Diagnosing COVID-19: diagnostic importance of detecting E gene of the SARS-CoV-2 genome

Aim: To evaluate the significance of E gene analysis in addition to N and RdRp genes of SARS-CoV-2, and to compare the specificity and sensitivity of targets. Material(s) and Method(s): We used two reverse transcription-PCR assays: one targeting N, E and RdRp and the other targeting N and RdRp genes and analyzed variation in threshold cycle (Ct) values. Result(s): Of the 155 samples, 70.32% tested positive: all three genes were detected in 45.87%, N and RdRp in 19.27% and only N in 34.86%. Patients negative for the E gene were tested after symptoms disappeared and Ct values were significantly higher. Conclusion(s): Samples negative for the E gene were potentially false positive and clinical conditions should be assessed while interpreting results.Copyright © 2023 Future Medicine Ltd.

Comparative transcriptomic analyzes of human lung epithelial cells infected with wild-type SARS-CoV-2 and its variant with a 12-bp missing in the E gene.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that caused a global outbreak of coronavirus disease 2019 (COVID-19) pandemic. To elucidate the mechanism of SARS-CoV-2 replication and immunogenicity, we performed a comparative transcriptome profile of mRNA and long non-coding RNAs (lncRNAs) in human lung epithelial cells infected with the SARS-CoV-2 wild-type strain (8X) and the variant with a 12-bp deletion in the E gene (F8). In total, 3,966 differentially expressed genes (DEGs) and 110 differentially expressed lncRNA (DE-lncRNA) candidates were identified. Of these, 94 DEGs and 32 DE-lncRNAs were found between samples infected with F8 and 8X. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyzes revealed that pathways such as the TNF signaling pathway and viral protein interaction with cytokine and cytokine receptor were involved. Furthermore, we constructed a lncRNA-protein-coding gene co-expression interaction network. The KEGG analysis of the co-expressed genes showed that these differentially expressed lncRNAs were enriched in pathways related to the immune response, which might explain the different replication and immunogenicity properties of the 8X and F8 strains. These results provide a useful resource for studying the pathogenesis of SARS-CoV-2 variants.

Estimation of primer efficiency in multiplex PCR for detecting SARS-Cov-2 variants

In periodontology, polymerase chain reaction (PCR) has emerged as a standard diagnostic and research instrument. According to the findings in research, its sensitivity and specificity make it an efficient and speedy method for detecting, identifying, and quantifying organisms. The spike (S) gene, the envelope (E) gene, the membrane (M) protein, the nucleocapsids (N) gene, and the open reading frame (ORF) were used for the molecular identification of various variations of SARS-Cov-2 infection since they are critical components in the viral genome. Using kits approved by the Iraqi Ministry of Health following the spread of the covid-19 pandemic, this study sought to shed light on the significance of primers in the identification of various SARS-COV-2 variations involving many genes such as spike (S), envelope (E), membrane (M) protein, nucleocapsid (N), and ORFab1. A total of (150) clinically diagnosed patients for COVID-19 were screened for this study to estimate the accuracy and efficacy of primers used in detecting these genes are precise in diagnosing the infection in correspondence with clinical findings. The findings revealed that 18 (22.666%) of the cases had severe symptoms such as fever, unproductive hacking, breathlessness, muscle aches, and weariness, Whereas 42 (28%) of patients had lesser symptoms, and 74 (49.333%) of cases had minor signs on their own.. Results showed that all genes were accurate and efficient in the molecular diagnosis of the virus during the period from the beginning of March 2021 up to the writing of this manuscript in December 2021;the conclusion indicates that primers used in detecting these genes are efficient and accurate in the diagnosis of different variations of SARS-CAV-2 and there is no alteration in the viral genome in those genes that can disturb the result. © 2022 by the authors.

Characterisation of ORF3, M, N and E Gene Sequences of Porcine Epidemic Diarrhoea Virus from Domestic Pigs in Poland.

