Virology Laboratory Research Articles

Coronavirus Disease 2019 (COVID-19) Real World Data Infrastructure: A Big-Data Resource for Study of the Impact of COVID-19 in Patient Populations With Immunocompromising Conditions.

We developed a United States-based real-world data resource to better understand the continued impact of the coronavirus disease 2019 (COVID-19) pandemic on immunocompromised patients, who are typically underrepresented in prospective studies and clinical trials. The COVID-19 Real World Data infrastructure (CRWDi) was created by linking and harmonizing de-identified HealthVerity medical and pharmacy claims data from 1 December 2018 to 31 December 2023, with severe acute respiratory syndrome coronavirus 2 virologic and serologic laboratory data from major commercial laboratories and Northwell Health; COVID-19 vaccination data; and, for patients with cancer, 2010 to 2021 National Cancer Institute Surveillance, Epidemiology, and End Results registry data. The CRWDi contains 4 cohorts: patients with cancer; patients with rheumatic diseases receiving pharmacotherapy; noncancer solid organ and hematopoietic stem cell transplant recipients; and people from the general population including adults and pediatric patients. The project successfully linked and harmonized longitudinal, de-identified data on 5.2 million unique patients using privacy-preserving record lineage techniques. The system was developed in early 2024 and rapidly deployed, enabling longitudinal analysis of patient healthcare over the full geography of delivery settings and exploration of novel questions for populations at high risk for adverse outcomes. The successful development of the CRWDi enables researchers to address unanswered questions that have arisen during the COVID-19 pandemic. By making the data broadly and freely available to academic researchers, this real-world data system represents an important complement to existing consortia and clinical trials that have emerged during the healthcare crisis and is readily reproducible for future purposing.

Current data on the circulation of the Q fever pathogen in the Republic of Guinea

Background. Q fever is the one of the best-studied zoonoses, which is widespread throughout almost the entire territory of Africa, excluding the territory of the Sahara. However, the current data on the incidence of coxiellosis and the circulation of Coxiella burnetii on this continent are limited and vary according different sources. In 1980–1990, the Soviet-Guinean Research Virology and Microbiology Laboratory conducted studies to estimate the distribution of the Q fever pathogen, assess the herd immunity in humans and identify specific antibodies in the sera of livestock. However, in subsequent years, the research was suspended. The aim of this study is to obtain up-to-date data on the distribution of C. burnetii in all landscape and geographical zones of the Republic of Guinea. Materials and methods. The study was carried out in the laboratory of the Russian-Guinean Center for Epidemiology and Prevention of Infectious Diseases (Kindia, Republic of Guinea). The study involved 332 sera of febrile patients and 3156 sera from practically healthy volunteers, 1074 blood samples of livestock, 1648 suspensions of ticks, 319 specimens of small mammals and 298 of bats. The study was carried out using ELISA and PCR methods, selected samples were subjected to in-depth genetic analysis. Results and discussion. The study of the distribution of C. burnetii on the territory of all landscape-geographical zones of the Republic of Guinea was carried out. For the first time, an officially registered case of human Q fever case has been identified. The role of livestock, small mammals and bats in the circulation of the pathogen has been established. It has been shown that the main vectors in Guinea are ixodid ticks of the Amblyomma variegatum, Hyalomma truncatum and Rhipicephalus decoloratus species. Employing molecular methods, C. burnetii strains carrying the QpH1 plasmid, capable of causing diseases in humans and animals were identified. For the first time, the complete nucleotide sequence of 16S rRNA of the Q fever pathogen (OQ152497–OQ152500) identified on the territory of Guinea was determined and registered in the GenBank database. Conclusion. Taking into account the high epidemiological significance of Q fever, the study of the specifics of C. burnetii circulation in Guinea remains an urgent task. Regular monitoring and assessment of risk factors for diseases caused by coxiella are necessary for the development of an algorithm for laboratory diagnosis and recommendations for clinicians.

