Nasopharyngeal Swab Specimens Research Articles

Accuracy of point-of-care SARS-CoV-2 detection using buccal swabs in pediatric emergency departments.

To optimize the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected children, specimen collection and testing method are crucial considerations. Ideally, specimen collection is easy and causes minimal discomfort, and the laboratory approach is simple, accurate, and rapid. In this prospective cohort study we evaluated the accuracy of a point-of care nucleic acid device using caregiver/patient self-collected buccal swabs. Participants were recruited in 14 Canadian tertiary care pediatric emergency departments. Children <18 years of age deemed to require SARS-CoV-2 testing were eligible. Caregivers or the patient-collected buccal swabs which were tested on the ABBOTT ID NOW. The reference standard was nasopharyngeal swab specimens collected by a healthcare provider tested via laboratory reverse transcription PCR (RT-PCR). We enrolled 2,640 study participants and 14.4% (381/2,640) were SARS-CoV-2 RT-PCR-positive. Eight percent (223/2,640) and 85.0% (2,244/2,640) were concordant test-positive and concordant test-negative, respectively. Sensitivity and specificity of the investigational approach were 58.5% [95% confidence interval (CI): 53.4, 63.5] and 99.3% (95% CI: 98.9, 99.6), respectively. Cycle threshold values were lower among concordant [median 17 (15, 21)] relative to discordant [median 30 (22, 35)] swabs (P < 0.001). Sensitivity was greatest among children <4 years of age, when caregivers performed the swabs, among unvaccinated children, and those with shorter symptom duration. Across multiple pain measures, less pain was associated with buccal swab testing. Although accuracy of the buccal swab point-of-care SARS-CoV-2 test was good and negative agreement was excellent, sensitivity was only 58.5%. Concordance was greater among those with higher viral loads, and the approach involving buccal swabs was less painful.IMPORTANCETo optimize the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected children, specimen collection and testing method are crucial considerations. Ideally, specimen collection is easy and causes minimal discomfort, and laboratory approach is simple, accurate, and rapid. We evaluated the accuracy and pain associated with buccal swab specimen collection by caregivers or children themselves who were tested using a point-of-care isothermal nucleic acid amplification SARS-CoV-2 test. This novel approach was compared to nasopharyngeal swab specimens tested using laboratory-based PCR tests. While negative agreement was excellent, positive percent agreement was less than 60%. Concordance was greater among those with higher viral loads, and thus, sensitivity is excellent when transmissibility is more likely to occur. Importantly, the approach involving buccal swabs was significantly less painful, and thus, children and their caregivers are more likely to agree to testing using such an approach.CLINICAL TRIALSRegistered at ClinicalTrials.gov (NCT05040763).

Detection of Enteroviruses in a Pediatric Oncology Patient Population

Abstract Enteroviruses are responsible for around 10 to 15 million infections and tens of thousands of hospitalizations annually. Infants, children, and people with weakened immune systems are more likely to get hospitalized and can have serious complications from enterovirus infection. Non-polio enteroviruses like EV-D68 can cause mild to severe symptoms like wheezing, difficulty breathing and acute flaccid myelitis (AFM). Therefore, it is crucial to continue to monitor the prevalence of enteroviruses, especially those that can potentially cause severe symptoms like EV-D68. This study focused on determining the prevalence of enteroviruses in a pediatric oncology patient population. Residual Nasopharyngeal swabs (NPS) specimens, collected from pediatric patients (&amp;lt;18 years old) presenting to a cancer hospital in New York between September 2022 and December 2022, and September 2023 and December 2023 for cancer care were included in the study. Rhinovirus/Enterovirus (RV/EV) was detected using the BioFire FilmArray Respiratory Panel (bioMerieux, Inc.) or the ePlex Respiratory Pathogen Panel (GenMark/Roche Molecular). For Enterovirus identification, RNA was extracted from the RV/EV positive NPS using the KingFisher™ Flex Magnetic Particle Processors (Thermo Scientific, USA), followed by RT-PCR in the ABI 7500 Fast real-time PCR instrument (ThermoFisher, USA) for enteroviruses and EV-D68 using pan-Enterovirus and EV-D68 specific primers, respectively. A total of 880 and 694 NPS samples from patients &amp;lt;18 years old were tested during the study period in 2022 and 2023, respectively. Of these, 238 (238/880; 27%) and 225 (225/694; 32%) RV/EV positives NPS were detected in 2022 and 2023 respectively. A total of 370 (370/463; 80%) RV/EV positive samples (182 samples from 2022 and 188 samples from 2023) were selected for additional testing and 14 samples (14/370; 4%) were positive for Enteroviruses. Of note, EV-D68 was not detected. In this study, we report a high prevalence of RV/EV which is consistent with the higher detection rate in the fall seasons, when our samples were collected. The low prevalence of EV suggests a high prevalence of RV, although this was not tested. Despite a nationwide surge in the USA in 2022, there was no EV-D68 circulating in these patients. Further analysis, using Whole genome sequencing of the enteroviruses detected in this study will provide additional information on specific circulating Enterovirus genotypes.

Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

BackgroundThe Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. ObjectiveWe aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Study designDiagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. ResultsSensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. ConclusionThese results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

Radiographically confirmed pneumonia in Malawian children and associated pneumococcal carriage after introduction of the 13-valent pneumococcal conjugate vaccine

BackgroundThe 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced in Malawi in 2011 with an expected impact of reducing pneumococcal pneumonia in children. We aimed to describe clinical characteristics and nasopharyngeal (NP) carriage of pneumococcus by serotype in children hospitalized with primary end-point pneumonia (PEP) between 2013 and 19 after the introduction of PCV-13.MethodsWe conducted a secondary analysis of children aged under-5-years hospitalized with acute respiratory illness (ARI) in Malawi. Chest radiographs conducted at admission were read by two independent clinicians according to WHO criteria for PEP, and a third reviewer resolved discordant diagnoses. NP swab specimens were processed and Streptococcus pneumoniae growth was serotyped. Multivariable regression analysis was conducted to assess the association between clinical characteristics, NP serotypes, and PEP.ResultsWe had complete radiographic and NP serotype data for 500 children, of which 54 isolates were vaccine-type (VT) (10.8%), 165 were non-VT (NVT; 33.0%), and 281 had no pneumococcal growth (56.2%). Among these, 176 (35.2%) had PEP on chest x-ray. Among those with PEP, pneumococcal carriage was documented in 43.8% of cases, and VT serotypes accounted for 10.8%. For children with PEP, we found no association between clinical characteristics and carrying either VT, NVT, or no pneumococcus.ConclusionCarriage of S. pneumoniae remains high among children hospitalized with ARI in Malawi, but children with VT carriage were no more likely to have PEP than children carrying no pneumococcus or those with NVT carriage. There were no differences in clinical characteristics between those carrying VT, NVT, or no pneumococcus.

A-279 Performance Evaluation of the Respiratory Viral for BD MAX™ System in Geriatric Population

Abstract Background Respiratory Viral Infection is one of the most common infection in the geriatric population and cause major concern in the Long-Term Care Facilities. Influenza A and B, SARS-CoV-2, and respiratory syncytial virus (RSV) are responsible for most of the hospitalization and deaths among elderly patients. Rapid identification, treatment, and isolation are among the most important steps to fight the spreading of the infection. Methods The BD Respiratory Viral Panel for BD MAX™ System is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT- PCR) test intended for the simultaneous, qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. The assay is targeting RNA from the nucleocapsid phosphoprotein gene (N1 and N2 regions) of the SARS-CoV-2, a conserved region of the matrix protein M1 gene for influenza A, conserved regions of the matrix protein M1 gene and HA gene for influenza B, conserved regions of the N and M genes for RSV, and the human RNase P gene. The assay was evaluated for: precision, reproducibility, accuracy, and correlation with BioGx for SARS-CoV-2, Solana (Quidel) for influenza A, influenza B and RSV; the percentage agreement for the evaluation steps was calculated. We ran 86 samples collected from patients residing in Long-Term Care Facilities. Statistical analyses were done using Analyse-it. Results The percentage agreement for precision, reproducibility, and accuracy were 100%. The patients’ correlation was 100% when compared to the existing molecular testing in the laboratory. Female patients accounted for 62.8% (54 female and 32 male); 2 patients were positive for both influenza A and RSV, and one patient was positive for both influenza A and SAR-CoV-2. Conclusions The Respiratory Viral panel for BD MAX™ System gave the benefit of a fully automated, high precision, accuracy and acceptable sensitivity assay; in addition, it offers easier collection (one swabs versus multiple swabs), faster turnaround time for four targets in less than three hours which allow for earlier isolation to prevent the spreading of the infection and initiating treatment when needed. As with every molecular assay avoiding contamination is very important to eliminated false positive results.