Introduction: Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen causing porcine epidemic diarrhoea and acute gastroenteritis in pigs of all ages. Previous analysis of the viral genome of PEDV in Poland was only based on the spike protein (S) gene sequences and no analysis of other genes has been performed. The aim of this study was to analyse the envelope (E), membrane (M) and nucleocapsid (N) protein and open reading frame 3 (ORF3) gene sequences. Material and Methods: Viral RNA from 18 Polish pig faecal samples that were quantitative reverse transcription PCR-positive for PEDV was analysed in four genomic regions (E, M, N and ORF3). Results: Phylogenetic analysis based on these regions' sequences revealed that Polish PEDV isolates were highly related and were clustered into group G2a across the four genes compared. Moreover, the Polish strains were located in distinct subclusters on the phylogenetic trees, which suggests the presence of at least three independently evolving PEDV genetic lines circulating in Poland. The occurrence of unique mutations in the sequences of Polish PEDV strains suggests that PEDV continues to undergo evolutionary processes, accumulating the mutations necessary for viral fitness in its natural hosts. The Polish PEDV strains differed genetically from the CV777 vaccine strain, suggesting the risk of relatively low vaccine efficacy if this strain is used. Conclusion: Our results promote a better understanding of the genetic diversity of PEDV field isolates in Poland and highlight the importance of molecular characterisation of PEDV field strains for the development of an effective vaccine against PEDV.

Evaluation of CCR5-Î"32 mutation and HIV-1 surveillance drug-resistance mutations in peripheral blood mononuclear cells of long-term non progressors of HIV-1-infected individuals

Aim: This study aimed to evaluate chemokine receptor 5 delta 32 (CCR5-δ32) mutation and HIV-1 surveillance drug-resistance mutations (SDRMs) in peripheral blood mononuclear cells of long-term non progressors (LTNPs) of HIV-1-infected individuals. Materials & methods: This research was performed on 197 treatment-naive HIV-1-infected patients. After follow-up, it was determined that 15 (7.6%) of these people were LTNPs. The PCR assay was performed to identify the CCR5 genotype and HIV-1 SDRMs. Results: One (6.7%) of the LTNPs was heterozygous (wt/δ32) for the CCR5 delta 32 (CCR5δ32). However, none of the individuals was homozygous for this mutation (δ32/δ32). Moreover, none of the LTNPs showed HIV-1 SDRMs. The CRF35-AD subtype was the most dominant subtype, with a percentage of 93.3%. Conclusion: Iranian elite controllers are negative for CCR5-delta 32 homozygous genotype and drug resistance against antiretroviral drugs.

Molecular Characterization and Phylogenetic Analysis of a Variant Recombinant Porcine Epidemic Diarrhea Virus Strain in China.

Since 2010, a variant of porcine epidemic diarrhea virus (PEDV) has re-emerged in several provinces of China, resulting in severe economic losses for the pork industry. Here, we isolated and identified a variant PEDV strain, SC-YB73, in Guangdong Province, China. The pathological observations of jejunum showed atrophy of villi and edema in the lamina propria. The sequence analysis of the viral genome identified a six-nucleotide insertion in the E gene, which has not previously been detected in PEDV strains. Furthermore, 50 nucleotide sites were unique in SC-YB73 compared with 27 other PEDV strains. The phylogenetic analysis based on the complete genome showed that SC-YB73 was clustered in variant subgroup GII-a, which is widely prevalent in the Chinese pig population. The recombination analysis suggested that SC-YB73 originated from the recombination of GDS47, US PEDV prototype-like strains TW/Yunlin550/2018, and COL/Cundinamarca/2014. In the present study, we isolated and genetically characterized a variant PEDV strain, thus providing essential information for the control of PED outbreaks in China.

Severe Acute Respiratory Syndrome Coronavirus 2 Is Detected in the Gastrointestinal Tract of Asymptomatic Endoscopy Patients but Is Unlikely to Pose a Significant Risk to Healthcare Personnel.

