|Year : 2017 | Volume
| Issue : 1 | Page : 35-43
Seroprevalence and clinical manifestations of Chikungunya virus infection in rural areas of Chandrapur, Maharashtra, India
Anuja P Kawle1, Amit R Nayak1, Shradha S Bhullar1, Smita R Borkar1, Sanjeev D Patankar2, Hatim F Daginawala1, Lokendra R Singh1, Rajpal S Kashyap1
1 Biochemistry Research Centre, Central India Institute of Medical Sciences, Nagpur, Maharashtra, India
2 Guru Nanak College of Science, Chandrapur, Maharashtra, India
|Date of Submission||07-Jul-2016|
|Date of Acceptance||25-Nov-2016|
|Date of Web Publication||9-Aug-2017|
Rajpal S Kashyap
Biochemistry Research Laboratory, Central India Institute of Medical Sciences, 88/2, Bajaj Nagar, Nagpur–440 010, Maharashtra
Source of Support: None, Conflict of Interest: None
Background & objectives: Chikungunya virus (CHIKV) infection has recently witnessed re-emergence, affecting rural areas of India with high morbidity rates. This prospective study was conducted to evaluate seroprevalence and clinical manifestation in targeted villages reporting cases of CHIKV infection.
Methods: A total of482 patients were recruited from Kalmana and Kothari villages of Ballarpur; Chandrapur district of Maharashtra state, India during CHIKV outbreaks in 2011–12. The serum samples from infected CHIKV patients were simultaneously screened through ELISA for detection of antigen and antibodies (IgM and IgG). Chi-square analysis was used to evaluate differences in seropositivity between age, gender and clinical manifestations of CHIKV.
Results: Out of 482 enrolled participants, 197 (41%) males and 285 (59%) females were aged between 5 and 92 yr. The clinical manifestations such as small joint pain (80%), neck stiffness (75%), fever (49%) and large joint pain (47%) were observed amongst CHIKV infected subjects. Mucocutaneous rashes (91%) on knees (71%), feet (56%), fingers and palms (54%) were also observed. Overall, seroprevalence of CHIKV infection was found to be 46% in infected participants during the epidemic period. Among risk factors, ageing and female gender was strongly associated with a raised seroprevalence of CHIKV infection along with symptoms such as rashes, small joints pain and neck stiffness.
Interpretation & conclusion: This study reported high seroprevalence rates of CHIKV infection in targeted populations, suggesting its re-emergence in rural India. Proper surveillance is, therefore, necessary to minimize re-emergence and in controlling these impending and sporadic outbreaks.
Keywords: Chikungunya; ELISA; epidemics; Maharashtra; peptide; seroprevalence
|How to cite this article:|
Kawle AP, Nayak AR, Bhullar SS, Borkar SR, Patankar SD, Daginawala HF, Singh LR, Kashyap RS. Seroprevalence and clinical manifestations of Chikungunya virus infection in rural areas of Chandrapur, Maharashtra, India. J Vector Borne Dis 2017;54:35-43
|How to cite this URL:|
Kawle AP, Nayak AR, Bhullar SS, Borkar SR, Patankar SD, Daginawala HF, Singh LR, Kashyap RS. Seroprevalence and clinical manifestations of Chikungunya virus infection in rural areas of Chandrapur, Maharashtra, India. J Vector Borne Dis [serial online] 2017 [cited 2022 Jan 26];54:35-43. Available from: https://www.jvbd.org/text.asp?2017/54/1/35/203167
| Introduction|| |
The re-emergence of chikungunya virus (CHIKV) infection in India during 2006 was life-threatening, which created an epidemic like situation affecting almost 210 districts in 13 states. The most affected states with crippling consequences of the disease were Andhra Pradesh, Karnataka, Maharashtra, Madhya Pradesh, Tamil Nadu, Gujarat and Kerala. Interestingly, these frequent epidemics and outbreaks were observed mostly in the metropolitan, urban and periurban areas probably due to urbanization and larger populations which bolstered the breeding pockets for mosquitoes.
CHIKV infection is generally described as a self limiting febrile illness with sudden onset, usually accompanied by headache, myalgia, rash, and characteristic arthralgia,,. An association of CHIKV infection with neurological complications has also been reported, but till date remains poorly documented,.