B-201 Clinical Performance Evaluation of IRON-qPCR™ RV, a Novel Molecular POCT system, for the Qualitative Detection of Respiratory Viruses in Nasopharyngeal Swab

Abstract Background The availability of Point-of-Care testing (POCT) based on molecular diagnostics to test simultaneously for multiple respiratory viruses is very limited though significant progress has been made in molecular testing for COVID-19 during the pandemic. Considering the similar symptoms presented by patients infected with various respiratory pathogens, it is crucial to have multiple POCT systems capable of detecting multiple respiratory pathogens while distinguishing SARS-CoV-2 from other pathogens. To meet the growing demand for multiplex testing for respiratory pathogens, a molecular POCT, IRON-qPCR™ RV has been developed. IRfON-qPCR™ RV is a multiplex real-time RT-PCR assay designed for the qualitative detection and differentiation of SARS-CoV-2, Influenza A, Influenza B, RSV-A, and RSV-B RNA from human nasopharyngeal swab specimens. The IRON-qPCR™ proceeds the entire process automatically from nucleic acid extraction to real-time RT-PCR within 40 minutes. The main goal of this study is to evaluate the clinical performance of IRON-qPCR™ RV in comparison with other assays and kits using nasopharyngeal swab samples. Methods To assess the clinical performance of the IRON-qPCR™ RV, specimen collection and evaluation were conducted at a clinical institution in France, utilizing a total of 980 nasopharyngeal swab samples. The comparators used were the Xpert® Xpress CoV-2/Flu/RSV plus Kit and the RealStar® RSV RT-PCR Kit 3.0. Since RSV subtypes are not distinguished by the Xpert kit, all RSV without subtype distinction were compared. Additionally, RealStar kit was used to compare the results of IRON-qPCR™ RV for RSV subtypes A and B. Clinical samples showing discrepancies between Xpert and RealStart, and the IRON-qPCR™ RV were further analyzed using Allplex™ SARS-CoV-2/FuA/FluB/RSV Assay and sequencing. Results Through the comparison of clinical performance of the IRON-qPCR™ RV with Xpert for SARS-CoV-2, Influenza A, and Influenza B in nasopharyngeal swab samples, the clinical sensitivity was analyzed to be over 95.5%, and the clinical specificity was over 99.6%. For RSV, compared to Xpert, the clinical sensitivity was analyzed to be 96.9%, and the clinical specificity was 98.9%. Furthermore, in comparison with RealStar, the clinical sensitivity and specificity of IRON-qPCR™ RV for RSV-A were determined to be 96.2% and 100%, respectively, and the clinical sensitivity and specificity for RSV-B were 96.0% and 100%. Additionally, discrepant samples in comparison with Xpert were further tested using the Allplex as a resolver kit. The result showed that the clinical sensitivity and specificity of IRON-qPCR™ RV for SARS-CoV-2, Influenza A, and Influenza B were over 98.3% and 100%. For RSV, the clinical sensitivity and specificity were 99.5% and 99.1%. Conclusions The clinical performance of IRON-qPCR™ RV was analyzed with the clinical sensitivity of over 95% and the clinical specificity of over 99%. Upon analysis including the results of the resolver kit for discrepant samples, increased clinical sensitivity and specificity were determined. The results of this study strongly support that the IRON-qPCR™ RV is a reliable POCT system for the simultaneous detection and differentiation of multiple respiratory viruses. Furthermore, it is applicable to district hospitals as well as clinical laboratories for accurate and timely testing.

