Objective: Acute hemorrhagic conjunctivitis (AHC), commonly called pink eye, saw an alarming increase in incidence from July to September 2023 in different parts of India. Pink eye occurrences had reportedly increased three to four times more than in prior years, raising concerns among the community and healthcare professionals. This study aimed to identify the aetiological agent associated with AHC in 2023, genetically characterize the agent and describe the clinical presentation. Methods: From July to September 2023, 300 ocular and throat swab samples were collected from patients with AHC across various regions of India, including Maharashtra, Daman & Diu, Delhi, Lucknow, and Hyderabad. These samples represented a diverse geographic spread of the condition. The swabs were examined for qRT-PCR analyses, to detect adenovirus and enterovirus. Following this, conserved regions within the enteroviral 5 '-UTR and VP2/3 C gene were further investigated for serotype identification. Results: Enterovirus was found in 52.6 % (158 out of 300) of the patients. Among the enterovirus-positive samples, coxsackievirus-A24 was present in every positive sample. Conclusion: The rise in AHC cases in India in 2023 was attributed to the Coxsackievirus-A24 strain GIV C5. (c) 2024 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Coxsackievirus-A6 (CV-A6) is responsible for more severe dermatological manifestations compared to other enteroviruses such as CV-A10, CV-A16, and EV-A71, causing HFMD in children and adults. Between 2005 and 2007, the recombinant subclade D3/RF-A started to expand globally, and a CV-A6 pandemic started. The study aimed to conduct whole-genome sequencing (WGS) of an isolated CV-A6 strain from currently circulating HFMD cases from India in 2022. Gene-specific RT-PCR and sequencing were used to perform molecular characterization of the isolated virus. Confirmation of these isolates was also performed by transmission electron microscopy and WGS. Among eleven positive clinical enterovirus specimens, eight CV-A6 strains were successfully isolated in the RD cell line. Isolates confirmed the presence of the CV-A6 strain based on VP1 and VP2 gene-specific RT-PCR. Sequences of isolates were clustered and identified as the novel CV-A6 strain of the D3/Y sub-genotype in India. The studies revealed that the D3/Y sub-genotype is being introduced into Indian circulation. The predicted putative functional loops found in VP1 of CV-A6 showed that the nucleotide sequences of the amino acid were a remarkably conserved loop prediction compatible with neutralizing linear epitopes. Therefore, this strain represents a potential candidate for vaccine development and antiviral studies.

Viral infection is frequently the cause for acute hemorrhagic conjunctivitis (AHC) epidemics. AHC can result from adenoviruses, with enterovirus 70 and coxsackievirus A24 being the primary agents. AHC was initially identified in Ghana in 1969, caused by enterovirus 70 and leading to a global pandemic. Since 2000, outbreaks of AHC linked to coxsackievirus A24 variant have been documented in Spain, Pakistan, Singapore, India, Korea, and China. A sudden surge of conjunctivitis cases reported in October 2022 in and out of the Hyderabad region. This infection presented with usual symptoms of redness of the eyes, discharge, pain in the eyes and crusting. Occular swab samples from 110 patients were collected in order to identify and characterize the virus that was causing the epidemic. We examined adenovirus, enterovirus, COVID-19 and Herpes Simplex Virus by using commercially kits available at the hospital. Conserved regions in the enteroviral 5'-UTR and VP2 gene were analyzed further for characterization of serotype at the National apex laboratory. None of them was found positive except Enterovirus in 16.36% (18/110) of the patients. From enterovirus-positive samples, the coxsackievirus A24 was observed in all 18 positive samples. These clinical isolates constitute a new lineage cluster associated with genotype IV-C5, according to additional sequencing of the full-length VP2 genes and subsequent phylogenetic analysis. In conclusion, the current outbreak of acute haemorrhagic conjunctivitis in Hyderabad, India was traced to the coxsackievirus A24 strain GIV C5.

