Search

There is always a concern about the quality of cell-based products in terms of the contamination of the cells and their lack of efficiency. Therefore, it is of prime importance to ensure these cells' identity, purity, efficacy, and suitability for the production of biological products and diagnostic uses. Hence, cells must be identified, evaluated, documented, and stored to be used consistently and efficiently. With these conditions, vaccine manufacturers have a suitable reserve of efficient and valuable cells for the production and quality control of biological products. In this review article, a strategic plan was drawn for cell-substrate well-characterization and identification according to scientific principles, the author's work experience, and regulatory guidance for the optimal use of that cell in research and diagnostic studies especially for the biological product production process. For this purpose, all aspects of cell identification, cell evaluation, and cell characterization are discussed. Because of the importance of cell identity in the competence of a cell substrate, in the cell identification section, all aspects of cell identification, including general cell information and specific cell characteristics, especially in terms of cell passage history, cell storage conditions, and cell coding and labeling, were studied. In the part of cell evaluation and determination of cell characteristics, all required tests to determine cell characteristics from various aspects, including determination of cell identity, cell growth conditions, cell quality, efficiency, and the possibility of cell contamination with adventitious agents, including cellular, viral, bacterial, mycoplasma, and mycobacterial agents were introduced. Due to the importance of endogenous virus contamination, this topic is specially discussed. In addition, the stability of the cell both from the aspect of genetic stability and from the aspect of stability of cell efficiency, were discussed. In the end, while reviewing the necessary documents to be under the control of the cell for use in the laboratory, based on the studies conducted, the certificate of the cell has been compiled. Therefore, on this basis, the studied cell can be used for research and diagnostic studies of virology, especially for the production and quality control of biological products. [ABSTRACT FROM AUTHOR]

The XVIII International Parvovirus Workshop took place in Rimini, Italy, from 14 to 17 June 2022 as an on-site event, continuing the series of meetings started in 1985 and continuously held every two years. The communications dealt with all aspects of research in the field, from evolution and structure to receptors, from replication to trafficking, from virus–host interactions to clinical and veterinarian virology, including translational issues related to viral vectors, gene therapy and oncolytic parvoviruses. The oral communications were complemented by a poster exhibition available for view and discussion during the whole meeting. The XVIII International Parvovirus Workshop was dedicated to the memory of our dearest colleague Mavis Agbandje-McKenna (1963–2021). [ABSTRACT FROM AUTHOR]

Purpose: To identify the bibliometric information of Human papillomavirus (HPV) genotype co-infection in certain literature database over the past two decades. Methods: Web of Science was used as the main database to identify all eligible articles focusing on HPV genotype co-infection at the date of October 16, 2022. From this journal database, we identified 463 articles on HPV genotype coinfection, conducted statistical analysis according to the author, journal, publication year and month, country or region, keyword and impact factor. Results: The articles included in our analysis were published between 1994 and 2022. The index of citations per year ranged from 170.4 to 13.1. These articles were from 78 countries or regions, with most publications from the United States (n=73), followed by China (n=65) and Italy (n=50). The journal that contributed the most publications on HPV heterotypic gene co-infection was PLOS ONE with a total of 29 articles, followed by JOURNAL OF MEDICAL VIROLOGY (n=28), INFECTIOUS AGENTS AND CANCER (n=14) and JOURNAL OF CLINICAL VIROLOGY (n=12). Among existing research in the field of HPV co-infection, we found that epidemiological distribution and infection mechanism has been the two major topics for scholars, and studies on detection methods for HPV multiple genotypes were also included. Conclusion: Over decades, epidemiological studies and mechanism investigationhas been the central topics when it comes to HPV genotypes coinfection. Studies on HPV co-infection remained relatively insufficient, mainly stays in qualitative level while detailed infection data and high quality literature publications were still lack of valuable discussion. [ABSTRACT FROM AUTHOR]

Over time, the SARS-CoV-2 virus has acquired several genetic mutations, particularly on the receptor-binding domain (RBD) spike glycoprotein. The Omicron variant is highly infectious, with enhanced immune escape activity, and has given rise to various sub-lineages due to mutations. However, there has been a sudden increase in COVID-19 reports of the Omicron subvariant BF.7 (BA.2.75.2), which has the highest number of reported cases, accounting for 76.2% of all cases worldwide. Hence, the present systematic review aimed to understand the viral mutations and factors associated with the increase in the reports of COVID-19 cases and to assess the effectiveness of vaccines and mAbs against the novel Omicron variant BF.7. The R346T mutation on the spike glycoprotein RBD might be associated with increased infection rates, severity, and resistance to vaccines and mAbs. Booster doses of COVID-19 vaccination with bivalent mRNA booster vaccine shots are effective in curtailing infections and decreasing the severity and mortality by enhancing the neutralizing antibodies (Abs) against the emerging Omicron subvariants of SARS-CoV-2, including BF.7 and future VOCs. • The SARS-CoV-2 Omicron (B.1.1.529) variant was identified later in November 2021, with enhanced immune escape. • Omicron subvariant BF.7 (BA.2.75.2) is a novel highly infectious subvariant of Omicron. • The R346T mutation on the spike glycoprotein RBD Omicron of BF.7 variant is associated with increased infectiousness, and serious illness. • Current evidence has shown that Additional booster dose vaccination, preferably with bivalent mRNA vaccines has been associated with an increased immunogenicity against highly infectious SARS-CoV-2 Omicron subvariants including BF-7, but are resistant to majority of currently available mAbs. [ABSTRACT FROM AUTHOR]

