Laboratory Diagnosis for SARS-CoV-2 Infection.

The optimal diagnostic test for SARS-CoV-2 infection should be selected based on a patient's clinical syndrome and presentation in relation to symptom onset. Molecular testing, most often reverse-transcriptase polymerase chain reaction, offers the highest sensitivity and specificity during acute infection, whereas antigen testing can also be useful for acute diagnosis when rapid turnaround of results is necessary or if molecular testing is unavailable. Serologic testing is often reserved for identifying individuals with prior or late COVID-19 infection.

Quantitative assessment of the effects of massive nucleic acid testing in controlling a COVID-19 outbreak.

BACKGROUND: From 20 July to 26 August 2021, local outbreaks of COVID-19 occurred in Nanjing City and Yangzhou City (Jiangsu Province, China). We analyzed the characteristics of these outbreaks in an effort to develop specific and effective intervention strategies. METHODS: Publicly available data on the characteristics of the COVID-19 outbreaks in Jiangsu Province were collected. Logistic regression was used to assess the association of age and sex with clinical severity. Analysis of onset dates, generation time distributions, and locations were used to estimate the mean transmission distance. A branching process model was used to evaluate different management strategies. RESULTS: From 20 July to 26 August 2021, 820 patients were diagnosed with COVID-19 in Jiangsu Province, with 235 patients (28.7%) from Nanjing, 570 (69.5%) from Yangzhou, and 15 (1.8%) from other cities. Overall, 57.9% of the patients were female, 13.7% were under 20 years-old, and 58.3% had moderate disease status. The mean transmission distance was 4.12 km, and closed-loop management of the area within 2.23 km of cases seemed sufficient to control an outbreak. The model predicted that the cumulative cases in Yangzhou would increase from 311 to 642 if the interval between rounds of nucleic acid amplification testing (NAAT) increased from 1 to 6 days. It also predicted there would be 44.7% more patients if the NAAT started 10 days (rather than 0 days) after diagnosis of the first case. The proportion of cases detected by NAAT would increase from 11.16 to 44.12% when the rounds of NAAT increased from 1 to 7 within 17 days. When the effective vaccine coverage was 50%, the outbreak would be controlled even when using the most relaxed non-pharmaceutical interventions. CONCLUSIONS: The model predicted that a timely closed-loop management of a 2.23 km area around positive COVID-19 cases was sufficient to control the outbreak. Prompt serial NAAT is likely to contain an outbreak quickly, and our model results indicated that three rounds of NAAT sufficiently controlled local transmission. Trial registration We did not involve clinical trial.

Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19.

Guidance for SARS-CoV-2 RNA-Based Molecular Assay Analytical Performance Evaluations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is frequently diagnosed through detection of viral RNA using nucleic acid amplification testing (NAAT) assays that are usually used in centralized settings. Following the publication of the SARS-CoV-2 genetic sequence, multiple diagnostic assays were launched in 2020. These assays require evaluation beyond manufacturer self-reported performance to determine whether they are suitable for use, meet country acceptance criteria, and are compatible with existing in-country platforms. In order to meet the demand for testing services, rapid yet robust assay performance evaluations are required. In our setting, these evaluation protocols required the use of residual patient specimens and reference materials, as typical clinical trials are time-consuming and limited by cost and the cyclical nature of SARS-CoV-2 infection. This protocol is designed to assist in the rapid and robust evaluation of nucleic acid-based assays for the detection of SARS-CoV-2 using limited specimens, reference materials, and test kits. While it is specific for RNA-based assays, it can be adapted for fully automated analyses. The preparation and processing of evaluation panels is described, followed by methods for analytical precision analysis and data visualization. Assay robustness and scalability are briefly discussed as these can be critical for implementation. This protocol is designed to be flexible and alternative options are provided throughout the text where possible.

Turnaround times for molecular testing of pediatric viral cerebrospinal fluid samples in United Kingdom laboratories.