IntroductionThere have been a few reports of viral load detection in stool and urine samples of patients with coronavirus disease 2019 (COVID-19), and the transmission of the virus through faecal oral route. For clinical diagnosis and treatment, the widely used reverse transcription-polymerase chain reaction (qRT-PCR) method has some limitations.MethodsThe aim of our study to assess the presence and concentration of SARS CoV-2 RNA in stool and urine samples from COVID-19 patients with mild, moderate, and severe disease, we compared a traditional qRT-PCR approach with a ddPCR. ddPCR and qRT-PCR-based target gene analysis were performed on 107 COVID-19-confirmed patients paired samples (N1 and N2). The MagMax magnetic beads base method was used to isolate RNA. Real-time qRT-PCR and dd PCR were performed on all patients.Results and DiscussionThe average cycle threshold (Ct) of qRT-PCR was highly correlated with the average copy number of 327.10 copies/l analyzed in ddPCR. In ddPCR, urine samples showed 27.1% positivity while for stool it was 100%.ConclusionThis study’s findings not only show that SARS CoV-2 is present in urine and faeces, but also suggest that low concentrations of the viral target ddPCR make it easier to identify positive samples and help resolve for cases of inconclusive diagnosis.

Human adenoviruses (HAdVs) are the viral agents responsible for a wide spectrum of acute and chronic diseases. HAdVs are the most important etiological agents of acute gastroenteritis (AGE) and are identified as the major contributor to the deaths of diarrheal children globally. The significant rise in HAdV infections in rotavirus-vaccinated children documented in multiple studies demands continuous monitoring of HAdV strains. After the inclusion of rotavirus vaccines in the immunization schedule of India, public health research regarding prevalence, etiology, and risk factors is highly necessary for evidence-based policies and their implementation to sustain diarrhea prevention programs. In the present study, children admitted for AGE between 2013 and 2016 in seven different hospitals in Maharashtra and Gujrat states of Western India were subjected for investigation. HAdVs were found in 5.2% of the fecal specimens with the dominance of species-F (52.4%) strains, followed by the occurrence of non-enteric adenoviruses of species A (17.4%), C (11.4%), B (8.2%), and D (3.2%). The species-F strains were predominant in Ahmadabad (78.5%), Mumbai (61.5%), and Surat (57.1%) cities, followed by species-A strains. In Pune city, species B strains were detected in all HAdV patients, with none of the species A strains. Clinically, patients infected with enteric and non-enteric HAdV strains were indistinguishable. However, a high viral load was observed in species-F specimens as compared to non-species-F. The present study on fecal specimens collected in the pre-rotavirus vaccination era from hospitalized AGE patients will be important for future comparative analysis to know the exact impact of vaccination in children of Western India.

Background: Hand, foot, and mouth disease (HFMD) is an enteroviral disease that occurs as outbreaks and sporadic cases in India. In this study, we investigated and characterized the aetiology of HFMD cases that occurred in Karnataka, South India from April to October 2022.Methods: Throat swabs, vesicular swabs, urine, and blood samples from suspected cases were analysed by reverse transcription polymerase chain reaction (RT-PCR) for the detection of enteroviruses. Molecular typing of the enterovirus-positive samples was carried out by amplifying the partial virion protein 1(VP1) gene sequence, followed by sequencing and phylogenetic analysis. Results: Out of the 187 samples received from 82 cases, 93 (50%) tested positive (55/82 cases, 67%) for enteroviruses, with the majority of the HFMD cases reported in paediatric population of less than 5 years (36/55, 65.4%), while 3 cases (3/55, 5.4%) were adults. Out of the 55 enterovirus-positive cases, 31 showed partial VP1 region amplification and 19 of these cases were typed as coxsackievirus A16 (CV-A16) (13/19, 68.4%) and CV-A6 (6/19, 31.6%). The CV-A16 strains identified belonged to subclade B1c while two CV-A6 strains belonged to subclade E2. On molecular testing for other viruses causing fever-rash symptoms, 4/27 (15%) enterovirusnegative cases were detected as herpes simplex virus (1 case) and varicella zoster virus (3 cases) positive.Conclusion: The main causative agent of HFMD in Karnataka in 2022 was CV-A16, followed by CV-A6. Apart from the common paediatric HFMD cases, adult cases were also reported during this period. Further studies involving laboratory and clinical investigations are essential for monitoring and managing HFMD in the community.

Outbreaks of HFMD in children aged <5 years have been reported worldwide and the major causative agents are Coxsackievirus (CV) A16, enterovirus (EV)-A71 and recently CVA6. In India, HFMD is a disease that is not commonly reported. The purpose of the study was to identify the enterovirus type(s) associated with large outbreak of Hand, foot, and mouth disease during COVID-19 pandemic in 2022. Four hundred and twenty five clinical samples from 196-suspected cases were collected from different parts of the country. This finding indicated the emergence of CVA6 in HFMD along with CVA16, soon after the gradual easing of non-pharmaceutical interventions during-pandemic COVID-19 and the relevance of continued surveillance of circulating enterovirus types in the post-COVID pandemic era.