Background and Aims: Recent evidence suggests that the gut is an additional target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, whether SARS-CoV-2 spreads via gastrointestinal secretions remains unclear. To determine the prevalence of gastrointestinal SARS-CoV-2 infection in asymptomatic subjects, we analyzed gastrointestinal biopsy and liquid samples from endoscopy patients for the presence of SARS-CoV-2. Methods: We enrolled 100 endoscopic patients without known SARS-CoV-2 infection (cohort A) and 12 patients with a previous COVID-19 diagnosis (cohort B) in a cohort study performed at a regional hospital. Gastrointestinal biopsies and fluids were screened for SARS-CoV-2 by polymerase chain reaction (PCR), immunohistochemistry, and virus isolation assay, and the stability of SARS-CoV-2 in gastrointestinal liquids in vitro was analyzed. Results: SARS-CoV-2 ribonucleic acid was detected by PCR in the colonic tissue of 1/100 patients in cohort A. In cohort B, 3 colonic liquid samples tested positive for SARS-CoV-2 by PCR and viral nucleocapsid protein was detected in the epithelium of the respective biopsy samples. However, no infectious virions were recovered from any samples. In vitro exposure of SARS-CoV-2 to colonic liquid led to a 4-log-fold reduction of infectious SARS-CoV-2 within 1 hour (P ≤ .05). Conclusion: Overall, the persistent detection of SARS-CoV-2 in endoscopy samples after resolution of COVID-19 points to the gut as a long-term reservoir for SARS-CoV-2. Since no infectious virions were recovered and SARS-CoV-2 was rapidly inactivated in the presence of colon liquids, it is unlikely that performing endoscopic procedures is associated with a significant infection risk due to undiagnosed asymptomatic or persistent gastrointestinal SARS-CoV-2 infections.

Melting Curve-based Assay as an Alternative Technique for the Accurate Detection of SARS-CoV-2.

Background: Early and cost-effective diagnosis and monitoring of the infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critically important to anticipate and control the disease. We aimed to set up a SYBR Green-based one-step real-time polymerase chain reaction (PCR) as a lower-cost alternative method to detect the virus. Materials and Methods: An in-house SYBR Green-based PCR assay targeting the envelope (E) and RNA-dependent RNA polymerase (RdRp) genes, was set up to diagnose the infection, and was compared with the reference probe-based PCR method. Results: When the commercial probe-based assay was considered as the reference method, SYBR Green-based PCR had a slightly lower sensitivity (81.98% and 86.25% for E and RdRp targets, respectively) and a good specificity (100% and 94.44% for E and RdRp targets, respectively). For both gene targets, three different melting temperature (Tm) patterns were found in the PCRs of the nasopharyngeal/oropharyngeal swab samples, but no size polymorphism was seen in agarose gel electrophoresis. Conclusion: Further studies to improvement of the assay are needed to make it an inexpensive and reliable tool for the diagnosis of COVID-19.

Application of digital PCR to determine the reliability of Xpert Xpress SARS-CoV-2 assay with envelope (E) gene negative and nucleocapsid (N2) gene positive results.

This study used digital polymerase chain reaction (dPCR) to determine whether envelope (E) gene-negative and nucleocapsid (N2) gene-positive (E-N+) results obtained with the Cepheid Xpert Xpress SARS-CoV-2 assay are reliable. Using droplet digital PCR results as a reference, 18 of 22 E-N+ samples with a low viral load (81.8%) were identified as true positives.

Detection of Missense and Frame Shift Mutations in Envelope Proteins using Molecular Phylogenetic SARS-COV-2

The envelope (E) protein, which is 76-109 amino acids long, is a structural viroporin identified in coronaviruses. On March 1, 2020, ten distinct E-proteins were selected out of a total of 50 at the general Health laboratory (Babil), and the partial E gene of SARS-CoV-2 genomes was sequenced and recoded in the GenBank with accession number MW827729, MW827730, MW827731, MW827732, MW827733, MW827734, MW827735,MW827736, MW827737, MW827738. The study looked for missense and frame shift mutations in the envelope proteins of different Covid 19 patients to determine the lineage of the direct envelope protein (SARS-CoV-2). A phylogenetic analysis of envelope proteins, which looks at sequence homology and amino acid conservation, provides even more evidence to the evolutionary origin. Frame-shift mutations were found in both the N and C terminals of the 10 known partial sequences of human SARS- COV2. genomes.

