Scrub typhus is an acute febrile zoonotic illness caused by Orientia tsutsugamushi, an obligate intracellular bacterium transmitted to humans through the bite of infected larval trombiculid mites (chiggers) [1]. The disease is endemic within the “tsutsugamushi triangle,” encompassing large parts of South and Southeast Asia, including India [2]. In recent years, scrub typhus has re-emerged as a major cause of acute undifferentiated febrile illness in India, contributing significantly to morbidity and mortality, particularly in rural and semi-urban populations [3]. The clinical presentation of scrub typhus is highly variable and often non-specific, making early diagnosis challenging. Common symptoms include fever, headache, myalgia, and malaise, while severe cases may progress to complications such as acute respiratory distress syndrome, acute kidney injury, meningoencephalitis, myocarditis, and multi-organ dysfunction syndrome [4]. Although the presence of an eschar at the site of the chigger bite is considered pathognomonic, it is inconsistently observed in Indian patients, thereby limiting its diagnostic value [5]. Due to overlapping clinical features with other endemic febrile illnesses such as dengue, malaria, enteric fever, and leptospirosis, scrub typhus is frequently underdiagnosed or misdiagnosed [6]. Laboratory confirmation plays a crucial role in the diagnosis of scrub typhus. While indirect immunofluorescence assay (IFA) is considered the reference standard, its routine use is limited in many Indian healthcare settings due to high cost, lack of standardization, and requirement for specialized infrastructure and technical expertise [7]. Consequently, a variety of serological tests are employed in routine diagnostic practice, each with inherent advantages and limitations. The Weil–Felix test (WFT) remains one of the most widely used screening tests for scrub typhus in resource-limited settings. Based on the cross-reactivity between rickettsial antibodies and Proteus OXK antigen, the test is inexpensive and easy to perform, making it accessible at peripheral healthcare centers [8]. However, its low sensitivity and specificity limit its reliability as a standalone diagnostic test, necessitating confirmation by more specific assays [9]. Immunochromatographic tests (ICT), or rapid card tests, detect IgM antibodies against Orientia tsutsugamushi and provide rapid results, facilitating early clinical decision-making and initiation of empirical therapy [10]. Despite their operational advantages, variability in diagnostic accuracy among commercially available kits and reduced sensitivity during the early phase of illness restrict their diagnostic utility [11]. IgM enzyme-linked immunosorbent assay (ELISA) is currently the most commonly used serological test for the diagnosis of scrub typhus in endemic regions of India. It offers higher sensitivity and specificity compared to WFT and ICT and is suitable for batch testing in tertiary care laboratories [12]. When interpreted in conjunction with clinical features and laboratory parameters, IgM ELISA provides reliable diagnostic confirmation and correlates well with disease severity [13]. Given the absence of a single ideal diagnostic test applicable to all healthcare settings, a combined diagnostic approach using Weil–Felix test, immunochromatographic card test, and IgM ELISA may improve diagnostic accuracy and reflect real-world laboratory practices in India [14]. Correlating the results of these serological tests with clinical manifestations can enhance early diagnosis, guide appropriate treatment, and reduce complications associated with delayed recognition. The present study was therefore undertaken to evaluate the utility of multiple serological tests and their clinical correlation in the diagnosis of scrub typhus among Indian patients.

Study Design This was a prospective observational study conducted to evaluate the role of serological tests and their clinical correlation in the diagnosis of scrub typhus. Study Duration The study was carried out over a period of two years, from December 2022 to November 2025. Study Setting The study was conducted in the Department of Pathology, Dr. Bhim Rao Ramji Ambedkar Government Medical College, Kannauj, in coordination with the Department of Medicine and other clinical departments. Study Population A total of 214 patients presenting with acute febrile illness and clinically suspected of scrub typhus were enrolled during the study period. Inclusion Criteria • Patients of either sex and all age groups • History of fever lasting ≥5 days • Clinical suspicion of scrub typhus based on symptoms such as headache, myalgia, rash, eschar, lymphadenopathy, or evidence of organ involvement • Patients who provided written informed consent Exclusion Criteria • Patients with laboratory-confirmed alternative diagnoses such as malaria, dengue, enteric fever, or leptospirosis • Patients who had received doxycycline, azithromycin, or chloramphenicol prior to sample collection • Inadequate, lipemic, or hemolyzed blood samples Sample Size Out of 214 suspected cases, 200 patients fulfilled the inclusion criteria and were included in the final analysis. Sample Collection Approximately 5 mL of venous blood was collected from each patient under aseptic conditions. Samples were allowed to clot and centrifuged at 3000 rpm for 10 minutes to separate serum. Serum samples were stored at 2–8°C until serological testing. Serological Tests Performed Weil–Felix Test The Weil–Felix test was performed using Proteus OXK antigen by tube agglutination method. Serum dilutions ranging from 1:20 to 1:640 were tested. A titre of ≥1:160 or a fourfold rise in paired sera was considered suggestive of scrub typhus. Immunochromatographic Test (ICT) All 200 serum samples were tested using a commercial rapid immunochromatographic card test for qualitative detection of IgM antibodies against Orientia tsutsugamushi. Results were interpreted within 15 minutes as per manufacturer’s instructions. IgM ELISA IgM ELISA for scrub typhus was performed on all samples using a commercial ELISA kit. Optical density values were measured using an ELISA reader at 450 nm, and results were interpreted according to kit-specific cut-off values. IgM ELISA positivity was considered confirmatory. Clinical and Laboratory Data Collection Detailed clinical data including duration of fever, presence of eschar, rash, lymphadenopathy, hepatosplenomegaly, and signs of organ involvement were recorded. Laboratory parameters analyzed included: • Complete blood count • Platelet count • Liver function tests (AST, ALT) • Renal function tests (serum urea, creatinine) Clinical–Serological Correlation Results of the Weil–Felix test, ICT, and IgM ELISA were correlated with clinical features and laboratory abnormalities to assess diagnostic accuracy and disease association. Statistical Analysis Data were entered into Microsoft Excel and analyzed using SPSS software (version 26). Descriptive statistics were expressed as mean, standard deviation, and percentages. Associations between serological test results and clinical parameters were evaluated using Chi-square test and Fisher’s exact test. A p-value <0.05 was considered statistically significant.

