Chikungunya virus (CHIKV) is an arthropod-borne alphavirus of the family Togaviridae that has emerged as a major public health concern in tropical and subtropical regions worldwide. Transmitted primarily by Aedes aegypti and Aedes albopictus, the virus is responsible for outbreaks of acute febrile illness characterized by severe polyarthralgia, myalgia, rash, and fatigue.[1] CHIKV is a positive-sense, single-stranded RNA virus with a genome of approximately 12 kb, encoding four nonstructural and five structural proteins. The envelope glycoproteins E1 and E2 play a critical role in viral attachment, membrane fusion, and host cell entry, while the capsid protein packages the viral RNA.[2,3] Following inoculation through a mosquito bite, the virus initially replicates in fibroblasts and macrophages and subsequently disseminates to joints, muscles, liver, and spleen via lymphatic and hematogenous spread.[4] Clinically, chikungunya is characterized by abrupt-onset high-grade fever and debilitating joint pain, which may persist long after the acute phase.[5] The term “chikungunya,” derived from the Makonde language meaning “that which bends up,” reflects the stooped posture caused by intense joint pain during infection.[6] The virus was first identified during an outbreak in Tanzania in 1952 and subsequently caused epidemics across Africa and Asia.[7] After a prolonged period of relative inactivity, CHIKV re-emerged dramatically during 2004–2006, leading to widespread outbreaks in the Indian Ocean islands and India, with over one million suspected cases reported.[8] In India, chikungunya has become endemic, with recurrent outbreaks reported across most states. Surveillance data from the National Centre for Vector Borne Disease Control indicate seasonal peaks, particularly during and following the monsoon, when vector breeding intensifies.[9] Factors such as rapid urbanization, inadequate water management, climate variability, and expansion of Aedes albopictus into peri-urban and rural areas have contributed to sustained transmission.[10,11] The disease typically progresses through an acute phase followed by a chronic phase marked by persistent or relapsing arthralgia. Approximately 30–50% of infected individuals develop chronic inflammatory arthritis lasting months to years.[12] The pathogenesis of chronic disease is attributed to prolonged immune activation, persistence of viral RNA in joint tissues, and elevated proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-α.[5,10] Diagnosis relies on clinical suspicion supported by laboratory testing. IgM ELISA remains the most practical diagnostic modality in routine settings, as antibodies usually appear within five days of symptom onset, while molecular assays such as RT-PCR are limited to early infection and resource-equipped laboratories.[4,13] Currently, no licensed antiviral treatment or vaccine is available for chikungunya, and management remains largely supportive. Preventive strategies therefore focus on vector control, environmental sanitation, and public awareness. Understanding regional seroprevalence and clinical patterns is essential for strengthening surveillance and guiding targeted public health interventions. Hence, the present study was undertaken to evaluate the seroprevalence and clinical profile of chikungunya virus infection among patients attending a tertiary care hospital in Kathua district. Aims and Objectives Primary Objective • To determine the seroprevalence of chikungunya virus infection among clinically suspected patients attending a tertiary care hospital in the Kathua district. Secondary Objectives • To assess the demographic distribution (age, gender, and area of residence) of chikungunya seropositive cases and identify potential epidemiological risk factors. • To analyze the clinical manifestations and outcomes of chikungunya infection, focusing on acute symptoms and chronic musculoskeletal complications.

The present cross-sectional study was conducted in the Department of Microbiology at a tertiary care Government Medical College and Hospital, Kathua, using secondary data that was maintained in the microbiology laboratory registers of clinically suspected dengue cases reported to various inpatient and outpatient departments of the hospital for a period of one year from June 2023 to March 2024. The clinical definition of a suspected case was as per the World Health Organization criteria, i.e., acute febrile illness with a body temperature of >38.5°C and severe arthralgia or arthritis that is not explained by other medical conditions.[14] A suspected (clinical) case was further labelled as a confirmed case of chikungunya in the presence of laboratory confirmation of infection. There were no exclusion criteria for processing of the collected samples. Ethical approval was obtained from the Institutional Ethics Committee of GMC, Kathua (IEC/GMCK/36; 29/05/2024). The study adhered to ethical guidelines. Collection and Processing About 2 ml to 3 ml of blood was collected from each patient using strict aseptic precautions. Serum was separated by centrifuging samples at 3000 rpm for five minutes and tested immediately. In case of delay in processing, sera were stored at a temperature of 2°C-8°C.[15] Chikungunya IgM Capture ELISA A 96-well plate has been coated with anti-human IgM antibodies. Add 50 µL of 1:100 diluted serum, and controls were added to the ELISA plate. The assay plate was incubated at 37°C for one hour and then washed. Add 50 µL of anti-chick antigen to each well of the plate and incubate for one hour. Now, 50 µL of anti-chick monoclonal antibody HbX was added to each well, followed by incubation for 1 hour, and all the wells were washed. 50 µL of avidin-HRP was added to each well and again incubated for 30 minutes. A 100-micron tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂) substrate solution was added to each well after washing and incubated for 10 minutes at room temperature. The OD was read within 30 minutes of the addition of 100 µL stop solution at the wavelength of 450 nm with 620 and 650 as references. Within 30 minutes of adding the substrate solution, the cut-off value was calculated using the formula (Mean Absorbance formula). Statistical Analysis The data was collected, tabulated, and analyzed. Pearson's chi-square test was used as a test of significance. A value of p<0.05 was considered significant.