Background: Vaccination against rotavirus was offered in Stockholm to children born on 1 March 2014 and onwards with 85% coverage after two years. We investigated changes in nosocomial diarrhoea 2010—2018 in children admitted to Astrid Lindgren Children's Hospital, Stockholm, Sweden. Methods: We retrospectively identified cases from diagnostic and virology department registers. Complications and chronic medical conditions were retrieved from the case records. Children <18 years of age who developed diarrhoea ≥48 h after admission for another diagnosis and had a faecal sample submitted to the virology department were included. Results: There were 474 episodes of nosocomial diarrhoea. Of these, 401 (85%) occurred in children with chronic medical conditions. In children <5 years the rates of nosocomial rotavirus gastroenteritis, with 95% confidence intervals, significantly decreased from 0.34 (0.25—0.45) per 100 admissions prevaccination to 0.09 (0.04—0.17) postvaccination and from 0,66 (0.48—0.88) to 0.16 (0.07—0.30) cases per 1000 hospital days. Postvaccination norovirus became the most frequent pathogen. Virus-positive cases were more common in young children and in winter months. Conclusions: Before the initiation of rotavirus vaccination, norovirus and rotavirus were equally common causes of nosocomial diarrhoea. Postvaccination, rotavirus was reduced by approximately 75% while the frequency of other viruses did not change. [ABSTRACT FROM AUTHOR]

As for the case of SARS-CoV-2, genome sequencing of influenza viruses is of potential interest to raise and address virological issues. Recently, false-negativity of real-time reverse transcription-PCR (qPCR) assays that detect influenza A/H3N2 virus RNA were reported and associated with two mutations (A37T and C161T) in the Matrix-encoding (M1) gene located on viral segment 7. This triggered a national alert in France. The present study sought to assess the association between the presence of these mutations and potential false negative results of influenza A/H3N2 virus RNA detection by commercialized qPCR assays at the clinical virology laboratory of our university hospitals in southern France. This study focused on the genetic diversity in the M1 gene and segment 7 of 624 influenza A/H3N2 virus genomes obtained from respiratory samples having tested qPCR-positive with M1 gene-targeting assays in our clinical virology laboratory. A total of 585 among the 624 influenza A/H3N2 virus genomes (93.7%) were of clade 3C.2a1b.2a.2, and 39 (6.3%) were of clade 3C.2a1b.1a. M1 gene substitutions A37T and C161T were both present in 582 (93.3%) genomes, only of clade 3C.2a1b.2a.2. Substitution A37T was present in 621 (99.5%) genomes. Substitution C161T was present in 585 genomes (93.8%), all of clade 3C.2a1b.2a.2. Moreover, 21 other nucleotide positions were mutated in ≥90% of the genomes. The present study shows that A37T/C and C161T mutations, and other mutations in the M1 gene and segment 7, were widely present in influenza A/H3N2 virus genomes recovered from respiratory samples diagnosed qPCR-positive with commercialized assays. [ABSTRACT FROM AUTHOR]

UK National Health Service (NHS) Clinical Virology Departments provide a repertoire of tests on clinical samples to detect the presence of viral genomic material or host immune responses to viral infection. In December 2019, a novel coronavirus (SARS-CoV-2) emerged which quickly developed into a global pandemic; NHS laboratories responded rapidly to upscale their testing capabilities. To date, there is little information on the impact of increased SARS-CoV-2 screening on non-SARS-CoV-2 testing within NHS laboratories. This report details the virology test requests received by the Leicester-based NHS Virology laboratory from January 2018 to May 2022. Data show that in spite of a dramatic increase in screening, along with multiple logistic and staffing issues, the Leicester Virology Department was mostly able to maintain the same level of service for non-respiratory virus testing while meeting the new increase in SARS-CoV-2 testing. [ABSTRACT FROM AUTHOR]

