Nasopharyngeal Swab Specimens Research Articles

Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses.

Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.

Isolation, characterization, and pathogenicity assay of Acanthamoeba and its endosymbionts in respiratory disorders and COVID-19 hospitalized patients, northern Iran

Acanthamoeba spp., are common free-living amoebae found in nature that can serve as reservoirs for certain microorganisms. The SARS-CoV-2 virus is a newly emerged respiratory infection, and the investigation of parasitic infections remains an area of limited research. Given that Acanthamoeba can act as a host for various endosymbiotic microbial pathogens and its pathogenicity assay is not fully understood, this study aimed to identify Acanthamoeba and its bacterial and fungal endosymbionts in patients with chronic respiratory disorders and hospitalized COVID-19 patients in northern Iran. Additionally, a pathogenicity assay was conducted on Acanthamoeba isolates. Urine, nasopharyngeal swab, and respiratory specimens were collected from two groups, and each sample was cultured on 1.5% non-nutrient agar medium. The cultures were then incubated at room temperature and monitored daily for a period of two weeks. Eight Acanthamoeba isolates were identified, and PCR was performed to confirm the presence of amoebae and identify their endosymbionts. Four isolates were found to have bacterial endosymbionts, including Stenotrophomonas maltophilia and Achromobacter sp., while two isolates harbored fungal endosymbionts, including an uncultured fungus and Gloeotinia sp. In the pathogenicity assay, five isolates exhibited a higher degree of pathogenicity compared to the other three. This study provides significant insights into the comorbidity of acanthamoebiasis and COVID-19 on a global scale, and presents the first evidence of Gloeotinia sp. as a fungal endosymbiont. Nevertheless, further research is required to fully comprehend the symbiotic patterns and establish effective treatment protocols.

Infectious respiratory pathogens among patients with acute exacerbation of idiopathic pulmonary fibrosis during the coronavirus disease 2019 pandemic in Japan

BackgroundFew studies have investigated the prevalence of pathogens in patients with acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF), specifically, the interactions between respiratory pathogens and AE-IPF during the coronavirus disease 2019 (COVID-19) pandemic. ObjectivesWe aimed to analyze pathogens in patients with AE-IPF between September 2020 and December 2022. MethodsThis retrospective observational study was conducted at our hospital between September 2020 and December 2022. In patients with AE-IPF, pre-hospitalization polymerase chain reaction (PCR) tests for respiratory pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were performed using multiplex PCR or Smart Gene assay with nasopharyngeal swab specimens. Microbiological assays, including Gram staining, sputum cultures, blood cultures, and urinary antigen tests for Streptococcus pneumoniae and Legionella pneumophila, were also performed. ResultsForty-nine patients with AE-IPF were included. The median age was 75 years old and 42 (86%) were male. Only one of the 49 patients (2%) was positive for SARS-CoV-2. Two of 28 patients (7%) were positive for human rhinovirus/enterovirus. No bacteria were detected in sputum culture, blood culture, or urinary antigen tests. ConclusionsThe detection frequency of SARS-CoV-2 infection in patients with AE-IPF was lower than that of human rhinovirus/enterovirus. Continuous analysis for the presence of pathogens is necessary for appropriate infection control because respiratory viruses may increase as the coronavirus pandemic subsides.

Molecular epidemiology and temporal dynamic of SARS-CoV-2 imported from Hong Kong to mainland China

Tracking the genome diversity and evolution of virus like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the hotspot of current researches, which were essential for the developing of prevention strategies and therapeutic vaccines. In the current study, a total of 206073 oral or nasopharyngeal swab specimens were collected in 2022 from entry populations originated from Hong Kong, among which 141 whole genome sequences were obtained through next-generation sequencing and analyzed. Two major Omicron sub-lineages were identified: BA.2.2 (70.21%, 99/141) and BA.5 (24.11% 34/141). An average of 53.22 mutations was emerged in the genome, with S protein and ORF1a harboring the highest number of mutations among structure proteins and non-structure proteins (p<0.001), respectively, making them the most variable regions. The temporal changes of the mutations among each protein were unique, indicating the distinct evolution pattern of these proteins. A novel mutation in the Nsp5 (3 C-like proteinase) was reported for the first time. As it may influence the viral replication and transcription, further monitoring is needed. The data reported here highlighted the importance of monitoring the SARS-CoV-2 genome and would be useful for the generation of evidence-based prevention programs and effective vaccines to COVID-19.

