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Background: Cytomegalovirus (CMV) causes significant morbidity and mortality in immunocompromised patients, particularly transplant recipients. Quantitation of CMV DNA in peripheral blood is used to monitor prophylactic and pre-emptive approaches to prevent CMV disease, whereas CMV DNA testing of non-plasma specimens may aid in the diagnosis of end-organ disease., Methods: The analytical performance of the FDA-approved Aptima CMV Quant Assay was evaluated using reference CMV (SeraCare) diluted in defibrinated human plasma, as well as negative bronchoalveolar lavage fluid and tissue. Agreement was determined using 100 clinical acid-citrate-dextrose (ACD) plasma specimens, 77 bronchoalveolar lavage (BAL) fluids, and 101 tissues previously tested using artus CMV qPCR., Results: Aptima CMV lower limit of detection (LLOD) was 169 IU/mL for ACD plasma, 100 IU/mL for BAL, and 50 IU/mL for tissue. Positive percent agreement (PPA) was 100.0% (50/50; 95% CI: 92.9% - 100.0%) and negative percent agreement (NPA) was 94.0% (47/50; 95% CI: 83.5% - 98.8%) for ACD plasma. Bland-Altman analysis revealed a bias of 0.20 log 10 IU/mL (Aptima - artus) with 95% limits of agreement of -0.53 to 0.93. For BAL fluids, PPA was 70.0% (14/20; 95% CI: 45.7% - 88.1%) and NPA was 82.4% (43/51; 95% CI: 69.1% - 91.6%). For tissues, PPA was 90.0% (45/50; 95% CI: 78.2% - 96.7%) and NPA was 94.0% (47/50; 95% CI: 83.5% - 98.8%)., Conclusions: The Aptima CMV Quant Assay demonstrates high analytical sensitivity and good overall agreement using clinical plasma and tissue specimens., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Benjamin Pinsky reports research contracts from Hologic, Inc. and Altona Diagnostics, as well as travel support from Altona., (Copyright © 2023. Published by Elsevier B.V.)

Background: Tests that sensitively detect the presence of actively replicating SARS-CoV-2 may improve patient care by allowing the safe and timely discontinuation of isolation. Correlates of active replication include nucleocapsid antigen and virus minus-strand RNA., Methods: Qualitative agreement of the DiaSorin LIAISON SARS-CoV-2 nucleocapsid antigen chemiluminescent immunoassay (CLIA) with minus-strand RNA was determined using 402 upper respiratory specimens from 323 patients previously tested using a laboratory-developed SARS-CoV-2 strand-specific RT-qPCR. Nucleocapsid antigen levels, minus-strand and plus-strand cycle threshold values, as well as virus culture, were used to evaluate discordant specimens. Receiver operating characteristic curves were also used to identify virus RNA thresholds for active replication, including values harmonized to the World Health Organization International Standard., Results: Overall agreement was 92.0% [95% confidence interval (CI): 89.0 - 94.5], positive percent agreement was 90.6% (95% CI: 84.4 - 95.0), and negative percent agreement was 92.8% (95% CI: 89.0 - 95.6). The kappa coefficient was 0.83 (95% CI: 0.77 - 0.88). Discordant specimens contained low levels of nucleocapsid antigen and minus-strand RNA. 84.8% (28/33) were negative by culture. Sensitivity-optimized plus-strand RNA thresholds for active replication were 31.6 cycles or 3.64 log 10 IU/mL; resulting in 100.0% sensitivity (95% CI: 97.6 to 100.0) and 55.9 specificity (95% CI: 49.7 to 62.0)., Conclusions: Detection of nucleocapsid antigen by CLIA performs equivalently to minus-strand detection via strand-specific RT-qPCR, though these methods may overestimate replication-competent virus compared to culture. Careful implementation of biomarkers for actively replicating SARS-CoV-2 has the potential to inform infection control decision-making and patient management., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier B.V.)