B-207 Clinical Evaluation of ExiStation™ FA 96 RV for the detection of human respiratory viruses in human nasopharyngeal swab specimen

Abstract Background There has been growing demand for fully automated multiplex testing of respiratory viruses in molecular diagnostics following the COVID-19 pandemic. ExiStation™ FA 96 RV developed by BIONEER is a multiplex real-time RT-PCR assay. It is designed for the simultaneous detection and differentiation of SARS-CoV-2, Influenza A, Influenza B, and RSV RNA in human nasopharyngeal swab specimens from individuals showing signs and symptoms of respiratory viral infection. ExiStation™ FA 96 automates the entire process from RNA extraction to real-time RT-PCR for up to 96 samples at once within 100 minutes. Moreover, the system is equipped to decap sample tubes, eliminating the need for handling sample transport media. In this study, the clinical performance of the ExiStation™ FA 96 RV assay was evaluated using nasopharyngeal swab samples. Methods Nasopharyngeal swab samples, totaling 859 samples, are collected according to the procedures by the clinical institution in France and preserved in Virus transport media. Once anonymized and randomly allocated, the swab transport media tube, without capping procedure, are directly placed onto the ExiStation™ FA 96. Subsequently, both extraction and real-time RT-PCR are automatically processed. Clinical sensitivity, specificity, and agreement are analyzed by comparing results with confirmatory reagents SARS-CoV-2 RT-qPCR Reagent Kit, PKAmp™ Respiratory SARS-CoV-2 RT-PCR Panel Kit, and Bosphore SARS-CoV-2/Flu/RSV Panel Kit, as well as comparator reagent Allplex™ Respiratory Panel 1 and Allplex™ SARS-CoV-2 Assay. Results The comparison between confirmatory reagents and the ExiStation™ FA 96 RV resulted in a clinical sensitivity of at least 96.4% (164/164 for SARS-CoV-2, 64/65 for Influenza A, 74/75 for Influenza B, and 53/55 for RSV) and a clinical specificity of 100% (500/500 for all targets). When compared with the comparator reagent, the overall agreement and Cohen's kappa value were both determined to be above 99.1% and 0.989, respectively (664/664 for SARS-CoV-2, 564/565 for Influenza A, 573/575 for Influenza B, and 550/555 for RSV). Additionally, any discrepant samples identified in comparison with the comparator reagent were verified through sequencing analysis. Upon sequencing analysis on a total of 8 samples and aligning the confirmed results with the analysis content, no alterations observed in the analysis of clinical sensitivity and specificity. Conclusions The evaluation of the ExiStation™ FA 96 RV assay showed outstanding clinical performance in detecting four respiratory viruses, SARS-CoV-2, Influenza A, Influenza B, and RSV, with high sensitivity and specificity. This study supports that the ExiStation™ FA 96 RV assay is a reliable tool for simultaneous molecular diagnostics of various respiratory infections in clinical laboratories.

Unveiling viral pathogens in acute respiratory disease: Insights from viral metagenomics in patients from the State of Alagoas, Brazil.

Respiratory illness affects individuals across all age demographics on a global scale, often precipitated by viral infections. The symptomatic manifestations of these diseases bear clinical resemblance, complicating the accurate determination of their etiological origins. Furthermore, the diagnostic panels for respiratory pathogens used within local medical practices, may not encompass the full spectrum of viral agents responsible for such ailments. Consequently, a significant number of clinically important viral pathogens may remain undetected. In the light of this, we conducted a metagenomic examination of 66 nasopharyngeal swab specimens, obtained from patients presenting with acute respiratory conditions yet tested negative by the standard diagnostic panels available locally. These specimens were obtained from the Public Health Laboratory, Maceio, State of Alagoas. Our findings indicate a predominant diagnostic escape of rhinoviruses and notably enterovirus D68. Moreover, our study identified a substantial quantity of sequence reads attributed to human respirovirus 3 (human parainfluenza 3) along with various herpresviruses including human herpesvirus-1, Epstein-Barr virus (Human herpesvirus-4), Human herpesviruses 6 and 7 and human parvovirus B19 (B19V). Notably, the metagenomic analysis uncovered a widespread presence of the emerging human vientovirus FB in most of sample pools, though its clinical importance remains to be elucidated. The obtained results in this study underscore the invaluable role of viral metagenomics in the identification of underrecognized viruses bearing clinical relevance. Furthermore, it offers insights into the dissemination of these pathogens within the studied area, thereby informing public health strategies aimed at enhancing diagnostic accuracy and improving patient care.