Development of a lateral flow immunoassay with HRP enhancement for spiked SARS-CoV-2 protein N detection in human saliva

The Coronavirus (SARS-CoV-2) is an enveloped RNA virus that caused a dangerous COVID-19 pandemic following an outbreak in Wuhan, China in 2019. In response to the pandemic, the development of lateral flow assays (LFAs) has been crucial for the detection of viruses, commonly targeting the spike (S) or nucleocapsid (N) protein in nasopharyngeal swab (NS) specimens. COVID-19, caused by the SARS-CoV-2 virus, predominantly manifests as a respiratory tract infection-like illness, characterized by symptoms such as fever, dry cough, upper airway congestion, runny nose, sore throat, myalgia, headache, and exhaustion. This study presents the development of an LFA targeting the N protein to detect the coronavirus in saliva specimens, using a sandwich format. The use of 80 nm gold nanoparticles (AuNPs) has been investigated, and the application of horseradish peroxidase (HRP) and TMB substrate has been employed to enhance the limit of detection (LOD). The results demonstrate a significant 200-fold improvement in LOD, from 10 to 50 pg/ml, for N protein spiked in saliva samples after the application of HRP-TMB. This finding highlights an important advancement towards the utilization of saliva samples in diagnostic applications.

Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs.

Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0-9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values.IMPORTANCEUnderstanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.

COVID-19 in Patients with Different Hematological Malignancies: Treatment Strategy and Outcome: An Iraqi Experience

Background: Patients with hematological malignancies may have higher rates of morbidity and mortality due to SARS-COVID-19 than those in the general population. Objectives: To evaluate the outcome of SARS-COVID-19 in patients with different hematological malignancies undergoing active or modified therapy. Settings: This was a retrospective cohort, multicenter study conducted on 167 patients diagnosed with SARS-COVID-19. The selected patients had been diagnosed with different hematological malignancies and had received treatment (chemotherapy or immunosuppressive). The study was carried out in different centers across Iraq between 1 December 2020 and 1 December 2021. Patients and Methods: This study was conducted in seven hematology centers across Iraq with 167 patients; of these, this study showed that 88 (52.7%) were males and 79 (47.3%) were females (mean age 51.9 ± 18.3 years). All patients had had a COVID-19 infection confirmed through RNA-specific fragments by real-time reverse transcriptase polymerase chain reaction (RT-PCR) of the nasopharyngeal swab specimens, and by computed tomography (CT) of the chest. Demographic data were obtained from patient files, which included the type of hematological malignancy, treatment (chemotherapy, immunotherapy, supportive), severity of COVID-19, modification of treatment and disease outcome for patients, whether alive or not. Patients identified as having benign or non-hematological malignancies were excluded. Results: The treatment protocol was modified in 124 patients (74.3%), while 43 patients (25.7%) continued on the same protocol. Regarding treatment outcomes, 77.2% of patients were alive at the end of the study and 22.8% had died. The survival of COVID-19 patients did not change significantly when the treatment protocol was modified compared to those who continued on treatment as per protocol. The death rate was significantly higher among patients with severe disease compared to those with mild and moderate disease (61.4% v. 0% and 5.0%, respectively) (p&lt;0.001). Conclusions: The management of patients with hematological malignancies during the COVID-19 pandemic may be challenging, with higher mortality in patients with concurrent hematological malignancies and COVID-19. The survival of COVID-19 patients did not change significantly when treatment was modified. Age groups, sex, type of hematological malignancy, and disease status did not affect the survival of these patients. The death rate was significantly higher among patients with hematological malignancies and severe COVID-19 compared with mild or moderate disease.

