A Cross-Sectional Study in Northeast India to Assess Malaria, Scrub Typhus, and Dengue in Patients with Acute Febrile Illness Using ELISA

Technical Summary: Cross-Sectional Study on AFI Etiology in Northeast India

Problem Statement
Acute Febrile Illness (AFI) remains a primary driver of hospitalization in tropical and subtropical regions, particularly in India. The clinical presentation of dengue, scrub typhus, and malaria often overlaps, creating significant diagnostic confusion that can delay appropriate treatment and increase morbidity and mortality. While molecular methods offer high specificity, they are often less practical for routine screening in resource-constrained settings. There is a specific need to evaluate the utility of Enzyme-Linked Immunosorbent Assay (ELISA) for the simultaneous early detection of these three pathogens in patients presenting with AFI, particularly within the critical first week of symptom onset.

Methodology
This study employed a cross-sectional design conducted at a tertiary care teaching hospital in Northeast India.

• Population: The study enrolled 200 patients (95 urban, 105 rural) presenting with AFI lasting 0–6 days.
• Sampling: A consecutive non-probability sampling strategy was used to include all eligible patients during the research period.
• Diagnostic Protocol: Venous blood samples (3–5 mL) were collected, and serum was separated via centrifugation. Commercial ELISA kits were utilized according to manufacturer instructions to detect:
  • Dengue: NS1 antigen (for early viraemic phase, 0–6 days) and IgM antibodies (for later presentation).
  • Scrub Typhus: IgM antibodies against Orientia tsutsugamushi using recombinant antigens.
  • Malaria: Plasmodium pLDH antigen via sandwich ELISA.
• Data Analysis: Demographic data, residential status, and fever duration were recorded. Statistical analysis was performed using PAST software, employing the Chi-square test to assess relationships between variables (p < 0.05 considered significant).

Key Results
The study revealed a nearly equal gender distribution (51% male, 49% female) and a slightly higher proportion of rural participants (53%) compared to urban (47%).

• Urban Cohort (n=95):
  • Dengue NS1 antigen positivity: 16% (15 patients).
  • Dengue IgM positivity: 7% (7 patients).
  • Scrub typhus IgM positivity: 12% (11 patients).
  • Malaria antigen positivity: 17% (16 patients).
• Rural Cohort (n=105):
  • Dengue serology positivity: 18% (19 patients).
  • Scrub typhus IgM positivity: 19% (20 patients).
  • Malaria antigen positivity: 22% (23 patients).
• Fever Duration: A correlation was observed between longer fever durations (6–14 days) and higher positivity rates. Rural patients presented with a higher burden of cases in the 6–14 day fever group compared to the 0–5 day group, suggesting delayed healthcare seeking in rural areas.
• Prevalence: Malaria (22% in rural) and scrub typhus (19% in rural) emerged as the leading causes of AFI in the rural population, while dengue was also prevalent but slightly less dominant than the other two in the rural cohort.

Key Contributions

• Comparative Urban-Rural Analysis: The study provides specific data contrasting the etiological burden of AFI in urban versus rural settings within Northeast India, highlighting a consistently higher positivity rate for all three pathogens in rural populations.
• Validation of ELISA in Early Detection: The research demonstrates the practical application of ELISA-based assays for the simultaneous screening of dengue, scrub typhus, and malaria. It confirms the efficacy of NS1 antigen detection in the early febrile phase (0–6 days) and IgM detection in later stages.
• Diagnostic Strategy: The findings support the use of ELISA as a scalable, sensitive, and specific tool for early etiological identification in endemic regions, potentially reducing the reliance on empirical therapy.

Significance and Claims
The paper asserts that ELISA-based diagnostics are effective tools for the early identification of AFI etiologies, facilitating timely clinical decision-making and appropriate treatment. The study emphasizes that the higher incidence of AFI in rural areas underscores the necessity for improved monitoring, earlier access to diagnostic services, and targeted public health interventions in these regions. By corroborating WHO guidelines for laboratory confirmation of febrile diseases, the authors conclude that integrating these serological and antigen-based assays into routine diagnostic workflows can significantly aid in managing the burden of dengue, scrub typhus, and malaria in resource-limited settings.