Validating saliva as a biological sample for cost-effective, rapid and routine screening for SARS-CoV-2.

PURPOSE: Compared to nasopharyngeal/oropharyngeal swabs (N/OPS-VTM), non-invasive saliva samples have enormous potential for scalability and routine population screening of SARS-CoV-2. In this study, we investigate the efficacy of saliva samples relative to N/OPS-VTM for use as a direct source for RT-PCR based SARS-CoV-2 detection. METHODS: We collected paired nasopharyngeal/oropharyngeal swabs and saliva samples from suspected positive SARS-CoV-2 patients and tested using RT-PCR. We used generalized linear models to investigate factors that explain result agreement. Further, we used simulations to evaluate the effectiveness of saliva-based screening in restricting the spread of infection in a large campus such as an educational institution. RESULTS: We observed a 75.4% agreement between saliva and N/OPS-VTM, that increased drastically to 83% in samples stored for less than three days. Such samples processed within two days of collection showed 74.5% test sensitivity. Our simulations suggest that a test with 75% sensitivity, but high daily capacity can be very effective in limiting the size of infection clusters in a workspace. Guided by these results, we successfully implemented a saliva-based screening in the Bangalore Life Sciences Cluster (BLiSC) campus. CONCLUSION: These results suggest that saliva may be a viable alternate source for SARS-CoV-2 surveillance if samples are processed immediately. Although saliva shows slightly lower sensitivity levels when compared to N/OPS-VTM, saliva collection is logistically advantageous. We strongly recommend the implementation of saliva-based screening strategies for large workplaces and in schools, as well as for population-level screening and routine surveillance as we learn to live with the SARS-CoV-2 virus.

Reclassification of respiratory syncytial virus genotypes in India.

Respiratory syncytial virus (RSV) is known to be the major cause of lower respiratory tract infections in infants and in the elderly. RSV was recently reclassified and simplified into three genotypes of the RSV-A subgroup (GA1-GA3) and into seven genotypes of the RSV-B subgroup (GB1-GB7). This classification strategy was not implemented globally. This study intended to reclassify the sequences that were submitted in GenBank till September 2021 from India. The gene sequences of the ectodomain region, second hypervariable region (SHR), and the partial second hypervariable region (PSHR) of the G gene were selected for the analysis. 25 ectodomain, 36 s hypervariable, and 19 partial second hypervariable regions of the RSV-A subgroup and 42-ectodomain, 49-s hypervariable region and 11-partial second hypervariable region of RSV-B subgroup were used for phylogenetic analysis. P-distance was calculated to support the genotype determination done by phylogenetic analysis. Phylogenetic analysis revealed that GA2.3.1, GA2.3.3, GA2.3.4, GA2.3.5, and GA2.3.6b lineages of GA2 genotype for RSV-A; and GB5.0.1, GB5.0.2, GB5.0.3, GB5.0.4a, GB5.0.4c, GB5.0.5a, GB5.0.5c lineages of GB5 genotype and GB7 genotype for RSV-B were that circulated in India. This work has implication for RSV vaccine research, and also for strategies for the prevention and control of RSV infection in humans. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00802-x.

Kinetics of human infection with Kyasanur Forest Disease Virus.

BACKGROUND: Kyasanur Forest disease (KFD) was first reported in 1957 and became an emerging tick-borne viral disease of public health importance in India. However, very little is known about the host-virus interaction and pathogenesis of KFD in humans. This study described the presence, duration, and kinetics of KFDV RNA in body fluids in infected human cases. METHODOLOGY: We enrolled 76 laboratory-confirmed KFD individuals and followed them up in the study. We obtained serial samples of blood, throat swabs in viral transport medium (VTM), urine, stool, and semen during the acute and convalescent phase of KFD illness. In addition, specimens were inactivated, and nucleic acid was extracted and tested for KFDV real-time reverse transcriptase -PCR. Clinical data was also obtained from the subjects. RESULT: The study provides evidence of KFD virus RNA in different biological body fluids of humans. The percentage positivity of KFDV RNA in blood was 100% during the first four days of illness. PCR became negative in most cases by 7-8 days; a subset of cases (14%) had prolonged viremia for up to 15 days post-onset of illness. Relatively low copies of KFDV RNA were also detected in throat swabs and urine in the first week of illness. In addition, we detected KFDV RNA in stool samples of cases of those who had diarrhea at an early stage of infection. CONCLUSION: The study provides evidence of KFDV RNA in different biological body fluids, which will help understand the pathogenesis, transmission pattern and develop diagnostic algorithms of KFDV in humans. In Kyasanur Forest disease infection, the blood has more RNA copies/ml than other body fluids, and viremia may last up to two weeks post-infection.