In vitro Antiviral Activity of Eucheuma cottonii against New Castle Disease Virus

Aims: To determine the antiviral activity of Eucheuma cottonii against the New Castle disease virus. Study Design: In vitro study. Place and Duration of the Study: The research was conducted in the Pharmaceutical Chemistry, Drug Design, and Pharmacognosy department at St. John’s University of Tanzania and Virology Laboratory at Sokoine University of Agriculture between April 2022 and August 2022. Methodology: The present study used an OVO assay and hemagglutination test to investigate the In vitro antiviral activity of E. cottonii collected from Zanzibar, Tanzania, against the Newcastle disease virus (NDV). Ovo assay was assessed using 60 embryonated eggs and a hemagglutination test was performed by collecting the allantoic fluid from treated eggs and serum from hatched chicks to detect antigen titer against NDV using 96 well plate diffusion method. Results: Results of the Ovo assay showed that all the 40 embryonated eggs used as test group were alive and thus confirmed that the extract of E. cottonii (0.32 g/mL) is strong enough to destroy the virus. In which the results from the serial dilution assay (hemagglutination test) confirmed a substantial decrease in the high level of NDV as observed in the allantoic fluid of the infected embryonated eggs to range from 10^-3 to 10^-8 serial dilutions. Conclusion: The OVO assay and hemagglutination results indicate that E. cottonii possesses antiviral activity against NDV. Still further studies like isolation and characterization of bioactive compounds are needed to apply E. cottonii as an alternative source of therapy to fight against NDV.

Survey of clinical microbiology and infectious disease testing capabilities among institutions in Africa.

Inadequate laboratory infrastructure and testing capabilities are a major impediment to addressing the infectious disease burden in Africa. Therefore, the aims of this study were to characterize the clinical microbiology/infectious disease laboratory capabilities among countries in Africa. A survey to assess the microbiological testing capabilities at hospitals, government laboratories, and free-standing public and private laboratories in African countries was developed by subject matter experts. Questions included institutional demographics and microbiology services in the broad categories of bacteriology, virology, mycology, parasitology, and rapid diagnostics/point-of-care testing. The survey was distributed using the American Society of Clinical Pathology email listserv between June and August 2022. In total, 131 unique institutions in 28 countries endorsed at least 1 type of microbiology service, with parasitology (80.9%, 106/131) and bacteriology (77.9%, 102/131) being most common, while mycology (45.0%, 59/131) and virology (45.8%, 60/131) laboratories were less prevalent. The most frequently performed bacteriology test was bacterial identification (90.2%, 92/102), followed by aerobic bacterial cultures and antimicrobial susceptibility testing (both 89.2%, 91/102). Among all clinical microbiology/infectious disease laboratories, the most commonly tested agents were HIV (90.8%, 119/131), Treponema pallidum (78.6%, 103/131), Plasmodium falciparum (76.3%, 100/131), Mycobacterium tuberculosis (76.3%, 100/131), and hepatitis C virus (74.8%, 98/131). These findings provide contemporary data regarding the availability of critical infectious disease testing capabilities among institutions in Africa. These results and future additional studies will be crucial for understanding where strategic investment in the laboratory and public health infrastructure is warranted.

Serological Profiles of Hepatitis B Virus in Patients With Crohn's Disease Undergoing Anti-Tumor Necrosis Factor Alpha (TNFα) Therapy at Ibn Sina University Hospital, Rabat.