Herpes simplex virus (HSV) 1 and 2, varicella zoster virus (VZV), and human herpesvirus 6 (HHV-6) cause severe infections in immunocompromised hosts. Interventions to optimize virus-specific adaptive immunity may have advantages over antivirals in the prophylaxis and treatment of these infections. We sought to review adaptive immune responses and methods for assessing and replenishing cellular and humoral immunity to HSV, VZV, and HHV-6 in solid organ transplant and hematopoietic cell transplant recipients. We searched PubMed for relevant studies on immune responses to HSV, VZV, and HHV-6 as well as studies describing methods for evaluating and restoring cell-mediated immunity to other double-stranded DNA viruses in transplant recipients. Recent studies, randomized controlled trials, and investigations highlighting key concepts in clinical virology were prioritized for inclusion. We describe the mechanisms of adaptive immunity to HSV, VZV, and HHV-6 and limitations of antivirals as prophylaxis and treatment for these infections in solid organ transplant and hematopoietic cell transplant recipients. We review methods for measuring and restoring cellular immunity to double-stranded DNA viruses; their potential applications to management of HSV, VZV, and HHV-6 in immunocompromised hosts; and barriers to clinical use. Vaccination and virus-specific T cell therapies are discussed in detail. The growing repertoire of diagnostic and therapeutic techniques focused on virus-specific adaptive immunity provides a novel approach to management of viral infections in transplant recipients. Investigations to optimize such interventions specifically in HSV, VZV, and HHV-6 are needed. [Display omitted] [ABSTRACT FROM AUTHOR]

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to C T values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of C T values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings. [ABSTRACT FROM AUTHOR]

Background: HSV-1, HSV-2 and VZV are alpha Herpesviruses, neurotropic viruses that are associated with various neurologic complications upon primary infection or reactivation. Cases of myelitis and radiculomyelitis are rare and appropriate etiologic diagnoses can be tricky. Case presentation: Here we describe the case of a young immunocompetent woman who developed painful and extended vesicular genital lesions, with subsequent radiculomyelitis. HSV-1/-2 PCRs in the cerebrospinal fluid were misleadingly negative, whereas HHV-6 PCR was positive. Positive anti-HSV-2 IgM and IgG in serum was consistent with HSV-2 primary infection. On the other hand, the detection of HHV-6 DNA was explained by inherited chromosomally integrated HHV-6. The clinical course was favorable with high-dose IV acyclovir and corticosteroids. Conclusion: HSV-2-related radiculomyelitis is a rare clinical entity, which can be difficult to diagnose. In this case report, the causative virus was not detected in the patient's CSF, whereas HHV-6 DNA, non-pathogenic in this situation, was paradoxically positive. The diagnosis was based on the clinical features typical for HSV-2 primary infection, confirmed by the serology results. The delay between the genital lesions and the appearance of the radiculomyelitis, along with the absence of HSV-2 detection in the CSF, suggests a possible immuno-mediated physiopathological process. As for the HHV-6 DNA detection in the patient's CSF, it was explained by inherited chromosomally integrated HHV-6. This case illustrates how both negative and positive clinical virology results need careful interpretation according to the clinical findings. [ABSTRACT FROM AUTHOR]

The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) directly or indirectly affects every individual worldwide. The fight against SARS-CoV-2 is based on the rapid and accurate diagnosis and subsequent isolation of infected individuals. Therefore, the demands for the scientific development of diagnostic methods for the confirmation of SARS-CoV-2 are enormous. Currently, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is the main method used for detecting viruses, including SARS-CoV-2, and is considered the gold standard for coronavirus disease 2019 (COVID-19) identification. However, various alternatives have been investigated due to the time and cost demands of this method or to shortages of reagents. In this review, we focus on matrix-assisted laser desorption and ionisation with time-of-flight analyser mass spectrometry (MALDI-TOF MS) techniques as potential tools for the diagnosis of viruses with an emphasis on SARS-CoV-2. MALDI-TOF is commonly used in clinical laboratories for bacterial characterization and identification, but in the field of clinical virology, MALDI-TOF remains only a promising technology for routine diagnosis. This review provides an overview of the development of clinical virology from the point of view of using MALDI-TOF for virus identification and as a possible diagnostic tool for SARS-CoV-2 detection. In addition, this review summarizes the current state of standard methods for virus diagnostics including the preparation of clinical samples. [ABSTRACT FROM AUTHOR]

