-359

[116] Gérardin P, LaBeaud AD, Ritz N. Chikungunya fever during pregnancy and in children: and overview on clinical and research perspectives. Intech Open Access. 2016. p. 19-41. DOI:

[117] Barr KL. Vaidhyanathan V, Chikungunya in infants and children: Is pathogenesis increasing? Viruses.

Diseases. 2009;**15**:329-331

[119] Ramful D, Carbonnier M,

Pasquet M, Bouhmani B, Ghazouani J, Noormahomed T, et al. Mother-to-child transmission of chikungunya virus infection. The Pediatric Infectious Disease Journal. 2007;**26**:811-815

[120] Maron BJ, Tajik AJ, Ruttenberg HD, Graham TP, Atwood GF, Victorica BE, et al. Hypertrophic cardiomyopathy in infants: Mother-to-child transmission of chikungunya. Circulation. 1982;**65**:7-17

[121] Sewell EK, Keene S. Perinatal care of infants with congenital birth defects. Clinics in Perinatology.

[122] Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L,

microevolution of chikungunya viruses causing the Indian ocean outbreak. PLoS Medicine. 2006;**3**:e263

[123] Jadhav M, Namboodripad M, Carman RH, Carey DE, Myers RM. Chikungunya disease in infants and children in Vellore: A report of clinical and haematological features

Vaney MC, et al. Genome

2018;**45**:213-230

[118] Lewthwaite P, Vasanthapuram R, Osborne JC, Begum A, Plank JL, Shankar MV, et al. Chikungunya virus and central nervous system infections in children, India. Emerging Infectious

2010;**16**:418-425

10.5772/64424

2019;**11**(3):23

**54**

## Clinical Features and Management of Chronic Chikungunya Arthritis

*Joshua Britton Bilsborrow, José Kennedy Amaral and Robert T. Schoen*

### **Abstract**

Chikungunya virus is a single-stranded RNA alphavirus transmitted to humans by *Aedes* species mosquitos, causing an acute illness known as chikungunya fever with maculopapular rash, headache, polyarthritis/arthralgias, and gastrointestinal symptoms. Up to half of affected patients develop a chronic disabling arthritis following resolution of the acute infection, which can last for months or even years. The pathophysiology of chronic chikungunya arthritis remains controversial; it may result from a dysregulated immune response or be caused by persistent viral infection. Treatment for patients with chronic chikungunya arthritis remains investigational. Limited data suggests that immunosuppressive therapies such as methotrexate and TNF alpha inhibitors may be beneficial, though randomized clinical trials are needed.

**Keywords:** chikungunya, alphavirus, arthritis, disability, DMARDs

#### **1. Introduction**

Chikungunya virus (CHIKV) is a small, single-stranded RNA alphavirus transmitted to humans by *Aedes* species mosquitoes, including *Aedes aegypti* and *Aedes albopictus*. CHIKV was initially isolated in Tanzania in 1952–1953 [1]. The word "chikungunya" means "that which bends up" or "to become contorted" in the Makonde language, referring to the prostrated appearance of affected patients [2].

Prior to its isolation, chikungunya (CHIK) was often misdiagnosed as dengue [3]. During the twentieth century, chikungunya fever (CHIKF) epidemics occurred sporadically and were limited to Africa and Asia, but in the twenty-first century CHIK has become a global disease. There was a major outbreak in coastal Kenya in 2004, which subsequently spread to Réunion in 2005 and throughout the Indian Ocean region [4]. Cases among travelers returning from endemic regions were reported in Italy in 2007 [5].

CHIKV reached the Western Hemisphere in 2013 with an outbreak on the island of Saint Martin. Since then, the virus has spread throughout the region with more than 2 million cases documented in the Americas by the end of 2016, though the actual number is likely much higher [3]. In the United States, cases involving travelers to endemic regions have been documented in 49 states, and locally-acquired cases have occurred in Florida and Texas [6].

Factors contributing to the spread of CHIK include increasing urbanization, overstrained health care infrastructures in developing countries, ease of international travel, and climate change with expansion of mosquito vectors [7].

There are three known genotypes of CHIKV: Asia, East/Central/South Africa, and West Africa. The 2004–2005 pandemic that originated in Kenya and subsequently spread throughout the Indian Ocean region involved the East/Central/ South Africa genotype. The pandemic that emerged in the Americas in 2013 originally involved the Asia genotype, although more recently the East/Central/South Africa lineage has been reported in the Western Hemisphere [8, 9].

### **2. Clinical manifestations**

#### **2.1 Acute chikungunya fever**

CHIKV infection results from transmission by the mosquito vector. Following an incubation period of 5–7 days, patients develop an acute febrile illness, chikungunya fever (CHIKF), characterized by high fevers, maculopapular rash, headaches, polyarthritis/arthralgias, myalgias, nausea, vomiting, and diarrhea [1]. Joint pain is often severe, and most often involves the metacarpal-phalangeal and interphalangeal joints of the hands, the wrists, the ankles, and the metatarsal-phalangeal joints of the feet. Less commonly involved but described joints include the shoulders, elbows, hips, knees, and inter-vertebral joints [10]. Acute CHIKF causes significant physical disability. For example, during the 2005 Comoros epidemic, an estimated 80% of affected patients were hospitalized or bed-bound due to severity of their symptoms [11].

More severely affected patients can develop neurological disease including meningoencephalitis, myelitis, radiculitis, and/or peripheral neuropathy, including reports of Guillain-Barré syndrome [12]. Rare ophthalmological manifestations reported include keratitis, episcleritis, optic neuritis, uveitis, and retinal detachment [13]. Uncommon but serious cardiac manifestations include arrhythmias, vasculopathy, myocarditis, and/or dilated cardiomyopathy [14, 15]. Patients with acute infection can have laboratory abnormalities including thrombocytopenia and leukopenia (lymphopenia is more common than neutropenia) [4].

Maternal-to-child transmission has also been reported, with up to 50% of neonates acquiring infection during childbirth if born within 5 days of maternal infection. Musculoskeletal manifestations are less prominent in newborns, with CHIKF being more notable for fevers, rash, cytopenias, hepatitis, and/or encephalitis [16].

Acute CHIKF typically resolves in 10–14 days, and has an overall reported case fatality rate of <1% based on epidemics in the Indian Ocean region and the Americas. However, the case fatality rate is higher among newborns, the elderly, and patients with underlying cardiovascular and pulmonary conditions [7, 17, 18]. Economopoulou and colleagues studied the case fatality rate among atypical cases of CHIKF in Réunion (defined as patients presenting with symptoms other than fevers and arthralgias); 65/610 patients from this group died for a case fatality rate of 10.6% [19].

During the widespread Indian Ocean region pandemic, a point mutation (A226V) in the E1 surface glycoprotein of CHIKV may have allowed better adaptation in *Aedes albopictus*, which had previously been a minor vector [20]. Along with low background immunity among populations residing in regions not historically affected, this may account for the rapid spread and high rates of infectivity seen for CHIKV in the twenty-first century.

