Distribution Patterns of Non-SARS-CoV-2 Respiratory Pathogens Among Patients With Influenza-Like Illness During the COVID-19 Pandemic: A Cross-Sectional Study From Bangladesh

Background: During the COVID-19 pandemic, diagnostic efforts were heavily concentrated on SARS-CoV-2, leaving patients who tested negative with limited etiological workup despite continuing to present with influenza-like illness. This study aimed to identify and characterize the non-SARS-CoV-2 respiratory pathogens responsible for illness in this group at a tertiary care hospital in Bangladesh. Methods: We conducted a descriptive cross-sectional study at the Department of Virology, Bangladesh Medical University (BMU), from July to December 2022, enrolling 165 adults with influenza-like symptoms who tested negative for SARS-CoV-2. Nasopharyngeal swabs were analyzed by multiplex real-time PCR (VIASURE Respiratory Panel III kit, CerTest Biotec, Zaragoza, Spain). Pathogen distribution was assessed using Chi-squared goodness-of-fit (GoF) as a global test, confirmed by exact Monte Carlo multinomial simulation (100,000 runs). Individual pathogens were compared using binomial exact tests with Benjamini-Hochberg (BH)-false discovery rate (FDR) correction. Co-infection independence was assessed with one-sided Fisher’s exact tests plus BH-FDR correction; effect sizes are reported as odds ratios (ORs) with 95% confidence intervals (CIs). Results: Of 165 participants (mean age 34.8 ± 12.7 years; 61.8% male), 59 (35.8%; 95% CI: 28.8%-43.3%) had at least one pathogen, yielding 46 viral and 29 bacterial pathogen detections. Both distributions deviated significantly from uniformity (viral: χ²(6) = 48.35, p < 0.001; bacterial: χ²(2) = 20.14, p < 0.001). Human rhinovirus was the most statistically significant viral pathogen (21/46; 45.7%; BH-adjusted p < 0.001; z = +6.08), and Streptococcus pneumoniae the most significant bacterial pathogen (21/29; 72.4%; BH-adjusted p < 0.001; z = +4.46). Co-infections occurred in 15 patients (9.1%); no pair reached significance after BH-FDR correction, though rhinovirus-influenza B (OR = 5.41; 95% CI: 1.39-21.12; BH p = 0.087) and S. pneumoniae-Haemophilus influenzae (OR = 11.83; 95% CI: 1.85-75.65; BH p = 0.087) showed the strongest trends. Conclusion: Non-SARS-CoV-2 respiratory pathogens like human rhinovirus and S. pneumoniae were among the causes of substantial illness in Bangladesh throughout the pandemic. Broad-spectrum multiplex PCR testing should be integrated into the clinical workup of SARS-CoV-2-negative patients with persistent respiratory symptoms.