Comparing Polymerase Chain Reaction Testing of Nasopharyngeal Swab and Lower Respiratory Tract Specimens for the Diagnosis of Pneumocystis jirovecii Pneumonia.

Using nasopharyngeal (NP) swab samples instead of lower respiratory tract specimens for polymerase chain reaction (PCR) to diagnose Pneumocystis jirovecii pneumonia (PJP) may be better tolerated and improve diagnostic accessibility. In this 2-year Australian retrospective cohort study of patients with clinically suspected PJP, P jirovecii PCR on NP swab samples had perfect specificity but low sensitivity (0.66).

Improving the detection capability and efficiency of SARS-CoV-2 RNA specimens by the specimen turn-around process with multi-department cooperation.

Improving the detection capability and efficiency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA specimens is very important for the prevention and control of the outbreak of Coronavirus disease 2019 (COVID-19). In this study, we evaluated the detection capability and efficiency of two outbreaks of COVID-19 before and after the process re-engineering in April and July 2022. This retrospective cross-sectional study involved 359,845 SARS-CoV-2 RNA specimens 2 weeks before and 2 weeks after the two outbreaks of COVID-19 in April and July. The number, transportation time and detection time of specimens, and the number of reports of more than 24 h were analyzed by SPSS software. While 16.84% of people chose nasopharyngeal swabs (NPS) specimens, 83.16% chose oropharyngeal swabs (OPS) specimens to detect SARS-CoV-2 RNA. There were significant upward trends in the percentage of 10 sample pooling (P-10) from April before process re-engineering to July after process re-engineering (p < 0.001). Compared with April, the number of specimens in July increased significantly not only 2 weeks before but also 2 weeks after the outbreak of COVID-19, with an increase of 35.46 and 93.94%, respectively. After the process re-engineering, the number of reports more than 24 h in the 2 weeks before and after the outbreak of COVID-19 in July was significantly lower than that in April before process re-engineering (0% vs. 0.06% and 0 vs. 0.89%, both p < 0.001). The present study shows that strengthening the cooperation of multi-departments in process re-engineering, especially using the P-10 strategy and whole process informatization can improve the detection capability and efficiency of SARS-CoV-2 RNA specimens.

Molecular epidemiology and clinical characteristics of respiratory syncytial virus in hospitalized children during winter 2021-2022 in Bengbu, China.

This study aimed to study the molecular epidemiology and clinical characteristics of respiratory syncytial virus (RSV) infection from hospitalized children with ARTI in Bengbu. One hundred twenty-four nasopharyngeal swab specimens and clinical data from children with ARTI cases were collected in Bengbu, China, during winter 2021-2022. The samples were detected by qPCR of 13 respiratory viruses. Phylogenetic analysis was constructed using MEGA 7.0. All analyses were performed using SAS software, version 9.4. In winter 2021-2022, URTI, NSCAP, SCAP, and bronchiolitis accounted for 41.03%, 27.35%, 17.09%, and 14.53% of hospitalized children in Bengbu, China. The detection rates of the top three were RSV (41.94%), ADV (5.65%), and FluB (5.65%) in hospitalized children through 13 virus detection. RSV is the main pathogen of hospitalized children under 2 years old. Forty-eight sequences of G protein of RSV were obtained through PCR amplification, including RSV-A 37 strains and RSV-B 11 strains. Phylogenetic analysis showed that all RSV-A and RSV-B were ON1 and BA9 genotypes, respectively. ON1 genotypes were further divided into two clades. The majority of ON1 strains formed a unique genetic clade with T113I, V131D, N178 G, and H258Q mutations. Furthermore, RSV infection was an independent risk factor for ventilator use (OR = 9.55, 95% CI 1.87-48.64). There was a high incidence of RSV among hospitalized children during winter 2021-2022 in Bengbu with ON1 and BA9 being the dominant strains. This study demonstrated the molecular epidemiological characteristics of RSV in children with respiratory infections in Bengbu, China.