Pneumococcal transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of serotype 19F in Indonesia

Genetic evolution of resistance due to mutations and transposon insertions is the primary cause of antimicrobial resistance in Streptococcus pneumoniae. Resistance to macrolide, tetracycline, and chloramphenicol is caused by the insertion of specific genes that carried by transposon (Tn). This study aims to analyze transposon profiling associated with macrolide, tetracycline, and chloramphenicol resistance from carriage isolates of S. pneumoniae serotype 19F in Indonesia. S. pneumoniae serotype 19F isolates were collected from nasopharyngeal swab specimens from different regions in Indonesia. Genomic DNA was extracted from sixteen isolates and whole genome sequencing was performed on Illumina platform. Raw sequence data were analyzed using de novo assembly by ASA3P and Microscope server. The presence of transposons was identified with detection of int and xis genes and visualized by pyGenomeViz. The genome size of S. pneumoniae ranges from 2,040,117 bp to 2,437,939 bp, with a GC content of around 39 %. ST1464 (4/16) and ST271 (3/16) were found as the predominant sequence type among isolates. Tn2010 was the most common transposon among S. pneumoniae serotype 19F isolates (7/16) followed by Tn2009 (4/16), and Tn5253 (3/16). We identified two deletion sites within the tetM gene (2 bp and 58 bp) that confer tetracycline susceptibility from one isolate. This study suggests that genomic analysis can be employed for the detection and surveillance of antimicrobial resistance genes among S. pneumoniae strains isolated from various regions in Indonesia.

SARS-CoV-2 Variants May Affect Saliva RT-PCR Assay Sensitivity.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants demonstrate predilection for different regions of the respiratory tract. While saliva-based reverse transcription-polymerase chain reaction (RT-PCR) testing is a convenient, cost-effective alternative to nasopharyngeal swabs (NPS), few studies to date have investigated whether saliva sensitivity differs across variants of concern. SARS-CoV-2 RT-PCR was performed on paired NPS and saliva specimens collected from individuals with acute coronavirus disease 2019 (COVID-19) symptoms or exposure to a COVID-19 household contact. Viral genome sequencing of NPS specimens and Los Angeles County surveillance data were used to determine the variant of infection. Saliva sensitivity was calculated using NPS-positive RT-PCR as the reference standard. Factors contributing to the likelihood of saliva SARS-CoV-2 RT-PCR positivity were evaluated with univariate and multivariable analyses. Between June 2020 and December 2022, 548 saliva samples paired with SARS-CoV-2 positive NPS samples were tested by RT-PCR. Overall, saliva sensitivity for SARS-CoV-2 detection was 61.7% (95% CI, 57.6%-65.7%). Sensitivity was highest with Delta infection (79.6%) compared to pre-Delta (58.5%) and Omicron (61.5%) (P = 0.003 and 0.01, respectively). Saliva sensitivity was higher in symptomatic individuals across all variants compared to asymptomatic cases [pre-Delta 80.6% vs 48.3% (P < 0.001), Delta 100% vs 72.5% (P = 0.03), Omicron 78.7% vs 51.2% (P < 0.001)]. Infection with Delta, symptoms, and high NPS viral load were independently associated with 2.99-, 3.45-, and 4.0-fold higher odds of SARS-CoV-2 detection by saliva-based RT-PCR (P = 0.004, <0.001, and <0.001), respectively. As new variants emerge, evaluating saliva-based testing approaches may be crucial to ensure effective virus detection.

CHAMP: A Centrifugal Microfluidics-Based CRISPR/Cas12b-Combined Real-Time LAMP One-Pot Method for Mycoplasma pneumoniae Infection Diagnosis.