Role of commercially available SARS-CoV-2 detection kits in pandemic of COVID-19 on the basis of N and E gene detection

C oronavirus December irresistible 2019 hasspecialistblowout introducing inworldwidea broad a genuine frompestilence general the timeis wellbeing vitalwhentoits emergency. therevelationachievement The in Hubei capacity ofProvince,isolate to recognize endeavorsChina an in notwithstanding the delicate and precise screening of expected instances of disease from patients in a clinical setting. Structural proteins the basic key role-playing in SARS-CoV2 identification include a spike, envelope membrane, nucleocapsid, and helper proteins. N-protein ties to the infection single positive-strand RNA that permits the infection to assume control over human cells and transform them into infection industrial facilities inside the capsid and E-protein shows a significant part in infection gathering, film permeability of the host cell, and infection has cell correspondence. Nucleic-Acid base testing presently offers the most touchy and early discovery of COVID-19. Notwithstanding, analytic advancements have explicit impediments and announced a few false negative and false positive cases, particularly during the beginning phases of contamination. Presently, more refined diagnostics are being created to improve the COVID-19 determination. This article presents an outline of diagnostic approaches to address a few inquiries and issues identified with the constraints of flow innovations and future innovative work difficulties to empower ideal, fast, minimal effort, and precise analysis of arising irresistible illnesses We depict purpose of-care diagnostics that are not too far off and urge scholastics to propel their advancements past origination. Creating fitting and-play diagnostics to deal with the SARS-CoV-2 flare-up would be valuable in forestalling forthcoming pandemics. © 2021. Advancements in Life Sciences. All rights reserved.

Accuracy of Real-Time Polymerase Chain Reaction in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (Ct) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC (N1: P = 0.0012, N2: P = 0.0013, N3: P = 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. IMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.

Identification of Hotspot Mutations in the N Gene of SARS-CoV-2 in Russian Clinical Samples That May Affect the Detection by Reverse Transcription-PCR.

Sensitive and reliable diagnostic test systems based on real-time PCR are of great importance in the fight against the ongoing SARS-CoV-2 pandemic. The genetic variability of the SARS-CoV-2 virus leads to the accumulation of mutations, some of which may affect the sensitivity of modern PCR assays. The aim of this study was to search in Russian clinical samples for new mutations in SARS-CoV-2 gene N that can affect the detection by RT-PCR. In this study, the polymorphisms in the regions of the target gene N causing failed or poor detection of the target N in the RT-PCR assay on 12 selected samples were detected. Sequencing the entire N and E genes in these samples along with other 195 samples that were positive for both target regions was performed. Here, we identified a number of nonsynonymous mutations and one novel deletion in the N gene that affected the ability to detect a target in the N gene as well a few mutations in the E gene of SARS-CoV-2 that did not affect detection. Sequencing revealed that majority of the mutations in the N gene were located in the variable region between positions 193 and 235 aa, inside and nearby the phosphorylated serine-rich region of the protein N. This study highlights the importance of the further characterization of the genetic variability and evolution of gene N, the most common target for detecting SARS-CoV-2. The use of at least two targets for detecting SARS-CoV-2, including one for the E gene, will be necessary for reliable diagnostics.

Low Represented Mutation Clustering in SARS-CoV-2 B.1.1.7 Sublineage Group with Synonymous Mutations in the E Gene.