#### **2.2 Chronic chikungunya arthritis**

Arthritis/arthralgia is a principal feature of CHIKF. Many patients recover within several weeks, but up to 50% develop chronic joint pain and swelling. When rheumatic disease persists for more than 12 weeks, we refer to these symptoms as

**57**

*Clinical Features and Management of Chronic Chikungunya Arthritis*

thritis, and 19.5% chronic tenosynovitis 15 months after CHIKF [23].

or joint symptoms may remit and then recur at a later time [21].

chronic chikungunya arthritis (CCA). Arthritic manifestations can last for weeks, months, or even years [21]. Rodriguez-Morales and colleagues retrospectively studied 283 patients from the 2015 epidemic in Risaralda Department, Colombia. At 26 weeks post-infection, 53.7% of the patients reported chronic musculoskeletal symptoms, including 49.5% with morning stiffness, 40.6% with joint swelling, and 16.6% with joint erythema [22]. Another large observational study from Kerala, India found that 57% of patients had chronic polyarthralgias, 22% chronic polyar-

The classical pattern of arthritis involves the small-to-medium sized joints in a peripheral and symmetric distribution [24]. This pattern can resemble rheumatoid arthritis (RA). Joint pain with/without synovitis can persist following acute CHIKF,

A prospective Mexican cohort study showed that greater severity of acute infection predicted development of chronic arthritis, as measured by the disease activity index 28 (DAS28), World Health Organization Disablement Assessment Schedule II (WHODAS-II), and serum IL-6 level [25]. Other risk factors for chronic disease include patient age >45 years and high viral load (>109

During acute CHIK infection, serum cytokines IL-1Ra, IL-1β, IL-6, IL-7, IL-8, IL-12, IL-15, and IFN-α increase, while RANTES (CCL5) decreases [27, 28]. With the transition to CCA, elevated levels of IL-6, GM-CSF, and IL-17 become predominant [28]. The IL-17 signature in particular may drive chronic joint inflammation, stimulating the upregulation of other pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α, matrix metalloproteinases, and RANK-RANKL leading to osteoclastogenesis and bone erosions [29]. Alphavirus infection of osteoblasts has been shown to perturb the RANKL-osteoprotegerin ratio, contributing to bone loss. This imbalance may also provide a mechanism for joint erosions in chronic disease [30]. CHIKV primarily infects human epithelial and endothelial cells, fibroblasts, and macrophages. Replication has not been observed in lymphocytes, monocytes, or monocyte-derived dendritic cells [31]. Viral tropism to the highly-vascularized synovial tissues of the joints may be responsible for the prominence of arthritis following acute infection. Whether CHIKV persists in synovial tissue during the chronic phase remains unclear, however, and there is ongoing debate about whether CCA arises secondary to immunological dysregulation or is due to persistent alpha-

Hoarau and colleagues demonstrated the presence of CHIKV RNA and viral proteins within perivascular synovial macrophages from one patient with CCA 18 months following acute infection [32]. This finding has not been replicated in other patients with chronic joint disease, however. Viral RNA has been isolated from knee synovial tissue of patients infected with a related alphavirus, Ross River virus [33]. In non-human primates, CHIKV can be recovered from muscle, synovial, lymphoid, and hepatic tissues following resolution of acute infection. Macrophages

In a cohort from the Réunion epidemic, 16 CCA patients were evaluated for persistence of viral infection. Synovial fluid (10 patients) and biopsied tissue (6 patients) was evaluated with reverse transcriptase polymerase chain reaction (RT-PCR) for CHIKV. All samples were negative, suggesting active viral replication

These findings were replicated in a Colombian cohort with CCA, evaluated during the 2014–2015 epidemic. In all patients, synovial fluid was aspirated from inflamed joints. CHIKV DNA was not recovered by RT-PCR, viral proteins were not detected by mass spectrometry, and viral cultures were also negative for all patients. The authors concluded that CCA is probably a post-infectious autoimmune process [36].

per ml)

*DOI: http://dx.doi.org/10.5772/intechopen.86486*

during acute infection [26].

virus infection of the synovial tissue.

have also been identified as viral reservoirs [34].

is not the cause of chronic articular disease [35].

#### *Clinical Features and Management of Chronic Chikungunya Arthritis DOI: http://dx.doi.org/10.5772/intechopen.86486*

*Current Topics in Neglected Tropical Diseases*

**2. Clinical manifestations**

**2.1 Acute chikungunya fever**

There are three known genotypes of CHIKV: Asia, East/Central/South Africa, and West Africa. The 2004–2005 pandemic that originated in Kenya and subsequently spread throughout the Indian Ocean region involved the East/Central/ South Africa genotype. The pandemic that emerged in the Americas in 2013 originally involved the Asia genotype, although more recently the East/Central/South

CHIKV infection results from transmission by the mosquito vector. Following an incubation period of 5–7 days, patients develop an acute febrile illness, chikungunya fever (CHIKF), characterized by high fevers, maculopapular rash, headaches, polyarthritis/arthralgias, myalgias, nausea, vomiting, and diarrhea [1]. Joint pain is often severe, and most often involves the metacarpal-phalangeal and interphalangeal joints of the hands, the wrists, the ankles, and the metatarsal-phalangeal joints of the feet. Less commonly involved but described joints include the shoulders, elbows, hips, knees, and inter-vertebral joints [10]. Acute CHIKF causes significant physical disability. For example, during the 2005 Comoros epidemic, an estimated 80% of affected patients were hospitalized or bed-bound due to severity of their symptoms [11]. More severely affected patients can develop neurological disease including meningoencephalitis, myelitis, radiculitis, and/or peripheral neuropathy, including reports of Guillain-Barré syndrome [12]. Rare ophthalmological manifestations reported include keratitis, episcleritis, optic neuritis, uveitis, and retinal detachment [13]. Uncommon but serious cardiac manifestations include arrhythmias, vasculopathy, myocarditis, and/or dilated cardiomyopathy [14, 15]. Patients with acute infection can have laboratory abnormalities including thrombocytopenia and

Africa lineage has been reported in the Western Hemisphere [8, 9].

leukopenia (lymphopenia is more common than neutropenia) [4].

Maternal-to-child transmission has also been reported, with up to 50% of neonates acquiring infection during childbirth if born within 5 days of maternal infection. Musculoskeletal manifestations are less prominent in newborns, with CHIKF being more notable for fevers, rash, cytopenias, hepatitis, and/or encephalitis [16]. Acute CHIKF typically resolves in 10–14 days, and has an overall reported case fatality rate of <1% based on epidemics in the Indian Ocean region and the Americas. However, the case fatality rate is higher among newborns, the elderly, and patients with underlying cardiovascular and pulmonary conditions [7, 17, 18]. Economopoulou

and colleagues studied the case fatality rate among atypical cases of CHIKF in

Réunion (defined as patients presenting with symptoms other than fevers and arthralgias); 65/610 patients from this group died for a case fatality rate of 10.6% [19]. During the widespread Indian Ocean region pandemic, a point mutation (A226V) in the E1 surface glycoprotein of CHIKV may have allowed better adaptation in *Aedes albopictus*, which had previously been a minor vector [20]. Along with low background immunity among populations residing in regions not historically affected, this may account for the rapid spread and high rates of infectivity seen for

Arthritis/arthralgia is a principal feature of CHIKF. Many patients recover within several weeks, but up to 50% develop chronic joint pain and swelling. When rheumatic disease persists for more than 12 weeks, we refer to these symptoms as

**56**

CHIKV in the twenty-first century.