We use cryoelectron microscopy (cryo-EM) as a sequence- and culture-independent diagnostic tool to identify the etiological agent of an agricultural pandemic. For the past 4 years, American insect-rearing facilities have experienced a distinctive larval pathology and colony collapse of farmed Zophobas morio (superworm). By means of cryo-EM, we discovered the causative agent: a densovirus that we named Zophobas morio black wasting virus (ZmBWV). We confirmed the etiology of disease by fulfilling Koch's postulates and characterizing strains from across the United States. ZmBWV is a member of the family Parvoviridae with a 5,542 nt genome, and we describe intersubunit interactions explaining its expanded internal volume relative to human parvoviruses. Cryo-EM structures at resolutions up to 2.1 Å revealed single-strand DNA (ssDNA) ordering at the capsid inner surface pinned by base-binding pockets in the capsid inner surface. Also, we demonstrated the prophylactic potential of non-pathogenic strains to provide cross-protection in vivo., Competing Interests: Declaration of interests Rutgers University filed US Provisional Application 63/591,484 “Method for inhibiting Z. morio black wasting disease” on behalf of J.J.P. and J.T.K. S.A.Y. is employed by REGENXBIO Inc. and holds shares in the company. J.T.K. receives a research grant from REGENXBIO Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: We aimed to determine the cutoff value of anti-hepatitis C virus (HCV) in the diagnosis of real positive patients based on the chemiluminescence immunoassay (CIA) test kit absorbance routinely used in our laboratory and to reveal the potential cost-effectiveness of confirmatory tests for false-positive samples. Methods: All anti-HCV CIA test results between 2016 and 2019 were retrospectively screened and sample/cutoff (S/Co) values of the patients were recorded. Among these, the results that were confirmed with HCV RNA polymerase chain reaction (PCR) test were included. Results: Of the 257 patients included in the study, 84 (32.68%) were positive for HCV RNA. The optimal S/Co value was 8.58 with sensitivity and specificity values being 95.24% and 85.55%, respectively. According to this 8.58 S/Co value, the anti-HCV test was reactive in 105 cases and 80 (76.2%) of these cases had active HCV infection. To prevent false negativity, the additional cost of using 1.0 S/Co value to our institution was 4114.64 USD. In our institution, approximately 6.25 working hours is spent to finalize the HCV RNA PCR test. The hours spent for S/Co of 1.0 and 8.58 was 1606.25 and 658.25, respectively. Conclusion: False-positive anti-HCV results are an economic burden on countries. At least, different S/Co values might be used in accordance with the purpose of screening and prevalence of HCV infection in different laboratories and different populations. [ABSTRACT FROM AUTHOR]

Using new and original nineteenth-century sources, we analysed the epidemiology, clinical features and virology of the 1889 pandemic, which was referred to at the time as 'Russian flu' or 'Asiatic flu'. However, we rejected this identification of the disease as an 'influenza', which we believe to have been based on insufficient knowledge of the causative agent and instead posit that the pandemic was caused by a coronavirus. We provide a new account of the 1889–1893 pandemic, with a more detailed chronology that included at least four epidemiological waves. At the end of 1889, a new virus appeared in Europe, which could be identified as the coronavirus HCoV-OC43, causing crude death rates of 1.3 per 1000 population in St Petersburg; 2.1 per 1000 in Paris; 2.8 per 1000 in Bilbao and on the French–Spanish border; between 2.9 and 5.2 per 1000 in small towns in the Basque Country; and 5.8 deaths per 1000 in Madrid, which had the highest death rate. The clinical features of the disease differed from classical influenza pandemics in terms of the latency phase, duration, symptomatology, convalescence, immunity, age and death rates. Another factor to be considered was the neurotropic capacity of the disease. The most frequent form of the 1889 pandemic was the 'nervous form', with specific symptoms such as 'heavy headache' (céphalalgie gravative), tiredness, fever and delirium. There are strong parallels between the 1889–1894 pandemic and the COVID-19 pandemic, and a better understanding of the former may therefore help us to better manage the latter. [ABSTRACT FROM AUTHOR]

• ddPCR showed a limit of detection of 4 copies/mL (<1IU/ml) by Probit analysis. • ddPCR showed a good linearity and a high intra- and inter-run reproducibility. • ddPCR quantified HBV-DNA in most patients with viremia not quantifiable by Real Time-PCR. • Highly sensitive assays can optimize the diagnosis of occult HBV infection. • Highly sensitive assays can improve therapeutic management in chronic HBV infection. Droplet digital PCR is an innovative and promising approach for highly sensitive quantification of nucleic acids that is being increasingly used in the field of clinical virology, including the setting of hepatitis B virus (HBV). Here, we comprehensively report a robust and reproducible ddPCR assay for the highly sensitive quantification of serum HBV-DNA. The assay showed a limit of detection of 4 copies/ml (<1IU/ml) by Probit analysis, showed a good linearity (R2 = 0.94) and a high intra- and inter-run reproducibility with differences between the values obtained in the same run or in two independent runs never exceeding 0.14logcopies/mL and 0.21logcopies/mL, respectively. By analysing serum samples from chronically HBV infected patients (mostly under antiviral treatment), ddPCR successfully quantified serum HBV-DNA in 89.8% of patients with detectable serum HBV-DNA < 20 IU/mL [equivalent to <112copies/ml] by classical Real-Time PCR assay, with a median (IQR) of 8(5–14)IU/mL [45(28–78)copies/ml], and in 66.7% of patients with undetectable serum HBV-DNA, with a median (IQR) of 5(4–9)IU/mL [28(20–50)copies/ml]. Similarly, by analysing serum samples from patients with a serological profile compatible with occult HBV infection (anti-HBc+/HBsAg-), ddPCR successfully quantified serum HBV-DNA in 40% of patients with a median (IQR) value of 1(1–2)IU/mL [5(5–11)copies/ml], in line with the extremely limited viral replication typically observed in occult HBV infection. Overall, the availability of assays for the highly sensitive quantification of serum HBV-DNA can provide an added value in optimizing the diagnosis of occult hepatitis B infection, improving the therapeutic management of chronically HBV infected patients, also in the light of innovative drugs (upcoming in clinical practise) aimed at achieving HBV functional cure. [ABSTRACT FROM AUTHOR]

