Human metapneumovirus (HMPV) is a common respiratory pathogen that causes respiratory tract infections. In India, HMPV has been identified as one of the leading causes of morbidity and mortality in infants and young children with respiratory tract infections. The most reported sublineages of HMPV in India are B1, B2, A2b and A2c. A retrospective study was conducted to determine the circulating genotypes of HMPV among SARI cases from January 2016 to December 2018. Positive throat swab samples were confirmed with real-time RT-PCR. Subsequently, these samples were analysed using semi-nested conventional RT-PCR targeting the G gene, followed by sequencing and phylogenetic analysis. Clinical data analysis was also performed using SPSS 15.0 software. All 20 samples from the SARI cases were classified under the A2c sublineage of HMPV. Phylogenetic analysis indicated that these strains were genetically related to those circulating in Japan, China, and Croatia. Among the samples, ten showed 111-nucleotide duplications, while the other ten had 180-nucleotide duplications. Clinical analysis showed that four cases had coinfections with other pathogens. Our extensive analysis of patient samples determined that HMPV, especially the A2c genotype, significantly contributed to SARI cases within our study population, which signifies the importance of considering HMPV as a probable aetiological agent when investigating SARI outbreaks.

The diagnosis of an aetiology is dependent on the collection, transport, and storage of the infectious sample. The transport of the sample plays a crucial role in the chain of diagnosis. It is important to maintain the biological integrity of the pathogen during the transport of the sample to achieve an accurate diagnosis. This is important, particularly for labile organisms like viruses that are inactivated easily compared to other microorganisms. Many transport media have been utilised to ensure the integrity of the virus during transport. While most of the transport media are focused on preserving the infectious properties of the virus, progress has been made to develop virus transport media to inactivate the virus and obtain the stability of the viral nucleic acid, enabling better molecular diagnosis of the virus aetiologies. This review summarises the various media used for the transport of virus samples and focuses on the need to develop virus transport media that inactivates the virus and preserves the viral nucleic acid.

Background: Human metapneumovirus (HMPV) belongs to the family Pneumoviridae. It is one of the emerging respiratory viruses causing both upper and lower respiratory tract illnesses. HMPV has two genotypes: A and B. These genotypes are classified into lineage A1, A2, B1 and B2. Lineage-A2 is further classified as A2a, A2b and A2c. Similarly, B2 is classified as B2a and B2b. Studies have shown the circulation of A2b, B1 and B2 lineages in India. However, a limited amount of data is available on the current circulating genotypes of HMPV in India. Methods: Throat swab samples positive for HMPV by real-time RT- PCR, archived at Manipal Institute of Virology as a part of a hospital-based acute febrile illness surveillance study, was used from April 2016 to August 2018 by purposive sampling method. We performed the conventional reverse transcriptase-polymerase chain reaction for twenty samples targeting the G gene and then subjected them to sequencing. Phylogenetic analysis was done using MEGA X software by the Maximum Likelihood method. Results: All the twenty sequences belonged to the A2c subgroup. Phylogenetic analysis showed that strains from the study have genetic relation with circulating strains in Japan, China and Croatia. Seven out of the twenty sequences showed 180-nucleotide duplication and eleven sequences showed 111-nucleotide duplication. Two sequences did not show any duplications. Conclusion: In the current study, we report that A2c is the sub-lineage in India from April 2016 to August 2018. This study is the first retrospective study reporting the circulation of the A2c sub-lineage among adults in India with 180- and 111-nucleotide duplications in the G gene of human metapneumovirus.