Correlation of lymphocyte subsets and inflammatory biomarkers with disease severity in COVID-19 patients.

To determine the correlation of lymphocyte subsets and soluble serum inflammatory biomarkers with disease severity in coronavirus disease-2019 infection. The retrospective study was conducted at the Department of Immunology, Sindh Institute of Urology and Transplantation (SIUI), Karachi, Pakistan from September 1 to November 30, 2021, and comprised data of patients admitted from June to July 2021 who tested positive for coronavirus disease-2019 on the basis of reverse transcription-polymerase chain reaction of nasopharyngeal swab specimens. The patients were categorised into severe group A and non-severe group B. Initial investigations included complete blood count, neutrophil-to-lymphocytes ratio, C-reactive protein, D-Dimers and serum ferritin levels. Lymphocyte subsets included cluster of differentiation-3+, cluster of differentiation-4+/ cluster of differentiation-3+, cluster of differentiation-8+ T lymphocytes, cluster of differentiation-19+B lymphocytes, cluster of differentiation-16+ cluster of differentiation-56+ Natural Killer cells and serum cytokine levels of interleukin-2, interleukin- 4, interleukin-6, interleukin-10, tumour necrosis factor-alpha and interferon gamma. They were correlated with disease severity. Data was analysed using SPSS 20. Of the 54 patients, 33(61.1%) were males and 21(38.9%) were females. There were 29(53.70%) patients in group A with median age 52 years (interquartile range: 43.5-65 years), and 25(46.29%) in group B with median age 50 years (interquartile range: 36.5-59 years) (p=0.241). Disease was significantly more severe in male patients compared to female (p=0.002). In group A, cluster of differentiation-3+ T cells were reduced in 21(72.4%) patients, cluster of differentiation-8+ T cells in 16(55.2%), cluster of differentiation-4+ T cells in 23(79.3%) and cluster of differentiation-19+ B cells in 8(27.6%). In group B, cluster of differentiation-3+ T cells were reduced in 10(40%) subjects, cluster of differentiation-8+ T cells in 7(28%), cluster of differentiation-4+ T cells in 12(48%) and cluster of differentiation-19+ B cells in 4(16%) patients. Serum cytokine levels were not significantly different between the groups (p>0.05). In group A, 7(24.13%) patients died, and in such cases, the neutrophil-to-lymphocytes ratio was significantly higher (p=0.037). Pro-inflammatory markers and cytokine levels increased, while lymphocyte subsets decreased with increasing severity of the disease.

Molecular epidemiology and characteristics of respiratory syncytial virus among hospitalized children in Guangzhou, China

BackgroundHuman respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection and hospitalization, especially in children. Highly mutagenic nature and antigenic diversity enable the RSV to successfully survive in human population. We conducted a molecular epidemiological study during 2017–2021 to investigate the prevalence and genetic characteristics of RSV.MethodsA total of 6499 nasopharyngeal (NP) swabs were collected from hospitalized children at Department of Pediatrics, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, China. All NP swab specimens were preliminary screened for common respiratory viruses and then tested for RSV using specific PCR assays. Partial G genes of RSV were amplified for phylogenetic analysis and genetic characterization.ResultsThe overall detection rate for common respiratory viruses was 16.12% (1048/6499). Among those, 405 specimens (6.20%, 405/6499) were found positive for RSV. The monthly distribution of RSV and other respiratory viruses was variable, and the highest incidence was recorded in Autumn and Winter. Based on the sequencing of hypervariable region of G gene, 93 RSV sequences were sub-grouped into RSV-A (56, 60.2%) and RSV-B (37, 39.8%). There was no coinfection of RSV-A and RSV-B in the tested samples. Phylogenetic analysis revealed that RSV-A and RSV-B strains belonged to ON1 and BA9 genotypes respectively, indicating predominance of these genotypes in Guangzhou. Several substitutions were observed which may likely change the antigenicity and pathogenicity of RSV. Multiple glycosylation sites were noticed, demonstrating high selection pressure on these genotypes.ConclusionThis study illustrated useful information about epidemiology, genetic characteristics, and circulating genotypes of RSV in Guangzhou China. Regular monitoring of the circulating strains of RSV in different parts of China could assist in the development of more effective vaccines and preventive measures.

