A Community Study of SARS-CoV-2 Detection by RT-PCR in Saliva: A Reliable and Effective Method.

Efficient, wide-scale testing for SARS-CoV-2 is crucial for monitoring the incidence of the infection in the community. The gold standard for COVID-19 diagnosis is the molecular analysis of epithelial secretions from the upper respiratory system captured by nasopharyngeal (NP) or oropharyngeal swabs. Given the ease of collection, saliva has been proposed as a possible substitute to support testing at the population level. Here, we used a novel saliva collection device designed to favour the safe and correct acquisition of the sample, as well as the processivity of the downstream molecular analysis. We tested 1003 nasopharyngeal swabs and paired saliva samples self-collected by individuals recruited at a public drive-through testing facility. An overall moderate concordance (68%) between the two tests was found, with evidence that neither system can diagnose the infection in 100% of the cases. While the two methods performed equally well in symptomatic individuals, their discordance was mainly restricted to samples from convalescent subjects. The saliva test was at least as effective as NP swabs in asymptomatic individuals recruited for contact tracing. Our study describes a testing strategy of self-collected saliva samples, which is reliable for wide-scale COVID-19 screening in the community and is particularly effective for contact tracing.

Pooling saliva samples as an excellent option to increase the surveillance for SARS-CoV-2 when re-opening community settings.

In many countries a second wave of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occurred, triggering a shortage of reagents needed for diagnosis and compromising the capacity of laboratory testing. There is an urgent need to develop methods to accelerate the diagnostic procedures. Pooling samples represents a strategy to overcome the shortage of reagents, since several samples can be tested using one reaction, significantly increasing the number and speed with which tests can be carried out. We have reported the feasibility to use a direct lysis procedure of saliva as source for RNA to SARS-CoV-2 genome detection by reverse transcription quantitative-PCR (RT-qPCR). Here, we show that the direct lysis of saliva pools, of either five or ten samples, does not compromise the detection of viral RNA. In addition, it is a sensitive, fast, and inexpensive method that can be used for massive screening, especially considering the proximity of the reincorporation of activities in universities, offices, and schools.

Unified platform for genetic and serological detection of COVID-19 with single-molecule technology.

The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.

Is it enough for COVID-19 screening test? Limitation of swab test and general characteristics of mild symptom patients.

The most common method for SARS-CoV-2 testing is throat or nasal swabbing by real-time reverse transcription polymerase chain reaction (RT-PCR) assay. In South Korea, drive-through swab test is used for screening system and community treatment centers (CTCs), which admit and treat confirmed COVID-19 patients with mild symptoms, are being used. This retrospective study was conducted on patients admitted to a CTC on March 6, 2020. A total of 313 patients were admitted. The nasal and throat swabs were collected from the upper respiratory tract, and a sputum test was performed to obtain lower respiratory samples. The positive rate of the first set of test, sputum test was higher than that of the swab test (p = 0.011). In the second set of test, 1 week after the first ones, the rate of positive swab tests was relatively high (p = 0.026). In the first set of test, 66 of 152 (43.4%) patients showed 24-h consecutive negative swab test results, when the sputum test results were considered together, that number fell to 29 patients (19.1%) (p < 0.001). Also, in the second set of test, 63 of 164 (38.4%) patients met the discharge criteria only when the swab test was considered; that number fell to 30 (18.3%) when the sputum test results were also considered (p < 0.001). Using the swab test alone is insufficient for screening test and discharge decision. Patients who may have positive result in the sputum test can be missed.

May viral load detected in saliva in the early stages of infection be a prognostic indicator in COVID-19 patients?

OBJECTIVE: This study aimed to investigate the prognostic value of viral load detected in the saliva of COVID-19 patients in the early stage of infection. STUDY DESIGN: Oro-nasopharyngeal swab and saliva samples were collected from all patients simultaneously in the early stage of COVID-19. Viral loads were determined by extracting viral RNAs from saliva samples of patients whose ONP swabs were positive for SARS-CoV-2 by RT-qPCR. The demographic information, comorbidities, cycle threshold values, and one-month clinical courses were recorded and compared. RESULTS: The patients' clinical course was evaluated for one month; 56 % of patients had mild disease, 26.4 % had moderate disease, 9.6 % had severe disease, and 8% had a critical/mortal disease. The average cycle threshold values of SARS-CoV-2 in saliva and ONP samples were measured as 22.28 and 24.19, respectively. Cycle threshold value of saliva was found to be significant in predicting disease severity (Eta coefficient 0.979). A statistically significant relationship was found between the disease's severity and the mean of ONP samples' Ct-values (p < 0.05). Gender, age, body mass index, and co-morbidities were compared with the severity of the disease; no statistically significant difference was found. CONCLUSION: Viral load detected in saliva in the early period of COVID-19 infection may have a prognostic value in showing the disease's course in patients over 45-year-old. Saliva is an easily obtainable, reliable material for COVID-19 screening.