A total of 200 patients with acute febrile illness clinically suspected of scrub typhus were included in the final analysis. Demographic Characteristics Out of 200 patients, 118 (59%) were males and 82 (41%) were females, with a male-to-female ratio of 1.4:1. The age of patients ranged from 6 to 72 years, with a mean age of 34.6 ± 14.2 years. The majority of cases were observed in the 21–40 years age group. Clinical Features Fever was present in 100% (200/200) of patients. Other common symptoms included headache (64%), myalgia (58%), and gastrointestinal symptoms (32%). Eschar was detected in 38 patients (19%). Table 1: Age and Gender Distribution of Study Population (n = 200) Age Group (years) Male (n) Female (n) Total (n) Percentage (%) ≤20 22 18 40 20.0 21–40 52 34 86 43.0 41–60 30 22 52 26.0 >60 14 8 22 11.0 Total 118 82 200 100 Table 2: Clinical Features of Patients (n = 200) Clinical Feature Number of Patients Percentage (%) Fever 200 100.0 Headache 128 64.0 Myalgia 116 58.0 Nausea/Vomiting 64 32.0 Rash 42 21.0 Eschar 38 19.0 Lymphadenopathy 46 23.0 Hepatosplenomegaly 34 17.0 Breathlessness 28 14.0 Laboratory Findings Thrombocytopenia was the most common laboratory abnormality, observed in 110 patients (55%). Elevated liver enzymes were noted in 98 patients (49%), while leukocytosis was seen in 72 patients (36%). Table 3: Laboratory Parameters in Study Population (n = 200) Laboratory Parameter Number of Patients Percentage (%) Thrombocytopenia (<150,000/µL) 110 55.0 Elevated AST/ALT 98 49.0 Leukocytosis 72 36.0 Leukopenia 18 9.0 Raised Serum Creatinine 26 13.0 Serological Test Results Out of 200 patients: • Weil–Felix test was positive in 68 patients (34%) • Immunochromatographic test (ICT) was positive in 82 patients (41%) • IgM ELISA was positive in 96 patients (48%) Table 4: Positivity Rate of Serological Tests (n = 200) Serological Test Positive (n) Negative (n) Positivity (%) Weil–Felix Test 68 132 34.0 ICT (IgM) 82 118 41.0 IgM ELISA 96 104 48.0 Comparison of Serological Tests IgM ELISA detected the highest number of positive cases, followed by ICT and Weil–Felix test. Among the 96 ELISA-positive cases, 68 (70.8%) were also positive by ICT and 52 (54.2%) were positive by Weil–Felix test. Table 5: Comparison of Serological Tests with IgM ELISA as Reference (n = 96) Test Positive Cases Percentage (%) ICT 68 70.8 Weil–Felix Test 52 54.2 Clinical–Serological Correlation Among the 96 IgM ELISA–positive patients, fever was present in 100%, thrombocytopenia in 72 patients (75%), elevated liver enzymes in 66 patients (68.7%), and eschar in 30 patients (31.2%). A statistically significant association was observed between IgM ELISA positivity and thrombocytopenia as well as elevated liver enzymes (p < 0.05). Table 6: Clinical and Laboratory Correlation in IgM ELISA–Positive Cases (n = 96) Parameter Number of Patients Percentage (%) Fever 96 100.0 Thrombocytopenia 72 75.0 Elevated AST/ALT 66 68.7 Eschar 30 31.2 Raised Serum Creatinine 22 22.9 Figure 1: Positivity rate of different serological tests for scrub typhus. IgM ELISA demonstrated the highest positivity rate (48%) compared to immunochromatographic test (41%) and Weil–Felix test (34%), indicating superior diagnostic yield. Figure 2: Distribution of clinical features among patients with suspected scrub typhus. Fever was present in all patients, while other manifestations such as headache, myalgia, rash, lymphadenopathy, and eschar showed variable distribution, highlighting the non-specific clinical presentation of scrub typhus.