Clinical and Laboratory Diagnosis of Exanthems Among Japanese Children Younger Than 6 Years Old in the Post-Measles-Rubella Vaccine Era.

Exanthems are a common reason for visits to the pediatric emergency department. However, epidemiological data in the post-measles-rubella vaccine era is limited. We sought to determine the recent causes of exanthems in children younger than 6 years old in the pediatric emergency department. A prospective single-center study was conducted in Japan from August 2019 to March 2020. Children younger than 6 years old with exanthems were enrolled. Exanthems were classified into 7 morphological patterns. Varicella, herpes zoster, impetigo, urticaria and Kawasaki disease were diagnosed clinically. Nasopharyngeal swab specimens were collected from patients with nonspecific exanthems and evaluated by polymerase chain reaction (PCR) assays capable of detecting 24 pathogens. The final diagnosis was made by discussion of 3 physicians based on clinical course and microbiology. There were 9705 pediatric visits, of which 296 (3%) had exanthems and were younger than 6 years old. Clinical diagnosis was possible for 160 (54%), including urticaria in 110 (37%), Kawasaki disease in 29 (10%), impetigo in 10 (3%), varicella or herpes zoster in 7 (2%) and group A Streptococcus in 4 (1%). Among the remaining 136 (46%) children, 75 (25%) underwent testing by PCR. One or more pathogens were detected in 49 (65%), specifically enterovirus in 14 (19%), cytomegalovirus in 13 (17%), human herpesvirus type-6 in 12 (16%), adenovirus in 11 (15%) and human herpesvirus type-7 in 8 (11%). Final infectious disease diagnoses were roseola infantum in 11 (15%), enterovirus in 9 (12%), adenovirus in 6 (8%), mixed virus infection in 5 (7%), group A Streptococcus in 3 (4%), parechovirus-A in 3 (4%) and influenza in 3 (4%). The most common causes of pediatric exanthems were noninfectious diseases and viral exanthema. PCR assay was instrumental for etiological diagnosis of nonspecific exanthems.

Tracking severe acute respiratory syndrome coronavirus 2 transmission and co-infection with other acute respiratory pathogens using a sentinel surveillance system in Rift Valley, Kenya.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been the most significant public health challenge in over a century. SARS-CoV-2 has infected over 765 million people worldwide, resulting in over 6.9 million deaths. This study aimed to detect community transmission of SARS-CoV-2 and monitor the co-circulation of SARS-CoV-2 with other acute respiratory pathogens in Rift Valley, Kenya. We conducted a cross-sectional active sentinel surveillance for the SARS-CoV-2 virus among patients with acute respiratory infections at four sites in Rift Valley from January 2022 to December 2022. One thousand two hundred seventy-one patients aged between 3 years and 98 years presenting with influenza-like illness (ILI) were recruited into the study. Nasopharyngeal swab specimens from all study participants were screened using a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for SARS-CoV-2, influenza A, influenza B and respiratory syncytial virus (RSV). The samples that tested positive for influenza A (n = 73) and RSV (n = 12) were subtyped, while SARS-CoV-2 (n = 177) positive samples were further screened for 12 viral and seven bacterial respiratory pathogens. We had a prevalence of 13.9% for SARS-CoV-2, 5.7% for influenza A, 2% for influenza B and 1% for RSV. Influenza A-H1pdm09 and RSV B were the most dominant circulating subtypes of influenza A and RSV, respectively. The most common co-infecting pathogens were Streptococcus pneumoniae (n = 29) and Haemophilus influenzae (n = 19), accounting for 16.4% and 10.7% of all the SARS-CoV-2 positive samples. Augmenting syndromic testing in acute respiratory infections (ARIs) surveillance is crucial to inform evidence-based clinical and public health interventions.