Out of the 539 clinically suspected cases of Chikungunya evaluated over the one-year study period, 96 patients were confirmed positive for anti-CHIKV IgM antibodies, yielding a seroprevalence of 17.81% (Figure 1). Gender-wise distribution revealed that females were disproportionately affected, accounting for 18.48% (n=61) of positivity compared to 16.74% (n=35) in males (Figure 2). Statistical analysis revealed no significant association between gender and Chikungunya positivity (X² = 0.05, p-value = 0.69, p > 0.05), indicating that gender had no significant influence on the occurrence of the infection. Age-wise analysis demonstrated that children below 14 years were the most affected group, contributing 38.5% (n=37) of all positive cases, followed by individuals aged 15–28 years (25%; n=24). The highest rate of positivity was observed among individuals aged >56 years (31.25%), followed by those aged 43-56 years (20%) and below 14 years (19.68%) (Table 1). However, statistical analysis showed no significant association between age and Chikungunya positivity (X² = 0.05, p-value = 0.71, p > 0.05), suggesting that age was not a determining factor in infection rates. Geographical distribution analysis indicated a marked rural predominance in Chikungunya cases. Out of a total of 539 suspected cases, 229 were from rural areas and 310 from urban areas. Among these, 68 cases (29.69%) were confirmed positive in the rural population, whereas only 28 cases (9.03%) were positive in urban settings. Overall, the total positivity rate was 17.81% (Figure 3). Fever with sudden onset was the most common presentation among the 539 symptomatic individuals who were examined (89.8%), followed by body pain (78%) and polyarthralgia (75.8%). Rash was recorded in 35.6% of cases, but joint swelling was seen in 67.6% of cases. Nearly half of the seropositive patients (48.2%) progressed to chronic arthritis, and 23.2% experienced persistent disability, highlighting the prolonged impact of infection beyond the acute phase (Table 2, Figure 4). 443 (82.19%) of the 539 clinically suspected cases had characteristic symptoms such as rash, arthralgia, myalgia, and high fever; however, they were seronegative (Table 3). Table 1: Age details of symptomatic enrolled patients Age in years Suspected Positives Rate of Positivity (%) <14 188 37 19.68 15-28 210 24 16.19 29-42 85 17 15.29 43-56 40 10 20.00 <56 16 8 31.25 Table 2. Clinical profile of symptomatic enrolled patients Clinical Presentation All (n=539, %) Fever with acute onset. 484, 89.8% Fever with insidious onset. 55, 10.2% Body rash 191, 35.6% Body pain 419, 78% Joint pain 407, 75.8% Joint swelling 363, 67.6% Chronic Arthritis 259, 48.2% Disability 125, 23.2% Table 3: Distribution of Symptomatic Patients with Negative Chikungunya IgM ELISA Parameter Number (n=443) Percentage (%) Acute-onset fever 392 88.5 Body pain / myalgia 341 77.0 Joint pain (arthralgia) 318 71.8 Joint swelling 266 60.0 Rash 151 34.1 Chronic joint symptoms (>3 months 112 25.3