The present study was carried out in villages of Kalmana and Kothari situated in Ballarpur Taluka of Chandrapur district, Maharashtra which were hit by CHIKV epidemic in 2011–12,. The possible reason behind CHIKV epidemic was poor socioeconomic status of the population, illiteracy, unawareness about the disease and unhygienic living practices. In addition to that, the earning populations of these villages are mostly farmers working in damp and shady agricultural fields having water potholes, dried husks, plants and weeds providing an ideal habitat for mosquito breeding.
The probable diagnosis of CHIKV can be made on the basis of clinical manifestations, where as confirmatory diagnosis of chikungunya can be made only by laboratory tests. Currently, used tests include RT-PCR for confirming the presence of CHIKV, while sensitive IgM antibody ELISA is used to distinguish the disease from dengue virus (DENV). There is a paucity on available data about chikungunya prevalence from the northwest side of Maharashtra state;hence, the present study was undertaken to understand the prevalence of CHIKV infection through serodiagnosis among patients presenting with acute febrile illness using inhouse developed ELISA kits. The aim of the present study was, therefore, to evaluate the seroprevalence and clinical manifestations of CHIKV infection in the targeted rural areas of Ballarpur Taluka for early detection of CHIKV infection through multi-diagnostic approach in order to reduce its widespread transmission.
| Material & Methods|| |
The study was approved by the Institutional Ethics Committee of Central India Institute of Medical Sciences (CIIMS), Nagpur, Maharashtra, India and is in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Written consent was obtained from each participant after a detailed oral explanation about the study. Since, the study was also conducted on children, a written informed consent was obtained from the immediate caretaker, or next of kin, prior to inclusion, on behalf of children participating in the study.
This was a prospective study carried out in the villages of Kalmana (19°49’ 13.386’N, 79°25’ 59.214Έ) and Kothari (19°47’ 27.6972’N, 79°29’ 44.5092Έ) located in the Taluka of Ballarpur; district Chandrapur, Maharashtra, India. The District of Chandrapur has a large number of coal mines, and cements industries and houses one of the largest paper manufacturing industries in the country. It was badly affected by CHIKV epidemic in the state during 2011-12,. The two neighbouring villages Kalmana and Kothari, where the outbreak occurred are situated at a distance of 6-8 km from each other. The entire area of the district falls in the Godavari basin where the altitude is 189 m above sea level. The study area of Chandrapur experiences hot and dry climate with temperatures ranging from 34 to 48°C, while monsoons mark the rainy season, lasting from June to September with an average annual rainfall of 1249.4 mm. Due to scorching hot and dry climate during summers and demographically vast, uncontrolled and unplanned industrialization and urbanization the city faces a scarcity of water. In addition to that, the villages lack proper sanitation, drainage systems and garbage disposal facilities due to which they are surrounded by water potholes and littered solid wastes in and around the streets.
Initially, a preliminary study was carried out in both the villages to estimate the sample size of the population to be surveyed. Door-to-door interviews were conducted, by visiting all houses of villages using a structured questionnaire prepared in the native language of villages. All participants in the study were included using predefined inclusion criteria. The structured questionnaire was prepared with the help of expert physicians and researchers to gather information about an individual (age and gender) and the symptoms experienced during the epidemic period. The patients with symptoms of CHIKV infection such as fever, small and large joints pain with or without swelling, neck stiffness, back pain, vomiting, headache, without haemorrhagic rash, etc. were included in this study. Any patients presenting pain clearly related to other etiologies such as rheumatologic pain, muscular pain, migraines, diabetes mellitus, psychiatric illness, entrapment neurology, etc. were excluded from the study. Participants who were <5 yr of age, pregnant females, HIV positive patients, who had a past history of dengue and patients whose blood was haemolyzed were excluded from the study. A total of 482 participants [Kalmana (n=243) and Kothari (n=239)] were finally enrolled in the study [Figure 1].
|Figure 1: Flow diagram of the study representing the inclusion/ exclusion criteria adopted for recruitment of the study participants. Out of 1200 selected participants, 482 were selected for the study.|
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Sample collection, storage, transportation and processing
For estimation of the panel of the CHIKV Ag and antibodies (IgM and IgG), 3 ml venous blood was collected from CHIKV infected patients in sterile vacutainer tubes. The blood samples were centrifuged for 5 min at 5000 rpm and serum was separated. All samples were stored at −80°C until experimental use. About, 50 age-gender matched subjects who had no clinical features of CHIKV and negative by both Ag and antibody tests were considered as a control group.
