Co-circulation of two Mumps virus genotypes in Assam, India.

Mumps is a vaccine-preventable disease, and research on the vaccine's efficacy has recently indicated declining efficacy that has failed to protect against primary infections or reinfections, leading to a global resurgence in nations that use mumps vaccine in their national immunization programmes (NIPs). Lack of reports on its infection, documentation and published studies prevents it from being recognized as a public health issue in India. The waning of immunity is ascribed to the changes between the circulating and vaccine strains. The goal of the current study was to describe the circulating MuV strains in the Dibrugarh district of Assam, India, from 2016 to 2019. Blood samples were examined for IgM antibodies, and throat swab samples were put through Taqman assay for molecular detection. The small hydrophobic (SH) gene was targeted for genotyping through sequencing, and its genetic variations and phylogenetic analysis were carried out. Mumps RNA was found in 42 cases, and Mumps IgM in 14, of which 60% (25/42) of the cases were male and 40% (17/42) were female mostly affecting children between the ages of 6 and 12. Sequence and phylogeny analyses of SH gene revealed Genotypes C (83%) and G (17%) were simultaneously circulating during the study period. The study offers crucial genetic baseline information for the creation of Mumps prevention and control measures. Therefore, based on the research, it is clear that developing an effective vaccination strategy should take into account all currently prevalent genotypes in order to provide better protection against the disease's comeback.

T cell responses in symptomatic moderate patients with pandemic 2009 H1N1 influenza A virus infection.

The response of the host immune system should be appropriate to fight against pandemic 2009 H1N1 (pH1N1) influenza A virus without causing damage to its self. T cells play an indispensable role in the fight against the virus, but have the potential to cause host immunopathological changes. A better understanding of the immunoregulation that occurs during pH1N1 infection is necessary for preventing severity of the disease. In this study, we found that a significantly higher percentage of Vδ1+ T cells and increased expression of activation markers in total T cells in patients with moderate pH1N1 infection could lead to its efficient fight against the virus. On the other hand, the percentages of total and CD4+ T cells were decreased along with an increased expression of exhaustion marker-Tim-3 on T cells that might suppress excessive T cell responses in the host. This tuning of T cell responses might be necessary in efficient combat against pH1N1 virus, without aggravating T cell mediated immunopathology in patients with moderate pH1N1-infection. Keywords: pH1N1; T cells; activation; exhaustion; Tim-3.

Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling.

BACKGROUND & OBJECTIVES: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. METHODS: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed. RESULTS: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ctvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. INTERPRETATION & CONCLUSIONS: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.

Geographical distribution of primary & secondary dengue cases in India - 2017: A cross-sectional multicentric study.

Background & objectives: Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India. Methods: A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases. Results: Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups. Interpretation & conclusions: Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme.

Polymorphism of HLA class I and class II alleles in influenza A(H1N1)pdm09 virus infected population of Assam, Northeast India.

Human leucocyte antigen (HLA) represents one of the most highly polymorphic systems which plays a central role in the immune response. Genetic polymorphism of HLA in influenza A(H1N1)pdm09 infected population may be an important factor in disease progression and severity that needs further probing. In this study, a total of 110 Influenza like illness patients were recruited from the population of Assam, Northeast India, from which 35 cases infected by A(H1N1)pdm09 viruses and 35 controls were typed for HLA-A, B and DRB1 locus by PCR-SSP method. A total of seven alleles of HLA-A, 16 alleles of HLA-B, and 11 alleles of HLA-DRB1 locus were identified. The most common alleles within each locus in cases were HLA-A*11 (85.71%, P = 0.046), HLA-B*35 (25%, P = 0.0001), and HLA-DRB1*15 (49.35%, P = 0.133) as compared to the controls, HLA-A*11 (40.82%), HLA-B*35 (0.00%), and HLA-DRB1*15 (67.53%). The frequency of HLA-A*11 and HLA-B*35 were significantly higher in cases as compared to the controls. In DRB1 locus, HLA-DRB1*10 was significantly higher in cases (20.78%, P = 0.005) than that of controls (0.00%). Whereas, HLA-DRB1*15 showed a higher frequency in controls than in cases. In addition, HLA-DRB3*01 (P = 0.053), DRB4*01 (P = 1.000), and DRB5*01(P = 0.591) were also identified along with HLA-DRB1 haplotype. From this preliminary study, it is suspected that there may be a role of HLA-A*11, HLA-B*35 and HLA-DRB1*10 in conferring susceptibility to influenza A(H1N1)pdm09 infection in the study population. A larger extended study on HLA polymorphism may explain the association between HLA and influenza A(H1N1)pdm09 infection and provide insights for HLA restricted peptide based vaccines.