Chikungunya virus (CHIKV), an arthropod-borne alphavirus transmitted mainly by Aedes aegypti and Aedes albopictus, continues to pose a major public health challenge in tropical and subtropical regions. The present study, conducted at Government Medical College, Kathua, represents one of the first investigations assessing both the seroprevalence and clinical profile of chikungunya infection. The study revealed a seroprevalence rate of 17.81%, providing significant evidence of local CHIKV transmission and establishing chikungunya as an emerging endemic disease in this part of Jammu and Kashmir. The seroprevalence observed in our study (17.81%) aligns closely with national and global data. Kumar et al. reported a seroprevalence of 18% in a large-scale national survey across India.[8] Similarly, Kang et al. documented pooled seroprevalence rates between 15% and 25% in endemic regions worldwide.[13] Studies from Maharashtra and Odisha reported positivity rates ranging from 10% to 24%, depending on seasonal and ecological variations.[16] These comparable findings indicate that chikungunya has become well-established across diverse Indian geographical zones, including northern states where it was previously underreported. Globally, comparable trends have been reported from Bangladesh (20%), Sri Lanka (18%), and sub-Saharan Africa (up to 25%), highlighting the virus’s ability to adapt to various climates and mosquito vectors.[17] Interestingly, our study observed a higher prevalence among females (63.5%) compared to males (36.4%). Similar gender patterns have been reported in studies from India and neighboring regions.[16,17] This may be attributed to behavioral exposure—since Aedes mosquitoes are day-biting species that breed in domestic water containers, women engaged in household activities are more likely to be bitten during daytime hours. Age-wise analysis revealed that individuals above 56 years and children below 14 years were more frequently affected, which mirrors findings from other epidemiological studies.[8,13] Children are biologically more vulnerable because of immature immune defenses and increased exposure during outdoor play, while older adults are prone to more severe disease due to immunosenescence and comorbidities. Similar bimodal patterns have been documented in outbreaks across Southeast Asia and Africa.[13,18] Moreover, rural areas accounted for a higher burden (70.8%) compared to urban areas (29.2%). This trend indicates a shifting epidemiological profile of chikungunya in India. Earlier outbreaks were largely confined to urban centers where Aedes aegypti thrives; however, the expansion of Aedes albopictus—a more adaptable species capable of breeding in rural and peri-urban settings—has broadened the virus’s ecological range.[9,19] Rural areas often have inadequate sanitation, uncovered water storage, and low awareness regarding mosquito control practices, which collectively enhance transmission potential. Similar rural predominance was observed in studies from Chandrapur, Maharashtra[16], and coastal Odisha.[8] These findings highlight the urgent need for vector control programs in rural areas, community-based awareness drives, and strengthening of primary healthcare surveillance networks. The classical triad of fever, polyarthralgia, and rash was observed in most patients, consistent with global literature. Fever with abrupt onset (89.8%), body pain (78%), and joint pain (75.8%) were the predominant features, while rash occurred in about one-third of cases. These symptoms correspond closely with reports from Simon et al. and Chow et al., who described similar clinical manifestations among confirmed CHIKV cases.[5,12] A noteworthy finding of this study is the high proportion of clinically suspected chikungunya cases that were IgM-negative. Similar observations have been reported from endemic regions, where a substantial fraction of symptomatic patients lack serological confirmation.[8,13] This may be attributed to early sample collection before IgM seroconversion, waning antibody levels in late presenters, or infection with other co-circulating arboviruses such as dengue and Zika, which share overlapping clinical features. A key finding of this study is that 48.2% of seropositive patients developed chronic arthritis and 23.2% experienced persistent disability, underlining the prolonged morbidity associated with CHIKV infection. Chronic chikungunya arthritis has been increasingly recognized as a post-viral inflammatory condition involving persistent viral RNA in joint tissues and sustained cytokine activation. Elevated levels of interleukin-6 (IL-6), TNF-α, and granulocyte macrophage colony-stimulating factor (GM-CSF) have been linked with chronic joint inflammation and tissue damage.[20,21] Histopathological studies reveal that CHIKV can infect macrophages, fibroblasts, and osteoblasts in joint tissues, leading to increased expression of RANKL and inhibition of osteoprotegerin (OPG), resulting in bone resorption and arthritic changes.[22] This explains the long-lasting joint pain and disability even after viral clearance. Clinicians must therefore differentiate post-chikungunya arthritis from autoimmune arthritis such as rheumatoid arthritis, as management strategies differ substantially. Limitations There are some drawbacks to this study. Because it is hospital-based, it might not accurately reflect transmission at the community level, especially in instances with no symptoms or very few symptoms. Because molecular diagnostics like RT-PCR were not used, reliance on IgM ELISA alone may have resulted in an underestimation of very early infections. Furthermore, the cross-sectional design made it more difficult to thoroughly evaluate long-term clinical outcomes and temporal trends.

The present study demonstrates a substantial burden of chikungunya virus infection among clinically suspected cases attending a tertiary care hospital in the Kathua district, confirming active transmission in this region. The observed seroprevalence highlights chikungunya as an emerging public health concern in an area where epidemiological data have been limited. Distinct demographic and geographic patterns were identified. A higher proportion of seropositive cases originated from rural areas, indicating increased exposure risk likely related to environmental conditions, vector breeding practices, and gaps in preventive measures. Infection was documented across all age groups, with relatively higher positivity among children and older adults, suggesting greater vulnerability in these populations. Clinically, chikungunya infection was predominantly characterized by abrupt-onset fever, polyarthralgia, and body pain. Importantly, a considerable proportion of patients progressed to chronic arthritis and persistent disability, underscoring the prolonged morbidity associated with the infection beyond the acute phase. Overall, the findings emphasize the need for heightened clinical awareness, strengthened laboratory surveillance, and focused vector control strategies, especially in rural areas. Early diagnosis and timely public health interventions are essential to reduce transmission, prevent long-term complications, and limit the growing burden of chikungunya in this region.

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