Background: Despite the sharp increase in mpox (formerly monkeypox) incidence and the wide geographic spread of mpox during the 2022 outbreak, the community prevalence of infection remains poorly characterized. This study is a retrospective epidemiologic survey to estimate mpox prevalence., Methods: Samples obtained for sexually transmitted infection (STI) testing from April to September 2022 in the public hospital and clinic system of San Mateo County, California were screened for mpox virus (MPXV) using polymerase chain reaction., Results: 16/1,848 samples from 11/1,645 individuals were positive for MPXV by qPCR. 4/11 individuals with positive MPXV testing were cisgender women, 2 of whom were pregnant at the time of sample collection. Both deliveries were complicated by chorioamnionitis. Anorectal and oropharyngeal samples were the most likely to be positive for MPXV (4/60 anorectal samples and 4/66 oropharyngeal samples compared with 5/1,264 urine samples and 3/445 vaginal samples)., Conclusions: Our study is one of the first epidemiologic surveys for MPXV infection outside of sexual health/STI clinic settings. Relatively high rates of MPXV from oropharyngeal and anorectal samples reinforces the importance of MPXV testing at various anatomic sites, particularly if patients are presenting with non-lesional symptoms (pharyngitis, proctitis). However, the United States Food and Drug Administration (FDA) has not yet authorized non-lesional MPXV testing. The identification of MPXV in women in our cohort suggests that the rates of mpox in women may have previously been underestimated and highlights the risk of pregnancy complications associated with mpox., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: SARS-CoV-2 variant surveillance informs vaccine composition and decisions to de-authorize antibody therapies. Though detailed genetic characterization requires whole-genome sequencing, targeted mutation analysis may complement pandemic surveillance efforts., Methods: This study investigated the qualitative performance of a multiplex oligonucleotide ligation assay targeting 19 spike mutations using 192 whole genome sequenced upper respiratory samples representing SARS-CoV-2 variants of concern., Results: Initial valid results were obtained from 95.8% [95% confidence interval (CI): 92.0 - 98.2; 184/192] of samples. All eight invalid samples were valid on repeat testing. When comparing SARS-CoV-2 oligonucleotide ligase assay SARS-CoV-2 variant calls with whole genome sequencing, overall positive percent agreement was 100% (95% CI: 98.1 - 100.0; 192/192), as was the positive and negative percent agreement for each of the tested variants; Gamma, Delta, Omicron BA.1, BA.2, and BA.4/BA.5., Conclusions: This multiplexed oligonucleotide ligation assays demonstrated accurate SARS-CoV-2 variant typing compared to whole genome sequencing. Such an approach has the potential to provide improved turnaround compared to sequencing and more detailed mutation coverage than RT-qPCR., Competing Interests: Declaration of Competing Interest FK, TJB, SBH, JNW, and GBS are MSD employees. Testing was performed at Stanford using reagents and instruments provided by MSD to BAP., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Seasonal outbreaks of infectious conjunctivitis remain a public health issue. Determination of outbreak etiologies in the context of a worldwide pandemic may provide useful information to guide public health strategies. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge., Methods: This prospective study was conducted from April 2021 to September 2021. All outpatients presenting to the Aravind Eye Center (Madurai, India) with signs and symptoms consistent with acute infectious conjunctivitis were eligible. Three swabs were obtained from each participant: one from each conjunctiva and one from the anterior nares. Samples were processed for metagenomic RNA deep sequencing (RNA-seq)., Results: Samples from 106 study participants were sequenced. The most common presenting symptoms were tearing (86%) and itching (71%). Preauricular lymphadenopathy was present in 38% of participants. 20% of participants had close contacts with similar symptoms. Systemic symptoms such as coughing, runny nose, vomiting or diarrhea were uncommonly reported. 60% of all participants used some medicated eye drops upon enrollment. 75% of study participants demonstrated infection with human adenovirus D (HAdV-D). 11% of conjunctivitis was associated with SARS-CoV-2. 15% had no definitive pathogen detected. 8% of all participants had codetection of more than one pathogen on RNA-seq., Conclusions: During the COVID-19 Delta surge in India, HAdV-D was the most common pathogen associated with infectious conjunctivitis. SARS-CoV-2 was the second most common associated pathogen. Seasonal surveillance may be necessary for the determination of emerging and reemerging pathogens responsible for infectious conjunctivitis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier B.V.)