Introduction Anti-tumor necrosis factor alpha (TNFα) therapies have revolutionized the management of Crohn's disease (CD). However, they increase the risk of viral reactivation, particularly hepatitis B virus (HBV). This study aims to define the HBV serological profiles of patients with CD who are candidates for biological therapy, identifying profiles at potential risk for reactivation or exacerbation following immunosuppressive treatment. Materials and methods This descriptive retrospective study included patients with CD, aged over 16 years, who were candidates for anti-TNFα treatment at Ibn Sina University Hospital Center (UHC) in Rabat, Morocco, from January 2015 to March 2023. The serological profiles of patients, including hepatitis B surface antigen (HBsAg), hepatitis B surface antibodies, and total hepatitis B core antibodies (HBcAb), were determined using microparticle chemiluminescence immunoassay with the ARCHITECT i2000sr or Alinity (Abbott Diagnostics, Chicago, Illinois, United States) automated systems at the Central Virology Laboratory (CVL) of Ibn Sina UHC Rabat. HBV DNA quantification was performed using the m2000 Abbott Diagnostic or GeneXpert system. Results Out of 249 patients with CD who were candidates for biological therapy, 131 (52.6%) received anti-TNFα treatment, including 39 (29.8%) with adalimumab and 92 (70.2%) with infliximab. The median age was 41 years, and the male-to-female ratio was 0.52. The overall HBV screening rate before starting biological therapy was 68.7%. HBV screening was conducted for 90 patients at the CVL, where serological marker analysis categorized five distinct profiles. A majority of patients (65, 72.2%) had negative serological profiles for HBV, while 10 (11.1%) were immunized via vaccination. Profiles at risk of viral reactivation or worsening following immunosuppressive therapy included 12 (13.3%) patients immunized by contact, two (2.3%) with isolated HBcAb, and one (1.1%) with active viral hepatitis (positive HBsAg and HBcAb), who was initiated on tenofovir 300 mg before starting combination therapy. No cases of primary infection or viral B reactivation were observed during the study. Conclusions In our study, 15 patients (16.7%) exhibited a potential risk of viral reactivation or worsening of HBV following the initiation of immunosuppressive therapy. The authors recommend precise patient selection, thorough pretreatment evaluation, and regular follow-up during therapy to minimize adverse events associated with anti-TNFα treatment. Additionally, a prophylactic or preemptive strategy should be considered. The risk of late reactivation after discontinuation of biological therapy should also be carefully monitored.

Epidemiology and genetic diversity of human respiratory syncytial virus in Belgium between 2011 and 2019

BackgroundHuman respiratory syncytial virus (HRSV) is worldwide one of the leading causes of acute respiratory tract infections in young children and the elderly population. Two distinct subtypes of HRSV (A and B) and a multitude of genotypes have been described. The laboratory of Clinical and Epidemiological Virology (KU Leuven/University Hospitals Leuven) has a long-standing history of HRSV surveillance in Belgium.MethodsIn this study, the seasonal circulation of HRSV in Belgium was monitored during 8 consecutive seasons prior to the SARS-CoV-2 pandemic (2011–2012 until 2018–2019). By use of a multiplex quantitative real time PCR panel, 27,386 respiratory samples were tested for HRSV. Further subtyping and sequencing of the HRSV positive samples was performed by PCR and Sanger sequencing. The prevalence and positivity rate were estimated in 4 distinct age groups and the circulating strains of each subtype were situated in a global context and in reference to the described genotypes in literature.ResultsHRSV circulated in Belgium in a yearly re-occurring pattern during the winter months and both HRSV subtypes co-circulated simultaneously. All HRSV-B strains contained the 60 nt duplication in the HVR2 region of the G gene. Strains of subtype HRSV-A with a 72 nt duplication in the HVR2 region were first observed during the 2011–2012 season and replaced all other circulating strains from 2014 to 2015 onwards.

Molecular Biology for Diagnosis of Congenital and Neonatal Infections in the Cerebrospinal Fluid of Newborns from a Brazilian Tertiary Hospital.

This was a prospective descriptive study involving the molecular analysis of 151 CSF samples from neonates, collected for cytological and biochemical diagnosis from 2017 to 2021. After the results and consent from the participants' caregivers were obtained, the leftover material was sent to the University's Virology Laboratory and submitted for DNA/RNA extraction and Nested-PCR/RT-PCR. A review of the patients' medical records and descriptive statistics was performed. This work was approved by the Ethics Committee (CAAE: 86760218.3.0000.5404). A total of 151 CSF samples were obtained, 16 of which were positive (10.6% [95% CI%: 6.18-16.63%]). Two of these were PCR-positive for HSV-1 (1.3%), four for VZV (2.6%), one for CMV (0.67%), two for Toxoplasmosis (1.3%), four for Parvovirus B-19 (2.6%), and four for Zika (2.6%). The proportion of positive PCR results was higher in the group that presented with malformations (25.0% vs. 8.4%, p = 0.040). The pathogens identified by PCR were mostly Zika virus, VZV, and B-19, and these were mainly found in newborns with malformations.

Human papillomavirus carriage in the semen of men consulting for infertility: prevalence and correlations with sperm characteristics.