**2.2 Chronic chikungunya arthritis**

chronic chikungunya arthritis (CCA). Arthritic manifestations can last for weeks, months, or even years [21]. Rodriguez-Morales and colleagues retrospectively studied 283 patients from the 2015 epidemic in Risaralda Department, Colombia. At 26 weeks post-infection, 53.7% of the patients reported chronic musculoskeletal symptoms, including 49.5% with morning stiffness, 40.6% with joint swelling, and 16.6% with joint erythema [22]. Another large observational study from Kerala, India found that 57% of patients had chronic polyarthralgias, 22% chronic polyarthritis, and 19.5% chronic tenosynovitis 15 months after CHIKF [23].

The classical pattern of arthritis involves the small-to-medium sized joints in a peripheral and symmetric distribution [24]. This pattern can resemble rheumatoid arthritis (RA). Joint pain with/without synovitis can persist following acute CHIKF, or joint symptoms may remit and then recur at a later time [21].

A prospective Mexican cohort study showed that greater severity of acute infection predicted development of chronic arthritis, as measured by the disease activity index 28 (DAS28), World Health Organization Disablement Assessment Schedule II (WHODAS-II), and serum IL-6 level [25]. Other risk factors for chronic disease include patient age >45 years and high viral load (>109 per ml) during acute infection [26].

During acute CHIK infection, serum cytokines IL-1Ra, IL-1β, IL-6, IL-7, IL-8, IL-12, IL-15, and IFN-α increase, while RANTES (CCL5) decreases [27, 28]. With the transition to CCA, elevated levels of IL-6, GM-CSF, and IL-17 become predominant [28]. The IL-17 signature in particular may drive chronic joint inflammation, stimulating the upregulation of other pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α, matrix metalloproteinases, and RANK-RANKL leading to osteoclastogenesis and bone erosions [29]. Alphavirus infection of osteoblasts has been shown to perturb the RANKL-osteoprotegerin ratio, contributing to bone loss. This imbalance may also provide a mechanism for joint erosions in chronic disease [30].

CHIKV primarily infects human epithelial and endothelial cells, fibroblasts, and macrophages. Replication has not been observed in lymphocytes, monocytes, or monocyte-derived dendritic cells [31]. Viral tropism to the highly-vascularized synovial tissues of the joints may be responsible for the prominence of arthritis following acute infection. Whether CHIKV persists in synovial tissue during the chronic phase remains unclear, however, and there is ongoing debate about whether CCA arises secondary to immunological dysregulation or is due to persistent alphavirus infection of the synovial tissue.

Hoarau and colleagues demonstrated the presence of CHIKV RNA and viral proteins within perivascular synovial macrophages from one patient with CCA 18 months following acute infection [32]. This finding has not been replicated in other patients with chronic joint disease, however. Viral RNA has been isolated from knee synovial tissue of patients infected with a related alphavirus, Ross River virus [33]. In non-human primates, CHIKV can be recovered from muscle, synovial, lymphoid, and hepatic tissues following resolution of acute infection. Macrophages have also been identified as viral reservoirs [34].

In a cohort from the Réunion epidemic, 16 CCA patients were evaluated for persistence of viral infection. Synovial fluid (10 patients) and biopsied tissue (6 patients) was evaluated with reverse transcriptase polymerase chain reaction (RT-PCR) for CHIKV. All samples were negative, suggesting active viral replication is not the cause of chronic articular disease [35].

These findings were replicated in a Colombian cohort with CCA, evaluated during the 2014–2015 epidemic. In all patients, synovial fluid was aspirated from inflamed joints. CHIKV DNA was not recovered by RT-PCR, viral proteins were not detected by mass spectrometry, and viral cultures were also negative for all patients. The authors concluded that CCA is probably a post-infectious autoimmune process [36].

Evidence for molecular mimicry between host tissues and CHIKV E1 glycoprotein has been postulated [37]. However, the specific mechanisms by which CHIK infection might lead to immunological dysregulation and autoimmunity are unknown.

### **2.3 Diagnosis of chronic chikungunya arthritis**

The diagnosis of CHIK depends on epidemiologic information, characteristic clinical features, the time course of the infection, and laboratory confirmation. Many patients live in or have had recent travel to an area with endemic transmission of *Aedes* mosquitos. Laboratory testing depends on the time course of infection. During acute disease, CHIK viremia lasts for 5–7 days. At this time, RT-PCR of serum can be diagnostic. Anti-CHIKV IgM antibodies appear at 3–8 days and remain positive for 1–3 months. Anti-CHIKV IgG antibodies are detectable at 4–10 days and remain positive for months to years [1].

CCA patients present with chronic debilitating joint symptoms ranging from morning stiffness and arthralgias to frank inflammatory synovitis. A classical pattern of small-and-medium joint peripheral involvement has been described, but mono- and oligoarthritis can also occur (**Figure 1**). In some patients, CCA presents clinically as an RA "mimic," but most patients have negative tests for rheumatoid factor and anti-cyclic citrullinated peptide antibodies [38]. Patients with CCA often meet diagnostic and/or clinical criteria for RA or spondyloarthritis [39]. The distinguishing clinical feature is a previous history of acute CHIKF, with laboratory confirmation of serum positivity for IgM and/or IgG anti-CHIK antibodies.

Radiographic imaging of involved joints may be normal, especially early in the disease, with progression to bone erosions in some patients over time. Magnetic resonance imaging has greater sensitivity for the detection of inflammatory changes, and can show synovial thickening, bone marrow edema, effusions, and/or tenosynovitis [38].

#### **Figure 1.**

*Patient with chronic chikungunya arthritis (CCA). A 50-year-old woman with CCA and synovitis of the right third PIP and left second PIP joints. She had acute CHIKF 3 years prior and subsequently developed CCA of the hand joints. Image courtesy of José Kennedy Amaral, M.D., Pernambuco, Brazil.*

**59**

*Clinical Features and Management of Chronic Chikungunya Arthritis*

Guidelines for the management of CCA emphasize symptomatic pain control with acetaminophen/paracetamol, codeine, and/or neuropathic medications such as gabapentin. Adjunctive treatment includes physical therapy, thermotherapy, and/or cryotherapy [16]. These approaches can relieve pain and improve function, but are

In an uncontrolled case series during the 2005–2006 Indian Ocean pandemic, short-term corticosteroid treatment improved arthritis and tenosynovitis, and reduced disability in patients with CCA [40]. Corticosteroid treatment led to greater pain relief and patient satisfaction compared to paracetamol, NSAIDs,

Padmakumar and colleagues performed a randomized-controlled trial evaluating the efficacy of single and combination NSAID and corticosteroid treatment regimens for CCA. Functional and pain assessments improved with corticosteroids (prednisolone 10 mg daily) in addition to the NSAID aceclofenac 200 mg daily, compared to those who received aceclofenac alone. The addition of hydroxychloro-

