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Comparative testing of different respiratory specimens using the Xpert Xpress SARS-CoV-2 test. (A) Percent positive rate and (B) N2 gene cycle threshold (Ct) values of samples from all participants with at least one SARS-CoV-2 positive sample (N=84 for all samples and N=37 for NP swab). NP=Nasopharyngeal: VTM=Viral transport medium; eNAT=eNAT transport media, Copan diagnostics. ns=not statistically different. ****P<0.0001; ***P<0.001, **P=0.02.
Source publication
Introduction. Non-invasive sample collection and viral sterilizing buffers have independently enabled workflows for more widespread COVID-19 testing by reverse-transcriptase polymerase chain reaction (RT-PCR).
Gap statement. The combined use of sterilizing buffers across non-invasive sample types to optimize sensitive, accessible, and biosafe sampl...
Contexts in source publication
Context 1
... shown in Fig. 2a, undiluted saliva added directly to the cartridge ('direct saliva') gave the highest detection rate at 90.5 % (76/84), followed by NP-VTM (86.5 %, 32/37) and saliva in eNAT buffer (84.5 %; 71/84), which were significantly higher compared to nasal or oral swabs (P<0.0001). Saliva in VTM (71.4 %; 60/84) also performed better than oral ...
Context 2
... were significantly higher compared to nasal or oral swabs (P<0.0001). Saliva in VTM (71.4 %; 60/84) also performed better than oral swabs in VTM (50 %; 42/84) or eNAT (58 %; 49/84), as well as nasal swabs in VTM (50 %; 42/84) or eNAT (67.8 %; 57/84). We further analysed N2-gene cycle threshold (Ct) values for all positive samples as shown in Fig. 2b. Average N2 gene Ct values were the earliest for NP-VTM (32±5.4) and saliva direct (Ct=34.2±5.8) and most delayed for oral-VTM (37.5±4.9). The Ct range difference was statistically significant between saliva direct and oral-VTM (P<0.0001), oral-eNAT (P=0.0003) and saliva-VTM (P=0.0026). However, there was no significant difference of ...
Context 3
... for oral-VTM (37.5±4.9). The Ct range difference was statistically significant between saliva direct and oral-VTM (P<0.0001), oral-eNAT (P=0.0003) and saliva-VTM (P=0.0026). However, there was no significant difference of N2-Ct range for NP-VTM (P=0.28), nasal-VTM (P=0.09), nasal-eNAT (P=0.82) and saliva-eNAT (P=0.26) compared to saliva direct (Fig. 2b). There were three negative NP specimens that were detected in saliva, which we observed to have N2 Ct values of 39.4, 40.3 and 36.1 (Fig. S1c), indicating below LOD level viral loads [20] possibly contributing to the discrepancy. Only one of the sample sets was positive by NP swab (Ct=35.4) but negative in saliva direct and both saliva ...
Context 4
... evaluated if the composition of different transport media, specifically VTM and eNAT, had any influence on the detection sensitivity. As described, nasal and oral swabs were collected in both VTM and eNAT whereas saliva was collected from patients in an empty sterile cup, then subsequently swabbed and stored in VTM and eNAT. As shown before in Fig. 2A, compared to VTM, eNAT increased the positivity rate by about 20 % (40/84 vs 57/84) for nasal swabs (P=0.008), followed by 12 % for saliva (60/84 vs 70/84, P=0.065) and 6 % for oral swabs (42/84 vs 47/84, P=0.43). When data from all sample types were combined to compare the two media, eNAT offered over 12 % advantage (142 vs 174 out of ...
Context 5
... to saliva swabbed into eNAT, direct saliva yielded an overall delayed SPC-Ct values in the Xpert test, indicating possible PCR inhibition and increased (>60 PSI) in-cartridge pressure values (Fig. S2b). Saliva diluted into eNAT at a ratio of 1 : 2 (N=43) yielded the second highest PCR positive rate (97.7 %, 42/43) after saliva direct (100 %, 43/43) (Fig. 3). Dilutions of 1 : 1 and 1 : 4 yielded 95 % (40/42) and 93 % (40/43) positive rate, respectively. Saliva swabs in eNAT showed the lowest sensitivity at 86 % (37/43, P>0.05; Fig. ...
