Pretreatment Methods for Human Nasopharyngeal Swabs to Increase the Signal to Noise Ratio of High Sensitivity Immunoassays.

Mucous samples collected through nasopharyngeal (NP) swabs are considered gold standard specimens for the detection of respiratory pathogens. Matrices of these highly viscous samples often cause significant background noises in immunoassays, especially immunoassays with high sensitivity. We demonstrated such nonspecific background signals in both a chemiluminescence enzyme-linked immunosorbent assay (ELISA) and a novel highly sensitive immunoassay called Microbubbling SARS-CoV-2 Antigen Assay (MSAA). We developed and demonstrated the effectiveness of two quick sample pretreatment methods, filtration and preadsorption, to decrease nonspecific signals and increase the signal-to-noise ratio (SNR). Using these pretreatment methods, the SNR (at 3.6 × 104 copies/mL of inactivated SARS-CoV-2) was increased by 42.4-fold (95% CI 41.0-43.8) and 67.1-fold (95% CI 57.9-76.3) in the MSAA, and 1.3-fold (95% CI 0.9-1.7) and 1.8-fold (95% CI 1.6-2.0) in the chemiluminescence ELISA assay. Sample pretreatment methods developed in this study are broadly adaptable for the development of immunoassays for highly viscous samples.

Facilitating Safe Discharge Through Predicting Disease Progression in Moderate Coronavirus Disease 2019 (COVID-19): A Prospective Cohort Study to Develop and Validate a Clinical Prediction Model in Resource-Limited Settings.

BACKGROUND: In locations where few people have received coronavirus disease 2019 (COVID-19) vaccines, health systems remain vulnerable to surges in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Tools to identify patients suitable for community-based management are urgently needed. METHODS: We prospectively recruited adults presenting to 2 hospitals in India with moderate symptoms of laboratory-confirmed COVID-19 to develop and validate a clinical prediction model to rule out progression to supplemental oxygen requirement. The primary outcome was defined as any of the following: SpO2 < 94%; respiratory rate > 30 BPM; SpO2/FiO2 < 400; or death. We specified a priori that each model would contain three clinical parameters (age, sex, and SpO2) and 1 of 7 shortlisted biochemical biomarkers measurable using commercially available rapid tests (C-reactive protein [CRP], D-dimer, interleukin 6 [IL-6], neutrophil-to-lymphocyte ratio [NLR], procalcitonin [PCT], soluble triggering receptor expressed on myeloid cell-1 [sTREM-1], or soluble urokinase plasminogen activator receptor [suPAR]), to ensure the models would be suitable for resource-limited settings. We evaluated discrimination, calibration, and clinical utility of the models in a held-out temporal external validation cohort. RESULTS: In total, 426 participants were recruited, of whom 89 (21.0%) met the primary outcome; 257 participants comprised the development cohort, and 166 comprised the validation cohort. The 3 models containing NLR, suPAR, or IL-6 demonstrated promising discrimination (c-statistics: 0.72-0.74) and calibration (calibration slopes: 1.01-1.05) in the validation cohort and provided greater utility than a model containing the clinical parameters alone. CONCLUSIONS: We present 3 clinical prediction models that could help clinicians identify patients with moderate COVID-19 suitable for community-based management. The models are readily implementable and of particular relevance for locations with limited resources.

Colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) and carbapenem-resistant Enterobacterales (CRE) in healthcare and community settings in Botswana: an antibiotic resistance in communities and hospitals (ARCH) study.

OBJECTIVES: Although extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) and carbapenem-resistant Enterobacterales (CRE) are a global challenge, data on these organisms in low- and middle-income countries are limited. In this study, we sought to characterize colonization data critical for greater antibiotic resistance surveillance efforts. METHODS: This study was conducted in three hospitals and six clinics in Botswana. We conducted ongoing surveillance of adult patients in hospitals and clinics and adults and children in the community. All participants underwent rectal swab sampling to identify ESCrE and CRE. RESULTS: Enrollment occurred from January 15, 2020, to September 4, 2020, but paused from April 2, 2020, to May 21, 2020, because of a countrywide COVID-19 lockdown. Of 5088 individuals approached, 2469 (49%) participated. ESCrE colonization prevalence was 30.7% overall (43% for hospital participants, 31% for clinic participants, 24% for adult community participants, and 26% for child community participants) (P <0.001). A total of 42 (1.7%) participants were colonized with CRE. CRE colonization prevalence was 1.7% overall (6.8% for hospital participants, 0.7% for clinic participants, 0.2% for adult community participants, and 0.5% for child community participants) (P <0.001). ESCrE and CRE prevalence varied substantially across regions and was significantly higher prelockdown versus postlockdown. CONCLUSIONS: ESCrE colonization was high in all settings in Botswana. CRE prevalence in hospitals was also considerable. Colonization prevalence varied by region and clinical setting and decreased after a countrywide lockdown.