Results of preoperative screening for COVID-19 correlate with the incidence of infection in the general population -a tertiary care experience.

Objectives: Many hospitals have recently instituted policies mandating preoperative COVID-19 testing. However, it is uncertain whether institutions can dictate such policies based on infection rates found in the general population. Therefore, the main aims of the study were to determine (1) what proportion of preoperative patients tested positive, (2) what percentage was asymptomatic, and (3) whether variations throughout time in numbers of positive patients reflected changes observed in our state.Methods: All COVID-19 preoperative screening tests (nasopharyngeal-swab RT-PCR testing) performed in our hospital between 04/13/2020 and 08/27/2020 were retrospectively reviewed. The unit of analysis was number of patients who tested negative/positive. Medical records of positive patients were reviewed to determine the presence of COVID-19 symptoms. A curve was created showing our number of positive patients per week and another one presenting the number of positive patients per day in Florida, both figures were compared.Results: A total of 7,213 patients from all specialties were preoperatively tested, out of which 85 were positive for an overall infection rate of 1.2%. In 18% (15/85) of positive patients, it was not possible to determine symptomatology. Among remaining patients, 49% (34/70) were asymptomatic while 51% (36/70) were symptomatic for COVID-19. Peak of positive cases occurred in mid-July in both curves, and the upward and downward tendencies in positive numbers mirrored each other.Conclusion: COVID-19 infection rate among our preoperative patients was very low. Nearly 50% of positive patients were asymptomatic. Our data suggest that a tertiary hospital can promulgate COVID-19 preoperative screening policies based on infection trends observed in the general population. However, in addition to the test, patients should be encouraged to self-quarantine for 14 days before surgery.

Positive Ratio of Polymerase Chain Reaction (PCR) and Validity of Pre-Screening Criteria at an Outpatient Screening Center during the Early Phase of the COVID-19 Epidemic in Japan.

Despite the increase in COVID-19 cases globally, the number of cases in Japan has been relatively low, and an explosive surge in the prevalence has not occurred. In March 2020, the Ministry of Health, Labour and Welfare (MHLW) in Japan recommended the original criteria for polymerase chain reaction (PCR) testing, although there was a lack of evidence for appropriate targets for COVID-19 testing. This study aimed to evaluate the COVID-19 positive ratio and pre-screening criteria in Tokyo immediately after the insurance-covered SARS-CoV-2 PCR testing became available in Japan. We subjected 277 individuals with mild symptoms in metropolitan Tokyo (positive: 9.0%) from March 9 to 29, 2020, to SARS-CoV-2 PCR testing. The results revealed that 25 (9.0%) of them were PCR-positive. The sensitivity and specificity of the MHLW criteria were 100% and 10.7%, respectively. When the criteria excluded nonspecific symptoms, fatigue, and dyspnea, the sensitivity slightly decreased to 92%, and the specificity increased to 22.2%. The specificity was highest when the fever criterion was ≥37.5°C for ≥4 days, and exposure/travel history, including age and underlying comorbidities, was considered. Our findings suggest that the MHLW criteria, including the symptoms and exposure/travel history, may be useful for COVID-19 pre-screening.

A SARS-CoV-2 Reference Standard Quantified by Multiple Digital PCR Platforms for Quality Assessment of Molecular Tests.

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 105 ± 6.5 × 104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.

COVID-19: changing patterns among neurosurgical patients from North India, efficacy of repeat testing, and inpatient prevalence.