Clinical comparison of three SARS-CoV-2 nucleic acid amplification tests for routine diagnostic testing

BackgroundCycle threshold (Ct) values from SARS-CoV-2 nucleic acid amplification tests have been used to estimate viral load for treatment decisions. Additionally, there is a need for high-throughput testing, consolidating a variety of assays on one random-access analyzer. ObjectivesIn this study, the clinical performance of the Alinity m SARS-CoV-2, RealTime SARS-CoV-2, and GeneXpert Xpress SARS-CoV-2/Flu/RSV assays was assessed. MethodsAlinity precision and detection rates were evaluated using a dilution series of the Alinity m SARS-CoV-2 positive control. In a retrospective study, 7 remnant external quality assessment (EQA) specimens and 200 remnant nasopharyngeal swab specimens (100 positive and 100 negative) were tested in the three assays. ResultsAlinity had 100 % detection rate at 50 copies/mL and high reproducibility (Ct value coefficient of variation ≤3.1 %). All three assays correctly detected positive and negative EQA samples with comparable Ct values (max difference 2.38) and high linearity. In patient samples, positive percent agreement was 95 % (95 % CI 89–98 %) and negative percent agreement was 100 % (95 % CI 96–100 %) for Alinity, compared to the other two assays. Four specimens detected on Alinity m but not RealTime or Xpert had Ct values above 40. Assay results were highly correlated (r ≥ 0.94). Ct values (after addition of 10 unread cycles to the reported Ct of RealTime) were comparable across the three assays. ConclusionsAlinity m had high precision and accuracy and Ct values comparable to those of the RealTime and Xpert assays. The assays could be used interchangeably, with no need for adjustment of patient management decisions based on Ct values from each assay.

Performance evaluation of the cobas SARS-CoV-2 Duo, a novel qualitative and quantitative assay, for the detection of SARS-CoV-2 RNA.

Viral load quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a critical measure for monitoring clinical responses of patients with COVID-19. The cobas SARS-CoV-2 Duo (Roche Molecular Systems, Inc.) uses the cobas 6800/8800 platform and is an automated real-time reverse transcription PCR (RT-PCR) assay for the qualitative detection of SARS-CoV-2 RNA (ORF1a and ORF1a/b non-structural regions) in nasal and nasopharyngeal specimens. This assay also performs quantitation of the SARS-CoV-2 RNA level in the collected specimen. The precision, accuracy, and linearity of the cobas SARS-CoV-2 Duo test were assessed and correlated with the cycle threshold (Ct) value of the cobas SARS-CoV-2 assay, and the cobas Liat SARS-CoV-2 and influenza A/B assay (Inf A/B) (cobas Liat) in 201 clinical nasopharyngeal swab specimens. The analytical measurement range of the cobas SARS-CoV-2 Duo has been verified to be 1.0E + 02 IU/mL to 1.0E + 09 IU/mL. The between- and within-run precision evaluation and the linearity results (R 2 = 0.9961) of the cobas SARS-CoV-2 Duo were acceptable based on the verification criteria. A total of 201 nasopharyngeal specimens were evaluated. The positive, negative, and total agreements between the cobas SARS-CoV-2 Duo and the cobas SARS-CoV-2 assay were 93.5% (157 of 168), 75% (33 of 44), and 92.5% (186 of 201), respectively. The qualitative results (Ct values) from the cobas SARS-CoV-2 and the cobas Liat demonstrated good agreement with the quantitative results from the cobas SARS-CoV-2 Duo assay. In conclusion, the diagnostic performance of the cobas SARS-CoV-2 Duo assay is appropriate. It is a precise, accurate, and sensitive method for the detection of SARS-CoV-2 RNA and is comparable to two qualitative assays. IMPORTANCE Quantitative SARS-CoV-2 tests for viral load are necessary to guide patient treatment, as well as to determine infection control measures and policies. Although the real-time RT-PCR assays can report the Ct value to estimate the viral load, there are several serious concerns regarding the use of Ct values. Importantly, Ct values can vary significantly among between- and within-run methods. The diagnostic performance of the cobas SARS-CoV-2 Duo is appropriate. It is a precise, accurate, and sensitive method for the detection of SARS-CoV-2 RNA and is comparable to two qualitative assays (the cobas SARS-CoV-2 and the Liat cobas SARS-CoV-2 and Inf A/B). In contrast, using the Ct value to estimate viral load is not reliable, and utilization of a quantitative detection test, such as the cobas SARS-CoV-2 Duo, to accurately measure the viral load is needed.