CHIKV infection was confirmed by a panel of inhouse developed ELISA tests based on Ag detection using nine antipeptide antibodies as well as IgM and IgG antibodies detection using nine synthetic peptides. Participants were categorized as seropositive or negative for CHIKV based on positivity/negativity for either or both tests. Patients were given supportive care as per the clinical symptoms along with the course of methylpred-nisolone 1 g daily for 3–5 days.
Estimation of IgM and IgG antibodies using inhouse developed peptide based ELISA
IgM and IgG were detected using inhouse developed peptide based ELISA protocol described elsewhere by Morey et al.
Estimation of CHIKV antigen using antipeptide antibodies based ELISA
Diluted 100 μl serum samples (1:200) from CHIKV patients and control subjects were added to the microti-tre wells, and incubated for 60 mins at 37°C. Plates were then blocked with 0.5% bovine serum albumin (BSA) in phosphate buffered saline-tween 20 (PBS-T) for 60 min. After blocking, nine antipeptide antibodies were added (1:10000) to the plates and incubated for 60 min. The wells were washed and goat-antihuman IgG-Horse Radish Peroxidase (HRP) antibody (1:10000) was added. After 45 min. of incubation, 100 μl of TMB/ H2O2 substrate solution was added and incubated for 10 min. This reaction was stopped by adding 100 μl of 2.5 N H2SO4 in each well. The absorbance of colour developed in each well was read at 450 nm (Readwell touch ELISA plate reader, Robonik India).
The sample size was calculated using Raosoft software (Raosoft, Inc.). Data analysis was performed using MedCalc statistical software (version 10.1.2.0). Differences in seropositivity between gender/age groups, and clinical manifestations were calculated by chi-square tests and p-value of <0.05 was considered statistically significant. Bar and line graphs were plotted using Graph Pad Prism software (version 6).
| Results|| |
The sample size of 482 subjects was calculated to obtain 7.8% absolute precision (with 95% confidence interval level) based on an estimated attack rate of 10% CHIKV infection. This sample size was distributed across the study areas in proportion to the population size. Out of 482 participants, 197 (41%) males and 285 (59%) females were aged between 5 and 92 yr. The typical CHIKV clinical manifestations such as fever (49%), arthralgia [small joints (80%) and large joints (47%)], neck stiffness (75%), vomiting (23%) and back pain (23%) were found to be present in the enrolled patients. Patients also exhibited maculopapular rashes (91%) all over the body, especially on the knees (71%), feet (56%), fingers and palms (54%), and shoulders (31%) [Figure 2].
|Figure 2: Depicts typical signs and symptoms of chikungunya infection marked all over the body.|
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Characteristics and seropositivity of CHIKV Ag in the study subjects are depicted in [Table 1]. Out of the 482 participants, CHIKV Ag was detected in 83 (17.2%) cases. The seropositivity of CHIKV Ag was found to increase with age group where 21% (39/190) of older participants aged >50 yr recorded higher positivity for CHIKV Ag as compared to other groups. Similarly, no significant difference was observed in seroprevalence among both the sexes, with males accounting slightly higher positive rates [18.2% (36/197)] as compared to females [16.5% (47/287)]. Among the clinical features recorded after the epidemic, fever[26% (61/239)] (p= 0.0001) and largejoint pain [13% (29/226)] (p<0.0228) were most frequently observed while other manifestations included small joints pain [17.7% (68/384)], back pain [22% (17/79)], vomiting [17% (19/113)] and rashes [20% (60/307)].
|Table 1. Characteristics and CHIKV Ag seropositivity of 482 study participants|
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The characteristics and seropositivity of CHIKV IgM antibody in the study population are listed in [Table 2]. Out of 482 study participants, 131 (27%) cases showed sero-positivity for CHIKV IgM antibody. Chi-square analysis revealed that CHIKV IgM seroprevalence was significantly (p = 0.0094) associated with increasing age groups. Adults between the age group of 19 and 49 yr showed highest IgM seroprevalence of 34% (85/251). There was no significant difference between the genders. The clinical manifestations such as back pain (42%), large joint pain (40%), rashes (23%) and fever (19%) were found to be significantly associated (p<0.05) with IgM seroposi-tivity. Similarly, IgM seropositivity was also observed in 28% of the study population with complaints of vomiting and small joint pain [Table 2].