Background: Viral conjunctivitis (pink eye) can be highly contagious and is of public health importance. There remains significant debate whether SARS-CoV-2 can present as a primary conjunctivitis. The aim of this study was to identify pathogens associated with outpatient infectious conjunctivitis during the COVID-19 Delta surge., Methods: This prospective study was conducted in the spring and summer months of 2021. 106 patients with acute conjunctivitis who presented to the Aravind Eye Center in Madurai, India were included. One anterior nasal swab and one conjunctival swab of each eye were obtained for each enrolled patient. Samples were subsequently processed for unbiased metagenomic RNA deep sequencing (RNA-seq). Outcomes included clinical findings and codetection of other pathogens with SARS-CoV-2 in patients with conjunctivitis., Results: Among the 13 patients identified with human coronavirus RNA fragments in their swabs, 6 patients had SARS-CoV-2 infection, 5 patients had coinfections of SARS-CoV-2 and human adenovirus (HAdV), 1 patient had a coinfection with human coronavirus OC43 and HAdV, and 1 patient had a coinfection of Vittaforma corneae and SARS-CoV-2. 30% had bilateral disease and symptoms on presentation. Petechial hemorrhage was noted in 33% of patients with SARS-CoV-2 infection. No patients with SARS-CoV-2 or SARS-CoV-2 and HAdV infections had subepithelial infiltrates on presentation. All patients denied systemic symptoms., Conclusions: Among the patients presented with conjunctivitis associated with human coronavirus infection, over 50% of the patients had co-infections with other circulating pathogens, suggesting the public-health importance of broad pathogen testing and surveillance in the outpatient conjunctivitis population., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Cycle threshold (Ct) values from SARS-CoV-2 reverse transcription quantitative PCR (RT-qPCR) tests are used to measure viral burden. Calibration to the First WHO International Standard for SARS-CoV-2 RNA may improve quantitative inter-assay agreement., Methods: WHO standard was tested using four emergency use authorized RT-qPCRs to generate calibration curves and evaluate Ct value differences. Harmonization of two assays, Cepheid Xpert Xpress SARS-CoV-2 targeting E and nucleocapsid (N2) [Xpert (E) and Xpert (N2)] and a laboratory-developed test targeting E [LDT (E)], was assessed using 93 positive upper respiratory samples. Platform (target) pairs were compared via Bland-Altman analysis and Passing-Bablok regression., Results: Ct values with the WHO standard were comparable across platforms and targets, except Xpert (N2) for which the mean difference was a median of 3.68 cycles (Interquartile Range, IQR = 3.23 to 3.76 cycles) greater than other platform (target) pairs. Using clinical samples, the mean difference of Xpert (N2) to LDT (E) was 3.64 cycles (95% Confidence Interval, CI =1.51 to 5.76). After calibration, the mean difference of Xpert (N2) to LDT (E) was 0.08 log 10 IU/mL (95% CI = -0.56 to 0.71) and the regression was y = 1.00x * 0.08 (95% CI slope = 0.93 to 1.07, 95% CI intercept = 0.28 to 0.42)., Conclusions: Calibration to the WHO standard resulted in the harmonization of two RT-qPCR tests, whereas analysis by Ct value alone may have led to erroneous quantitation. Harmonization to the WHO standard has the potential to improve the generalizability of clinical associations with SARS-CoV-2 RNA levels., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Significant overlap exists between the symptoms of SARS-CoV-2 and other respiratory viruses. This poses a serious challenge to clinical diagnosis, laboratory testing, and infection control programs., Objectives: To evaluate the performance of the Hologic Panther Fusion Respiratory Assays (RA) compared to the GenMark ePlex Respiratory Pathogen Panel (RPP) and to assess the ability of the Panther Fusion to perform parallel testing of SARS-CoV-2 and other respiratory viruses from a single sample., Study Design: A diagnostic comparison study was carried out using 375 clinical nasopharyngeal specimens. Assay performance was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient., Results: Overall agreement between the Fusion RA and ePlex RPP was 97.3 % (95 % CI 96.3-98.0), positive percent agreement was 97.2 % (95 % CI 93.0-99.2), negative percent agreement was 97.3 % (95 % CI 96.3-98.0), and the kappa coefficient was 0.85 (95 % CI 0.81-0.89). Forty additional viruses in 30 specimens were detected by Fusion that were not detected by ePlex. The maximum specimen throughput for parallel testing of the Fusion Respiratory Assays with SARS-CoV-2 was 275 samples in 20.7 h for Fusion SARS-CoV-2 and 350 samples in 20.0 h for Aptima Transcription Mediated Amplification SARS-CoV-2., Conclusion: Fusion RA demonstrated substantial agreement compared to the ePlex RPP. However, the Fusion detected respiratory viruses not identified by ePlex, consistent with higher clinical sensitivity. Workflows for parallel testing of respiratory pathogens and SARS-CoV-2 demonstrate that the Panther Fusion instrument provides a flexible, moderate to high throughput testing option for pandemic and seasonal respiratory viruses., (Copyright © 2021. Published by Elsevier B.V.)