We aim to study the semen carriage of human papillomavirus (HPV) and evaluate its association with patient characteristics. We conduct a single-center cohort study at Amiens University Hospital Center (Amiens, France). From May 1 to October 31, 2021, 461 men consulting for infertility and with semen analysis data were included. Each participant gave his written informed consent for the use of laboratory, demographic, clinical, and lifestyle data. A proportion of the semen samples were sent to a virology laboratory for HPV screening in a polymerase chain reaction (PCR) assay. In univariate and multivariate analyses with a logistic regression model, HPV+ and HPV- groups were compared with regard to semen characteristics (including the DNA fragmentation index and the sperm decondensation index) and demographic, clinical, and lifestyle variables. Semen HPV carriage was detected in 22.3% of the patients. High-oncogenic-risk HPV genotypes were predominant (57.6%). Multivariate analysis showed that HPV carriage was significantly associated with the presence of at least one abnormal spermogram dinging (according to the 6th World Health Organization criteria), with an adjusted odds ratio (OR) of 4.10 (95% confidence interval [CI]: 2.32-7.25, P < 0.001). A statistically significant association was also found for the type of infertility (OR: 1.61, 95% CI: 1.00-2.57, P = 0.05), the presence of varicocele (OR: 3.99, 95% CI: 1.48-10.71, P = 0.01), and a history of cryptorchidism, testicular ectopia, or monorchidism (OR: 3.54, 95% CI: 1.07-11.66, P = 0.04). Infection with a single HPV genotype or multiple HPV genotypes was significantly associated with at least one abnormal spermogram finding for all HPV oncogenic risk groups (OR: 3.93, 95% CI: 2.08-7.41, P < 0.001; and OR: 4.11, 95% CI: 1.58-10.68, P = 0.01, respectively). The association between sperm HPV carriage and the risk of infertility was statistically significant in a multivariate analysis (OR: 5.63, 95% CI: 3.16-10.01, P < 0.001) and after adjustment for the propensity score (OR: 6.10, 95% CI: 3.33-11.21, P < 0.001). Our results suggest that semen HPV carriage has an impact on male fertility. Sperm screening for HPV might be a useful addition to the work-up for male infertility.

Validation, implementation and quality control of a Torque Teno Virus qPCR in a multinational clinical trial

BackgroundImmunosuppressive medication after organ transplantation is usually dosed through therapeutic drug monitoring. Trough levels of antirejection medication however, do not adequately predict rejection or infections. The TTVguideIT trial is a multinational clinical trial evaluating the safety of Torque Teno Virus (TTV) load assessed by qPCR, as an alternative to trough level tacrolimus dosing. MethodsPrior to, and during the clinical trial, the inter-and intra-laboratory variability, accuracy, and precision of the TTV R-GENE® assay was evaluated through analysis of internal quality control (IQC), external quality assessment (EQA) and linearity panels performed by the thirteen participating clinical virology laboratories, each using their standard testing platforms. ResultsIQC samples with a target of 4 log10 copies/mL (cp/mL) were tested by the participating laboratories 130 times during the implementation phase and 987 times during the trial phase. They showed excellent accuracy, with an inter-laboratory standard deviation (SD) of 0.17 log10 cp/mL, and an intra-laboratory SD of 0.03 to 0.20 log10 cp/mL during the implementation phase, and an inter-laboratory SD of 0.19 log10 cp/mL, and an intra-laboratory SD 0.07 to 0.18 log10 cp/mL during the trial phase. Three EQA panels and three linearity panels showed similarly small variability during implementation as well as within the trial phase. ConclusionThis data shows that TTV load measurement can be standardized for use in a multinational clinical trial. By using IQC, LP and EQA samples, the quality and integrity of the assay can be compared between laboratories and precise and accurate results can be generated.

Building the clinical workforce of pediatric infectious diseases in Bolivia for better care of children with cancer