Exposure to Plasmodium vivax is associated with the increased expression of exhaustion markers on γδ T lymphocytes.

Gamma delta (γδ) T cells exhibit potent anti-Plasmodium activity but are also implicated in the immunopathology of malaria. It is currently poorly understood how γδ T cells are affected in human suffering from Plasmodium vivax infection or in symptomless individuals living in an endemic region. We examined both the percentages and expression of markers associated with immune exhaustion in γδ T cells in individuals living in a P. vivax endemic region by flow cytometry. The percentage of γδ T cells in the blood was significantly higher both in acute P. vivax-positive patients and in individuals from an endemic region in comparison with control uninfected adults. The frequency of the expression of the exhaustion markers-Tim-3, Lag-3, CTLA-4 and PD-1 was higher in γδ and total T cells from P. vivax-infected patients than in those populations from control uninfected adults. Individuals from a P. vivax endemic region showed elevated percentages of Tim-3-, Lag-3- and CTLA-4-positive γδ T cells and an increased percentage of Tim-3-positive total T cells. The phenotypic exhaustion of these cells might be a protective mechanism preventing the immunopathology associated with activated T cells and may provide a rationale for targeted manipulation of this process in diseases such as malaria.

Cutaneotrichosporon (Trichosporon) debeurmannianum: A Rare Yeast Isolated from Blood and Urine Samples.

Cutaneotrichosporon (Trichosporon) debeurmannianum is a rarely isolated yeast from clinical samples. Nine isolates of this yeast were identified from clinical samples within a period of 3 years from June 2012 to May 2015. These isolates were from blood and urine samples sent to a clinical mycology laboratory of a tertiary care hospital in Assam, North East India. Clinically, the patients were diagnosed as septicemia and urinary tract infection. The age of the patients ranged from 2 to 50 years. Identification was made by sequencing the ITS region of ribosomal RNA gene. Antifungal susceptibility test by disk diffusion method (CLSI, M44-A) showed all the isolates to be sensitive to fluconazole and voriconazole. Vitek 2 compact commercial yeast identification system misidentified this yeast as Cryptococcus laurentii and low discrimination Cryptococcus laurentii/Trichosporon mucoides. This species was originally named as Trichosporon debeurmannianum. In 2015, this yeast has been included into new genera Cutaneotrichosporon based on an integrated phylogenetic classification of the Tremellomycetes. To the best of our knowledge, this is the first report of identification of this species from blood and urine samples of clinically suspected cases. We are reporting these isolates because of their rarity in clinical samples. The pathogenic potential and epidemiological relevance of this yeast remains to be seen.

Outbreak of influenza-like illness investigated during August 2013 near Indo-China border of Arunachal Pradesh, Northeast India.

During August 2013, an outbreak of influenza-like illnesses (ILI) was investigated in Monigong area, near Indo-China border of Arunachal Pradesh, Northeast India. Influenza type A/H3N2 was detected by RT-PCR in 33.3% (8/24) of ILI cases. Sequence analysis of HA and NA genes revealed eight and five amino acid substitutions, respectively in Monigong H3N2 (Mo/H3N2) strains as compared to vaccine strain A/Victoria/361/2011. Four non-synonymous substitutions, three localizing at antigenic sites T144A, A; R158G, B; L173S, D, and one H9Y in close proximity to a potential glycosylation site aa8 in HA1 domain along with the substitution T329N in NA are likely to influence the antigenicity/virulence of Mo/H3N2 viruses. J. Med. Virol. 88:1999-2003, 2016. © 2016 Wiley Periodicals, Inc.

Seroprevalence of Toxoplasma gondii Among Pregnant Women in Northeast India.