Chikungunya fever is a viral disease spread by Aedes mosquitoes, reported in over 110 countries. In India, it was first detected in Kolkata in 1963 and is now widespread. Diagnosis often relies on detecting anti-chikungunya IgM antibodies, but these may not be present during the acute phase as viremia can last up to 8 days, leading to underreporting. The present study aims to assess the diagnostic use of real-time RT-PCR for detecting chikungunya-specific nucleic acid in serum samples during the early stages of infection. A retrospective cross-sectional study was conducted using archived samples collected as a part of 'the hospital-based Acute Febrile Illness (AFI) surveillance project'. AFI cases having fever ≤8 days and arthralgia without any aetiology between 2016 and 2018 from Karnataka, Kerala, and Tamil Nadu were included in the study. Samples were subjected to nucleic acid extraction followed by chikungunya real-time RT-PCR using a standardized in-house protocol. The samples that tested positive for Chikungunya by real-time RT-PCR were further tested for detecting anti-Chikungunya IgM antibodies using enzyme-linked immunosorbent assay. Demographic characterization of the cases was performed using SPSS version 20 and GraphPad Prism version 10. Out of a total of 646 samples tested, 31 samples (4.79%) were positive by real-time RT-PCR for chikungunya virus, 20 of which had a Ct value of <30, indicating a relatively high viral load. Among the 31 serum samples tested for anti-Chikungunya IgM antibodies, only one showed a positive result. Demographic analysis showed that 67% of cases were male and 32% were female, respectively. Clinical data analysis showed that most of the cases presented with cough (87%), headache (80.6%), myalgia (77.4%), coryza (70.9%), vomiting (58%), and abdominal pain (38.7%). The current study findings highlight the importance of screening patients with fever for up to 8 days and arthralgia for not only detecting IgM antibodies against chikungunya using ELISA but also for chikungunya virus-specific nucleic acid through real-time PCR/nucleic acid amplification techniques, or other methods. Not performing laboratory tests to screen for chikungunya-specific nucleic acid or antigen may result in underreporting of chikungunya cases, thereby impacting effective control measures and management of cases.

The comprehensive identification of viruses using metagenomic next-generation sequencing (mNGS) has revolutionized the infectious disease diagnosis. Accurate detection of viruses on time is crucial to effectively control the outbreak. This review has meticulously analyzed the use of mNGS to identify viruses from respiratory samples, emphasizing how sample enrichment and amplification methods affect viral detection efficiency and genome coverage. A total of 66 studies were considered to evaluate sequencing workflows and their alignment with routine molecular techniques. Among these studies, the most frequently used method to enrich viruses was centrifugation (50%), followed by enzymatic treatment (45%). Sequence-independent single-primer amplification (SISPA) and switching mechanism at the 5’ end of RNA template (SMART) was most commonly used amplification methods, which produced better genome coverage for most of the viruses. While mNGS showed better detection capabilities, they showed variable agreement with routine diagnostic methods, with sensitivity varying from 62.5% to 95%. Rhinovirus was detected mainly in lower respiratory tract infections (68%) and respiratory syncytial viruses in upper respiratory tract infections (60%). Although mNGS has the potential, its routine use is hampered by various technical issues. Overcoming these limitations through optimized sequencing protocols and greater cost-effectiveness will be pivotal in integrating mNGS into clinical applications.

Kyasanur Forest Disease (KFD) is a tick-borne viral haemorrhagic illness endemic to southern India. Early identification of patients at risk for poor outcomes remains a clinical challenge. The study aimed to evaluate the role of cycle threshold (Ct) values in predicting outcomes. We conducted a retrospective cohort study of adults with PCR-confirmed KFD admitted to a tertiary care hospital in South India between January 2019 and December 2024. The primary outcome was 28-day all-cause mortality. Univariate and multivariable logistic regression analyses were performed to identify independent predictors of mortality. Receiver operating characteristic (ROC) analysis was used to assess the prognostic performance of Ct values. Among 213 patients, 20 (9.4%) died. Mortality was independently associated with lower Ct values, older age, lower respiratory tract involvement, altered sensorium, and elevated white blood cell counts on multivariable analysis. ROC analysis yielded an area under the curve of 0.675 for Ct value alone. A Ct cut-off ≥27.75 predicted survival with 68.4% sensitivity and 70.0% specificity. Lower Ct values are independently associated with increased mortality in KFD and may serve as a pragmatic tool for early risk stratification, especially in resource-limited settings.