Abstract Background Infections in children with cancer can be fatal if not appropriately managed. Access to trained and competent professionals in infection care and prevention (IC&amp;P) improve care outcomes. Bolivia expects about 1700 new pediatric cancer cases annually. In 2023, 2371 hematological neoplasms were reported in children, cared mainly at 4 public hospitals where, until 2016, 5 pediatric infectious disease (PID) specialists were working in 2 of these 4 hospitals. For better PID clinical workforce for critically ill children, we built a PID medical residency training. In 2017, the training accepted its first trainees at Hospital del Niño “Dr. Ovidio Aliaga Uría” (Hospital del Niño), a public hospital in La Paz. Here, we describe the program, how we built it, and its outcomes. Methods The PID training goal is to train leaders in PID for clinical observations, PID literature interpretation, and to transform them into concepts that can be applied to ID pathogenesis or treatment. At the end of the 2-year PID training program, we expect trainees will gain expertise in PID clinical practice and clinical or translational science research. Our PID program was based on other PID specialty training programs from Mexico, Spain, and the USA of which, we adapted standard topics and formats of training. The content has routine and prevalent PIDs of Bolivia and the region, including on immunocompromised patients. We expected that the trainee dedicate full time to the PID training program plus caring for assigned patients and those requested consultations in the wards and clinics of Hospital del Niño; review articles; participate in clinical discussions; attend the Infection Control Course of St. Jude Children’s Research Hospital; attend and / or present in the PID case-based learning international sessions; visit the virology, bacteriology, parasitology and immunology laboratories of the national reference laboratory; and attend international rotations at participating hospitals. Results Since 2017, 5 new PID pediatricians graduated from our program, 3 work in public hospitals that care for children with cancer and 2 work in government insurance hospitals. They are working in hospitals in La Paz (n=2), Cochabamba (n=1), Santa Cruz (n=1), and Tarija (n=1). These graduates are leaders in ICP, antimicrobial stewardships, and education of hospital staffs. As per December 2023, they produced 5 articles for local journals, 2 for international journals, 7 abstracts for scientific meetings. Currently, 2 pediatricians participate in the PID training program, and post-graduation, they plan to work in departments of Potosí and Sucre where there are no PID specialists. Challenges to establish PID specialists in Bolivia are lack of value attributed to PID and its role in care quality. Conclusion Creating local expertise in PID are essential to improve and sustain the optimum care for children with cancer in Bolivia especially in places where these personnel did not exist before. However, for best investment return of PID trained professionals, having PID job descriptions and paid positions in hospitals will absorb PID specialists to benefit the largest population of children with cancer in Bolivia.

Reaction Times in Modern Virology: "This is not a Drill".

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of interest BA.2.87.1 has not driven any Coronavirus disease 2019 (COVID-19) pandemic wave. Nevertheless, it has served to test the reaction times of modern virology laboratories. In this commentary, we highlight how fast the reaction has been at characterizing this sublineage, leading at an unprecedented pace to almost as many papers as the number of viral sequences.

Analysis of HSV1/2 Infection Reveals an Association between HSV-2 Reactivation and Pregnancy.

The herpes simplex viruses consist of the strains, HSV-1 and HSV-2, which are prevalent worldwide and lack a definitive cure. We aimed to explore the specific characteristics of HSV 1 and 2 infections, such as differences between gender assigned at birth, age at infection, site of infection, comorbidities, and effect of pregnancy, through a data analysis. Between 2011 and 2018, the Israeli Central Virology Laboratory diagnosed 9189 samples using multiplexed real-time PCR. In addition, we extracted all of the medical data for 287 females hospitalized at the Sheba Medical Center with HSV-1 (161) or HSV-2 (126) genital infections. HSV-2 was almost absent in the orofacial samples from both genders, while in other lesion sites, HSV-2 was significantly more abundant in females than in males (p < 0.05,). HSV-2 was initially detected at puberty. In the hospitalized females' malignancies, both HSV-1 and HSV-2 were found with a non-significant difference. Simultaneously, pregnancies were more common in females who were HSV-2-positive compared with those who were HSV-1-positive (27.8% vs. 12.4%, respectively, p < 0.01). Primary infections occur more with HSV-1 than with HSV-2 (15.6% vs. 3.2%, respectively). Our findings demonstrate that genital HSV-2 infection episodes are more frequent during pregnancy, suggesting that pregnancy may serve as a risk factor for HSV-2 reactivation or infection.