Multi-center clinical evaluation of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab specimens from symptomatic individuals.

The symptomology is overlapping for respiratory infections due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A/B viruses, and respiratory syncytial virus (RSV). Accurate detection is essential for proper medical management decisions. This study evaluated the clinical performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab (NPS) specimens from individuals of all ages with signs and symptoms of respiratory infection consistent with COVID-19, influenza, or RSV. Retrospective known-positive and prospectively obtained residual NPS specimens were collected during two respiratory seasons in the USA. Clinical performance was established by comparing Panther Fusion SARS-CoV-2/Flu assay results to a three-molecular assay composite comparator interpretation for SARS-CoV-2 and to the FDA-cleared Panther Fusion Flu A/B/RSV assay results for all non-SARS-CoV-2 targets. A total of 1,900 prospective and 95 retrospective NPS specimens were included in the analyses. The overall prevalence in prospectively obtained specimens was 20.7% for SARS-CoV-2, 6.7% for influenza A, and 0.7% for RSV; all influenza B-positive specimens were retrospective specimens. The positive percent agreement of the Panther Fusion assay was 96.9% (378/390) for SARS-CoV-2, 98.0% (121/123) for influenza A virus, 95.2% (20/21) for influenza B virus, and 96.6% (57/59) for RSV. The negative percent agreement was ≥98.5% for all target viruses. Specimens with discordant Panther Fusion SARS/Flu/RSV assay results all had cycle threshold values of ≥32.4 (by comparator or by Panther Fusion SARS/Flu/RSV assay). Only five co-infections were detected in the study specimens. The Panther Fusion SARS-CoV-2/Flu/RSV assay provides highly sensitive and specific detection of SARS-CoV-2, influenza A virus, influenza B virus, and RSV in NPS specimens.

Diagnostic performance of saliva RT-PCR test as a diagnostic tool and its utility in the detection of SARS-CoV-2 shedding with different patient characteristics: Prospective observational study

BackgroundSalivary shedding of SARS-CoV-2 is a known entity and its role has been established in transmission of the disease. The present study was performed to evaluate the duration of viral shedding in saliva in COVID-19 patients and its variation among symptomatic and asymptomatic patients with or without co-morbidities. MethodsThe present prospective observational study was conducted at the COVID-19 care hospital associated with primary to tertiary care in New Delhi, India. A total of 124 COVID-19 confirmed cases enrolled in two phases (January–March 2021; April–June 2021) who consented for 48hrly saliva and nasopharyngeal swab (NPS) specimens till discharge from the hospital for SARS-CoV-2 detection were included. The specimens obtained were tested for SARS-CoV-2 by Real-Time PCR. ResultsThe sensitivity and the specificity of RT-PCR on saliva were 81.7 ​% and 85.0 ​%, respectively. The sensitivity of saliva-based PCR was comparable in symptomatic and asymptomatic patients (81.6 ​% vs 82.1 ​%). The sensitivity of saliva-based PCR markedly increased in the second phase of enrollment as compared to the first phase (92.6 ​% vs 78.5 ​%) indicating higher level of salivary shedding by the delta variant of SARS-CoV-2. The sensitivity of PCR on saliva was the highest up to day seven of illness. The median duration of RNA shedding in saliva was comparable among the symptomatic and asymptomatic patients. The severity of the disease was not associated with the duration of SARS-CoV-2 shedding in saliva. ConclusionsSARS-CoV-2 shedding in saliva continued till seven days in large number of patients including asymptomatic patients. Saliva is non-inferior to NPS specimen in the diagnosis of SARS-CoV-2. Saliva specimen is recommended as a good alternate to NPS for SARS-CoV-2 testing.