Molecular epidemiology of respiratory syncytial virus among children and adults in India 2016 to 2018.

Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections among children less than 5 years of age and the elderly. This study intended to determine the circulating genotypes of RSV among severe acute respiratory illness (SARI) cases during the period 2016-2018 in India, among hospitalized acute febrile illness cases of age ranging from 1 to 65 years. Throat/nasopharyngeal swab samples were subjected for testing RSV and subgroups by real-time reverse transcriptase polymerase chain reaction (RT-PCR), further sequencing and phylogenetic analysis were performed for the second hypervariable region of the G gene. RSV-A and B subtypes co-circulated during the years 2016, 2017, and 2018, with RSV-A as the dominant subtype in 2016, and RSV-B as the dominant subgroup in 2017 and 2018. Phylogenetic analysis revealed that the circulating genotypes of RSV were GA2 (16/16), of RSV-A, and GB5 (23/23) of RSV-B in the South, North, and Northeast region of India during the period between 2016 and 2018. Here we report the first study comprising the distribution of RSV-A and B genotypes in the different geographic regions of India among children and adults during the year 2016 to 2018. We also report GA2.3.7 lineage of GA2 genotype for the first time in India to the best of our knowledge.

Dynamics of human B and T cell adaptive immune responses to Kyasanur Forest disease virus infection.

Kyasanur Forest disease (KFD) is a tick-borne, acute, febrile viral illness endemic in southern India. No major studies have been done to understand the adaptive immune response during KFDV infection in humans. In this study, KFDV-positive patients were prospectively enrolled, and repeated peripheral blood collections were performed. Clinical and virologic characterization of these samples is reported along with phenotypic analysis of cellular immunity and quantitation of humoral immunity. We noted robust T and B cell responses, particularly of CD8 T cells, during KFDV infection in most of the patients. Virus clearance from the blood coincided with peak CD8 T cell activation and the appearance of KFDV-specific IgG. Increased frequency of plasmablasts and very few activated B cells were observed in the acute phase of KFD infection. Notably, only humoral immunity and activated B cell frequency in the acute phase correlated with prior KFDV vaccination, and only with 2 or more doses. This novel work has implications in KFD vaccine research as well as in understanding the pathogenesis.

The prevalence of anti-hepatitis C antibody among acute febrile illness cases in Idar Taluk, Gujarat, West India.

Purpose: The major cause of chronic hepatitis is infections with hepatitis B virus and hepatitis C virus (HCV) globally. However, there exists sparse epidemiological data regarding the prevalence of HCV infection from India. Methodology: We carried out a cross-sectional study to estimate the prevalence of anti-HCV antibody among acute febrile illness cases aged between 1 and 65 years in Idar Taluk, Sabarkantha district, Gujarat state located in West India. A total of 702 serum samples collected from the study area during the year 2017, were screened for anti-hepatitis C IgG by enzyme-linked immunosorbent assay. The serum samples screened positive were then subjected to molecular testing for confirmation. Results: Among the 702 study participants screened, 16 cases were reported to be anti-HCV IgG positive with an estimated seroprevalence rate of 2.3% (95% confidence interval: 1.4%-3.7%). Out of the 16 cases, two samples were confirmed positive by molecular testing indicating active infection. When analysed phylogenetically, one strain was genotyped as HCV1b genotype, and the other one was clustered along with HCV3a genotype. Both the patients with hepatitis C infection were observed to be having a probable 1-year survival rate of 100% and a 2-year survival rate of 85% when the Child-Turcotte-Pugh classification was applied. Conclusion: The estimated seroprevalence of hepatitis C in Idar Taluk, Sabarkantha district, west India was 2.3%. HCV genotypes 1b and 3a were observed to be circulating in the study area.