Scrub typhus has re-emerged as an important cause of acute undifferentiated febrile illness in India and other parts of the Asia–Pacific region. The present study evaluated the diagnostic utility of commonly employed serological tests and their clinical correlation in Indian patients, reflecting real-world laboratory practices in resource-limited healthcare settings. In the present study, males constituted 59% of the study population, with a male-to-female ratio of 1.4:1, and the majority of cases were observed in the 21–40-year age group. Similar demographic trends have been reported in Indian studies by Rathi and Rathi and Varghese et al., who attributed the higher incidence among young adult males to increased outdoor occupational exposure and agricultural activities [15,16]. Comparable age and gender distributions have also been documented in studies from Southeast Asia, including Thailand, indicating a consistent epidemiological pattern across endemic regions [17]. Fever was the most consistent clinical manifestation, present in 100% of patients, which is in agreement with findings from multiple Indian and international studies [18,19]. Other commonly observed symptoms such as headache and myalgia were comparable to those reported by Chrispal et al. and Mahajan [18,19]. The prevalence of eschar in the present study was 19%, which falls within the range reported in Indian studies (7–35%) [20,21]. The relatively low detection rate of eschar in Indian patients may be due to darker skin pigmentation, atypical anatomical locations, or delayed presentation, thereby limiting its reliability as a diagnostic marker. Laboratory abnormalities such as thrombocytopenia (55%) and elevated liver enzymes (49%) were frequently observed in the study population. Among IgM ELISA–positive patients, thrombocytopenia and transaminitis were present in 75% and 68.7% of cases, respectively, showing a statistically significant association with scrub typhus infection (p < 0.05). These findings are consistent with observations by Varghese et al. and Kim et al., who highlighted thrombocytopenia and hepatic dysfunction as important laboratory indicators aiding early clinical suspicion of scrub typhus [16,22]. With respect to serological testing, IgM ELISA demonstrated the highest positivity rate (48%), followed by immunochromatographic test (41%) and Weil–Felix test (34%). This finding supports previous Indian studies that have consistently shown superior diagnostic performance of IgM ELISA compared to Weil–Felix test and rapid diagnostic assays [23,24]. IgM ELISA is currently regarded as the most reliable serological test for routine diagnosis of scrub typhus in endemic regions. The Weil–Felix test, though inexpensive and widely available, demonstrated limited sensitivity in the present study. Only 54.2% of IgM ELISA–positive cases were detected by Weil–Felix test, reinforcing its inadequacy as a standalone diagnostic tool. Similar limitations of Weil–Felix test have been reported by Aggarwal et al. and other investigators, who recommend its use only as a preliminary screening test in conjunction with clinical findings and more specific assays [25]. The immunochromatographic test detected 70.8% of IgM ELISA–positive cases, performing better than Weil–Felix test but remaining inferior to ELISA. Previous studies from India and other endemic countries have reported variable sensitivity of ICT kits, largely influenced by differences in antigen composition and the timing of sample collection during the course of illness [26,27]. Despite these limitations, ICT remains a valuable tool for rapid bedside diagnosis and early initiation of empirical therapy. The combined use of Weil–Felix test, ICT, and IgM ELISA in the present study represents a pragmatic, tiered diagnostic approach suitable for Indian healthcare settings. This strategy enhances overall diagnostic yield, reduces the likelihood of missed diagnoses, and supports timely initiation of appropriate antibiotic therapy, thereby preventing severe complications. Such an integrated diagnostic approach has been recommended by global health authorities and supported by international studies on rickettsial diseases [28]. The limitations of the present study include the absence of confirmatory indirect immunofluorescence assay or molecular diagnostic methods and its single-center design. However, the strengths of the study lie in its prospective nature, adequate sample size, and practical applicability to routine diagnostic laboratories in endemic regions.

Scrub typhus remains a significant and underdiagnosed cause of acute febrile illness in India. The present study highlights the importance of integrating clinical evaluation with serological testing for accurate and timely diagnosis. Among the diagnostic modalities assessed, IgM ELISA demonstrated the highest diagnostic yield, while immunochromatographic tests and Weil–Felix test served as useful adjuncts, particularly in resource-limited settings. Clinical features such as fever, thrombocytopenia, and elevated liver enzymes showed strong correlation with seropositivity and can aid in early clinical suspicion. A combined, tiered diagnostic approach using multiple serological tests improves diagnostic confidence, facilitates early initiation of appropriate therapy, and may reduce morbidity and mortality associated with scrub typhus. Strengthening laboratory diagnostic capacity and increasing clinician awareness are essential to improve patient outcomes in endemic regions.

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