Outbreaks and sporadic cases of japanese encephalitis in the state of Odisha, India: Outcome of 7 years of laboratory surveillance.

Acute encephalitis syndrome (AES) is a major public health challenge in India. We report here the epidemiology of sporadics and outbreaks of Japanese Encephalitis (JE) in Odisha state during 2012-2018. A total of 4235 AES cases (sporadics - 3394, outbreak cases - 841) recorded including 42 outbreaks; majority (n = 18) of which were during 2016. Overall JE virus (JEV) positivity was 11.78% (outbreak cases - 24.5%, sporadic cases - 8.6%). Age ≤15 years were largely affected during outbreaks, while 16-60 years population was dominant among sporadics. The major outbreak (2016) involved 336 patients from a tribal dominated district, spread over 173 villages. JEV seropositivity was high (43.45%) with 28.57% mortality. Epidemiological linkage with pig rearing was documented through JEV neutralizing antibodies in 50% of pig serum samples. Although the postvaccination period (2017-18) showed increase in AES case reporting but low JE proportion. Ongoing surveillance and preparedness of the health system would be of importance, especially in tribal-dominated districts.

A comparison of SARS-CoV-2 rapid antigen testing with realtime RT-PCR among symptomatic and asymptomatic individuals.

BACKGROUND: Identification of SARS-CoV-2 positive patients with rapid and cost-effective test methods is the key for isolating infected individuals, interrupting the transmission chain, and thus, containment of the CoVID-19 disease. In this regard, Rapid Antigen Test (RAT) plays an important role at point of care testing but the low sensitivity attributing towards escape of positive cases is reported as a major disadvantage of RAT which led us to evaluate a RAT kit among symptomatic and asymptomatic individuals suspected of CoVID-19. METHODS: We analyzed 329 parallel nasopharyngeal swabs for RAT (Zydus Cadila, India) at the point of collection in a hospital-based facility and RealTime RT-PCR in the laboratory. The performance parameters were analyzed by evaluating the specificity, sensitivity, Negative Predictive Value (NPV), Positive Predictive Value (PPV), and Kappa coefficient. RESULTS: The sensitivity and specificity were found to be 75.17% and 98.89% respectively. Positive Predictive value was 98.25% and the negative predictive value was 82.79%. The accuracy between the two techniques was found to be 88.14% with a kappa coefficient of 0.756 (SE: 0.036 and CI at 95%: 0.686 to 0.826) with a good strength of agreement (0.61-0.80) between the two testing techniques. Among the false-negative cases, 22 (59.5%) were asymptomatic having the Cycle Threshold (Ct) range 27 to 32.9 including 12 cases with a history of close contact with the known positive cases (i.e. household contact). The remaining 15 cases (40.5%) were symptomatic having low to moderate Ct values. CONCLUSION: It is observed from the results that the false negative result for symptomatic individuals is a matter of concern as it was noted in 4 cases of our study subjects who required hospitalisation later. Also the positives among asymptomatic contacts are important from epidemiological point of view for isolation and curtailing the infection from spreading in a community. These results support the fact that RAT showing sensitivity below 80% can be used for mass screening purposes with provision for additional testing in case of false negative with symptomatic individuals. Also false-negative results should be interpreted cautiously considering the epidemiological link as well as the clinical condition of the patients.

Expansion of the measles and rubella laboratory network, India.

Objective: To expand the measles and rubella laboratory network of India by integrating new laboratories. Methods: In collaboration with the World Health Organization (WHO), the Indian government developed a 10-step scheme to systematically expand the number of laboratories performing serological and molecular testing for measles and rubella. The Indian Council of Medical Research and WHO identified suitable laboratories based on their geographical location, willingness, preparedness, past performance and adherence to national quality control and quality assurance mechanisms. The 10-step scheme was initiated with training on measles and rubella diagnostic assays followed by testing of both measles and rubella serology and molecular unknown panels, cross-verification with reference laboratories and ended with WHO on-site accreditation. Findings: After extensive training, technical support, funding and monitoring, all six selected laboratories attained passing scores of 90.0% or more in serological and molecular proficiency testing of measles and rubella. Since 2018, the laboratories are a part of the measles and rubella network of India. Within 12 months of initiation of independent reporting, the six laboratories have tested 2287 serum samples and 701 throat or nasopharyngeal swabs or urine samples. Conclusion: The process led to strengthening and expansion of the network. This proficient laboratory network has helped India in scaling up serological and molecular testing of measles and rubella while ensuring high quality testing. The collaborative model developed by the Indian government with WHO can be implemented by other countries for expanding laboratory networks for surveillance of measles and rubella as well as other infectious diseases.

Breakthrough SARS-CoV-2 infections among BBV-152 (COVAXIN®) and AZD1222 (COVISHIELDTM ) recipients: Report from the eastern state of India.