Cytotoxicity and Apoptotic DNA Fragmentation Evaluation of Ethanolic Extract of Phyllanthus niruri L. in Vero and Primary Feline Testicular Cells

Phyllanthus niruri or Dukung Anak is native to Southeast Asia, particularly in Malaysia and has been used as traditional preparations and as health supplements. However, previous studies have found that P. niruri can impair the male reproductive functions. With booming population of stray animals within the community, a new controlling method that is more affordable and safer must be developed. This study was aimed to explore the safety of P. niruri as a male herbal contraceptive to be used in veterinary medicine as one of the non-surgical sterilisation methods. For the preliminary part of this ongoing research, we have investigated the cytotoxicity activity of P. niruri ethanolic extract on Vero and primary feline testicular cells (FTC) and DNA apoptotic activity in FTC. Phyllanthus niruri extract was prepared using cold maceration method in 50% ethanol. The FTC were prepared from feline testes obtained from post-routine castration from local private veterinary clinics, while Vero cells were obtained from the archived cells of Virology Laboratory, Faculty of Veterinary Medicine Universiti Malaysia Kelantan (FPV UMK). The MTT assay was performed to evaluate the cytotoxicity of these plant extracts on FTC and Vero cells. Apoptosis was assessed using DNA laddering assay on extracted DNA of the treated FTC cells and observed the DNA patterns in gel electrophoresis. The 50% cytotoxic concentration (CC50) of the ethanolic extract of P. niruri was a dose dependent. The DNA laddering assay revealed that the plant extract does not induce apoptosis in FTC. Therefore, the study outcomes indicated that the ethanolic extract of P. niruri is potentially safe for animal use. However, in vivo studies are required to confirm these findings.

Comprehensive Analysis of SARS-CoV-2 Dynamics in Bangladesh: Infection Trends and Variants (2020-2023).

The first case of COVID-19 was detected in Bangladesh on 8 March 2020. Since then, the Government of Bangladesh (GoB) has implemented various measures to limit the transmission of COVID-19, including widespread testing facilities across the nation through a laboratory network for COVID-19 molecular testing. This study aimed to analyze the dynamics of SARS-CoV-2 in Bangladesh by conducting COVID-19 testing and genomic surveillance of the virus variants throughout the pandemic. Nasopharyngeal swabs were collected from authorized GoB collection centers between April 2020 and June 2023. The viral RNA was extracted and subjected to real-time PCR analysis in icddr,b's Virology laboratory. A subset of positive samples underwent whole-genome sequencing to track the evolutionary footprint of SARS-CoV-2 variants. We tested 149,270 suspected COVID-19 cases from Dhaka (n = 81,782) and other districts (n = 67,488). Of these, 63% were male. The highest positivity rate, 27%, was found in the >60 years age group, followed by 26%, 51-60 years, 25% in 41-50 years, and the lowest, 9% in under five children. Notably, the sequencing of 2742 SARS-CoV-2 genomes displayed a pattern of globally circulating variants, Alpha, Beta, Delta, and Omicron, successively replacing each other over time and causing peaks of COVID-19 infection. Regarding the risk of SARS-CoV-2 infection, it was observed that the positivity rate increased with age compared to the under-5 age group in 2020 and 2021. However, these trends did not remain consistent in 2022, where older age groups, particularly those over 60, had a lower positivity rate compared to other age groups due to vaccination. The study findings generated data on the real-time circulation of different SARS-CoV-2 variants and the upsurge of COVID-19 cases in Bangladesh, which impacted identifying hotspots and restricting the virus from further transmission. Even though there is currently a low circulation of SARS-CoV-2 in Bangladesh, similar approaches of genomic surveillance remain essential for monitoring the emergence of new SARS-CoV-2 variants or other potential pathogens that could lead to future pandemics.