Large-scale SARS-CoV-2 molecular testing coupled with whole genome sequencing in the diagnostic laboratories is instrumental for real-time genomic surveillance. The extensive genomic, laboratory, and clinical data provide a valuable resource for understanding cases of reinfection versus prolonged RNA shedding and protracted infections. In this study, data from a total of 22,292 clinical specimens, positive by SARS-CoV-2 molecular diagnosis at Johns Hopkins clinical virology laboratory between March 11th 2020 to September 23rd 2021, were used to identify patients with two or more positive results. A total of 3,650 samples collected from 1,529 patients who had between 2 and 20 positive results were identified in a time frame that extended up to 403 days from the first positive. Cycle threshold values (Ct) were available for 1,622 samples, the median of which was over 30 by 11 days after the first positive. Extended recovery of infectious virus on cell culture was notable for up to 70 days after the first positive in immunocompromised patients. Whole genome sequencing data generated as a part of our SARS-CoV-2 genomic surveillance was available for 1,027 samples from patients that had multiple positive tests. Positive samples collected more than 10 days after initial positive with high quality sequences (coverage >90% and mean depth >100), were more likely to be from unvaccinated, or immunosuppressed patients. Reinfections with viral variants of concern were found in 3 patients more than 130 days from prior infections with a different viral clade. In 75 patients that had 2 or more high quality sequences, the acquisition of more substitutions or deletions was associated with lack of vaccination and longer time between the recovered viruses. Our study highlights the value of integrating genomic, laboratory, and clinical data for understanding the biology of SARS-CoV-2 as well as for setting a precedent for future epidemics and pandemics. [ABSTRACT FROM AUTHOR]

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea—rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/μL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis. [ABSTRACT FROM AUTHOR]

Background: Accurate population estimates of disease incidence and burden are needed to set appropriate public health policy. The capture–recapture (C‐R) method combines data from multiple sources to provide better estimates than is possible using single sources. Methods: Data were derived from clinical virology test results and from an influenza vaccine effectiveness study from seasons 2016–2017 to 2018–2019. The Petersen C‐R method was used to estimate the population size of influenza cases; these estimates were then used to calculate adult influenza hospitalization burden using a Centers for Disease Control and Prevention (CDC) multiplier method. Results: Over all seasons, 343 influenza cases were reported in the clinical database, and 313 in the research database. Fifty‐nine cases (17%) reported in the clinical database were not captured in the research database, and 29 (9%) cases in the research database were not captured in the clinical database. Influenza hospitalizations were higher among vaccinated (58%) than the unvaccinated (35%) in the current season and were similar among unvaccinated (51%) and vaccinated (49%) in the previous year. Completeness of the influenza hospitalization capture was estimated to be 76%. The incidence rates for influenza hospitalizations varied by age and season and averaged 307–309 cases/100,000 adult population annually. Conclusion: Using C‐R methods with more than one database, along with a multiplier method with adjustments improves the population estimates of influenza disease burden compared with relying on a single‐data source. [ABSTRACT FROM AUTHOR]

Multiplexed SARS-CoV-2 sequencing: Existing SARS-CoV-2 diagnostic tests use RNA extraction followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), which limit their ability to quantify and sequence multiple variants in a single test. Chappleboim et al. describe a multiplexed next-generation sequencing (NGS)–based method called ApharSeq in which samples are pooled early via hybridization of barcoded primers. This allows for hundreds of pooled samples to undergo multiplexed reverse transcription, PCR, and sequencing to detect and classify variant sequences with high sensitivity and negligible contamination. The ApharSeq method was validated on clinical samples, demonstrating that dozens of samples can be pooled early to markedly reduce costs while still providing per-sample variant information. Most severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic tests have relied on RNA extraction followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays. Whereas automation improved logistics and different pooling strategies increased testing capacity, highly multiplexed next-generation sequencing (NGS) diagnostics remain a largely untapped resource. NGS tests have the potential to markedly increase throughput while providing crucial SARS-CoV-2 variant information. Current NGS-based detection and genotyping assays for SARS-CoV-2 are costly, mostly due to parallel sample processing through multiple steps. Here, we have established ApharSeq, in which samples are barcoded in the lysis buffer and pooled before reverse transcription. We validated this assay by applying ApharSeq to more than 500 clinical samples from the Clinical Virology Laboratory at Hadassah hospital in a robotic workflow. The assay was linear across five orders of magnitude, and the limit of detection was Ct 33 (~1000 copies/ml, 95% sensitivity) with >99.5% specificity. ApharSeq provided targeted high-confidence genotype information due to unique molecular identifiers incorporated into this method. Because of early pooling, we were able to estimate a 10- to 100-fold reduction in labor, automated liquid handling, and reagent requirements in high-throughput settings compared to current testing methods. The protocol can be tailored to assay other host or pathogen RNA targets simultaneously. These results suggest that ApharSeq can be a promising tool for current and future mass diagnostic challenges. [ABSTRACT FROM AUTHOR]