In this study, we attempted to record the breakthrough cases reported through passive and voluntary reporting at various healthcare facilities from different districts of Odisha, their clinical presentation, requirement of hospitalization postinfection, and antibody titer against spike antigen. Nasopharyngeal swab and serum samples alongwith demographic, clinical presentation and requirement of hospitalization postinfection were collected from vaccinated individuals through passive and voluntary reporting to various healthcare facilities of Odisha state to detect the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection and quantitative estimation of antibody titers. A total of 274 samples were found to be positive after 14 days of receiving complete doses of the vaccines. More than 83.2% of the individuals were found to be symptomatic with 9.9% of those required hospitalization. The seropositivity in individuals receiving Covishield (96.7%) was significantly higher than in Covaxin (77.1%). Hospitalized patients were having less median antibody titers than individuals in home isolation. The median age for breakthrough infection among the referred cases was 47.0 years (interquartile range [IQR]: 28.0) with a significantly older age group among Covishield recipients. The median spike receptor binding domain IgG titer values for Covaxin and Covishield recipients were 213.5 AU/ml (IQR: 537.5) and 647.5 AU/ml (IQR: 1645.1), respectively. The results reported here highlight the need for systematic data capture for the breakthrough infections to monitor the emergence of any vaccine escape variants and to plan the next steps in the coronavirus disease-19 (COVID-19) vaccine development by understanding the link between clinical protection and measured immunity against SARS-CoV-2 infection.

COVID-19 in children in Odisha state, India: a retrospective review.

We retrospectively analysed the swab samples tested for COVID-19 from 7 March 2020 to 17 August 2021 at the Indian Council of Medical Research-Regional Medical Research Centre, Bhubaneswar, Odisha. 553 763 nasopharyngeal swabs were collected from individuals suspected with COVID-19 in Odisha state. 75 190 (13.6%) samples were positive by reverse transcription-PCR. There were 5988 (8%) cases in children and young people under 18 years old. Odisha reported 996 153 COVID-19 cases which resulted in 6985 deaths in adults and 36 in children and young people under 18 years old.

Viral characteristics and clinical presentation in dengue co-infection- Findings from a facility based observational study in Odisha, India.

BACKGROUND: Dengue has affected many countries globally. Two-fifths part of the world is at risk, which can be affected by dengue disease. In India, the dengue incidence has increased in the recent past and emerged as an important health problem in many states including Odisha. Dengue disease presents with atypical clinical symptoms when associated with other co-infections. MATERIALS AND METHODS: A facility-based longitudinal study was carried out over a period of 1 year to determine the dengue co-infection and its outcome. The suspected cases were clinically assessed following a standard case report format and serological investigations including serotyping were carried out. RESULTS: 33.6% samples were dengue positive of which 78.5% were positive for NS1 Ag, 26.6% positive for dengue IgM and 5.1% to both. Among the dengue positive cases, 60.9% were male and mean age was 31.52 (±17.03) years. High occurrence of cases was during May to November with maximum in August. Among the 975 dengue positives, 57 (5.8%) were found to have co-infection. Chikungunya was the most common co-infection in 71.9%, followed by herpes simplex (HSV) (7%) and other diseases. Fever was the most common presenting symptom (98.2%), followed by myalgia (91.2%), retro orbital pain (91.2%), pain abdomen (12.3%), rash/lesion (8.8%), burning micturition (5.3%), petechiae (1.7%) and pruritus (1.7%) among the co-infected cases. CONCLUSIONS: All the four dengue serotypes were found to be circulating with DEN 2 as the most predominant one. About 5.8% of dengue cases have co-infection (mainly with Chikungunya) and clinically present with atypical signs and symptoms.

Yielding quality viral RNA by using two different chemistries: a comparative performance study.

Purity and integrity are two important criteria for any RNA extraction process to qualify the RNA for meaningful gene expression analysis. This study compares four commercially available RNA extraction kits using silica membrane and magnetic bead separation methods. The performance was evaluated in terms of both quantity (total RNA amount in µg/µl) and purity (260/280 ratio). The concentration and purity of each kit was significantly different from those of the others (p < 0.001). Although quantity obtained from Mag MAX is comparatively lower than QIAGEN, the quality is comparable as evident from real-time PCR performance. This study suggests that there are practical differences between these RNA extraction kits that should be taken into account while isolating RNA required for gene expression analysis.

Prevalence and trend of emerging and re-emerging arboviral infections in the state of Odisha.