|Table 2. Characteristics and CHIKV IgM seropositivity of 482 study participants|
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The characteristics and seroprevalence of CHIKV IgG antibody amongst the study population are shown in [Table 3]. The seroprevalence of CHIKV IgG antibody in targeted population was found to be 15%, affecting 73 cases. Similar significant trend as observed in IgM (p = 0.0094) was also observed in IgG (p = 0.0080) se-ropositivity amongst 19-49 yr of age group. The clinical symptoms such as back pain (25%), large joint pain (19%), and vomiting (8%) were significantly observed.
|Table 3. Characteristics and CHIKV IgG seropositivity of 482 study participants|
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[Figure 3] represents the percent positivity through the combination of ELISA based Ag and antibody diagnostic tests for the detection of CHIKV infection during the epidemic of 2011-12. Total seroprevalence of CHIKV infection was found to be 46%, whereas individual assays showed seroprevalence of 27% for IgM followed by 17.2% for Ag and 15% by IgG assays.
|Figure 3: Diagrammatic representation of percentage positivity (%) by combination of diagnostic tests in chikungunya virus infected samples (n=482). The bar graph represents a combinatorial approach of tests (individual and total) for the detection of chikungunya virus infection in serum samples by Ag and IgM and IgG antibodies using antipeptide antibodies and synthetic peptide based ELISA assays respectively.|
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In the current study, females comprised almost 59% as compared to 41% of males from the recruited participants of Kalmana and Kothari areas of the Chandrapur district. [Figure 4] demonstrates percent seropositivity of CHIKV Ag and antibody (IgM and IgG) in females with respect to age. The results indicated a rising trend in seropositivity of CHIKV among females with descending age wherein females of 46-65 yr of age showed high seroprevalence by both CHIKV Ag as well as antibody (IgM and IgG) tests.
|Figure 4: Age and female sex specific CHIKV Ag, IgM and IgG seroprevalence (%). The line graph demonstrates percent (%) seropositivity of Ag, IgM and IgG antibodies in females with respect to age.|
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| Discussion|| |
CHIKV is a flaviviral infection transmitted to humans through the bite of an infected female mosquito of the genus Aedes. Since, its first identification in the year 1952-53, both sporadic cases and major epidemics of CHIKV infection have been reported in Africa, In-dia, Southeast Asia and the Western Pacific. After the quiescence of more than three decades, India again faced re-emergence of CHIKV infection in 2006, where an estimated 13 developing cities encountered its lethal and long-lasting effects.
In the present study, the rural populations of Kalmana and Kothari villages from Ballarpur Taluka of Chandrapur district, Maharashtra were surveyed for disease prevalence which experienced CHIKV infection throughout 2011-12,. High rates of symptomatic manifestations like arthralgia followed by fever, large joint pain, back pain and vomiting were observed among the participants. Mucocutaneous manifestations (rashes) were mostly observed on body extremities like trunk, fingers and palms, feet, knees and shoulders. Interestingly, neck stiffness (75%) was also noticed in CHIKV infected patients who suggested that these arboviruses might cause neuroinvasive diseases like meningoencephalitis, acute flaccid paralysis (AFP), etc by crossing the blood brain barrier (BBB),.
During the outbreaks, total seroprevalence rate observed was 46% (222/482). The elevated rates of CHIKV were detected using a combination of either or both seropositive tests such as Ag detection using antipeptide antibodies or IgM or IgG antibody detection using synthetic peptides. This multi-combinatorial approach provides a platform for timely detection of CHIKV infection using a simple, cost-effective and easily accessible technique like ELISA which can predict stage specific infection in a patient.
It was observed that the percentage of Ag seropositiv-ity was slightly more in males (18.2%) as compared to females (16.5%) while taking gender into consideration. Fever (p <0.0001) and large joints pain (p = 0.0228) were statistically significant, suggesting detection of the symptomatic onset of high grade fever in the acute stage.