HIV-1 genotypic resistance testing using single molecule real-time sequencing

BackgroundHIV-1 resistance testing is recommended in clinical management and next-generation sequencing (NGS) methods are now available in many virology laboratories. ObjectivesTo evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing (Sequel, PacBio) for HIV-1 polymerase genotyping. Study design111 prospective clinical samples (83 plasma and 28 leukocyte-enriched blood fraction) were analyzed for routine HIV-1 resistance genotyping using Sanger sequencing, Vela NGS, and SMRT sequencing. We developed a SMRT sequencing protocol and a bio-informatics pipeline to infer antiretroviral resistance on both haplotype and variant calling approaches. ResultsThe polymerase was successfully sequenced by the three platforms in 98 % of plasma RNA samples for viral loads above 4 log copies/mL. The success rate decreased to 83 % using Sanger or Vela sequencing and to 67 % using SMRT sequencing for viral loads of 3 to 4 log copies/mL. Sensitivities of 50 %, 54 % and 61 % were obtained using SMRT, Vela, and Sanger sequencing, respectively, in cellular DNA from patients with prolonged undetectable plasma HIV-1 RNA. Ninety-eight percent of resistance-associated mutations (RAMs) identified with Sanger sequencing were detected using SMRT sequencing. Furthermore, 91 % of RAMs (> 5 % threshold) identified with Vela NGS were detected using SMRT sequencing. RAM quantification using Vela and SMRT sequencing was well correlated (Spearman correlation ρ = 0.82; P < 0.0001). ConclusionsSMRT sequencing of the full-length HIV-1 polymerase appeared performant for characterizing HIV-1 genotypic resistance on both RNA and DNA clinical samples. Long-read sequencing is a new tool for mutation haplotyping and resistance analysis.

Epstein-Barr virus qPCR testing on bronchoalveolar lavage fluid from immunocompromised patients

BackgroundEpstein-Barr Virus (EBV) is associated with lung disease in immunocompromised patients, particularly transplant recipients. EBV DNA testing of lower respiratory tract specimens may have diagnostic utility. MethodsThis was a retrospective, observational study of all patients with bronchoalveolar lavage (BAL) fluids submitted for EBV qPCR testing from February 2016 to June 2022 at the Stanford Clinical Virology Laboratory. ResultsThere were 140 patients that underwent 251 EBV qPCR BAL tests (median 1; range 1 – 10). These patients had a mean age of 15.9 years (standard deviation, 15.1 years) and 50 % were female. Transplant recipients accounted for 67.1 % (94/140) of patients, including 67.0 % (63/94) solid organ transplant (SOT) and 33.0 % (31/94) hematopoietic cell transplant. Diagnostic testing was performed more commonly than surveillance testing [57.0 % (143/251) v. 43.0 % (108/251)]; 96.2 % (104/108) of surveillance samples were from lung transplant recipients. Excluding internal control failures, 34.7 % (83/239) of BAL had detectable EBV DNA, encompassing a wide range of viral loads (median=3.03 log10 IU/mL, range 1.44 to 6.06). Overall agreement of EBV DNA in BAL compared to plasma was 74.1 % [117/158; 95 % confidence interval (CI): 66.5 % to 80.7 %], with a kappa coefficient of 0.44 (95 % CI: 0.30 to 0.57). Only 20.1 % (48/239) of results were discussed in a subsequent clinical note, and one result (0.4 %; 1/239) changed clinical management. ConclusionsEBV qPCR testing on BAL offers limited clinical impact. Additional biomarkers are required to improve the diagnosis of EBV-associated lung diseases.

SARS-CoV-2 positivity rates in tear secretions of inpatient COVID-positive individuals in an Indian tertiary care setting.

In the third wave of the coronavirus disease 2019 (COVID-19) pandemic, despite progressive vaccination coverage, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was rapidly spreading and the possibility for ocular transmission of COVID-19 was most relevant. This study aimed to determine the presence of SARS-CoV-2 RNA in tears as detected by real-time polymerase chain reaction (RT-PCR), among individuals who tested COVID-positive during the third wave of the COVID pandemic. Among 90 patients admitted with mild to severe COVID-19 infection (ranging from asymptomatic to severe symptoms), separate conjunctival samples were obtained from both eyes without topical anesthesia taking precautions to avoid contamination. Inferior fornices of the eyes were swept slowly with sterile Dacron swabs for 10 s or until visibly wet. They were inoculated directly in a viral lysis buffer containing carrier RNA and transported to the Virology Laboratory for the detection of SARS-CoV-2 RNA by RT-PCR. Conjunctival swabs were declared positive based on cycle threshold values of <37. Seven of 180 conjunctival swabs were positive (3.9%). Five patients had unilateral positive and one patient had bilateral positive conjunctival swabs. Thirty-six (40%) patients had their conjunctival swabs taken on the same day of detection of the first positive nasopharyngeal swab, while the rest (54) had their conjunctival swabs taken up to 3 days later. Overall, 20 (22.2%) patients were symptomatic for COVID-19. Considering the contagious nature of SARS-CoV-2 in the third wave, there is a possibility that ocular surface fluid might have been a contributing factor along with other modes of transmission of COVID-19.