The Mycoplasma pneumoniae outbreak poses health risks to community residents. However, it still has limitations for current clinical diagnostic methods (qPCR nucleic acid assay or IgM immunoassay), including specialized handling, expensive equipment, prolonged turnaround time, and false positives and negatives, highlighting the need to improve clinical diagnostic methods. Herein, we present a novel centrifugal microfluidics-based method for rapidly diagnosing M. pneumoniae infections (CHAMP system). This user-friendly method combines CRISPR/Cas12b and real-time loop-mediated isothermal amplification (LAMP) in a one-pot reaction, offering high sensitivity, specificity, and simplicity for methodology. By adding fully automated nucleic acid magnetic bead-extracted samples to a prepackaged centrifugal microfluidics chip, 48 samples can be automated tested simultaneously within 15 to 60 min at 60 °C. 427 clinical nasopharyngeal swab specimens were used for validation, demonstrating good positive and negative predictive values and good diagnostic sensitivity, specificity, and significant time savings. This method is particularly suitable for detecting low nucleic acid copies of M. pneumoniae samples.

A retrospective study revealing complex viral diversity and a substantial burden of HPV infection in SARS-CoV-2 positive individuals, Sierra Leone

BackgroundThe COVID-19 pandemic has underscored the critical role of sequencing technology in disease control and outbreak response. However, resource limitations and challenging environments often impede such efforts in low and middle-income countries. This study aimed to investigate the spectrum of viral co-infections, particularly with human viral pathogens, in SARS-CoV-2 positive individuals in Sierra Leone using metagenomic sequencing, evaluating the feasibility of utilizing this technology for epidemiological and evolutionary surveillance of pathogens related to public health in low-income environments.MethodsWe retrospectively collected and analyzed 98 nasopharyngeal swab specimens from SARS-CoV-2 positive individuals in Sierra Leone. Samples were pre-processed locally and transferred to China via FTA cards for metagenomic sequencing, which was performed using the Novaseq platform. The study focused on the identification of nasopharyngeal viruses co-infecting with SARS-CoV-2, with a deeper analysis of significant human viral pathogens such as HPV.ResultsThe study identified 22 viral taxa from 20 families, including 4 human viruses. Notably, 19.4% of samples showed HPV co-infection with 34 distinct types, predominantly beta and gamma HPVs. Multiple HPV types were found in individual samples, indicating a high complexity of viral co-infections.ConclusionsThe identification of a wide range of co-infecting viruses, particularly multiple HPV genotypes, highlights the complexity of viral interactions and their potential implications for public health. These findings enhance our understanding of viral co-infections and provide valuable insights for public health interventions in Sierra Leone. Further research is needed to explore the clinical significance of these findings and their impact on disease outcomes.

Prevalence, serotype distribution, and antimicrobial susceptibility profile of Streptococcus pneumoniae in Sea Nomad children under 5 years of age in Wakatobi, Southeast Sulawesi, Indonesia: A cross-sectional study

ObjectiveIndonesia commenced the nationwide introduction of pneumococcal conjugate vaccine (PCV) in 2022. Pre-vaccine Streptococcus pneumoniae data from across the country could be critical to enable vaccine impact evaluation in the future. This study evaluates colonization prevalence, factors associated with colonization, serotype distribution, and the antimicrobial susceptibility profile of S. pneumoniae. MethodsChildren under 5 years of age were enrolled from Bajau tribe settlements in Wakatobi, southeast Sulawesi, Indonesia, from October 2018 to February 2019. Nasopharyngeal swab specimens were analysed by culture, and isolates were serotyped using sequential multiplex polymerase chain reaction. Antibiotic susceptibility was performed by the disk diffusion method. Multivariable logistic regression was performed for risk factor analysis. ResultsA total of 499 NP swab specimens were collected; 61.9% were colonized with S. pneumoniae and 48.9% of the isolates were of PCV13-vaccine type. The most common serotypes were 23F, 6B, 19F, and 6A at 13.2%, 9.8%, 8.9%, and 8.0%, respectively. Exposure to cigarette smoke in the household and runny nose were significant risk factors for colonization, with aORs of 1.6 (95% confidence interval: 1.1–2.3) and 2.1 (95% confidence interval: 1.4–3.3), respectively. ConclusionsThe findings of this study may contribute to baseline pre-vaccine data in Indonesia that would be critical for the impact evaluation of vaccines.