Background: Measles virus (MeV) is an important cause of acute febrile illness and pediatric mortality globally, with recent U.S. outbreaks associated with under-vaccination. MeV is highly contagious and timely diagnosis is critical to limit spread. RNA detection is the most sensitive method for acute measles diagnosis; however, MeV nucleic acid amplification assays are not widely available., Methods: We performed a diagnostic accuracy study of a triple-target, real-time RT-PCR (rRT-PCR) assay for simultaneous detection of MeV N, H, and L genes., Results: The MeV triple-target rRT-PCR was tested against serial dilutions (7.0-2.0 log 10 copies/mL) of five MeV isolates representing circulating genotypes, and detected 98.7% (74/75) of nasopharyngeal (NP) swab dilutions, 100% (75/75) of plasma dilutions, and 85.3% (64/75) of urine dilutions. MeV RNA detection in urine was markedly improved with the addition of a nucleic acid stabilizing agent. A 95% lower limit of detection (LLOD) of < 3.0 log 10 copies/mL was established in each specimen matrix. No cross-reactivity with relevant viruses or interfering substances were identified in specificity studies. The MeV triple-target rRT-PCR detected all three gene targets in a clinical NP swab from an individual with confirmed measles infection. Furthermore, pooled testing from 798 influenza A/B/RSV-negative pediatric NP swabs identified two specimens positive for MeV RNA, confirmed by N gene sequencing to represent shedding of the vaccine-type measles virus., Conclusions: The MeV triple-target rRT-PCR assay showed high analytic sensitivity across circulating MeV genotypes in three clinically-relevant matrices. Implementation of this assay in the clinical laboratory may facilitate timely diagnosis of acute measles infection and implementation of appropriate infection control interventions., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Molecular diagnostics such as pathogen-directed PCRs have transformed testing for ocular infections since the late 1990s. Although these assays remain important diagnostic tools for samples with low biomass, the lack of diagnostic range motivates alternative molecular approaches for ocular infections. The aim of this study was to determine the performance of a high-throughput RNA sequencing approach, RNA-seq, to detect infectious agents in ocular samples from patients with presumed ocular infections., Methods: We compared the performance of RNA-seq to pathogen-directed PCRs using remnant nucleic acids from 41 aqueous or vitreous samples of patients with presumed ocular infections. Pathogen-directed PCRs were performed at the CLIA-certified Stanford Clinical Virology Laboratory. RNA-seq was performed in a masked manner at the Proctor Foundation at the University of California San Francisco. Percent positive and negative agreement between the two testing approaches were calculated. Discordant results were subjected to orthogonal testing., Results: The positive percent agreement between RNA-seq and pathogen-directed PCRs was 100% (95% confidence interval (CI): 78.5%-100%). The negative percent agreement was 92.6% (95% CI: 76.6%-97.9%). RNA-seq identified pathogens not on the differential diagnosis for 9.7% (4/41) of the samples. Two pathogens solely identified with RNA-seq were confirmed with orthogonal testing., Conclusions: RNA-seq can accurately identify common and rare pathogens in aqueous and vitreous samples of patients with presumed ocular infections. Such an unbiased approach to testing has the potential to improve diagnostics although practical clinical utility warrants additional studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Interleukin-6 (IL-6) plays an important role in chronic inflammation. Thus, we aimed to investigate the effects of IL-6 polymorphisms on predicting the progression of hepatitis B virus (HBV)-r elated liver cirrhosis., Methods: A cross-sectional study was conducted to analyse IL-6 polymorphisms and serum levels of IL-6 in HBV-infected patients at different clinical phases and in healthy controls. IL-6 polymorphisms were detected by the TaqMan PCR method, and plasma IL-6 levels were assessed by ELISA., Results: Our analysis included 182 chronic hepatitis B (CHB) patients, 190 HBV-infected liver cirrhosis cases, 125 inactive HBsAg carriers, and 246 healthy controls. Seven SNPs in IL-6 including rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992, and rs2069852 were analysed. In a haplotype analysis between HBV-infected liver cirrhosis cases and CHB patients, inactive HBV carriers or healthy controls, haplotype CT in block 1 and haplotype GGCGG in block 2 were associated with liver cirrhosis (P <0.05). Moreover, the genotype or allele frequencies were significantly different in IL-6 rs10499563 and rs2069837 when HBV-infected liver cirrhosis patients were compared with CHB patients, inactive HBV carriers or healthy controls. A further study found that compared with that in the healthy controls, inactive HBV carriers or CHB patients, plasma IL-6 was elevated in HBV-infected liver cirrhosis patients., Conclusion: In conclusion, the IL-6 rs10499563 and rs2069837 polymorphisms are associated with incidence of liver cirrhosis may through their effects on IL-6 expression and these two single nucleotide polymorphisms can be used as potential prognostic markers of HBV-related liver cirrhosis., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interest exists., (Copyright © 2020. Published by Elsevier B.V.)