Despite positive results, long-term use of corticosteroids is not advised due to well-known risks of infection, cataracts, glaucoma, hyperglycemia and diabetes mellitus, and osteopenia/osteoporosis associated with chronic cortico-

An open-label pilot study of chloroquine (CQ ) treatment for CCA in South Africa showed improvement in patient and physician disease activity assessments, though this trial was not blinded [43]. Brito and colleagues recommended HCQ at a dose of 6 mg/kg daily as first-line treatment for CCA, as part of a regimen potentially escalating to triple therapy with sulfasalazine (SSZ) and methotrexate

However, a randomized-controlled trial comparing short-term treatment with CQ to placebo for acute CHIK arthritis found no differences between-group in the duration of arthralgias or viremia, but increased rates of chronic arthralgias in the treated group [45]. Chopra and colleagues performed a comparative effectiveness trial between CQ 250 mg daily and meloxicam 7.5 mg daily in patients with CCA

In the open-label randomized trial by Ravindran and Alias, combination therapy (MTX 15 mg weekly, SSZ 1000 mg daily, and HCQ 400 mg daily) was superior to HCQ monotherapy [47]. The trial did not include a placebo group, precluding the

Overall, most current evidence suggests that antimalarials such as CQ and HCQ

The Ravindran and Alias trial included SSZ 1000 mg daily in combination with HCQ and MTX as part of triple therapy, compared to HCQ [47]. While combination therapy was more efficacious, the contribution of SSZ separate from MTX could

**3.1 Corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs)**

**3. Treatment of chronic chikungunya arthritis**

*DOI: http://dx.doi.org/10.5772/intechopen.86486*

medicinal herbs, and physical exercise [41].

quine (HCQ ) did not provide added benefit [42].

**3.2 Chloroquine and hydroxychloroquine**

and found no difference in efficacy [46].

possibility of determining the efficacy of HCQ.

are not effective for the treatment of CCA.

not disease-modifying.

steroid use.

(MTX) [44].

**3.3 Sulfasalazine**

not be established.

## **3. Treatment of chronic chikungunya arthritis**

*Current Topics in Neglected Tropical Diseases*

**2.3 Diagnosis of chronic chikungunya arthritis**

4–10 days and remain positive for months to years [1].

Evidence for molecular mimicry between host tissues and CHIKV E1 glycoprotein has been postulated [37]. However, the specific mechanisms by which CHIK infection

The diagnosis of CHIK depends on epidemiologic information, characteristic clinical features, the time course of the infection, and laboratory confirmation. Many patients live in or have had recent travel to an area with endemic transmission of *Aedes* mosquitos. Laboratory testing depends on the time course of infection. During acute disease, CHIK viremia lasts for 5–7 days. At this time, RT-PCR of serum can be diagnostic. Anti-CHIKV IgM antibodies appear at 3–8 days and remain positive for 1–3 months. Anti-CHIKV IgG antibodies are detectable at

CCA patients present with chronic debilitating joint symptoms ranging from morning stiffness and arthralgias to frank inflammatory synovitis. A classical pattern of small-and-medium joint peripheral involvement has been described, but mono- and oligoarthritis can also occur (**Figure 1**). In some patients, CCA presents clinically as an RA "mimic," but most patients have negative tests for rheumatoid factor and anti-cyclic citrullinated peptide antibodies [38]. Patients with CCA often meet diagnostic and/or clinical criteria for RA or spondyloarthritis [39]. The distinguishing clinical feature is a previous history of acute CHIKF, with laboratory

confirmation of serum positivity for IgM and/or IgG anti-CHIK antibodies.

Radiographic imaging of involved joints may be normal, especially early in the disease, with progression to bone erosions in some patients over time. Magnetic resonance imaging has greater sensitivity for the detection of inflammatory

changes, and can show synovial thickening, bone marrow edema, effusions, and/or

*Patient with chronic chikungunya arthritis (CCA). A 50-year-old woman with CCA and synovitis of the right third PIP and left second PIP joints. She had acute CHIKF 3 years prior and subsequently developed CCA of* 

*the hand joints. Image courtesy of José Kennedy Amaral, M.D., Pernambuco, Brazil.*

might lead to immunological dysregulation and autoimmunity are unknown.

**58**

**Figure 1.**

tenosynovitis [38].

Guidelines for the management of CCA emphasize symptomatic pain control with acetaminophen/paracetamol, codeine, and/or neuropathic medications such as gabapentin. Adjunctive treatment includes physical therapy, thermotherapy, and/or cryotherapy [16]. These approaches can relieve pain and improve function, but are not disease-modifying.

#### **3.1 Corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs)**

In an uncontrolled case series during the 2005–2006 Indian Ocean pandemic, short-term corticosteroid treatment improved arthritis and tenosynovitis, and reduced disability in patients with CCA [40]. Corticosteroid treatment led to greater pain relief and patient satisfaction compared to paracetamol, NSAIDs, medicinal herbs, and physical exercise [41].

Padmakumar and colleagues performed a randomized-controlled trial evaluating the efficacy of single and combination NSAID and corticosteroid treatment regimens for CCA. Functional and pain assessments improved with corticosteroids (prednisolone 10 mg daily) in addition to the NSAID aceclofenac 200 mg daily, compared to those who received aceclofenac alone. The addition of hydroxychloroquine (HCQ ) did not provide added benefit [42].

Despite positive results, long-term use of corticosteroids is not advised due to well-known risks of infection, cataracts, glaucoma, hyperglycemia and diabetes mellitus, and osteopenia/osteoporosis associated with chronic corticosteroid use.

#### **3.2 Chloroquine and hydroxychloroquine**

An open-label pilot study of chloroquine (CQ ) treatment for CCA in South Africa showed improvement in patient and physician disease activity assessments, though this trial was not blinded [43]. Brito and colleagues recommended HCQ at a dose of 6 mg/kg daily as first-line treatment for CCA, as part of a regimen potentially escalating to triple therapy with sulfasalazine (SSZ) and methotrexate (MTX) [44].

However, a randomized-controlled trial comparing short-term treatment with CQ to placebo for acute CHIK arthritis found no differences between-group in the duration of arthralgias or viremia, but increased rates of chronic arthralgias in the treated group [45]. Chopra and colleagues performed a comparative effectiveness trial between CQ 250 mg daily and meloxicam 7.5 mg daily in patients with CCA and found no difference in efficacy [46].

In the open-label randomized trial by Ravindran and Alias, combination therapy (MTX 15 mg weekly, SSZ 1000 mg daily, and HCQ 400 mg daily) was superior to HCQ monotherapy [47]. The trial did not include a placebo group, precluding the possibility of determining the efficacy of HCQ.

Overall, most current evidence suggests that antimalarials such as CQ and HCQ are not effective for the treatment of CCA.

### **3.3 Sulfasalazine**

The Ravindran and Alias trial included SSZ 1000 mg daily in combination with HCQ and MTX as part of triple therapy, compared to HCQ [47]. While combination therapy was more efficacious, the contribution of SSZ separate from MTX could not be established.