A new strategy of multiplex real-time RT-qPCR assay for differentiating Omicron variants from other SARS-CoV-2 lineages

The global dissemination of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) Omicron variants has been characterized by a distinct 9-base pair (bp) deletion within the nucleocapsid (N) gene, a genomic feature absent in previously circulating variants. In response to this unique characteristic, we have developed an innovative amplicon-competition strategy to differentiate Omicron lineages from other SARS-CoV-2 variants using a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay. This novel methodology demonstrates high specificity and sensitivity, effectively detecting viral loads ranging from 1500 to 1.5x106 copies per milliliter in nasopharyngeal swab specimens. The potential applications of this approach extend beyond current needs, providing a robust framework for the genomic characterization of future variants exhibiting unique genetic signatures.

Pathogenic investigation of human respiratory syncytial virus infection in kindergarten children in Tongzhou District, Beijing City in 2023

The study focused on individuals with influenza-like symptoms (fever, cough, sore throat, runny nose, and other respiratory symptoms) in three kindergartens in Tongzhou District, Beijing City, in April 2023. Nasopharyngeal swab specimens were collected, and real-time fluorescent quantitative PCR was used to detect common respiratory pathogens in the collected specimens. Positive specimens were subjected to sequencing analysis of the highly variable region of human respiratory syncytial virus (HRSV) G protein, homology analysis and phylogenetic tree analysis. A total of 25 fever cases were collected from 3 kindergartens, aged 3-8 years old, with an age M (Q1, Q3) of 4 (3.5, 5) years old. Ten confirmed cases of HRSV positive were screened and detected using the fluorescent quantitative PCR method, with a total detection rate of 40% (10/25). Typing identification and sequencing analysis confirmed that the main epidemic type was HRSV subtype B, which was highly homologous and closely related to previous epidemic strains in the region. Through pathogen investigation and analysis, it was preliminarily determined that this epidemic was dominated by HRSV subtype B.

Comparison of RT-PCR cycle threshold values between individual and pooled SARS-CoV-2 infected nasopharyngeal swab specimens.

The molecular reverse transcription-polymerase chain reaction (RT-PCR) testing of respiratory tract swabs has become mandatory to confirm the diagnosis of coronavirus disease 2019 (COVID-19). However, RT-PCR tests are expensive, require standardized equipment, and relatively long testing times, and the sample pooling method has been introduced to solve this issue. The aim of this study was to compare the cycle threshold (Ct) values of the individual sample and pooled sample methods to assess how accurate the pooling method was. Repeat RT-PCR examinations were initially performed to confirm the Ct values for each sample before running the pooled test procedure. Sample extraction and amplification were performed in both assays to detect ORF1ab, N, and E genes with a cut-off point value of Ct <38. Overall, there was no difference in Ct values between individual sample and pooled sample groups at all concentrations (p=0.259) and for all pooled sizes. Only pooled size of five could detect the Ct value in the pooled samples for all concentration samples, including low-concentration sample (Ct values 36 to 38). This study highlighted that pooled RT-PCR testing strategy did not reduce the quality of individually measured RT-PCR Ct values. A pool size of five could provide a practical technique to expand the screening capacity of RT-PCR.

Evaluating diagnostic accuracy of an RT-PCR test for the detection of SARS-CoV-2 in saliva