BACKGROUND: Rapid molecular testing has revolutionized the management of suspected viral meningitis and encephalitis by providing an etiological diagnosis in < 90 min with potential to improve outcomes and shorten inpatient stays. However, use of molecular assays can vary widely. AIM: To evaluate current practice for molecular testing of pediatric cerebrospinal fluid (CSF) samples across the United Kingdom using a structured questionnaire. METHODS: A structured telephone questionnaire survey was conducted between July and August 2020. Data was collected on the availability of viral CSF nucleic acid amplification testing (NAAT), criteria used for testing and turnaround times including the impact of the coronavirus disease 2019 pandemic. RESULTS: Of 196/212 (92%) microbiology laboratories responded; 63/196 (32%) were excluded from final analysis as they had no on-site microbiology laboratory and outsourced their samples. Of 133 Laboratories included in the study, 47/133 (35%) had onsite facilities for viral CSF NAAT. Hospitals currently undertaking onsite NAAT (n = 47) had much faster turnaround times with 39 centers (83%) providing results in ≤ 24 h as compared to those referring samples to neighboring laboratories (5/86; 6%). CONCLUSION: Onsite/near-patient rapid NAAT (including polymerase chain reaction) is recommended wherever possible to optimize patient management in the acute setting.

Healthcare personnel early return-to-work program after higher-risk SARS-CoV-2 exposure: A learning health system quality improvement project.

BACKGROUND: Incidence of health care personnel (HCP) with a higher-risk SARS-CoV-2 exposure and subsequent 14-day quarantine period adds substantial burden on the workforce. Implementation of an early return-to-work (RTW) program may reduce quarantine periods for asymptomatic HCP and reduce workforce shortages during the COVID-19 pandemic. METHODS: This observational quality improvement study included asymptomatic HCP of a multi-facility health care system with higher-risk workplace or non-household community SARS-CoV-2 exposure ≤4 days. The program allowed HCP to return to work 8 days after exposure if they remained asymptomatic through day 7 with day 5-7 SARS-CoV-2 nucleic acid amplification test result negative. RESULTS: Between January 4 and June 25, 2021, 384 HCP were enrolled, 333 (86.7%) remained asymptomatic and of these, 323 (97%) tested negative and were early RTW eligible. Mean days in quarantine was 8.16 (SD 2.40). Median day of early RTW was 8 (range 6-9, IQR 8-8). Mean days saved from missed work was 1.84 (SD 0.52). A total of 297 (92%) HCP did RTW ≤10 days from exposure and days saved from missed work was 546.48. CONCLUSIONS: Implementing an HCP early RTW program is a clinical approach for COVID-19 workplace safety that can increase staffing availability, while maintaining a low risk of SARS-CoV-2 transmission.

A study of quality assessment in SARS-CoV-2 pathogen nucleic acid amplification tests performance; from the results of external quality assessment survey of clinical laboratories in the Tokyo Metropolitan Government external quality assessment program in 2020.

INTRODUCTION: The Tokyo Metropolitan Government (TMG) conducted an external quality assessment (EQA) survey of pathogen nucleic acid amplification tests (NAATs) as a TMG EQA program for SARS-CoV-2 for clinical laboratories in Tokyo. METHODS: We diluted and prepared a standard product manufactured by Company A to about 2,500 copies/mL to make a positive control and distribute it with a negative control. The participants reported the use of the NAATs methods for SARS-CoV-2, the name of the real-time RT-PCR kit, the name of the detection device, the target gene(s), nucleic acid extraction kit, Threshold Cycle value in the case of RT-PCR and the Threshold time value and Differential calculation value in the case of Loop-Mediated Isothermal Amplification (LAMP) method. RESULTS: As a result, 17 laboratories using fully automated equipment and 34 laboratories using the RT-PCR method reported generally appropriate results in this EQA survey. On the other hand, among the laboratories that adopted the LAMP method, there were a plurality of laboratories that judged positive samples to be negative. CONCLUSION: The false negative result is considered to be due to the fact that the amount of virus genome contained in the quality control reagent used this time was below the detection limit of the LAMP method combined with the rapid extraction reagent for influenza virus. On the other hand, false positive results are considered to be due to the non-specific reaction of the NAATs. The EQA program must be continued for the proper implementation of the pathogen NAATs.