SARS-CoV-2 can be shed in feces and can enter sewage systems. In order to implement effective control measures and identify new channels of transmission, it is essential to identify the presence of infectious virus particles in feces and sewage. In this study, we attempt to utilize Molecular techniques, cell cultures and animal models to find out the infectivity of SARS-CoV-2 in the feces of COVID-19 patients. Our findings exclude the presence of infectious virus particles, suggesting that fecal-oral transmission may not be the main mode of transmission. Larger-scale initiatives are nevertheless required, particularly considering the emergence of new viral strains.

There have been several reports across the globe regarding the presentation of a severe multi-system hyperinflammatory syndrome, resembling Kawasaki disease (KD), in the pediatric population during the SARS-CoV-2 pandemic. The exact pathophysiology is still unclear; however, children typically demonstrate multi-organ dysfunction and less respiratory system involvement compared to adults. The limited literature is available at present for the identification and management of such patients. In this study, we investigated four cases in children ages 11–15 years that fulfilled the case definition for the pediatric multi-system inflammatory syndrome. All were found negative for SARS-CoV-2 from oropharyngeal swabs and stool. As they were having symptoms of diarrhea, tests for bacterial and enteric viral infections were performed after SARS-CoV-2 testing. Molecular analysis revealed that all the children were infected with enterovirus (Echovirus-18). Early and exact diagnosis is vital for timely, effective, and potentially life-saving management of such cases.

The main route of the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are through respiratory pathways and close contact of human-to-human. While information about other modes of transmission is comparatively less, some published literature supporting the likelihood of a fecal-oral mode of transmission has been accumulating. The diagnosis of SARS-COV-2 infected cases is based on the real-time reverse transcription-PCR (RT-PCR). The fecal excretion of SARS-COV-2 has been reported frequently, however, the role of fecal viral load with the severity of disease is not yet clear. Our study focused on the investigation of SARS-CoV-2 shedding in the fecal samples of patients with coronavirus disease 2019 (COVID-19). A total of 280 RT-PCR-positive patients were enrolled, among them 15.4% had gastrointestinal (GI) symptoms. It was shown that 62% of the patients were positive for SARS-CoV-2 RNA in fecal specimens. This positivity was not related to the presence of GI symptoms and the severity of disease. The next generation sequencing [NGS] of SARS-CoV-2 from fecal samples of patients was performed to analyze mutational variations. Findings from this study not only emphasized the potential presence of SARS-CoV-2 in feces, but also its continuing mutational changes and its possible role in fecal-oral transmission.