Serological techniques are an essential part of the diagnostic tools used in clinical virology. Among these techniques, antibody indexes are not novel, but do require specific expertise. Their niche has expanded substantially in recent years due to increasing evidence of their performance to diagnose viral infections. This narrative review describes the background and clinical applications of antibody indexes. The first objective is to provide an overview of the theoretical background, insights for implementation, limitations and pitfalls. The second objective is to review the available evidence for the diagnostic performance, with a specific focus on viral encephalitis and uveitis. A comprehensive literature search was performed in PubMed, including original studies and reviews, with no time limit on the studies included. The following search terms were used: antibody index, Goldmann–Witmer coefficient, Reibergram, viral encephalitis, viral uveitis, herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein–Barr virus, rubella virus, measles virus, enterovirus, influenza virus, flaviviruses. Antibody indexes can support the diagnosis of a spectrum of viral infections in immune privileged sites such as the central nervous system and the eye, through the demonstration of virus-specific intrathecal or intraocular antibody production. This is especially useful in situations where PCR has a lower positivity rate: infections with rapid viral clearance due to natural immunity or treatment and chronic stages of viral infections. Antibody indexes expand the clinical microbiologist's diagnostic toolbox. Careful interpretation of the results of these assays is crucial and further standardization of methods is required to improve interchangeability of results between laboratories. [ABSTRACT FROM AUTHOR]

Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rear-rangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology. [ABSTRACT FROM AUTHOR]

Hepatitis B core-related antigen (HBcrAg) is a composite measure of the serum levels of hepatitis B e antigen, hepatitis B core antigen, and a 22-kDa precore protein. It has been shown to reflect the level and transcriptional activity of covalently closed circular DNA in the liver. Longitudinal cohort studies have improved our understanding of the role of this novel viral marker in the natural history of chronic hepatitis B. HBcrAg kinetics reflect the response to peginterferon, and its role in defining guidelines for stopping peginterferon therapy has been evaluated. HBcrAg is a marker of intrahepatic viral activity, which may influence the risk of hepatocellular carcinoma. In this article, we review the virology and role of HBcrAg in defining phases of chronic hepatitis B. Furthermore, the function of HBcrAg in predicting treatment outcomes and its role in monitoring response to novel antiviral agents will be discussed. [ABSTRACT FROM AUTHOR]

Sensitive and reproducible diagnostics are fundamental to containing the spread of existing and emerging pathogens. Despite the reliance of clinical virology on qPCR, technical challenges persist that compromise their reliability for sustainable epidemic containment as sequence instability in probe-binding regions produces false-negative results. We systematically violated canonical qPCR design principles to develop a Pan-Degenerate Amplification and Adaptation (PANDAA), a point mutation assay that mitigates the impact of sequence variation on probe-based qPCR performance. Using HIV-1 as a model system, we optimized and validated PANDAA to detect HIV drug resistance mutations (DRMs). Ultra-degenerate primers with 3' termini overlapping the probe-binding site adapt the target through site-directed mutagenesis during qPCR to replace DRM-proximal sequence variation. PANDAA-quantified DRMs present at frequency ≥5% (2 h from nucleic acid to result) with a sensitivity and specificity of 96.9% and 97.5%, respectively. PANDAA is an innovative advancement with applicability to any pathogen where target-proximal genetic variability hinders diagnostic development. Iain MacLeod et al. develop the Pan-Degenerate Amplification and Adaptation (PANDAA) point mutation assay to detect HIV drug resistance mutations. This method can be applied to any pathogen where target-proximal genetic variability hinders diagnostic development. [ABSTRACT FROM AUTHOR]

Objective: Hepatitis B virus (HBV) infection causes substantial harm to mitochondrial activity, which hinders the development of effective treatments for chronic hepatitis B (CHB). The discovery of the mitochondrial-derived short peptide MOTS-c, which possesses multiple bioactivities, offers a promising new approach in treating HBV infection. This study aims to explore the diagnostic and therapeutic potential of MOTS-c in HBV-related diseases and its molecular mechanism., Design: In total, 85 healthy subjects and 404 patients with HBV infection, including 20 clinical treatment cohorts, were recruited for this study. MOTS-c levels were measured by ELISA and its diagnostic value was evaluated by receiving operating characteristic curve analysis. The therapeutic effect of MOTS-c was observed in multiple HBV-infected mice and cells through various techniques, including transcriptomic sequencing, flow cytometry, immunofluorescence and electron microscopy. Additionally, MOTS-c's potential interaction with myosin-9 (MYH9) and actin was predicted using immunoprecipitation, proteomics and target prediction software., Results: MOTS-c negatively correlates with HBV DNA expression (R=-0.71), and its AUC (the area under the curve) for distinguishing CHB from healthy controls is 0.9530, and IA (immune reactive) from IC (inactive HBV carrier) is 0.8689. Inhibition of HBV replication (with a 50-70% inhibition rate) was observed alongside improved liver function without notable toxicity in vitro or in vivo. MOTS-c was found to promote mitochondrial biogenesis and enhance the MAVS (mitochondrial antiviral signalling protein) signalling pathway. The impact is dependent on MOTS-c's ability to regulate MYH9-actin-mediated mitochondrial homeostasis., Conclusion: MOTS-c has the potential to serve as a biomarker for the progression of HBV infection while also enhancing antiviral efficacy. These findings present a promising innovative approach for effectively treating patients with CHB. Furthermore, our research uncovers a novel role for MOTS-c in regulating MYH9-actin-mediated mitochondrial dynamics and contributing to mitochondrial biogenesis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Establishing the diagnosis of viral diseases often needs sophisticated settings, equipment, expertise, and strict laboratory methods. In Sudan, as in most developing countries, viral diseases are mostly diagnosed by clinical presentation. As most viral infections are self-limiting and there is no specific treatment for most of them, laboratory diagnosis has not been a focus for improvement, particularly in public sector until the current pandemic of COVID-19. During this pandemic, the vital need for well-equipped clinical virology laboratories is urged. The aim of this work is to highlight the various diagnostic methods and to describe the current situation of clinical virology diagnostics in Sudan. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa. [ABSTRACT FROM AUTHOR]