Overall CHIKV IgM seropositivity was found to be 27%. The majority of the cases were from 19-49 yr of age group. The highest IgM seroprevalence was found in females 29% (82/285) as compared to males 21% (46/239). These findings were in agreement with the study patterns shown by Balasubramaniam et al, Dwibedi et al and Mohanty et al. Contrasting reports, however, suggest that males are more susceptible to CHIKV infection as compared to females,. This inconsistency could be linked to gender differences in exposure to infection due to community-specific habits, customs or behaviour patterns. CHIKV IgG assay showed lowest seropositivity (15%) as compared to Ag (17.2%) and IgM (27%) antibody suggesting that the patients recruited in this study were probably in the acute phase of the CHIKV infection.
Chikungunya fever is characterized by a mild febrile disease manifesting clinical symptoms such as fever, incapacitating arthralgia, myalgia, headache, vomiting, backache and diffused maculopapular rashes,. In this study, a similar pattern of symptoms was observed with high rates of skin rashes (91%) being the dominant symptom, followed by small joints pain (80%), neck stiffness (75%) and fever (49%). Till now only a few case reports have been published suggesting that chikungunya fever can be exceptionally associated with neurological complications such as meningoencephalitis, myeloradiculitis, myelitis and myeloneuropathy7, 15, 23. These reports are in agreement with study findings of Kashyap et al, stating that meningoencephalitis appears to represent the most common clinical manifestation in CSF, which transpires either simultaneously or within few days of onset of systemic symptoms, along with neck stiffness during the period of viraemia. It was reported that the infection had affected more adults than children while morbidity was more among females in comparison to males.
In this study, there was no mortality, however; the morbidity rates were high amongst adults between the age groups of 19-49 yr and working on farms and agricultural fields. The virus is now spreading to new areas in this part of the state, as there seems to be no herd immunity to the virus. High vector abundance of Aedes mosquito and improved modes of transportation could be one of the main reasons thereby facilitating the movement of virae-mic individuals from one place to another, spreading the virus to new locations. In the Indian setting, due to low socioeconomic conditions, high population density, poor sanitary conditions, illiteracy and ignorance; the presence of the Aedes vector species contributes to the spread of CHIKV virus to wider areas.
In the current study, a multi-diagnostic approach using indirect as well as direct ELISA was adopted for the detection of CHIKV Ag and antibodies (IgM and IgG). CHIKV Ag assay was performed using antipeptide antibodies by indirect ELISA, while CHIKV IgM and IgG antibodies were detected using synthetic peptides,. These small scale laboratory based assays are easy to handle and perform, have high sensitivity and specificity and are cost-effective. Hence, the use of multiple immunoassays could give an edge over other advanced techniques for the detection of CHIKV infection of the acute phase, subclinical stage to circulating antibodies in rural populations. Out of482 suspected samples ofCHIKV infection, seropreva-lence ofAg, IgM and IgG was found to be 17, 27 and 15%, respectively, while 46% seropositivity was observed by using a combination of positivity of either of the tests (Ag or IgM or IgG). This population based study is one of the first to investigate seroprevalence and clinical manifestations of CHIKV infection in the newly exposed community of rural areas using multiple diagnostic approaches.
Such type of surveillance using appropriate and reliable cost-effective diagnostic measures is essential for monitoring of epidemics, and would be helpful in managing the disease outbreak situations especially in rural areas of developing countries.
This study had a few limitations. The data presented were of one-year and collected only from two villages during CHIKV outbreak in 2011-12, thus it does not represent the total prevalence rate of CHIKV infection in Chandrapur district. Nevertheless, this study gives a brief idea of the seroprevalence rate since the targeted villages were the largest in the Chandrapur district. A second limitation was the lack of follow up data which would help to predict the repercussions of this disease. Despite of the above limitations, the results obtained in the current study were very encouraging and, therefore, demands further validation of developed methods in comparison with a commercial kit to reconfirm the findings.
| Conclusion|| |
In conclusion, seroprevalence of CHIKV in the study (46%) was higher in the rural areas of Kalmana and Ko-thari of Ballarpur Taluka, Maharashtra in India during 2011–12 CHIKV epidemics, suggesting its continuance as a major health threat in present scenario. A multi-diagnostic approach is proposed for the timely detection of CHIKV infection using cost-effective and reliable pep-tide and antipeptide ELISA based assays. The virological surveillance of CHIKV and other vector-borne diseases should therefore be given utmost attention that will in turn help in the prediction, prevention and control of impending and sporadic outbreaks in developing countries.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]