OBJECTIVE: COVID-19 has affected surgical practice globally. Treating neurosurgical patients with the restrictions imposed by the pandemic is challenging in institutions with shared patient areas. The present study was performed to assess the changing patterns of neurosurgical cases, the efficacy of repeated testing before surgery, and the prevalence of COVID-19 in asymptomatic neurosurgical inpatients. METHODS: Cases of non-trauma-related neurosurgical patients treated at the Postgraduate Institute of Medical Education and Research (PGIMER) before and during the COVID-19 pandemic were reviewed. During the pandemic, all patients underwent a nasopharyngeal swab reverse transcription-polymerase chain reaction test to detect COVID-19 at admission. Patients who needed immediate intervention were surgically treated following a single COVID-19 test, while stable patients who initially tested negative for COVID-19 were subjected to repeated testing at least 5 days after the first test and within 48 hours prior to the planned surgery. The COVID-19 positivity rate was compared with the local period prevalence. The number of patients who tested positive at the second test, following a negative first test, was used to determine the probable number of people who could have become infected during the surgical procedure without second testing. RESULTS: Of the total 1769 non-trauma-related neurosurgical patients included in this study, a mean of 337.2 patients underwent surgery per month before COVID-19, while a mean of 184.2 patients (54.6% of pre-COVID-19 capacity) underwent surgery per month during the pandemic period, when COVID-19 cases were on the rise in India. There was a significant increase in the proportion of patients undergoing surgery for a ruptured aneurysm, stroke, hydrocephalus, and cerebellar tumors, while the number of patients seeking surgery for chronic benign diseases declined. At the first COVID-19 test, 4 patients (0.48%) tested were found to have the disease, a proportion 3.7 times greater than that found in the local community. An additional 5 patients tested positive at the time of the second COVID-19 test, resulting in an overall inpatient period prevalence of 1%, in contrast to a 0.2% national cumulative caseload. It is possible that COVID-19 was prevented in approximately 67.4 people every month by using double testing. CONCLUSIONS: COVID-19 has changed the pattern of neurosurgical procedures, with acute cases dominating the practice. Despite the fact that the pandemic has not yet reached its peak in India, COVID-19 has been detected 3.7 times more often in asymptomatic neurosurgical inpatients than in the local community, even with single testing. Double testing displays an incremental value by disclosing COVID-19 overall in 1 in 100 inpatients and thus averting its spread through neurosurgical services.