The Utility of Urine-Based Sampling for Cervical Cancer Screening in Low-Resource Settings.

Background: WHO has recommended Visual Inspection with Acetic acid (VIA) or Human Papillomavirus (HPV) DNA testing if feasible, for cervical cancer screening in low income countries. However, the number of women undergoing screening is very low as a result of limited information, inadequate infrastructure and invasive nature of sampling. Methods: A cross sectional study was carried out comparing HPV DNA detection by Polymerase Chain Reaction (PCR) in paired cervical and urine samples procured from histologically confirmed cervical cancer cases. Results: Amongst the samples collected from 114 cervical cancer cases, HPV DNA was tested positive in cervical samples of 89 (78.1%) and urine samples of 55 (48.2%) patients. The agreement between the two sampling methods was 66.7% and the kappa value was 0.35 indicating a fair agreement. The sensitivity of HPV detection using urine samples was 59.6% (95% confidence interval 49.16%-69.15%) and the specificity was 92% (95% confidence interval 75.0%-97.8%). Conclusion: Even though not acceptable as an HPV DNA screening tool due to low sensitivity, the urine sampling method is inexpensive and more socially acceptable for large epidemiological surveys in developing countries to estimate the burden.

Spatial distribution of Haemaphysalis species ticks and human Kyasanur Forest Disease cases along the Western Ghats of India, 2017-2018.

Kyasanur Forest Disease (KFD) is a viral haemorrhagic fever, transmitted to humans and other hosts by a tick vector of genus Haemaphysalis. It affects 400-500 people annually in the Western Ghats region of India through spring to summer season. To understand the species composition, distribution, and abundance of Haemaphysalis ticks in endemic taluks (sub-districts) of India, a surveillance for ticks was conducted between October 2017 and January 2018. In total 105 sites were selected based on grid sampling from five taluks representing five KFD endemic states in south India. A sum of 8373 ticks were collected by using standard flagging method. The study showed a wide distribution of host seeking tick species among the selected taluks, wherein Haemaphysalis spinigera was predominant in 3/5 taluks, Haemaphysalis bispinosa in 1/5 taluks, and both the species in 1/5 taluks. Further, the H. spinigera abundance was categorised and compared with the incidence of human cases during the same season. The grids with very high and high H. spinigera abundance had 70% of the 205 human cases reported. This method of tick surveillance could be efficiently used as a standard model for KFD transmission risk assessment and prediction of impending outbreaks.

Adaptive Immune Responses in Humans During Nipah Virus Acute and Convalescent Phases of Infection.

BACKGROUND: Nipah virus (NiV) is 1 of 10 potential causes of imminent public health emergencies of international concern. We investigated the NiV outbreak that occurred in May 2018 in Kerala, India. Here we describe the longitudinal characteristics of cell-mediated and humoral immune responses to NiV infection during the acute and convalescent phases in 2 human survivors. METHODS: Serial blood samples were obtained from the only 2 survivors of the NiV outbreak in Kerala. We used flow cytometry to determine the absolute T-lymphocyte and B-lymphocyte counts and the phenotypes of both T and B cells. We also detected and quantitated the humoral immune response to NiV by virus-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assay. RESULTS: Absolute numbers of T lymphocytes remained within normal limits throughout the period of illness studied in both survivors. However, a marked elevation of activated CD8 T cells was observed in both cases. More than 30% of total CD8 T cells expressed Ki67, indicating active proliferation. Proliferating (Ki-67+) CD8 T cells expressed high levels of granzyme B and PD-1, consistent with the profile of acute effector cells. Total B-lymphocyte, activated B-cell, and plasmablast counts were also elevated in NiV survivors. These individuals developed detectable NiV-specific IgM and IgG antibodies within a week of disease onset. Clearance of NiV RNA from blood preceded the appearance of virus-specific IgG and coincided with the peak of activated CD8 T cells. CONCLUSIONS: We describe for the first time longitudinal kinetic data on the activation status of human B- and T-cell populations during acute NiV infection. While marked CD8 T-cell activation was observed with effector characteristics, activated CD4 T cells were less prominent.

Currently circulating genotypes of hepatitis E virus in India, 2014-2018.