Ganu and Ganu evaluated a cohort of 16 patients with persistent arthritis following acute CHIKF, comparing treatment with SSZ and MTX to SSZ alone. Improvement was noted in 71.4% of patients receiving combination therapy compared to 12.5% of patients receiving SSZ monotherapy [48]. A significant limitation of this trial was the lack of a control group; it remains questionable whether SSZ was any more effective than a placebo response. In addition, a majority of the patients had anti-cyclic citrullinated peptide antibodies, suggesting they had RA rather than CCA.

Overall, there is very limited data suggesting efficacy of SSZ monotherapy for the treatment of CCA, but it may be efficacious in combination with other medications such as MTX.

#### **3.4 Methotrexate**

Pandya treated 149 Indian patients with CCA with MTX 15–20 mg weekly in combination with HCQ. At 16 weeks, ACR20 responses were achieved in 48.9%, ACR50 in 18.8%, and ACR70 in 4.0%. Clinical response was less robust as measured by DAS28-ESR, with just 1/149 patients achieving clinical remission (DAS28-ESR <2.6) and only 4/149 with a good clinical response (DAS28-ESR <3.2) [49]. One important limitation of the study was that the diagnosis of CCA was made entirely on clinical grounds, without serological confirmation of anti-CHIKV antibodies. As such, the patient population may have been heterogeneous, including non-CHIK inflammatory arthritis syndromes.

In the trial by Ravindran and Alias, the combination therapy regimen including MTX 15 mg weekly (along with SSZ 1000 mg daily and HCQ 400 mg daily) was superior to HCQ alone (DAS28-ESR <3.2 at 24 weeks, 84% versus 14% respectively). Both groups also received prednisolone 7.5 mg daily, but this was tapered off by 6 weeks [47]. In another trial by Ganu and Ganu, patients with an inadequate treatment response to combination SSZ and HCQ were escalated to treatment with MTX 15–20 mg weekly versus placebo. The MTX group achieved a superior clinical response versus SSZ/HCQ (71.4% versus 12.5%) [48].

Javelle and colleagues reported on treatment of a Réunion cohort with CCA following the 2005-2006 epidemic. Among patients treated with MTX 7.5–25 mg weekly, 54/72 (75%) achieved a good clinical response [39].

Bouquillard and Combe treated patients with acute CHIKF who were subsequently diagnosed with RA. Among 19 patients treated with MTX, 13 had a good clinical response (68.4%). Among these patients, 54.1% were positive for rheumatoid factor, and 28.6% had anti-cyclic citrullinated peptide antibodies [50]. As such, many of the patients were diagnosed with seronegative RA, which can closely mimic the signs and symptoms of CCA, and which might respond to similar treatments.

Amaral and colleagues treated 48 patients with CCA with open-label MTX initiated at 7.5 mg weekly, with dose escalations for refractory symptoms at 4 weeks. The final mean MTX dose was 9.2 ± 3.2 mg per week. MTX therapy was combined with prednisone at a mean daily dose of 6.1 ± 2.2 mg for nine patients (18%). Two patients received HCQ (400 mg daily) with MTX, and one also received SSZ (1000 mg daily). At the first visit, the mean value for pain by visual analog scale was 7.7 ± 2.0. The mean values for pain at 4 and 8 weeks, compared to baseline, decreased to 3.0 and 2.6 respectively [24].

Overall, MTX has shown promise in the treatment of CCA, though previous trials have either combined MTX with HCQ and SSZ, or have been unblinded. Further randomized trials are needed to evaluate MTX monotherapy.

**61**

*Clinical Features and Management of Chronic Chikungunya Arthritis*

therapy with monoclonal antibodies for the treatment of CCA.

the treatment of CCA, in particular to prevent joint erosions.

decreasing T cell migration into joints might be beneficial.

patients with CCA could have a different outcome.

No human trials have yet been conducted to evaluate the efficacy of biologic

Ross River virus (RRV) is an alphavirus phylogenetically related to CHIKV. In RRV infected mice, treatment with the TNF-α inhibitor etanercept resulted in decreased weight gain, increased viral titers, and increased inflammatory cell recruitment and tissue damage [51]. This study suggests that etanercept treatment of human patients with acute CHIKF might be detrimental, though treatment of

Bouquillard and Combe treated patients with acute CHIKF followed by the diagnosis of RA (not categorized as CCA) with TNF-α inhibitors. These patients had been refractory to initial therapy with MTX. 6/6 patients had a good clinical response (four with etanercept, two with adalimumab) [50]. The majority of the patients had been diagnosed with seronegative RA, which was not distinguished

Treatment of CHIKV acutely-infected mice with the anti-CTLA-4 monoclonal antibody abatacept showed decreased T cell infiltration of joint tissues without affecting viral replication [52]. There is currently no data for its use in humans, nor

Pentosan polysulfate is a novel glycosaminoglycan-like molecule developed for the treatment of alphavirus infections. Treatment of CHIKV-infected mice with pentosan polysulfate reduced cartilage thinning and immunological infiltration of joints [53]. Intra-articular levels of the pro-inflammatory cytokines IL-6, IL-9, CCL2, and G-CSF were decreased, and levels of the anti-inflammatory IL-10 were increased through unclear mechanisms [30]. While developed for the treatment of acute CHIK infection, it remains unclear if pentosan polysulfate could be used for

Fingolimod is a sphingosine 1-phosphate receptor agonist developed for the treatment of multiple sclerosis. In CHIKV-infected mice, fingolimod treatment decreased the migration of CD4+ T cells into joints without affecting viral replication [54]. While the utility of fingolimod for treatment of CCA remains unknown,

CCA is an emerging chronic and disabling rheumatological syndrome which can persist for weeks, months, or years after acute CHIKF. With the global spread of CHIKV in past decades, increasing numbers of patients from developing countries in particular have acquired or are at risk for this chronic disabling rheumatic

The pathophysiology of the disease remains uncertain, though the weight of evidence suggests that the syndrome is caused by a post-viral autoimmune process,

There is limited clinical trial evidence for the use of disease modifying therapeutics for patients with CCA. Most previous trials have been open-label or of limited quality. Empiric treatment courses with corticosteroids, NSAIDs, antimalarials, and SSZ can be considered. However, given similarities between CCA and RA, it is our

*DOI: http://dx.doi.org/10.5772/intechopen.86486*

**3.5 Biologics**

from CCA.

for its use in treating CCA.

**3.6 Novel agents**

**4. Conclusions**

syndrome.

which follows viral clearance.

*Clinical Features and Management of Chronic Chikungunya Arthritis DOI: http://dx.doi.org/10.5772/intechopen.86486*

#### **3.5 Biologics**

*Current Topics in Neglected Tropical Diseases*

rather than CCA.

tions such as MTX.

**3.4 Methotrexate**

inflammatory arthritis syndromes.

response versus SSZ/HCQ (71.4% versus 12.5%) [48].

decreased to 3.0 and 2.6 respectively [24].

weekly, 54/72 (75%) achieved a good clinical response [39].

randomized trials are needed to evaluate MTX monotherapy.