Swish and gargle saliva sampling is a patient-friendly and comparable alternative to nasopharyngeal swabs to detect SARS-CoV-2 in outpatient settings for adults and children.

Nasopharyngeal swabs (NPS) are considered the standard specimen for SARS-CoV-2 detection by PCR. We aimed to compare detection performance and patient experience of NPS with swish and gargle saliva (SGS) to determine its potential as an accurate, easy-to-collect, and comfortable alternative. We conducted a prospective study from March to May 2021 and recruited pediatric and adult outpatients seeking COVID-19 testing. Paired NPS and 5-mL SGS were collected from each participant during the same visit and tested in parallel using validated rRT-PCR assays. Variant of concern (VOC) analysis was performed for positive NPS specimens. The participant completed the surveys regarding their experience with the two collection methods. We included 238 participants in the SARS-CoV-2 detection performance analysis. Thirty-two participants tested positive for both NPS and SGS. In comparison to NPS, the sensitivity and specificity of SGS to detect SARS-CoV-2 were 94.1% (95%CI; 80.3%-99.3%) and 97.1% (95%CI; 93.7%-98.9%), respectively. Six participants tested positive only with SGS and only two with NPS. NPS is an imperfect gold standard. Thus, when compared to the COVID-19 case, that is, a patient with positive NPS and/or SGS, the sensitivity for NPS and SGS were 85.0% and 95.0%, respectively. VOC results (n = 26) revealed that 92% of the cases were alpha. From the 238 surveys, 89.9% of the participants described SGS collection as comfortable or very comfortable compared to 15.1% for NPS; 90.2% of the participants were likely or very likely to return for SGS collection compared to 59.3% for NPS. SGS is an alternative to NPS for SARS-CoV-2 detection in adult and pediatric outpatients as it is more patient-friendly while still maintaining comparable performance.IMPORTANCEWidespread and frequent testing for COVID-19 was an important strategy to identify infected patients to isolate and control the spread of the disease during the pandemic. The nasopharyngeal swab (NPS) global supply chain and access to trained healthcare professionals for standard NPS collection were often compromised. Patient discomfort and limited access challenged health systems to reach large numbers for testing in adult and pediatric populations. Our study revealed that swish and gargle saliva (SGS) was comparable to NPS in detecting SARS-CoV-2 and more patient-friendly than NPS. Patients were more likely to repeat the test with SGS. SGS was amenable to self-collection instead of relying on skilled professionals. This comprehensive evaluation highlights the challenges of comparing the accuracy of new methods to imperfect gold standards and identifies additional patient-centric factors that should be considered when defining such standards. Thus, SGS is an advantageous alternative specimen collection for outpatient en masse testing.

Elevated A-to-I RNA editing in COVID-19 infected individuals.

Given the current status of coronavirus disease 2019 (COVID-19) as a global pandemic, it is of high priority to gain a deeper understanding of the disease's development and how the virus impacts its host. Adenosine (A)-to-Inosine (I) RNA editing is a post-transcriptional modification, catalyzed by the ADAR family of enzymes, that can be considered part of the inherent cellular defense mechanism as it affects the innate immune response in a complex manner. It was previously reported that various viruses could interact with the host's ADAR enzymes, resulting in epigenetic changes both to the virus and the host. Here, we analyze RNA-seq of nasopharyngeal swab specimens as well as whole-blood samples of COVID-19 infected individuals and show a significant elevation in the global RNA editing activity in COVID-19 compared to healthy controls. We also detect specific coding sites that exhibit higher editing activity. We further show that the increment in editing activity during the disease is temporary and returns to baseline shortly after the symptomatic period. These significant epigenetic changes may contribute to the immune system response and affect adverse outcomes seen in post-viral cases.