Genomic epidemiology of SARS-CoV-2 in Senegal in 2020-2021.

In Senegal, molecular diagnosis was widely used for the detection and management of COVID-19 patients. However, genomic surveillance was very limited in the public sector. This study aimed to share the experience of a Senegalese public sector laboratory in response to the COVID-19 pandemic, and to describe the distribution of variants circulating in 2020 and 2021. From July 2020 to December 2021, SARS-CoV-2 qRT-PCR was performed on nasopharyngeal samples from travelers and symptomatic patients at the Bacteriology and Virology Laboratory (LBV) of the Aristide le Dantec University Teaching Hospital. Samples with a cycle threshold (Ct) ≤ 30 were selected for whole-genome sequencing (WGS) using the Nanopore technology. In-house scripts were developed to study the spatial and temporal distribution of SARS-CoV-2 variants in Senegal, using our sequences and those retrieved from the GISAID database. Of 8,207 patients or travelers screened for SARS-CoV-2, 970 (11.8%) were positive and 386 had a Ct ≤ 30. WGS was performed on 133 samples. Concomitantly with high-quality sequences deposited in the GISAID database covering nine cities in Senegal in 2020 and 2021 (n = 1,539), we observed a high circulation of the 20A (B.1, B.1.416 and B.1.620) and 20B (B.1.1.420) lineages in 2020, while most of the samples belonged to Delta variants (AY34 and AY.34.1, 22%) in 2021. Despite its late involvement, COVID-19 diagnosis was routinely performed in LBV, but genomic characterization remained challenging. The genomic diversity of SARS-CoV-2 strains in Senegal reflected that observed worldwide during the first waves of the pandemic.

An Integrative Approach to the Study of Cognitive Abilities in a Non-Human Primate Model in a Virology Laboratory Environment.

Non-human primates, due to their similarities in immune response to humans, are the preferred model for studying infectious processes and any associated cognitive impairments. Behavioral tests are indispensable for investigating pathogenesis in neuroinfections, especially those that do not manifest with noticeable clinical symptoms, as well as in the transition to a chronic form of the disease. Modeling viral infection requires specialized experimental conditions. Our work describes techniques for investigating mnemonic functions, tiredness, attentional focus, quick-wittedness, and basic behavioral responses in primates under the assumed conditions for infections with viruses that do not have an airborne route of transmission. It also outlines approaches to the training and selection of primates for virological research, as well as analyzing gender differences in learning abilities, the impact of housing conditions on the results, and the correlation between training success and behavioral test scores. These methods will allow a more detailed study of non-human primates as a model for researching cognitive and behavioral impairments under infectious and immune stress, as well as the design of less energy-intensive experiments for evaluating the efficacy and safety of therapeutic and prophylactic strategies at early stages of infection.

Increase in measles cases in a City hospital, Istanbul, Turkey.

Measles cases are increasing remarkably in our country as well as all over the world. In this study, it was aimed to examine the epidemiological and clinical characteristics of measles cases detected in our hospital, as well as the measles seroprevalence in our region. A total of 7,452 individuals whose measles IgG and/or IgM antibodies were studied between December 2021 and March 2023 in the Medical Virology Laboratory in Başakşehir Çam and Sakura City Hospital were included in this retrospective study. Measles IgG and IgM antibodies were analysed by enzyme-linked immunosorbent assay. Demographic information, clinical symptoms and laboratory data of the participants were obtained from the hospital's electronic medical records. A total of 102 measles cases were identified between December 2021 and March 2023. Of these cases, 77 (75.5%) patients were ≤ 18 years old. Of the 73 measles cases with vaccination information, 90% were unvaccinated. The measles seroprevalence rate was 72.8%. The lowest seroprevalence rate (4.8%) among the age groups was found in 8-11-month-old babies, the highest cases rate (35.7%) was detected in this age group. It was determined that measles immunity increased with age (r = 0.276, p < 0.001) and was over 89.3% over the age of 30. Measles immunity is insufficient in our region and measles remains an important public health problem until the age of 18. The recent increase in measles cases in our country and around the world shows that current vaccination programmes need to be implemented more decisively and strictly.