Ganu and Ganu evaluated a cohort of 16 patients with persistent arthritis following acute CHIKF, comparing treatment with SSZ and MTX to SSZ alone. Improvement was noted in 71.4% of patients receiving combination therapy compared to 12.5% of patients receiving SSZ monotherapy [48]. A significant limitation of this trial was the lack of a control group; it remains questionable whether SSZ was any more effective than a placebo response. In addition, a majority of the patients had anti-cyclic citrullinated peptide antibodies, suggesting they had RA

Overall, there is very limited data suggesting efficacy of SSZ monotherapy for the treatment of CCA, but it may be efficacious in combination with other medica-

Pandya treated 149 Indian patients with CCA with MTX 15–20 mg weekly in combination with HCQ. At 16 weeks, ACR20 responses were achieved in 48.9%, ACR50 in 18.8%, and ACR70 in 4.0%. Clinical response was less robust as measured by DAS28-ESR, with just 1/149 patients achieving clinical remission (DAS28-ESR <2.6) and only 4/149 with a good clinical response (DAS28-ESR <3.2) [49]. One important limitation of the study was that the diagnosis of CCA was made entirely on clinical grounds, without serological confirmation of anti-CHIKV antibodies. As such, the patient population may have been heterogeneous, including non-CHIK

In the trial by Ravindran and Alias, the combination therapy regimen including MTX 15 mg weekly (along with SSZ 1000 mg daily and HCQ 400 mg daily) was superior to HCQ alone (DAS28-ESR <3.2 at 24 weeks, 84% versus 14% respectively). Both groups also received prednisolone 7.5 mg daily, but this was tapered off by 6 weeks [47]. In another trial by Ganu and Ganu, patients with an inadequate treatment response to combination SSZ and HCQ were escalated to treatment with MTX 15–20 mg weekly versus placebo. The MTX group achieved a superior clinical

Javelle and colleagues reported on treatment of a Réunion cohort with CCA following the 2005-2006 epidemic. Among patients treated with MTX 7.5–25 mg

Bouquillard and Combe treated patients with acute CHIKF who were subsequently diagnosed with RA. Among 19 patients treated with MTX, 13 had a good clinical response (68.4%). Among these patients, 54.1% were positive for rheumatoid factor, and 28.6% had anti-cyclic citrullinated peptide antibodies [50]. As such, many of the patients were diagnosed with seronegative RA, which can closely mimic the signs and symptoms of CCA, and which might respond to similar

Amaral and colleagues treated 48 patients with CCA with open-label MTX initiated at 7.5 mg weekly, with dose escalations for refractory symptoms at 4 weeks. The final mean MTX dose was 9.2 ± 3.2 mg per week. MTX therapy was combined with prednisone at a mean daily dose of 6.1 ± 2.2 mg for nine patients (18%). Two patients received HCQ (400 mg daily) with MTX, and one also received SSZ (1000 mg daily). At the first visit, the mean value for pain by visual analog scale was 7.7 ± 2.0. The mean values for pain at 4 and 8 weeks, compared to baseline,

Overall, MTX has shown promise in the treatment of CCA, though previous trials have either combined MTX with HCQ and SSZ, or have been unblinded. Further

**60**

treatments.

No human trials have yet been conducted to evaluate the efficacy of biologic therapy with monoclonal antibodies for the treatment of CCA.

Ross River virus (RRV) is an alphavirus phylogenetically related to CHIKV. In RRV infected mice, treatment with the TNF-α inhibitor etanercept resulted in decreased weight gain, increased viral titers, and increased inflammatory cell recruitment and tissue damage [51]. This study suggests that etanercept treatment of human patients with acute CHIKF might be detrimental, though treatment of patients with CCA could have a different outcome.

Bouquillard and Combe treated patients with acute CHIKF followed by the diagnosis of RA (not categorized as CCA) with TNF-α inhibitors. These patients had been refractory to initial therapy with MTX. 6/6 patients had a good clinical response (four with etanercept, two with adalimumab) [50]. The majority of the patients had been diagnosed with seronegative RA, which was not distinguished from CCA.

Treatment of CHIKV acutely-infected mice with the anti-CTLA-4 monoclonal antibody abatacept showed decreased T cell infiltration of joint tissues without affecting viral replication [52]. There is currently no data for its use in humans, nor for its use in treating CCA.

#### **3.6 Novel agents**

Pentosan polysulfate is a novel glycosaminoglycan-like molecule developed for the treatment of alphavirus infections. Treatment of CHIKV-infected mice with pentosan polysulfate reduced cartilage thinning and immunological infiltration of joints [53]. Intra-articular levels of the pro-inflammatory cytokines IL-6, IL-9, CCL2, and G-CSF were decreased, and levels of the anti-inflammatory IL-10 were increased through unclear mechanisms [30]. While developed for the treatment of acute CHIK infection, it remains unclear if pentosan polysulfate could be used for the treatment of CCA, in particular to prevent joint erosions.

Fingolimod is a sphingosine 1-phosphate receptor agonist developed for the treatment of multiple sclerosis. In CHIKV-infected mice, fingolimod treatment decreased the migration of CD4+ T cells into joints without affecting viral replication [54]. While the utility of fingolimod for treatment of CCA remains unknown, decreasing T cell migration into joints might be beneficial.

#### **4. Conclusions**

CCA is an emerging chronic and disabling rheumatological syndrome which can persist for weeks, months, or years after acute CHIKF. With the global spread of CHIKV in past decades, increasing numbers of patients from developing countries in particular have acquired or are at risk for this chronic disabling rheumatic syndrome.

The pathophysiology of the disease remains uncertain, though the weight of evidence suggests that the syndrome is caused by a post-viral autoimmune process, which follows viral clearance.

There is limited clinical trial evidence for the use of disease modifying therapeutics for patients with CCA. Most previous trials have been open-label or of limited quality. Empiric treatment courses with corticosteroids, NSAIDs, antimalarials, and SSZ can be considered. However, given similarities between CCA and RA, it is our

opinion that management with MTX should be further evaluated. Over time, new treatments including biologics and novel agents (pentosan polysulfate, fingolimod) may also emerge as treatment options.

## **Conflicts of interest**

None of the authors have any conflicts of interest to report.

## **Funding source**

None.

## **Author details**

Joshua Britton Bilsborrow1 , José Kennedy Amaral<sup>2</sup> and Robert T. Schoen1 \*

1 Section of Rheumatology, Allergy and Immunology, Yale University School of Medicine, New Haven, Connecticut, United States of America

2 Department of Infectious Diseases and Tropical Medicine, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

\*Address all correspondence to: robert.schoen@yale.edu

© 2019 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.

**63**

*Clinical Features and Management of Chronic Chikungunya Arthritis*

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[18] Freitas ARR et al. Excess mortality related to chikungunya epidemics in the context of co-circulation of

[13] Mahendradas P, Avadhani K, Shetty R. Chikungunya and the eye: A review. Journal of Ophthalmic Inflammation

and pathogenesis. The Open

2007;**76**(6):1189-1193

*DOI: http://dx.doi.org/10.5772/intechopen.86486*

[3] Silva LA, Dermody TS. Chikungunya virus: Epidemiology, replication, disease mechanisms, and prospective

[4] Staples JE, Breiman RF, Powers AM. Chikungunya fever: An epidemiological review of a re-emerging infectious disease. Clinical Infectious Diseases.