Newly emerging or re-emerging infections are posing continuous threat to both public health system and clinical care globally. The emergence of infections especially caused by arboviruses can be linked to several mechanisms which include geographical expansion linked to human development and transportation, global warming, enhanced transmission in peridomestic area and close proximity of human habitations to domestic as well as wild animals. The co-circulation of Dengue, Chikungunya and Zika is a matter of public health priority due to the fact that they are transmitted by the same vector as well as increase in the number of reported cases of severe dengue, post-chikungunya chronic joint disease and microcephaly related to Zika virus disease. The study was designed to estimate the prevalence of these arboviral infections in Odisha. About 5198 cases presenting with common clinical symptoms of fever, arthralgia, headache, myalgia and malaise were screened during 2016-2019. A total of 42.2% patients tested positive for dengue NS1 antigen (n = 4154), 30.2% for dengue IgM (n = 2161) and 14.3% for chikungunya IgM (n = 1816). A total of 1684 samples were subjected to Zika RT-PCR and none was tested positive. Peak in the numbers of dengue/ chikungunya cases was evident in the post-monsoon months of July - October. Circulation of all four serotypes of dengue i.e. DEN 1, 2, 3, and 4 was noticed in the state. Molecular investigation of suspected Chik cases in early phases showed circulation of Eastern Central Southern African genotype (E1:226A). There is dearth of knowledge about disease severity during arbovirus co-infections and importance of adequate management of patients at an early stage residing in risk areas. It is the first study in Odisha to study the pattern and status of these three arboviral diseases Dengue, Chikungunya and Zika. The outcome of this study will help in focusing and improvement of existing surveillance systems and vector control tools, as well as on the development of suitable antiviral agents and formulating candidate vaccine.

Burden of Rubella virus infection among females attending tertiary care hospitals of Odisha, India: a need for adult women vaccination.

Rubella is a contagious disease caused by rubella virus leading to adverse outcomes among pregnant women including abortions, low birth weight, stillbirths and congenital rubella syndrome (CRS) in the baby. If not pregnant, the clinical manifestations are mild and self-limiting. In this hospital based cross-sectional study, 1985 blood samples were collected from females attending outpatient services of various hospitals to serologically detect Rubella infection. Rubella antibodies namely Immunoglobulin M (IgM)/Immunoglobulin G (IgG) were detected through enzyme linked immunosorbent assay (ELISA) or by identifying virus through polymerase chain reaction (PCR). From the total enrolled participants, 1951 samples were tested with age ranging from 16 to 38 years. Among the positive samples, about 60% patients had IgG antibodies as compared to less than 1% IgM and 0.40% by PCR. Out of 1951 samples, 7/849 (0.82%) and 651/1070 (60.8%) had IgM & IgG rubella antibodies respectively. The odds of having abortion was [OR-13.14 (4.94-34.97)] among anti-rubella positive and primi-gravida [OR-43.6 (5.9-322)] women. Therefore, vaccination of women against rubella before planning of pregnancy or at adolescence seems to be the need of hour to avoid the ill consequences during pregnancy as well as for the new born baby.

Sequence analysis of Indian SARS-CoV-2 isolates shows a stronger interaction of mutant receptor-binding domain with ACE2.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected the whole world, including Odisha, a state in eastern India. Many people have migrated to the state from different countries as well as other states during this SARS-CoV-2 pandemic. The aim of this study was to analyse the receptor-binding domain (RBD) sequence of the spike protein from isolates collected from throat swab samples of COVID-19-positive patients and further to assess the RBD affinity for angiotensin-converting enzyme 2 (ACE2) of different species, including humans. METHODS: Whole-genome sequencing for 35 clinical SARS-CoV-2 isolates from COVID-19-positive patients was performed by ARTIC amplicon-based sequencing. Sequence analysis and phylogenetic analysis were performed for the spike region and the RBD region of all isolates. The interaction between the RBD and ACE2 of five different species was also analysed. RESULTS: The spike region of 32 isolates showed one or multiple alterations in nucleotide bases in comparison with the Wuhan reference strain. One of the identified mutations, at position 1204 (Ref A, RMRC 22 C), in the RBD coding region of the spike protein showed stronger binding affinity for human ACE2. Furthermore, RBDs of all the Indian isolates showed binding affinity for ACE2 of different species. CONCLUSION: As mutant RBD showed stronger interaction with human ACE2, it could potentially result in higher infectivity. The binding affinity of the RBDs for ACE2 of all five species studied suggests that the virus can infect a wide variety of animals, which could also act as natural reservoir for SARS-CoV-2.