Accuracy of saliva and nasopharyngeal sampling for detection of SARS-CoV-2 in community screening: a multicentric cohort study.

Nasopharyngeal sampling for nucleic acid amplification testing (NAAT) is the standard diagnostic test of coronavirus disease 2019. Our objectives were to assess, in real-life conditions, the diagnostic accuracy of a nasopharyngeal point-of-care antigen (Ag) test and of saliva NAAT for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in ambulatory care. This was a prospective cohort study from 19 October through 18 December 2020 in two community COVID-19 screening centers in Paris, France. Two nasopharyngeal swabs and one saliva sample were simultaneously collected. Diagnostic accuracies of nasopharyngeal Ag testing and of three saliva NAAT methods were assessed as compared to nasopharyngeal NAAT. A total of 1452 ambulatory children and adults were included. Overall, 129/1443 (9%) participants tested positive on nasopharyngeal NAAT (102/564 [18%] in symptomatic and 27/879 [3%] in asymptomatic participants). Sensitivity was 94%, 23%, 96%, and 94% for the three different protocols of saliva NAAT and for the nasopharyngeal Ag test, respectively. Estimates of specificity were above 95% for all methods. Diagnostic accuracy was similar in symptomatic and asymptomatic individuals. Diagnostic accuracy of nasopharyngeal Ag testing and of saliva NAAT is similar to that of nasopharyngeal NAAT, subject to compliance with specific protocols for saliva. Registration number: NCT04578509.

Detection of SARS-CoV-2 at the point of care.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of COVID-19. Testing for SARS-CoV-2 infection is a critical element of the public health response to COVID-19. Point-of-care (POC) tests can drive patient management decisions for infectious diseases, including COVID-19. POC tests are available for the diagnosis of SARS-CoV-2 infections and include those that detect SARS-CoV-2 antigens as well as amplified RNA sequences. We provide a review of SARS-CoV-2 POC tests including their performance, settings for which they might be used, their impact and future directions. Further optimization and validation, new technologies as well as studies to determine clinical and epidemiological impact of SARS-CoV-2 POC tests are needed.

High concentrations of hypochlorous acid-based disinfectant in the environment reduced the load of SARS-CoV-2 in nucleic acid amplification testing.

During the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic, chlorine-containing disinfectants have been widely used in nucleic acid amplification testing laboratories. Whether the use of disinfectants affect the results of viral nucleic acid amplification is unknown. We examined the impact of different hypochlorous acid (HOCl) concentrations on the quantitative results of SARS-CoV-2 by real-time reverse-transcription polymerase chain reaction (RT-PCR). We also explored the mechanisms and models of action of chlorine-containing disinfectants that affected the detection of SARS-CoV-2. The results showed that different HOCl concentrations and different action times had an impact on the SARS-CoV-2 results. High concentrations of ambient HOCl have a greater impact than low concentrations, and this effect will increase with the extension of the action time and with the increase in ambient humidity. Compared with the enzymes or the extracted RNA required for RT-PCR, the impact of HOCl on the SARS-CoV-2 detection is more likely to be caused by damage to primers and probes in the PCR system. The false negative result still existed after changing the ambient disinfectant to ethanol but not peracetic acid. The use of HOCl in the environment will have an unpredictable impact on the nucleic acid test results of SARS-CoV-2. In order to reduce the possibility of false negative of SARS-CoV-2 nucleic acid test and prevent the spread of epidemic disease, environmental disinfectants should be used at the beginning and end of the experiment rather than during the experimental operation.

Evaluating Differences in Whole Blood, Serum, and Urine Screening Tests for Zika Virus, Puerto Rico, USA, 2016.

We evaluated nucleic acid amplification testing (NAAT) for Zika virus on whole-blood specimens compared with NAAT on serum and urine specimens among asymptomatic pregnant women during the 2015-2016 Puerto Rico Zika outbreak. Using NAAT, more infections were detected in serum and urine than in whole blood specimens.