In late May the Government still planned to reopen schools on 1 June. While some schools did open, the school system in the end did not. At that time scientists still did not know a lot about the SARS-CoV-2 virus and the COVID-19 disease that it causes, especially about the long-term consequences of catching it. This lack of certainty is reflected in the research papers produced by the Government's Scientific Advisory Group for Emergencies (SAGE) and its subcommittees and by the then new Independent SAGE. This article considers a number of papers published over the last few days of May 2020 that policy makers and school and college leaders had to consider. They are Modelling and Behavioural Science Responses to Scenarios for Relaxing School Closures, produced by the Interdisciplinary Task and Finish Group on the Role of Children in Transmission (referred to as the ITFGRCT report), which is part of SAGE and is itself a collaborative analysis conducted by SPI-M (the Scientific Pandemic Influenza Group on Modelling) and SPI-B (the Scientific Pandemic Influenza Group on Behavioural Science) to explore the modelling outcomes and behavioural science aspects of nine school closure scenarios identified by the Department for Education (DfE), from schools either staying shut (Baseline 1), to remaining fully open (Baseline 2); the Department for Education's Explanatory Note on SAGE Paper: Interdisciplinary Task and Finish Group on the Role of Children in Transmission: Modelling and behavioural science responses to scenarios for relaxing school closure; Interdisciplinary Task and Finish Group on the Role of Children in Transmission Modelling and behavioural science responses to scenarios for relaxing school closures; Initial Impact Assessment on the Strategic Framework for Reopening Schools and Early Learning and Childcare Settings in Scotland from the Scottish Government; The Independent SAGE Report 2: Should Schools Reopen? Interim Findings and Concerns. Draft Document for Public Discussion from the Independent Scientific Advisory Group for Emergencies (SAGE); and COVID-19: Epidemiology, virology and clinical features, from Public Health England. [ABSTRACT FROM AUTHOR]

Signal amplification methods are the most preferred non-PCR molecular tools in diagnostic virology. The aim of signal amplification tests is to amplify and measure the signals obtained from the target DNA or RNA in clinical samples, instead of directly copying these molecules. With these characteristics, signal amplification methods do not pose any risk of contamination of the test samples, laboratory devices or working environment with amplified products. Approved signal amplification tests have been commercially available for detection and quantitation of HIV-1, HCV and HPV nucleic acids, as well as genotyping analysis of HPV. Signal amplification tests were first used in the early 1990s and have different working principles and special designs. These tests are now combined with new diagnostic techniques such as real-time PCR, the rolling circle method, luminex xMAP, DNA biosensor technology and sequence analysis, in the detection and genotyping of viral agents, multiplex analysis and the discovery of new and unknown viruses. In addition, approved signal amplification tests are used as the standard control methods for newly developed test designs. In this review, the working principles of signal amplification-based tests and their use in diagnostic virology are discussed in detail and examples of their use in other platforms are presented. [ABSTRACT FROM AUTHOR]

Marseilleviridae is a family of viruses which have only been propagated in acanthamoeba. Marseillevirus sequences have been recently detected in different human matrices by viral metagenomics. Single‐center studies worldwide have estimated a low prevalence of marseillevirus both in symptomatic patients and in healthy donors but, to date, no informations are available on the prevalence of this giant virus in Italy. By a polymerase chain reaction targeting the ORF152 viral sequence, we tested sera from 197 immunosuppressed patients and 285 healthy donors, and 63 and 30 respiratory and cerebrospinal fluid samples, respectively, of patients with various clinical conditions and referring the Virology Division for diagnostic purposes. We observed no evidence of Marseillevirus DNA in all 575 samples tested. Marseillevirus probably does not cause infection in human. Highlight: We observed no evidence of Marseillevirus DNA in immunocompetent and immunocompromised Italian patients.Marseillevirus probably does not cause infection in human. [ABSTRACT FROM AUTHOR]