OBJECTIVE: Toxoplasma gondii infection is primarily asymptomatic and one third of world's population is estimated to be infected by this protozoan parasite. This study was carried out to determine the seroprevalence of T. gondii infection among pregnant women from north east India, where data on this important parasitic infection is scanty. METHODS: A total of 1141 serum archival samples collected from antenatal clinic attendees in 2007-09, were screened for T. gondii IgG by ELISA and analyzed with their socio demographic information. RESULTS: The median age of the subjects were 25 years with an overall IgG seroprevalence of 48% (95% CI=45% to 51%). Seroprevalence was significantly associated with geographical location (p=0.000), among Mongoloids compared to Caucasoid (p=0.005), regular employees (p=0.003) or unskilled labors (p=0.04) compared to housewives, high or middle income group (p=0.003) compared to low income group and with increasing gravida (p=0.04). However on logistic regression analysis, only significant association was with geographical location (p=0.000) and occupation (unskilled labor) (p=0.04). Highest prevalence of 66.7% was noted among subjects with history of blood transfusion and lowest among Rh negative blood group (14.3%). CONCLUSIONS: T. gondii infection prevalence is high among pregnant women living in hilly states of northeast India, which warrants a detail investigation on congenital toxoplasmosis as well as its economic impact.

First isolation and characterization of SARS-CoV-2 from COVID-19 patient of North East India.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) responsible for the current pandemic has resulted in over 5 million deaths globally. More than a year has passed, still SARS-CoV-2 panic the public life. Virus isolation is of paramount importance for development of vaccines, in-vitro screening of antiviral compounds, pathogenesis studies, etc., Many cell lines were studied for amplification and replication of SARS-CoV-2 and Vero cells were found to be ideal cell lines for isolation. In May 2020, ICMR-Regional Medical Research Centre, NE region, India, successfully established the SARS-CoV-2 culture system in Vero CCL-81 cell lines. Phylogenetic analyses of the whole genome sequences of the SARS-CoV-2 isolate (EPI_ISL_2501532 | 2020-05-19) showed monophyletic clade G and lineage B.1.1.

An outbreak of acute hemorrhagic conjunctivitis due to Coxsackievirus A24 in a residential school, Naharlagun, Arunachal Pradesh: July 2023.

PURPOSE: An acute conjunctivitis outbreak was investigated at a residential school in Naharlagun, Arunachal Pradesh, Northeast India, in July 2023. We aimed to identify the etiological agent and assess any complications in follow-up cases. METHODS: We used a structured questionnaire to record clinical findings and followed up with cases one-month post-conjunctivitis. Sixty-one cases were examined and eight conjunctival and oropharyngeal swab samples were collected after obtaining informed consent from guardians/school authorities. We screened for 33 viral and bacterial pathogens using an IVD-approved Real-time PCR assay. Further, the samples were subjected to nucleic acid sequencing. RESULTS: Among 465 screened students and staff, 80 individuals (approximately 17.2%) showed acute hemorrhagic conjunctivitis symptoms among which 61 cases were available for clinical examination. We identified the Enterovirus responsible by targeted sequencing using next-generation sequencing. The etiological agent was found to be Coxsackievirus A24, a member of Enterovirus C, in seven out of eight samples subjected to sequencing. Common symptoms included conjunctival hyperemia and foreign body sensation (100%), bilateral eye involvement (73.8%), eye pain (70%), watery discharge (49.2%), and eyelid swelling (38%). Only 6.5% had purulent discharge. Most cases resolved within 5-6 days, with only 9.8% reporting abdominal symptoms post-conjunctivitis. No serious complications occurred within one month. Throat swabs aided in diagnosing enterovirus infections alongside eye swabs. CONCLUSIONS: The outbreak of acute conjunctivitis was caused by Coxsackievirus A24, a member of Enterovirus C. Cases resolved spontaneously within 6-7 days, with no severe complications. Collecting oropharyngeal swabs alongside conjunctival swabs could improve enteroviral conjunctivitis diagnosis.

Molecular characterization of human respiratory syncytial virus NA1 and GA5 genotypes detected in Assam in northeast India, 2009-2012.