Analysis of Indian SARS-CoV-2 Genomes Reveals Prevalence of D614G Mutation in Spike Protein Predicting an Increase in Interaction With TMPRSS2 and Virus Infectivity.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has emerged as a global pandemic worldwide. In this study, we used ARTIC primers-based amplicon sequencing to profile 225 SARS-CoV-2 genomes from India. Phylogenetic analysis of 202 high-quality assemblies identified the presence of all the five reported clades 19A, 19B, 20A, 20B, and 20C in the population. The analyses revealed Europe and Southeast Asia as two major routes for introduction of the disease in India followed by local transmission. Interestingly, the19B clade was found to be more prevalent in our sequenced genomes (17%) compared to other genomes reported so far from India. Haplotype network analysis showed evolution of 19A and 19B clades in parallel from predominantly Gujarat state in India, suggesting it to be one of the major routes of disease transmission in India during the months of March and April, whereas 20B and 20C appeared to evolve from 20A. At the same time, 20A and 20B clades depicted prevalence of four common mutations 241 C > T in 5' UTR, P4715L, F942F along with D614G in the Spike protein. D614G mutation has been reported to increase virus shedding and infectivity. Our molecular modeling and docking analysis identified that D614G mutation resulted in enhanced affinity of Spike S1-S2 hinge region with TMPRSS2 protease, possibly the reason for increased shedding of S1 domain in G614 as compared to D614. Moreover, we also observed an increased concordance of G614 mutation with the viral load, as evident from decreased Ct value of Spike and the ORF1ab gene.

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.

High Prevalence of Hepatitis C Virus Infection in Primitive Tribes of Eastern India and Associated Sociobehavioral Risks for Transmission: A Retrospective Analysis.

Purpose: The primitive tribal groups (PTGs) need special attention because of their low population growth: declining population size with high mortality rates. Scanty reports are available on the prevalence of hepatitis C virus (HCV) infection in primitive tribes of the country emphasizing their cultural and social practices associated with transmission of the disease. Methods: The study was conducted on 1765 tribal individuals covering 5 PTGs, namely Lodha, Saora, Khadia, Juanga, and Mankidia, from 6 districts of Odisha. Serum samples were tested for the anti-HCV antibody using commercially available enzyme immunoassays. HCV RNA was detected based on the 5' NCR region and genotyping was done by direct sequencing of the core region. Potential risk factors for HCV transmission were collected using a questionnaire and subjected to regression analysis through SPSS, version 17.0. Results: Antibody to HCV was detected in 0%, 3.3%, 5.7%, 8.5%, and 13.4% in Saora, Lodha, Khadia, Juanga, and Mankidia tribes, respectively. HCV RNA was detected in 8.6% (11/127) of the samples tested. Genotyping of HCV isolates in all HCV RNA-positive samples revealed genotype 1b. Sharing of razors and shaving by the village barber were found to be significantly (p<0.05) associated with HCV transmission in males, whereas tattooing and multiple injections were found to be significant risk factors for females. Conclusion: This study indicated a high prevalence of HCV infection in Mankidia and Juanga tribes in comparison with the national scenario, which needs public health attention.

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.

An outbreak of hepatitis E virus infection caused by genotype 1 in an urban setting in eastern India: a probe into risk factors for transmission.

The study was undertaken to find out the cause and etiology of an outbreak presumed to be due to water contamination that caused high morbidity in the western part of the state of Odisha during May, 2014. In this investigation 56 blood samples were collected and tested for HEV IgM through ELISA. Blood sample of 22 patients collected within 1 weeks post onset of symptoms and were subjected to RT-PCR and sequencing followed by phylogenetic analysis. Water samples were also analyzed for viral and bacterial contamination. A total of 290 individuals were examined for suspected jaundice. Out of 56 blood samples in 41 (73.2%) IgM for HEV was found. 12 samples out of 22 early phase samples were positive for RT-PCR and through phylogenetic analysis all were found to be of Genotype 1 and subtype A. This large outbreak was confirmed due to Hepatitis E virus and transmission was fast due to contamination of drinking water sources and lack of hygienic practices. The outcome of this investigation has created alertness among state health and municipal authorities to be prepared for the similar kind of situation for other part of the state.

Hepatitis E infection in Odisha, India: A descriptive analysis.

Up to 25% of hepatitis E virus (HEV)-infected pregnant women in their third trimester die. Despite HEV being an important cause of viral hepatitis, no robust surveillance exists in India. We reviewed jaundice outbreaks records and hospital records from jaundiced individuals seeking treatment and linked those records to laboratory results (HEV immunoglobulin M enzyme-linked immunosorbent assay) for January 2012 to September 2013 in Odisha state. A total of 14 HEV confirmed outbreaks were identified, of which 33% of 139 jaundiced cases were HEV positive. There were two deaths. An additional 495 jaundiced cases were identified through hospital records, of which 18% were HEV positive. Among HEV-positive women (n = 35), 34% were of childbearing age. While one may not be able to generalize our results, this finding suggests HE is widespread in Odisha and may represent hidden disease burden in this region. The policymakers should monitor HEV infections in similar geographical areas, especially among population of childbearing age women to initiate evidence-based control measures.