[5] Rezza G. Chikungunya is back in Italy: 2007-2017. Journal of Travel Medicine. 2018;**25**(1). DOI: 10.1093/

[6] Chikungunya virus in the United States. 2018. Available from: https:// www.cdc.gov/chikungunya/geo/ united-states.html [Accessed: 24

[7] Yactayo S et al. Epidemiology of chikungunya in the Americas. The Journal of Infectious Diseases. 2016;**214**(suppl 5):S441-S445

[8] Zeller H, Van Bortel W, Sudre B. Chikungunya: Its history in Africa and Asia and its spread to new regions in 2013-2014. The Journal of Infectious Diseases. 2016;**214**(suppl 5):S436-S440

[9] Charlys da Costa A et al. Spread of chikungunya virus east/central/ south African genotype in Northeast Brazil. Emerging Infectious Diseases.

2017;**23**(10):1742-1744

[1] Suhrbier A, Jaffar-Bandjee MC, Gasque P. Arthritogenic alphaviruses:

An overview. Nature Reviews Rheumatology. 2012;**8**(7):420-429

[2] Ganesan VK, Duan B, Reid SP. Chikungunya virus: Pathophysiology, mechanism, and modeling. Viruses. 2017;**9**(12):368

intervention strategies. The Journal of Clinical Investigation.

2017;**127**(3):737-749

2009;**49**(6):942-948

jtm/tay004

February 2019]

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*Clinical Features and Management of Chronic Chikungunya Arthritis DOI: http://dx.doi.org/10.5772/intechopen.86486*

## **References**

*Current Topics in Neglected Tropical Diseases*

may also emerge as treatment options.

**Conflicts of interest**

**Funding source**

**Author details**

Joshua Britton Bilsborrow1

None.

opinion that management with MTX should be further evaluated. Over time, new treatments including biologics and novel agents (pentosan polysulfate, fingolimod)

None of the authors have any conflicts of interest to report.

, José Kennedy Amaral<sup>2</sup>

Medicine, New Haven, Connecticut, United States of America

Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

provided the original work is properly cited.

\*Address all correspondence to: robert.schoen@yale.edu

1 Section of Rheumatology, Allergy and Immunology, Yale University School of

2 Department of Infectious Diseases and Tropical Medicine, Federal University of

© 2019 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,

and Robert T. Schoen1

\*

**62**

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[3] Silva LA, Dermody TS. Chikungunya virus: Epidemiology, replication, disease mechanisms, and prospective intervention strategies. The Journal of Clinical Investigation. 2017;**127**(3):737-749

[4] Staples JE, Breiman RF, Powers AM. Chikungunya fever: An epidemiological review of a re-emerging infectious disease. Clinical Infectious Diseases. 2009;**49**(6):942-948

[5] Rezza G. Chikungunya is back in Italy: 2007-2017. Journal of Travel Medicine. 2018;**25**(1). DOI: 10.1093/ jtm/tay004

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[9] Charlys da Costa A et al. Spread of chikungunya virus east/central/ south African genotype in Northeast Brazil. Emerging Infectious Diseases. 2017;**23**(10):1742-1744

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[29] Miossec P, Korn T, Kuchroo VK. Interleukin-17 and type 17 helper T cells. The New England Journal of Medicine. 2009;**361**(9):888-898

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[33] Soden M et al. Detection of viral ribonucleic acid and histologic analysis of inflamed synovium in Ross River virus infection. Arthritis and Rheumatism. 2000;**43**(2):365-369

[34] Labadie K et al. Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages. The Journal of Clinical Investigation. 2010;**120**(3):894-906

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*Clinical Features and Management of Chronic Chikungunya Arthritis*

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[43] Brighton SW. Chloroquine phosphate treatment of chronic chikungunya arthritis. An open pilot study. South African Medical Journal.

[44] Brito CA et al. Pharmacologic management of pain in patients with chikungunya: A guideline. Revista da Sociedade Brasileira de Medicina Tropical. 2016;**49**(6):668-679

[45] De Lamballerie X et al. On chikungunya acute infection and chloroquine treatment. Vector Borne and Zoonotic Diseases.

[46] Chopra A, Saluja M, Venugopalan A. Effectiveness of chloroquine and inflammatory cytokine response in patients with early persistent musculoskeletal pain and arthritis following chikungunya virus infection. Arthritis & Rhematology.

[47] Ravindran V, Alias G. Efficacy

2009;**4**(3):94-101

1984;**66**(6):217-218

2008;**8**(6):837-839

2014;**66**(2):319-326

of combination DMARD therapy vs. hydroxychloroquine monotherapy in chronic persistent chikungunya arthritis: A 24-week randomized controlled open label study. Clinical Rheumatology.

2017;**36**(6):1335-1340

2011;**59**:83-86

[48] Ganu MA, Ganu AS. Postchikungunya chronic arthritis—Our experience with DMARDs over two year follow up. The Journal of the Association of Physicians of India.

[49] Pandya s. Methotrexate and hydroxychloroquine combination therapy in chronic chikungunya

*DOI: http://dx.doi.org/10.5772/intechopen.86486*

chikungunya virus infection: A study of 307 patients with 32-month follow-up (RHUMATOCHIK study). Joint, Bone,

[35] Bouquillard E et al. Rheumatic manifestations associated with

[36] Chang AY et al. Chikungunya arthritis mechanisms in the Americas: A cross-sectional analysis of chikungunya arthritis patients twenty-two months after infection demonstrating no detectable viral persistence in synovial fluid. Arthritis & Rhematology.

[37] Reddy V et al. Molecular mimicry between chikungunya virus and host components: A possible mechanism for the arthritic manifestations. PLoS Neglected Tropical Diseases.

[38] Manimunda SP et al. Clinical progression of chikungunya fever during acute and chronic arthritic stages and the changes in joint morphology as revealed by imaging. Transactions of the Royal Society of Tropical Medicine and Hygiene.

[39] Javelle E et al. Specific management

of post-chikungunya rheumatic disorders: A retrospective study of 159 cases in Reunion Island from 2006-2012. PLoS Neglected Tropical Diseases.

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[41] Sissoko D et al. Post-epidemic chikungunya disease on Reunion Island: Course of rheumatic manifestations and associated factors over a 15-month period. PLoS Neglected Tropical

Spine. 2018;**85**(2):207-210

2018;**70**(4):585-593

2017;**11**(1):e0005238

2010;**104**(6):392-399

2015;**9**(3):e0003603

2007;**86**(3):123-137

Diseases. 2009;**3**(3):e389

*Clinical Features and Management of Chronic Chikungunya Arthritis DOI: http://dx.doi.org/10.5772/intechopen.86486*

[35] Bouquillard E et al. Rheumatic manifestations associated with chikungunya virus infection: A study of 307 patients with 32-month follow-up (RHUMATOCHIK study). Joint, Bone, Spine. 2018;**85**(2):207-210

*Current Topics in Neglected Tropical Diseases*

[26] Zaid A et al. Chikungunya arthritis: Implications of acute and chronic inflammation mechanisms on disease management. Arthritis & Rhematology.