Purpose: Hepatitis E virus (HEV) is an emerging pathogen causing acute viral hepatitis worldwide. Clinical manifestations often occur in young adults with an increased mortality rate among pregnant women. HEV genotypes 1 and 4 are mainly reported among humans and swines, respectively. Aims: The aim was to study the currently circulating genotypes of HEV in India. Materials and Methods: A retrospective cross-sectional study was carried out at Manipal Institute of Virology to know the circulating genotypes of hepatitis E, spanning over 5 years from August 2014 to September 2018. The serum samples screened serologically positive and confirmed positive for active infection by real-time reverse transcriptase-polymerase chain reaction (PCR) (Real Star® HEV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany) were further subjected to nested conventional PCR targeting the RdRp gene of non-structural ORF1 region. The purified PCR product was sequenced in BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Thermo Fisher Scientific, USA). The chromatograms obtained by sequencing were analysed using Sequencher 5.4.6, and HEV FASTA sequences were compared with reference sequences for HEV in GenBank Nucleotide Blast. Results: During the study period, there were 317 cases of laboratory-confirmed cases of acute viral hepatitis comprising 202, 70, 43 and 2 cases of hepatitis A, E, B and C, respectively. Serum samples of 70 acute hepatitis cases were positive for anti-hepatitis E IgM. According to the clinical case classification, there were 66 cases of acute viral hepatitis and four cases of fulminant hepatic liver failure. The mean age of the patients was 30.3 years (standard deviation = 12.5). The samples from various parts of India were genotyped as 1a. Conclusion: The HEV genotypes 1a was observed to be the currently circulating strain in the regions studied.

Detection of Genital HPV Infection Using Urine Samples: a Population Based Study in India.

BACKGROUND: Cervical cancer is the second commonest cancer among Indian women and its association with human papilloma virus (HPV) is well established. This preventable cancer accounts for the maximum number of cancer related deaths among rural Indian women. Unlike in developed countries there are no organized cervical cancer screening programmes in India due to lack of resources and manpower. OBJECTIVE: To detect genital HPV infection using urine samples among asymptomatic rural women in the age group of 18-65 years. MATERIALS AND METHODS: The study area chosen was Perdoor village in Udupi Taluk, Karnataka State and all the women in the age group of 18-65 years formed the study cohort. A cross sectional study was conducted by house visits and 1,305 women were enrolled in the study. After taking written informed consent a data sheet was filled and early stream random urine samples were collected, transported to a laboratory at 4OC and aliquoted. Samples were tested using nested HPV PCR with PGMY09/11 and GP5+/6+ primers. Positive cases were genotyped by sequence analysis. RESULTS: Study participants included 1,134 sexually active and 171 unmarried women with a mean age at marriage of 22.1 (SD=3.9) years. Study area showed high female literacy rate of 86.6%. Five urine samples tested positive for HPV DNA (0.4%). CONCLUSIONS: We found very low genital HPV infection rate among women from monogamous community. This is the first major population based study carried out among asymptomatic rural women to detect genital HPV infectio from Karnataka using urine samples.

Herbal plants and plant preparations as remedial approach for viral diseases.

Herbal plants, plant preparations and phytoconstituents have proved useful in attenuating infectious conditions and were the only remedies available, till the advent of antibiotics (many being of plant origin themselves). Among infectious diseases, viral diseases in particular, remain the leading cause of death in humans globally. A variety of phytoconstituents derived from medicinal herbs have been extensively studied for antiviral activity. Based on this rationale, an online search was performed, which helped to identify a large number of plant species harboring antiviral molecules. These herbal sources have been reported individually or in combinations across a large number of citations studied. Activities against rabies virus, Human immunodeficiency virus, Chandipura virus, Japanese Encephalitis Virus, Enterovirus, Influenza A/H1N1 and other influenza viruses were discovered during the literature search. This review includes all such plant species exhibiting antiviral properties. The review also encompasses composition and methodologies of preparing various antiviral formulations around the globe. An elaborate section on the formulations filed for patent registration, along with non-patented formulations, has also been included in this article. To conclude, herbal sources provide researchers enormous scope to explore and bring out viable alternatives against viral diseases, considering non-availability of suitable drug candidates and increasing resistance to existing drug molecules for many emerging and re-emerging viral diseases.