Does a humoral correlate of protection exist for SARS-CoV-2? A systematic review.

BACKGROUND: A correlate of protection (CoP) is an immunological marker associated with protection against infection. Despite an urgent need, a CoP for SARS-CoV-2 is currently undefined. OBJECTIVES: Our objective was to review the evidence for a humoral correlate of protection for SARS-CoV-2, including variants of concern. METHODS: We searched OVID MEDLINE, EMBASE, Global Health, Biosis Previews and Scopus to January 4, 2022 and pre-prints (using NIH iSearch COVID-19 portfolio) to December 31, 2021, for studies describing SARS-CoV-2 re-infection or breakthrough infection with associated antibody measures. Two reviewers independently extracted study data and performed quality assessment. RESULTS: Twenty-five studies were included in our systematic review. Two studies examined the correlation of antibody levels to VE, and reported values from 48.5% to 94.2%. Similarly, several studies found an inverse relationship between antibody levels and infection incidence, risk, or viral load, suggesting that both humoral immunity and other immune components contribute to protection. However, individual level data suggest infection can still occur in the presence of high levels of antibodies. Two studies estimated a quantitative CoP: for Ancestral SARS-CoV-2, these included 154 (95% confidence interval (CI) 42, 559) anti-S binding antibody units/mL (BAU/mL), and 28.6% (95% CI 19.2, 29.2%) of the mean convalescent antibody level following infection. One study reported a CoP for the Alpha (B.1.1.7) variant of concern of 171 (95% CI 57, 519) BAU/mL. No studies have yet reported an Omicron-specific CoP. CONCLUSIONS: Our review suggests that a SARS-CoV-2 CoP is likely relative, where higher antibody levels decrease the risk of infection, but do not eliminate it completely. More work is urgently needed in this area to establish a SARS-CoV-2 CoP and guide policy as the pandemic continues.

Implementation of an Extraction-Free COVID Real-Time PCR Workflow in a Pediatric Hospital Setting.

BACKGROUND: This study outlines the development, implementation, and impact of a laboratory-developed, extraction-free real-time PCR assay as the primary diagnostic test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a pediatric hospital. METHODS: Clinical specimens from both upper and lower respiratory tract sources were validated, including nasopharyngeal aspirates, nasopharyngeal swabs, anterior nares swabs, and tracheal aspirates (n = 333 clinical samples). Testing volumes and laboratory turnaround times were then compared before and after implementation to investigate effects of the workflow changes. RESULTS: Compared to magnetic-bead extraction platforms, extraction-free real-time PCR demonstrated ≥95% positive agreement and ≥97% negative agreement across all tested sources. Implementation of this workflow reduced laboratory turnaround time from an average of 8.8 (+/-5.5) h to 3.6 (+/-1.3) h despite increasing testing volumes (from 1515 to 4884 tests per week over the reported period of testing). CONCLUSIONS: The extraction-free workflow reduced extraction reagent cost for SARS-CoV-2 testing by 97%, shortened sample handling time, and significantly alleviated supply chain scarcities due to the elimination of specialized extraction reagents for routine testing. Overall, this assay is a viable option for laboratories to increase efficiency and navigate reagent shortages for SARS-CoV-2 diagnostic testing.

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

BACKGROUND: High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. METHODS: We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. RESULTS: Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. CONCLUSIONS: The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

Reduction in Sporadic Norovirus Infections Following the Start of the COVID-19 Pandemic, 2019-2020, Philadelphia.