Professor Demetrios A. Spandidos, Professor Emeritus of Clinical Virology at the University of Crete School of Medicine in Crete, Greece, is a pioneer in world research in Virology, Oncology and medical education. He has been the first Professor of Clinical Virology at the University of Crete School of Medicine, the only School of Medicine with a separate Department of Clinical Virology in Greece. According to Professor Spandidos, the understanding of the interactions of viruses with human host cells is what enables the development of new vaccines and new therapies against a wide range of diseases in humans, including cancer. Over the past two decades, the expansion of new knowledge, treatments, prevention and management options and the emerging needs on neonatal and paediatric viral infections have made the role of future paediatric virologists more than necessary. For this reason, he strongly believes that Paediatric Virology should be recognized as a new paediatric subspecialty. Professor Spandidos declares that medical education is a continuous sequence from undergraduate to postgraduate/specialty/subspecialty medical training and continuing learning. He also supports the concept that the future of medical education represents the future of Medicine. [ABSTRACT FROM AUTHOR]

Diagnosis of gastrointestinal infections has been revolutionized by the development of in vitro diagnostic (IVD) multiplex molecular panels for the detection of viral nucleic acids. In addition to a high degree of accuracy, these panels are commercially available and relatively simple to perform in the clinical laboratory. However, use of these panels must be carefully considered owing to the laboratory costs of the test, limited reimbursement, and potential for overuse. In this review from the Pan American Society for Clinical Virology, we focus on the viral components of GI multiplex panels (GIPs), presenting a brief overview of pathogens included on most panels and a discussion of advantages and challenges of the inclusion of viral targets on GIPs that should be considered before implementation in the clinical laboratory. [ABSTRACT FROM AUTHOR]

• This review article describes the amplification chemistries that are commonly used in the clinical virology laboratory. • The article includes different techniques for target, signal, and probe amplification chemistries. • Major features and limitations of each chemistry are described. • Commercial applications of these chemistries for viral pathogens that infect humans are included. Molecular diagnostic methods have evolved and matured considerably over the last several decades and are constantly being evaluated and adopted by clinical laboratories for the identification of infectious pathogens. Advancement in other technologies such as fluorescence, electronics, instrumentation, automation, and sensors have made the overall diagnostic process more accurate, sensitive, and rapid. Nucleic acid based detection procedures, which rely on the fundamental principles of DNA replication have emerged as a popular and standard diagnostic method, and several commercial assays are currently available based on different nucleic acid amplification techniques. This review focuses on the major amplification chemistries that are used for developing commercial assays and discusses their application in the clinical virology laboratory. [ABSTRACT FROM AUTHOR]

Summary: Prisons are a key demographic in the drive to eradicate hepatitis C virus (HCV) as a major public health threat. We have assessed the impact of the recently introduced national opt‐out policy on the current status of HCV testing in 14 prisons in the East Midlands (UK). We analysed testing rates pre‐ and post‐introduction of opt‐out testing, together with face‐to‐face interviews with prison healthcare and management staff in each prison. In the year pre‐opt‐out, 1972 people in prison (PIP) were tested, compared to 3440 in the year following opt‐out. From July 2016 to June 2017, 2706 people were tested, representing 13.5% of all prison entrants (median 16.6%, range 7.6%‐40.7%). Factors correlating with testing rates were as follows: pre‐admission location of the PIP (another prison or the community, OR 2.2, 95% CI 1.9‐2.3, P < 0.001); whether the PIP could access health care independently of prison officers (OR 1.7, 95% CI 1.5‐1.8, P < 0.001); the absence of out‐reach services for HCV treatment (OR 1.3, 95% CI 1.2‐1.5, P < 0.001), whether >50% of PIP reported ease of access to a nurse (OR 2.0, 95% CI 1.8‐2.2, P < 0.001), and whether prison health care was supplied by private or NHS providers (OR 1.3, 95% CI 1.2‐1.5, P < 0.001). Testing rates remained far below the minimum national opt‐out target of 50%. Inadequacy of healthcare facilities and constraints imposed by adherence to prison regimens were cited by healthcare and management staff at all prisons. Without radical change, the prison estate may be intrinsically incapable of supporting NHSE to deliver the HCV elimination strategy. [ABSTRACT FROM AUTHOR]

Background We analysed the laboratory surveillance data generated by the network of virology laboratories from January 2016 to July 2018 to describe the epidemiology of chikungunya in India. Methods Suspected patients reporting to virology laboratories are investigated for the presence of immunoglobulin M (IgM) antibodies against chikungunya virus (CHIKV) as evidence of recent infection. We analysed the data to describe the distribution of seropositive individuals by time, place and demographic characteristics. Results Of the 49 380 sera tested from suspected patients, 20.5% were seropositive. CHIKV IgM positivity was seen throughout the year, with a peak between September and December. The laboratories diagnosed 28 outbreaks of chikungunya during the study period. Conclusions Laboratory surveillance data indicate continued transmission of CHIKV in many regions of India. [ABSTRACT FROM AUTHOR]