Starting in 2019, the COVID-19 pandemic is a global threat that is difficult to monitor. SARS-CoV-2 is known to undergo frequent mutations, including SNPs and deletions, which seem to be transmitted together, forming clusters that define specific lineages. Reverse-Transcription quantitative PCR (RT-qPCR) has been used for SARS-CoV-2 diagnosis and is still considered the gold standard method. Our Eukaryotic Host Pathogens Interaction (EHPI) laboratory received six SARS-CoV-2-positive samples from a Sicilian private analysis laboratory, four of which showed a dropout of the E gene. Our sequencing data revealed the presence of a synonymous mutation (c.26415 C > T, TAC > TAT) in the E gene of all four samples showing the dropout in RT-qPCR. Interestingly, these samples also harbored three other mutations (S137L-Orf1ab; N439K-S gene; A156S-N gene), which had a very low diffusion rate worldwide. This combination suggested that these mutations may be linked to each other and more common in a specific area than in the rest of the world. Thus, we decided to analyze the 103 sequences in our internal database in order to confirm or disprove our "mutation cluster hypothesis". Within our database, one sample showed the synonymous mutation (c.26415 C > T, TAC > TAT) in the E gene. This work underlines the importance of territorial epidemiological surveillance by means of NGS and the sequencing of samples with clinical and or technical particularities, e.g., post-vaccine infections or RT-qPCR amplification failures, to allow for the early identification of these SNPs. This approach may be an effective method to detect new mutational clusters and thus to predict new emerging SARS-CoV-2 lineages before they spread globally.

Reliable Diagnostics of SARS-CoV-2 Infections Using One- and Two-Gene Molecular Tests for a Viral RNA Detection-Results Questioning Previous Observations.

SARS-CoV-2 is a new virus from the Coronaviridae family and its rapid spread is now the most important medical problem worldwide. Currently used tests vary in the number and selection of SARS-CoV-2 target genes. Meanwhile, the choice of the appropriate target gene may be important in terms of a reliable detection of a viral RNA. As some researchers questioned the sensitivity of the monogenic VIASURE SARS-CoV-2 S gene Real Time PCR Detection Kit (CerTest Biotec, Zaragoza, Spain) in mid-2020, the aim of the study was to evaluate the usefulness of this kit, used along with the BD MAX™ System (Becton Dickinson, East Rutherford, NJ, USA), and compare the results with two-gene Bosphore Novel Coronavirus (2019-nCoV) Detection Kit v1 (Anatolia Diagnostics and Biotechnology Products Inc., Istanbul, Turkey). Both tests were carried out on 306 nasopharyngeal/oropharyngeal swabs. The consistent results (72 positive and 225 negative results found simultaneously in both kits) were obtained for 297 (97.1%) samples altogether, while discrepancies between the results of the evaluated tests were observed for nine (2.9%) specimens. There were no statistically significant differences between the method used and the frequency of positive results. Both tests, targeted at detecting one and two genes, are effective in SARS-CoV-2 RNA detection.

Wastewater-based SARS-CoV-2 environmental monitoring for Piedmont, Italy.

The experience gained over the last hundred years clearly indicates that two groups of viruses represent the main risk for the development of highly transmissible epidemics and pandemics in the human species: influenza viruses and coronaviruses (CoV). Although the search for viruses with pandemic potential in the environment may have an important predictive and monitoring role, it is still based on empirical methodologies, mostly resulting from the clinic and not fully validated for environmental matrices. As far as the SARS-CoV-2 pandemic, currently underway, is concerned, environmental monitoring activities aiming at checking the presence of SARS-CoV-2 in wastewater can be extremely useful to predict and check the diffusion of the disease. For this reason, the present study aims at evaluating the SARS-CoV-2 diffusion by means of a wastewater-based environmental monitoring developed in Piedmont, N-W Italy, during the second and third pandemic waves. Wastewater sampling strategies, sampling points sample pre-treatments and analytical methods, data processing and standardization, have been developed and discussed to give representative and reliable results. The following outcomes has been highlighted by the present study: i) a strong correlation between SARS-CoV-2 concentration in untreated wastewater and epidemic evolution in the considered areas can be observed as well as a predictive potential that could provide decision-makers with indications to implement effective policies, to mitigate the effects of the ongoing pandemic and to prepare response plans for future pandemics that could certainly arise in the decades to come; ii) moreover, the data at disposal from our monitoring campaign (almost 500 samples analysed in 11 months) confirm that SARS-CoV-2 concentrations in wastewater are strongly variable and site-specific across the region: the highest SARS-CoV-2 concentration values have been found in sewer networks serving the most populated areas of the region; iii) normalization of viral concentrations in wastewater through Pepper Mild Mottle Virus (a specific faecal marker) has been carried out and commented; iv) the study highlights the potential of wastewater treatment plants to degrade the genetic material referable to SARS-CoV-2 as well. In conclusion, the preliminary data reported in the present paper, although they need to be complemented by further studies considering also other geographical regions, are very promising.