Background and objectiveSaliva has been proposed as a potential more convenient, cost-effective, and easier sample for diagnosing SARS-CoV-2 infections, but there is limited knowledge of the impact of saliva volumes and stages of infection on its sensitivity and specificity.MethodsIn this study, we assessed the performance of SARS-CoV-2 testing in 171 saliva samples from 52 mostly mildly symptomatic patients (aged 18 to 70 years) with a positive reference standard result at screening. The samples were collected at different volumes (50, 100, 300, and 500 µl of saliva) and at different stages of the disease (at enrollment, day 7, 14, and 28 post SARS-CoV-2 diagnosis). Imperfect nasopharyngeal (NP) swab nucleic acid amplification testing was used as a reference. We used a logistic regression with generalized estimating equations to estimate sensitivity, specificity, PPV, and NPV, accounting for the correlation between repeated observations.ResultsThe sensitivity and specificity values were consistent across saliva volumes. The sensitivity of saliva samples ranged from 70.2% (95% CI, 49.3–85.0%) for 100 μl to 81.0% (95% CI, 51.9–94.4%) for 300 μl of saliva collected. The specificity values ranged between 75.8% (95% CI, 55.0–88.9%) for 50 μl and 78.8% (95% CI, 63.2–88.9%) for 100 μl saliva compared to NP swab samples. The overall percentage of positive results in NP swabs and saliva specimens remained comparable throughout the study visits. We observed no significant difference in cycle number values between saliva and NP swab specimens, irrespective of saliva volume tested.ConclusionsThe saliva collection offers a promising approach for population-based testing.

Evaluation of the FPMC respiratory panel for detection of respiratory tract pathogens in nasopharyngeal swab and sputum specimens

ObjectivesThe performance of the new Respiratory Pathogen panel (fluorescent probe melting curve, FPMC) for the qualitative detection of 12 organisms (chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, etc.) was assessed.MethodsProspectively collected nasopharyngeal swab (NPS) and sputum specimens (n = 635) were detected by using the FPMC panel, with the Sanger sequencing method as the comparative method.ResultsThe overall percent concordance between the FPMC analysis method and the Sanger sequencing method was 100% and 99.66% for NPS and sputum specimens, respectively. The FPMC testified an overall positive percent concordance of 100% for both NPS and sputum specimens. The FPMC analysis method also testified an overall negative percent concordance of 100% and 99.38% for NPS and sputum specimens, respectively.ConclusionsThe FPMC analysis method is a stable and accurate assay for rapid, comprehensive detecting for respiratory pathogens.

Respiratory Viruses Co-detection on a Multiplex RT-PCR Panel: a Comparative Clinical Study.

The FilmArray Respiratory Panel RP 2.1 plus (FilmArray RP) is a point-of-care syndromic panel for respiratory pathogens. Although highly valuable in the clinical settings, the co-detection of pathogens in FilmArray RP may confound result interpretation. Nasopharyngeal swab specimens collected from patients with respiratory symptoms were analyzed by comparing co-detection results from FilmArray RP with those of Allplex Respiratory Panels (Allplex RP: Power-Chek for SARS-CoV-2). Out of 765 FilmArray RP tests, 143 (18.7%) showed co-detections (two: 122 (85.3%), three: 18 (12.6%), four: 2 (1.4%), and five viruses: 1 (0.7%). The most frequent co-detection was human rhinovirus/enterovirus (HRV/HEV) with respiratory syncytial virus (RSV) (22.3%, 32/143). The overall discordance rate between Film-Array RP and other tests was 32.9%. Notably, discordance in detecting adenovirus (AdV) was significant, with cases detected by FilmArray often not appearing in Allplex RP. Discordances were varied by virus combination. It is advisable to perform additional confirmatory testing based on clinical relevance.

COVID-19 Vaccines and COVID-19 in People Living with HIV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly around the world, and COVID-19 and HIV co-infection also became common. In this study, we aimed to investigate the impact of vaccination preferences and vaccination rates on the severity of COVID-19 in patients with HIV co-infection. People living with HIV who were followed in our hospital during the COVID-19 pandemic (January 2020- December 2022) were retrospectively included in the study. The diagnosis of COVID-19 was made by detecting SARS-CoV-2 RNA in nasopharyngeal swab specimens using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). Patients requiring hospital admission were classified as severe. The patient's demographics and vaccination status were collected from the hospital data system. Our study included 205 patients using antiretroviral therapy for HIV. The mean day count between the last vaccine date and SARS-CoV-2 PCR positivity was 163 days in the Comirnaty® group,149 days in the CoronaVac® group, and 154 days in the mixed-vaccinated group. Those vaccinated with Comirnaty® were statistically significantly less infected with COVID-19 after vaccination (p<0.05). The course and the outcomes of COVID-19 among SARS-CoV-2 vaccinated people living with HIV, especially with well-controlled HIV infection, seem to be similar to people living without HIV.