[27] Ng KW et al. Clinical features and epidemiology of chikungunya infection in Singapore. Singapore Medical Journal. 2009;**50**(8):785-790

[28] Chow A et al. Persistent arthralgia induced by chikungunya virus infection is associated with interleukin-6 and granulocyte macrophage colonystimulating factor. The Journal of Infectious Diseases. 2011;**203**(2):149-157

[29] Miossec P, Korn T, Kuchroo VK. Interleukin-17 and type 17 helper T cells. The New England Journal of Medicine.

[30] Chen W et al. Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss. Proceedings of the National Academy of Sciences of the United States of America.

[31] Sourisseau M et al. Characterization of reemerging chikungunya virus. PLoS

[32] Hoarau JJ et al. Persistent chronic inflammation and infection by chikungunya arthritogenic alphavirus in spite of a robust host immune response. Journal of Immunology.

2009;**361**(9):888-898

2014;**111**(16):6040-6045

Pathogens. 2007;**3**(6):e89

2010;**184**(10):5914-5927

[33] Soden M et al. Detection of viral ribonucleic acid and histologic analysis of inflamed synovium in Ross River virus infection. Arthritis and Rheumatism. 2000;**43**(2):365-369

[34] Labadie K et al. Chikungunya disease in nonhuman primates

involves long-term viral persistence in macrophages. The Journal of Clinical Investigation. 2010;**120**(3):894-906

2018;**70**(4):484-495

[19] Economopoulou A et al. Atypical chikungunya virus infections: Clinical manifestations, mortality and risk factors for severe disease during the 2005-2006 outbreak on Reunion. Epidemiology and Infection.

[20] Schuffenecker I et al. Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak. PLoS Medicine. 2006;**3**(7):e263

[21] Schilte C et al. Chikungunya virus-associated long-term arthralgia: A 36-month prospective longitudinal study. PLoS Neglected Tropical Diseases. 2013;**7**(3):e2137

[22] Rodriguez-Morales AJ et al. Postchikungunya chronic inflammatory

retrospective follow-up study of 283 adult and child cases in La Virginia, Risaralda, Colombia. F1000Research.

[23] Mathew AJ et al. Rheumaticmusculoskeletal pain and disorders in a naive group of individuals 15 months following a chikungunya viral epidemic in South India: A population based observational study. International Journal of Clinical Practice. 2011;**65**(12):1306-1312

[24] Amaral JK, Bingham CO 3rd, Schoen RT. Successful methotrexate treatment of chronic chikungunya arthritis. Journal of Clinical Rheumatology. 2018. DOI: 10.1097/

RHU.0000000000000943

2017;**36**(3):695-699

[25] Sepulveda-Delgado J et al. Inflammatory biomarkers, disease activity index, and self-reported disability may be predictors of chronic arthritis after chikungunya infection: Brief report. Clinical Rheumatology.

rheumatism: Results from a

2016;**5**:360

2009;**137**(4):534-541

other arboviruses in Brazil. PLoS Currents. 2017;**9**:ecurrents.outbreaks. 14608e586cd321d8d5088652d7a0d884

**64**

[36] Chang AY et al. Chikungunya arthritis mechanisms in the Americas: A cross-sectional analysis of chikungunya arthritis patients twenty-two months after infection demonstrating no detectable viral persistence in synovial fluid. Arthritis & Rhematology. 2018;**70**(4):585-593

[37] Reddy V et al. Molecular mimicry between chikungunya virus and host components: A possible mechanism for the arthritic manifestations. PLoS Neglected Tropical Diseases. 2017;**11**(1):e0005238

[38] Manimunda SP et al. Clinical progression of chikungunya fever during acute and chronic arthritic stages and the changes in joint morphology as revealed by imaging. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2010;**104**(6):392-399

[39] Javelle E et al. Specific management of post-chikungunya rheumatic disorders: A retrospective study of 159 cases in Reunion Island from 2006-2012. PLoS Neglected Tropical Diseases. 2015;**9**(3):e0003603

[40] Simon F et al. Chikungunya infection: An emerging rheumatism among travelers returned from Indian Ocean islands. Report of 47 cases. Medicine (Baltimore). 2007;**86**(3):123-137

[41] Sissoko D et al. Post-epidemic chikungunya disease on Reunion Island: Course of rheumatic manifestations and associated factors over a 15-month period. PLoS Neglected Tropical Diseases. 2009;**3**(3):e389

[42] Padmakumar B et al. Comparative evaluation of four therapeutic regimes in chikungunya arthritis: A prospective randomized parallel-group study. Indian Journal of Rheumatology. 2009;**4**(3):94-101

[43] Brighton SW. Chloroquine phosphate treatment of chronic chikungunya arthritis. An open pilot study. South African Medical Journal. 1984;**66**(6):217-218

[44] Brito CA et al. Pharmacologic management of pain in patients with chikungunya: A guideline. Revista da Sociedade Brasileira de Medicina Tropical. 2016;**49**(6):668-679

[45] De Lamballerie X et al. On chikungunya acute infection and chloroquine treatment. Vector Borne and Zoonotic Diseases. 2008;**8**(6):837-839

[46] Chopra A, Saluja M, Venugopalan A. Effectiveness of chloroquine and inflammatory cytokine response in patients with early persistent musculoskeletal pain and arthritis following chikungunya virus infection. Arthritis & Rhematology. 2014;**66**(2):319-326

[47] Ravindran V, Alias G. Efficacy of combination DMARD therapy vs. hydroxychloroquine monotherapy in chronic persistent chikungunya arthritis: A 24-week randomized controlled open label study. Clinical Rheumatology. 2017;**36**(6):1335-1340

[48] Ganu MA, Ganu AS. Postchikungunya chronic arthritis—Our experience with DMARDs over two year follow up. The Journal of the Association of Physicians of India. 2011;**59**:83-86

[49] Pandya s. Methotrexate and hydroxychloroquine combination therapy in chronic chikungunya

arthritis: A 16 week study. Indian Journal of Rheumatology. 2008;**3**(3):93-97

[50] Bouquillard E, Combe B. A report of 21 cases of rheumatoid arthritis following chikungunya fever. A mean follow-up of two years. Joint, Bone, Spine. 2009;**76**(6):654-657

[51] Zaid A et al. Disease exacerbation by etanercept in a mouse model of alphaviral arthritis and myositis. Arthritis and Rheumatism. 2011;**63**(2):488-491

[52] Miner JJ et al. Therapy with CTLA4-Ig and an antiviral monoclonal antibody controls chikungunya virus arthritis. Science Translational Medicine. 2017;**9**(375):eaah3438

[53] Herrero LJ et al. Pentosan polysulfate: A novel glycosaminoglycanlike molecule for effective treatment of alphavirus-induced cartilage destruction and inflammatory disease. Journal of Virology. 2015;**89**(15):8063-8076

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**67**

Section 2

Zoonotic Diseases