Extractionless nucleic acid detection: a high capacity solution to COVID-19 testing.

We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS-CoV-2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID-19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS-CoV-2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E- and N1-gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID-19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for >2-fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID-19 detection and significantly reduce the turn-around time of reporting COVID-19 test results.

Rapid adaptation and continuous performance evaluation of SARS-CoV-2 envelope gene (E-gene) real-time RT-PCR assays to support the hospital surge in test demand.

We describe the timely adaption of both published WHO E-gene protocol and commercially available LightMix Modular E-gene assay to the test platform (ABI 7900 Fast real-time analyzer and TaqMan Fast One-step Virus Master Mix) available in an accredited tertiary hospital laboratory with an on-going evaluation to ensure the provision of quality service within the time constraint. The LightMix Modular E-gene was slightly more sensitive when compared to the WHO E-gene, both analytically and diagnostically. The assay was recommended for screening of SARS-CoV-2 infection. With the availability of technically competent staff through continuous training, the provision of round-the-clock service is feasible despite the test is of high complexity. The thermal cycling duration of the adapted LightMix E-gene and WHO E-gene is shortened by half and one hour respectively and allows the number of runs to double when 24-h round-the-clock service is provided. An increase in testing capacity could support surges in testing demand, which is essential to control the current SARS-CoV-2 pandemic, to prevent potential overwhelming of the healthcare system, and to optimize utilization of the isolation beds.

Haemophilus influenzae and SARS-CoV-2: Is there a role for investigation?

During the current pandemic of COVID-19, the authors observed that during screening test for SARS-CoV-2 targeting the E-gene by qRT-PCR, few nasopharyngeal/oropharyngeal samples showed amplification signals at late cycle threshold (CT-value) > 35 despite being negative for other confirmatory target genes. Thirty such samples (taken as cases) showing detectable CT of > 35 cycle in E-gene which were negative for other target genes of SARS-CoV-2 and 30 samples with undetectable fluorescence in E-gene were taken as controls for investigation. An in-vitro diagnostic approved commercial qRT-PCR multiplex kit detecting 33 respiratory pathogens which can also detect Haemophilus influenzae was used for screening the samples. It was observed that out of the 30 samples showing detectable CT> 35 in E-gene, 11 samples were positive for Haemophilus influenzae whereas in the controls only three samples were positive for H. influenzae (p-value: 0.03) which was statistically significant. Further, the probes and primers were screened against H. influenzae for matches in the genome. It was observed that all primers and probes for the E-gene of SARS-CoV-2 had over 13 bp long sequences matching 100% with multiple sites across the H. influenzae genome. This qRT-PCR primer & probes are being used extensively across India, and laboratories using them should be aware of the cross-reactivity of primers & probes with the H. influenzae genome. Further, the authors observed that 95.9% (5415/5642) of COVID-19 positive cases detected in their laboratory were asymptomatic at the time of collection of samples. This warrants further investigations.

SARS-CoV-2 in environmental perspective: Occurrence, persistence, surveillance, inactivation and challenges.

The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.