INTRODUCTION: Norovirus infections are common in the USA and worldwide. Detection of norovirus in fecal samples is now common in routine tests for enteric pathogens using molecular methods. We observed a change in positivity rates for norovirus after the beginning of the coronavirus disease 2019 (COVID-19) pandemic in our laboratory and performed a more detailed analysis of testing results. METHODS: We reviewed the positivity rates for detection of common enteric pathogens from stool samples submitted to an academic medical center laboratory pre (2016-2019) and post the start of the COVID-19 pandemic (2020). RESULTS: In contrast to other enteric pathogens, norovirus positivity rates dropped dramatically from a yearly average of 3.9% in 2016-2019 to 0.76% from March 2020 through the end of 2020. CONCLUSION: A sustained reduction in norovirus positivity rates was temporally associated with COVID-19 mitigation processes in the Philadelphia area, while positivity rates for other common enteric pathogens were only intermittently reduced.

Quantifying the Impact of Nasopharyngeal Specimen Quality on Severe Acute Respiratory Syndrome Coronavirus 2 Test Performance.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse-transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) has been used to estimate quantitative viral load, with the goal of targeting isolation precautions for individuals with coronavirus disease 2019 (COVID-19) and guiding public health interventions. However, variability in specimen quality can alter the Ct values obtained from SARS-CoV-2 clinical assays. We sought to define how variable nasopharyngeal (NP) swab quality impacts clinical SARS-CoV-2 test sensitivity. METHODS: We performed amplification of a human gene target (ß-actin) in parallel with a clinical RT-PCR targeting the SARS-CoV-2 ORF1ab gene for 1282 NP specimens collected from patients with clinical concern for COVID-19. We evaluated the relationship between NP specimen quality, characterized by late Ct values for the human gene target ß-actin Ct, and the probability of SARS-CoV-2 detection via logistic regression, as well as the linear relationship between SARS-CoV-2 and ß-actin Ct. RESULTS: Low-quality NP swabs are less likely to detect SARS-CoV-2 (odds ratio, 0.607 [95% credible interval {CrI}, .487-.753]). We observed a positive linear relationship between SARS-CoV-2 and ß-actin Ct values (slope, 0.181 [95% CrI, .097-.264]), consistent with a reduction in detection of 0.181 cycles for each additional cycle of the ß-actin target. COVID-19 disease severity was not associated with ß-actin Ct values. CONCLUSIONS: Variability in NP specimen quality significantly impacts the performance of clinical SARS-CoV-2 assays, and caution should be taken when interpreting quantitative SARS-CoV-2 Ct results. If unrecognized, low-quality NP specimens, which are characterized by a low level of amplifiable human DNA target, may limit the successful application of SARS-CoV-2 Ct values to direct infection control and public health interventions.

Copan eNAT Transport System To Address Challenges in COVID-19 Diagnostics in Regions with Limited Testing Access.

Community-based health care clinics and hospital outreach services have the potential to expand coronavirus disease 2019 (COVID-19) diagnostics to rural areas. However, reduced specimen stability during extended transport, the absence of a cold chain to centralized laboratories, and biosafety concerns surrounding specimen handling have limited this expansion. In the following study, we evaluated eNAT (Copan Italia, Brescia, Italy) as an alternative transport system to address the biosafety and stability challenges associated with expanding COVID-19 diagnostics to rural and remote regions. In this study, we demonstrated that high-titer severe acute respiratory virus syndrome coronavirus 2 (SARS-CoV-2) lysate placed into eNAT medium cannot be propagated in cell culture, supporting viral inactivation. To account for off-site testing in these settings, we assessed the stability of contrived nasopharyngeal (NP) specimens stored for up to 14 days in various transport media (eNAT, eSwab, viral transport medium [VTM], saline, and phosphate-buffered saline [PBS]) at 4°C, 22 to 25°C, and 35°C. The molecular detection of SARS-CoV-2 was unaffected by sample storage temperature over the 2 weeks when stored in eNAT or PBS (change in cycle threshold, ≤1). In contrast, variable stability was observed across test conditions for other transport media. As eNAT can inactivate SARS-CoV-2, it may support COVID-19 diagnostics at the point of care. Evaluation of compatibility of eNAT with Cepheid Xpert Xpress SARS-CoV-2 assay demonstrated diagnostic accuracy and sensitivity equivalent to those of VTM. Taken together, these findings suggest that the implementation of eNAT as a collection device can expand COVID-19 testing to areas with limited health care access.