Sir, Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in December 2019 in Wuhan, China. The pandemic of SARS-CoV-2 as of March 31, 2021 has affected 220 countries and territories around the world including India1. The number of reported SARS-CoV-2 cases in India is 12,039,644 with 1,62,114 deaths2. Pune city remains one of the hotspot cities of SARS-CoV-2 from western India. There are reports from China on SARS-CoV-2 infection in a family setting with person-to-person transmission34. There are no studies on family cluster reported from India which is the leading mode of human-to-human transmission345. In the present study, we report the clinical and laboratory findings, and a transmission pattern of four patients in a family cluster from Pune city. All four members presented with different clinical features after being infected with COVID-19. The index case contracted this infection from the hospital and subsequently infected other members in the family. The present study was a part of a hospital-based study initiated at the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, to investigate the SARS-CoV-2 shedding in excreta of COVID-19 patients during treatment and after recovery. The study was approved by the ethics committees of the institute and the hospital. The family that had four members in total was admitted for COVID-19 treatment. Written informed consent was obtained before the study. The index case was a 37 yr old male (index), a healthcare worker, who had worked in the COVID-19 ward. He was living with his wife (34 yr old) and two children (8 and 6 yr old). Being a healthcare worker, index had been exposed to multiple laboratory-confirmed COVID-19 patients. The index patient presented with fever, nasal discharge and generalized weakness on the onset of symptoms. He was admitted in the hospital and diagnosed with COVID-19 by real-time reverse transcriptase–polymerase chain reaction (rRT-PCR) using throat/nasal swabs as per the protocol published by the WHO6. On the fourth day of onset of symptoms, his wife (case 1) developed sore throat and rhinorrhoea. Case 1 along with two children (cases 2-3) was admitted on the sixth day of onset of symptoms in the index case, as they were confirmed positive for COVID-19 by rRT-PCR. Both the children were asymptomatic throughout the disease. An asymptomatic case was defined as a laboratory-confirmed COVID-19 infection case who was afebrile and well. None of the patients had diarrhoea as a part of their symptoms. All family members were discharged from the hospital by day 14 of the admission of the index case. Stool and urine samples were collected from all these patients on admission to check if the virus could be detected in specimens from non-respiratory sites and 14 days after collection of the first specimens (based on the incubation period of the virus). Additional stool samples were collected from the index case on 21, 32, 40, 48 and 55 days after the onset of symptoms to determine the duration of excretion of this novel virus in faeces. The viral nucleic acids were extracted from 30 per cent (w/v) suspensions in phosphate-buffered saline (pH 7.2-7.4) using spin columns (Qiagen, Hilden, Germany) as per manufacturer's instructions and determined by rRT-PCR targeting the genes, E (envelope), RdRp (RNA dependent RNA polymerase), ORF-1b-nsp14 (Open Reading Frame) and RNaseP (human RNase P) gene as internal control7. Viral copy numbers were quantified using E quantitative PCR standards developed at ICMR-NIV in ten-fold serial dilutions to generate a standard curve8. The family cluster of four (index case and cases 1-3) was infected with SARS-CoV-2. The timeline of contact and the duration of transmission of disease within the family as measured by rRT-PCR are indicated in Fig. 1. All the cases showed positivity in stool samples on days 8, 14 and 21 where higher viral loads corresponding to lower Ct values for E and ORF-1b-nsp14 genes were observed and no significant difference in Ct values of symptomatic and asymptomatic patients was observed (Fig. 2). However, on day 32, cases 2 and 3 became negative and case 1 was negative on the fortieth day. Furthermore, the index patient continued to excrete virus in stool until 48 days despite throat/nasal swab was found negative (Fig. 3). The viral shedding profile (Fig. 3) showed that the shedding increased slightly on day 6, peaked on the day 14 after the onset of illness and then dropped gradually to lower levels on day 55. Urine specimens of all the patients were negative. In general, the Ct values of ORF-1b-nsp14 gene for all specimens were lower compared to that of RdRp gene confirming earlier studies9 that ORF-1b-nsp14-based assay performed well as a confirmatory assay as compared to RdRp-based assays.Fig. 1: Time course of real-time reverse transcriptase–polymerase chain reaction (RT-PCR) test results for viral RNA in throat and stool specimens of COVID-19 patients.Fig. 2: Cycle threshold values for all cases within the family, positive for COVID-19 from stool specimen.Fig. 3: Copy number of E gene of SARS-CoV-2 from stool specimens collected at different time intervals from the index case using real-time (RT-PCR).The clinical features were diverse across the family; both the children were asymptomatic, the wife with mild symptoms and the index case had more severe respiratory symptoms. It was observed in this study that the transmission of SARS-CoV-2 infection occurred during the incubation period. A study done by Zou et al10 found that the viral loads of symptomatic and asymptomatic patients were similar and asymptomatic patients could infect others. Correlation of extended viral shedding in faeces in severe cases needs to be determined. Our observations are in concordance with reports that children develop mild or even asymptomatic illness compared to adults and elderly persons, and they may fare better when they have contracted the virus4511. Viral RNA was not detectable in urine specimens of these patients. However, improved methods of testing of urine samples are warranted. A rather surprising finding from the study was detection of viral RNA from the faecal specimens of the patients, though none of them had diarrhoea as part of their symptoms. Our study had a few limitations. SARS-CoV-2 was not isolated by the culture method from the samples that tested positive for the SARS-CoV-2 E gene by rRT-PCR. Real-time PCR detects viral RNA genome which can represent a replicating or a non-viable non-replicating virus. Angiotensin-converting enzyme 2, the receptor for spike protein of SARS-CoV-2, is expressed in large numbers on the brush border of intestinal enterocytes. Experimental models using human small intestinal organoids have demonstrated that SARS-CoV-2 can infect, replicate and produce significant titres of infectious viral particles in enterocytes12. Gastrointestinal symptoms in COVID-19 patients can be explained by this. In summary, our findings showed consistent person-to-person transmission of this novel coronavirus in hospital and family settings and also prolonged viral excretion by COVID-19 patients after negative conversion of pharyngeal swabs. The presence of SARS-CoV-2 RNA was demonstrated in the faeces of COVID-19 patients and suggested the possibility of SARS-CoV-2 transmission via the faecal-oral route. Faeces of the patients can be a potential source of transmission of the virus that may have important public health implications. Further studies are needed to ascertain if this would be a possible route of transmission. Financial support & sponsorship: The authors acknowledge the ICMR-NIV, Pune and ICMR, New Delhi, for financial support. Conflicts of Interest: None. Acknowledgment: The authors thank Dr Priya Abraham, Director ICMR-NIV, Pune, for the support during the study. The authors are grateful to Dr M L Chaudhary (Influenza Group, ICMR-NIV, Pune) for coordination with hospital laboratory authority for clinical sample from known SARS-CoV-positive patient. The assistance provided by Servshri P.S. Jadhav and Santosh (Deenanath Mangeshkar Hospital, Pune) during sample collection from the hospital and technical help by Shri R. Doiphode is duly acknowledged. The authors are thankful to NIC Team for supplying reagents for testing. Smt Veena Vipat is acknowledged for providing technical support for viral load detection.