Universal screening for SARS-CoV-2 infection: a rapid review.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by the novel betacoronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Most people infected with SARS-CoV-2 have mild disease with unspecific symptoms, but about 5% become critically ill with respiratory failure, septic shock and multiple organ failure. An unknown proportion of infected individuals never experience COVID-19 symptoms although they are infectious, that is, they remain asymptomatic. Those who develop the disease, go through a presymptomatic period during which they are infectious. Universal screening for SARS-CoV-2 infections to detect individuals who are infected before they present clinically, could therefore be an important measure to contain the spread of the disease. OBJECTIVES: We conducted a rapid review to assess (1) the effectiveness of universal screening for SARS-CoV-2 infection compared with no screening and (2) the accuracy of universal screening in people who have not presented to clinical care for symptoms of COVID-19. SEARCH METHODS: An information specialist searched Ovid MEDLINE and the Centers for Disease Control (CDC) COVID-19 Research Articles Downloadable Database up to 26 May 2020. We searched Embase.com, the CENTRAL, and the Cochrane Covid-19 Study Register on 14 April 2020. We searched LitCovid to 4 April 2020. The World Health Organization (WHO) provided records from daily searches in Chinese databases and in PubMed up to 15 April 2020. We also searched three model repositories (Covid-Analytics, Models of Infectious Disease Agent Study [MIDAS], and Society for Medical Decision Making) on 8 April 2020. SELECTION CRITERIA: Trials, observational studies, or mathematical modelling studies assessing screening effectiveness or screening accuracy among general populations in which the prevalence of SARS-CoV2 is unknown. DATA COLLECTION AND ANALYSIS: After pilot testing review forms, one review author screened titles and abstracts. Two review authors independently screened the full text of studies and resolved any disagreements by discussion with a third review author. Abstracts excluded by a first review author were dually reviewed by a second review author prior to exclusion. One review author independently extracted data, which was checked by a second review author for completeness and accuracy. Two review authors independently rated the quality of included studies using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool for diagnostic accuracy studies and a modified form designed originally for economic evaluations for modelling studies. We resolved differences by consensus. We synthesized the evidence in narrative and tabular formats. We rated the certainty of evidence for days to outbreak, transmission, cases missed and detected, diagnostic accuracy (i.e. true positives, false positives, true negatives, false negatives) using the GRADE approach. MAIN RESULTS: We included 22 publications. Two modelling studies reported on effectiveness of universal screening. Twenty studies (17 cohort studies and 3 modelling studies) reported on screening test accuracy. Effectiveness of screening We included two modelling studies. One study suggests that symptom screening at travel hubs, such as airports, may slightly slow but not stop the importation of infected cases (assuming 10 or 100 infected travellers per week reduced the delay in a local outbreak to 8 days or 1 day, respectively). We assessed risk of bias as minor or no concerns, and certainty of evidence was low, downgraded for very serious indirectness. The second modelling study provides very low-certainty evidence that screening of healthcare workers in emergency departments using laboratory tests may reduce transmission to patients and other healthcare workers (assuming a transmission constant of 1.2 new infections per 10,000 people, weekly screening reduced infections by 5.1% within 30 days). The certainty of evidence was very low, downgraded for high risk of bias (major concerns) and indirectness. No modelling studies reported on harms of screening. Screening test accuracy All 17 cohort studies compared an index screening strategy to a reference reverse transcriptase polymerase chain reaction (RT-PCR) test. All but one study reported on the accuracy of single point-in-time screening and varied widely in prevalence of SARS-CoV-2, settings, and methods of measurement. We assessed the overall risk of bias as unclear in 16 out of 17 studies, mainly due to limited information on the index test and reference standard. We rated one study as being at high risk of bias due to the inclusion of two separate populations with likely different prevalences. For several screening strategies, the estimates of sensitivity came from small samples. For single point-in-time strategies, for symptom assessment, the sensitivity from 12 cohorts (524 people) ranged from 0.00 to 0.60 (very low-certainty evidence) and the specificity from 12 cohorts (16,165 people) ranged from 0.66 to 1.00 (low-certainty evidence). For screening using direct temperature measurement (3 cohorts, 822 people), international travel history (2 cohorts, 13,080 people), or exposure to known infected people (3 cohorts, 13,205 people) or suspected infected people (2 cohorts, 954 people), sensitivity ranged from 0.00 to 0.23 (very low- to low-certainty evidence) and specificity ranged from 0.90 to 1.00 (low- to moderate-certainty evidence). For symptom assessment plus direct temperature measurement (2 cohorts, 779 people), sensitivity ranged from 0.12 to 0.69 (very low-certainty evidence) and specificity from 0.90 to 1.00 (low-certainty evidence). For rapid PCR test (1 cohort, 21 people), sensitivity was 0.80 (95% confidence interval (CI) 0.44 to 0.96; very low-certainty evidence) and specificity was 0.73 (95% CI 0.39 to 0.94; very low-certainty evidence). One cohort (76 people) reported on repeated screening with symptom assessment and demonstrates a sensitivity of 0.44 (95% CI 0.29 to 0.59; very low-certainty evidence) and specificity of 0.62 (95% CI 0.42 to 0.79; low-certainty evidence). Three modelling studies evaluated the accuracy of screening at airports. The main outcomes measured were cases missed or detected by entry or exit screening, or both, at airports. One study suggests very low sensitivity at 0.30 (95% CI 0.1 to 0.53), missing 70% of infected travellers. Another study described an unrealistic scenario to achieve a 90% detection rate, requiring 0% asymptomatic infections. The final study provides very uncertain evidence due to low methodological quality. AUTHORS' CONCLUSIONS: The evidence base for the effectiveness of screening comes from two mathematical modelling studies and is limited by their assumptions. Low-certainty evidence suggests that screening at travel hubs may slightly slow the importation of infected cases. This review highlights the uncertainty and variation in accuracy of screening strategies. A high proportion of infected individuals may be missed and go on to infect others, and some healthy individuals may be falsely identified as positive, requiring confirmatory testing and potentially leading to the unnecessary isolation of these individuals. Further studies need to evaluate the utility of rapid laboratory tests, combined screening, and repeated screening. More research is also needed on reference standards with greater accuracy than RT-PCR. Given the poor sensitivity of existing approaches, our findings point to the need for greater emphasis on other ways that may prevent transmission such as face coverings, physical distancing, quarantine, and adequate personal protective equipment for frontline workers.