A significant number of children die each year from acute respiratory tract infections especially in developing countries. Human respiratory syncytial virus (RSV) is the most common virus identified in such cases. Genetic characterization and the circulation pattern of RSV is important for future selection of appropriate vaccine strains. Limited information is available on the circulation of RSV in developing countries including India. The present study aimed to provide baseline information on the genetic variability of RSV in the Dibrugarh district of Assam, northeast India. Clinical specimens collected from children aged ≤6 years for routine influenza surveillance in the Dibrugarh district of Assam during the period 2009-2012, were screened for RSV by real-time reverse transcription-polymerase chain reaction. Genotyping was based on partial sequencing of the RSV attachment glycoprotein gene. RSV was detected in 7.9% (39/493) of cases. Only RSV group A viruses were detected during the study period with predominance of NA1 genotypes (89%). Two RSV GA5 genotypes were found to be co-circulating during 2012. The specific amino acid substitutions characteristics of the NA1 genotypes were distinct from RSV strains reported from the rest of India. It is concluded that the circulating genotypes of RSV in Assam, northeast India are NA1 and GA5. To our knowledge this is the first report of circulation of the NA1 genotype in India.

Rapid diagnosis of COVID-19 using disposal paper capacitive sensor.

Accurate and rapid detection and isolation become indispensable to restrict the spread of COVID-19. Since the start of COVID-19 pandemic in December 2019, many indisposal diagnostic tools are being developed incessantly. Out of all presently used tools, the gold standard rRT- PCR tool having very high sensitivity and specificity is a time consuming complicated molecular technique having requirements of special expensive equipment. Here, the main focus of this work is to develop rapid disposal paper capacitance sensor having simple and easy detection. We discovered a strong interaction between limonin and Spike-glycoprotein of SARS-COV-2 in comparison to its interaction with other similar viruses such as HCOV-OC43, HCOV-NL63, HCOV-HKU1, Influenza B and A viruses. The antibody free capacitive sensor having comb electrode structure was fabricated on whatman paper with drop coating of limonin (extracted using green method from pomelo seeds) and calibrated with known swab samples. The Blind test with unknown swab samples shows high sensitivity of 91.5% and high specificity of 88.37%. Requiring low sample volume and detection time and using biodegradable materials in the sensor fabrication assure the potential application as a point of care disposal diagnostic tool.

Norovirus gastroenteritis in children under-five years hospitalized for diarrhea in two cities of northeast India: A retrospective study.

PURPOSE: Norovirus gastroenteritis, known to cause 'winter vomiting disease' is increasingly being identified as a major cause of viral gastroenteritis worldwide. The impact and prevalence of this viral disease are lacking in many parts of India including northeast India. This study aimed to determine the prevalence and association of norovirus gastroenteritis among under-five-year-old hospitalized children in two cities in northeast India (Dibrugarh in Assam & Dimapur in Nagaland). MATERIALS AND METHODS: A retrospective analysis of 407 randomly selected diarrheal stool samples was conducted using a commercial multiplex probed-based real-time RT-PCR assay capable of detecting six-viral gastroenteritis pathogens including Norovirus GI, Norovirus GII, Rotavirus, Human Adenovirus, Human Astrovirus, and Sapovirus. RESULTS: Results showed that norovirus was detected in 18.4% of the samples (75/407; 95% CI: 14.8%-22.5%), with norovirus genogroup II being the predominant group in 97.3% of norovirus cases. A significant association of norovirus diarrhea was found with seasonality, with higher prevalence in colder months compared to warmer months (22.4% vs 9.1%, p-value:0.002). Additionally, 66.7% (50/75) of cases of norovirus gastroenteritis had reported vomiting as the major symptom and had a shorter duration of diarrhea (p-value 0.03). Co-infections with other viral pathogens were seen in 45.9% (187/407) of the cases. The detection of rotavirus was 67.1% (273/407), human adenovirus (45.9%; 187/407), sapovirus and astrovirus (5.9%, 24/407 each), and norovirus GI (0.5%, 2/407) among the cases. CONCLUSION: This study reports the prevalence of norovirus gastroenteritis in northeast India and further highlights that norovirus gastroenteritis is responsible for substantial cases of hospitalization of under-five years children in the region.

Presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli isolates recovered from an apex medical institute in North India.

PURPOSE: To detect the presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli (UPEC) isolates recovered from patients coming to the out patient department (OPD) of our hospital. METHODS: E. coli isolates recovered from patients who had symptoms of urinary tract infection (UTI) were processed for the presence of fimH and iss genes. DNA was extracted using an in house method after which conventional PCR using forward and reverse primers targeting the four genes was carried out. The amplified products were electrophoresed and visualized in a gel documentation imager. Relevant demographic details of the patients were recorded on a pre-designed pro-forma and antimicrobial susceptibility testing of the isolates was done by disc diffusion method. RESULTS: fimH was present in 87.5% of UPEC isolates whereas iss type 1 was seen in 7.3%, type 2 in 4.2% and iss type 3 in 71.9% isolates. Age of the patients ranged from 3 months to 82 â€‹yrs (mean 43.5 SD â€‹± â€‹18.20). UTI was more common in females (60.2%) as compared to males patients (39.8%). Dysuria (66.7%) was the most common symptom in the studied subjects and diabetes mellitus (42.6%) the most common co-morbidity. A total of 56.5% patients gave a history of prior antibiotic intake. The UPEC isolates were resistant to most of the antibiotics tested. However all the isolates were sensitive to polymyxin B and colistin. Fosfomycin resistance was seen in 9.5% of the UPEC isolates harbouring fimH gene. CONCLUSION: This is the first study that highlights the presence of iss type 3 gene in UPEC isolates along with the fimH and iss type 1 and 2 genes. The results of this study can serve as a stepping stone for future in depth research into the significance of the iss genes in causing UTI.

First Report of Rubber Collection Bowls & Plastic and Bamboo Water Containers as the Major Breeding Source of Ae. albopictus with the Indigenous Transmission of Dengue and Chikungunya in Rural Forested Malaria-Endemic Villages of Dhalai District, Tripura, India: The Importance of Molecular Identification.

BACKGROUND: With the reports of indigenous cases of dengue and chikungunya in the forest-covered rural tribal malaria-endemic villages of Dhalai District, Tripura, India, an exploratory study was undertaken to identify the vector breeding sites. METHODS: From June 2021 to August 2022, mosquito larvae were collected from both natural and artificial sources in the villages, house premises, and their nearby forested areas outside of the houses. Other than morphological characterisation, Aedes species were confirmed by polymerase chain reaction targeting both nuclear (ITS2) and mitochondrial genes (COI) followed by bidirectional Sanger sequencing. RESULTS: Aedes albopictus was abundantly found in this area in both natural and artificial containers, whereas Ae. aegypti was absent. Among the breeding sources of molecularly confirmed Ae. albopictus species, rubber collection bowls were found to be a breeding source reported for the first time. Plastic and indigenously made bamboo-polythene containers for storing supply water and harvesting rainwater in the villages with a shortage of water were found to be other major breeding sources, which calls for specific vector control strategies. Natural sources like ponds and rainwater collected on Tectona grandis leaves and Colocasia axil were also found to harbour the breeding, along with other commonly found sources like bamboo stumps and tree holes. No artificial containers as a breeding source were found inside the houses. Mixed breeding was observed in many containers with other Aedes and other mosquito species, necessitating molecular identification. We report six haplotypes in this study, among which two are reported for the first time. However, Aedes aegypti was not found in the area. Additionally, rubber collection bowls, ponds, and water containers also showed the presence of Culex quinquefasciatus and Culex vishnui, known JE vectors from this area, and reported JE cases as well. Different Anopheles vector spp. from this known malaria-endemic area were also found, corroborating this area as a hotbed of several vectors and vector-borne diseases. CONCLUSIONS: This study, for the first time, reports the breeding sources of Aedes albopictus in the forested areas of Tripura, with rubber collection bowls and large water storage containers as major sources. Also, for the first time, this study reports the molecular characterisation of the Ae. albopictus species of Tripura, elucidating the limitations of morphological identification and highlighting the importance of molecular studies for designing appropriate vector control strategies. The study also reports the co-breeding of JE and malaria vectors for the first time in the area reporting these vector-borne diseases.