Four gastroenteritis viruses were responsible for 54% of the acute gastroenteritis (AGE) cases in children hospitalized between May 2017 and December 2019 in Pune city of Maharashtra state, Western India. The majority (79%) of the children were <2 years of age. The prevalence of Rotavirus A (RVA) was 30.5% followed by 14.3% for norovirus, 8.4% for adenovirus, and 5.5% for astrovirus. The severity of the disease was highest in patients with coinfections compared with the patients with a single infection or negative for all (p = 0.024). Genotyping analysis showed that the majority of the RVA-positive samples (66%) could be typed as G3P[8], 63.6% of the norovirus as GII.4 Sydney [P16], 44% of the adenovirus as type 41%, and 56.2% of the astrovirus as astrovirus type 1. The almost equivalent prevalence of rotavirus and nonrotaviruses and acute gastroenteritis (AGE) cases without known etiology in around 46% of the cases was noted in the present study. Our data highlight that after the recent inclusion of rotavirus vaccines as a part of the National Immunization schedule in India, conducting extensive AGE surveillance in children should include nonrotaviruses such as norovirus.

Globally, there are nearly 1.7 billion cases and 1.6 million deaths due to diarrhoeal diseases every year. The burden is greatest in low-income populations with poor access to safe water, sanitation and urgent medical care1. In India, the problem is widespread with diarrhoeal diseases being the most common among the outbreaks reported across the country2. Among the causative agents, diarrhoeal outbreaks due to enteric viruses, namely rotavirus A, B and C, (RVA, RVB, RVC), norovirus, adenovirus and astrovirus, are frequently reported3-8. Among the As Cryptosporidium is a parasite please modify bacterial to non-viral agents, Vibrio cholerae, Cryptosporidium spp., Shigella spp., non-typhoidal Salmonella spp. and diarrhoeagenic Escherichia coli have been documented1,9.

An acute gastroenteritis outbreak at Devli Karad village, Maharashtra, India with an attack rate of 22.6% affected mainly adolescent and adult population. The viral investigations conducted on fecal specimens of patients hospitalized indicated the presence of rotavirus B (RVB) using RNA polyacrylamide gel electrophoresis and reverse transcription polymerase chain reaction. The samples collected from the source of drinking water also showed the presence of the only RVB. Absence of other viral agents and identification of RVB of genotype G2 as the etiological agent of the acute gastroenteritis outbreak highlights, the necessity of monitoring RVB, the viral agent known for its large outbreak potential.

Acute gastroenteritis outbreak occurred at Pargaon, Maharashtra, India in 1789 cases with an attack rate of 32.5% between November to December 2015. The stool specimens (n = 32) were investigated for different enteric viral agents using conventional methods. Transmission electron microscopy and RNA polyacrylamide gel electrophoresis respectively identified morphologically distinct rotavirus particles in 28% and RNA migration pattern of Group B Rotavirus (GBR) in 72% of the specimens. Reverse transcription polymerase chain reaction and nucleotide sequencing confirmed presence of GBR in 97% of the samples analyzed. The predominance of GBR infections and absence or insignificant presence of other agents confirmed GBR as an etiological agent of the gastroenteritis outbreak occurred in Maharashtra, India.