Background: Numerous nucleic acid amplification tests, including real-time, reverse transcription PCR (rRT-PCR) and isothermal amplification methods, have been developed to detect SARS-CoV-2 RNA, including many that have received emergency use authorization (EUA). There is a need to assess their test performance relative to one another., Objectives: The aim of this study was to compare the test performance of a high complexity laboratory-developed rRT-PCR EUA from Stanford Health Care (SHC) targeting the SARS-CoV-2 envelope (E) gene with other tests: the Atila isothermal amplification assay targeting the nucleocapsid (N) gene and open reading frame 1ab (ORF1ab), the Altona E and spike (S) multiplex, real-time RT-PCR, and the US Centers for Disease Control and Prevention (CDC) N1 and N2 rRT-PCRs., Study Design: A diagnostic comparison study was performed by testing nasopharyngeal samples from persons under investigation for coronavirus disease 2019 (COVID-19). Assay performance was assessed by percent agreement and Cohen's kappa coefficient., Results: Positive percent agreement with the SHC EUA reference assay was 82.8 % (95 % confidence interval (CI) 65.0 to 92.9) for Atila, 86.7 % (95 % CI 69.7 to 95.3) for the Altona E and S targets, and 86.7 % (95 % CI 69.7 to 95.3) and 90.0 % (95 % CI 73.6 to 97.3), for the CDC N1 and N2 targets, respectively. All assays demonstrated 100 % negative percent agreement. Kappa coefficients ranged from 0.86 to 0.92, indicating excellent agreement., Conclusions: Performance was comparable among the SARS-CoV-2 nucleic acid amplification methods tested, with a limited number of discrepancies observed in specimens with low viral loads., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Numerous nucleic acid amplification assays have recently received emergency use authorization (EUA) for the diagnosis of SARS-CoV-2 infection, and there is a need to assess their test performance relative to one another., Objectives: The aim of this study was to compare the test performance of the Hologic Panther Fusion SARS-CoV-2 assay targeting two regions of open reading frame 1ab (ORF1ab) to a high complexity molecular-based, laboratory-developed EUA from Stanford Health Care (SHC) targeting the SARS-CoV-2 envelope (E) gene., Study Design: We performed a diagnostic comparison study by testing nasopharyngeal samples on the two assays. Assay agreement was assessed by overall percent agreement and Cohen's kappa coefficient., Results: A total of 184 nasopharyngeal samples were tested using the two assays, of which 180 showed valid results and were included for the comparative analysis. Overall percent agreement between the assays was 98.3 % (95 % confidence interval (CI) 95.2-99.7) and kappa coefficient was 0.97 (95 % CI 0.93-1.0). One sample was detected on the SHC laboratory developed test (LDT) and not on the Panther Fusion, and had a Ct of 35.9. Conversely, 2 samples were detected on the Panther Fusion and not on the LDT, and had Ct values of 37.2 and 36.6., Conclusion: The Panther Fusion SARS-CoV-2 assay and the SHC LDT perform similarly on clinical nasopharyngeal swab specimens. Other considerations, including reagent availability, turnaround time, labor requirements, cost and instrument throughput should guide the decision of which assay to perform., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)