Context 6
... 41.7 with saliva direct, respectively, indicating the influence of Poisson distribution for viral loads considerably below the limit of detection. Whereas the average N2-Ct values were similar (ca. 33-34) for all saliva conditions tested (P>0.05), the SPC-Ct values were earlier with saliva in eNAT compared to saliva direct (31.25±1.74, P<0.001, Fig. S2c), suggesting that PCR inhibition was mitigated by the addition of eNAT to an appreciable ...
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... Since many samples are processed at sites remote from where they were taken, the conditions of transport to the laboratory are important. In addition, how a sample is handled throughout this process affects the integrity of viral RNA [20]. ...
The COVID-19 pandemic highlighted the crucial role of diagnostic testing in managing infectious diseases, particularly through the use of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) tests. RT-qPCR has been pivotal in detecting and quantifying viral RNA, enabling the identification and management of SARS-CoV-2 infections. However, despite its widespread use, there remains a notable gap in understanding fundamental diagnostic metrics such as sensitivity and specificity among many scientists and healthcare practitioners. This gap is not merely academic; it has profound implications for interpreting test results, making public health decisions, and affecting patient outcomes. This review aims to clarify the distinctions between laboratory- and field-based metrics in the context of RT-qPCR testing for SARS-CoV-2 and summarise the global efforts that led to the development and optimisation of these tests during the pandemic. It is intended to enhance the understanding of these fundamental concepts among scientists and healthcare professionals who may not be familiar with the nuances of diagnostic test evaluation. Such knowledge is crucial for accurately interpreting test results, making informed public health decisions, and ultimately managing infectious disease outbreaks more effectively.
... The genomic replication occurs at cytoplasmic membranes. The transcript proteins are introduced into endoplasmic reticulum (ER), Golgi, after which translated RNA is crammed inside the capsid, as explained in Figure 3 N, E, M, S parts of genome form capsid, envelope, membrane, and spike [10,11]. Understanding genomics and proteomics of viruses is crucial for developing molecular diagnostic techniques which are elaborated in this paper. ...
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... Studies comparing specimens collected at different times starting from the onset of symptoms, two-sided specimen collection versus on-sided collection, the amount of virus recovered through various methods, and evaluation of swabs collected from children with COVID-19 by parents would be extremely useful (21). eNAT (Copan Diagnostics Murrieta, CA) sterilizing buffer and saliva can be used safely and accurately for sensitive detection of the coronavirus by point-of-care GeneXpert instruments (30). New methods of self-collection and self-testing can provide more convenient, efficient, safe, and potentially cost-effective healthcare delivery for disease diagnosis and treatment (31). ...
From the beginning of the COVID-19 epidemic, clinical laboratories around the world have been involved with tests for detection of SARS-CoV-2. At present, RT-PCR (real-time reverse transcription polymerase chain reaction assay) is seen as the gold standard for identifying the virus. Many factors are involved in achieving the highest accuracy in this test, including parameters related to the pre-analysis stage. Having instructions on the type of sample, how to take the sample, and its storage and transportation help control the interfering factors at this stage. Studies have shown that pre-analytical factors might be the cause of the high SARS-CoV-2 test false-negative rates. Also, the safety of personnel in molecular laboratories is of utmost importance, and it requires strict guidelines to ensure the safety of exposed individuals and prevent the virus from spreading. Since the onset of the outbreak, various instructions and guidelines have been developed in this field by the institutions and the Ministry of Health of each country; these guidelines are seriously in need of integration and operation. In this study, we try to collect all the information and research done from the beginning of this pandemic in December 2019 - August 2022 concerning biosafety and protective measures, sample types, sampling methods, container, and storage solutions, sampling equipment, and sample storage and transportation for molecular testing of SARS-CoV-2.
... For example, using saliva for molecular testing had a similar accuracy to nasopharyngeal swabs 24 and would be less resourceintensive in terms of personnel and cost. 25 Swabbed saliva transported in sterilising buffer also had good accuracy, 26 an approach that might have some merit for sputum. ...
Background:
Integrated molecular testing could be an opportunity to detect and provide care for both tuberculosis and COVID-19. Many high tuberculosis burden countries, such as Peru, have existing GeneXpert systems for tuberculosis testing with GeneXpert Xpert MTB/RIF Ultra (Xpert Ultra), and a GeneXpert SARS-CoV-2 assay, GeneXpert Xpert Xpress SARS-CoV-2 (Xpert Xpress), is also available. We aimed to assess the feasibility of integrating tuberculosis and COVID-19 testing using one sputum specimen with Xpert Ultra and Xpert Xpress in Lima, Peru.
Methods:
In this cross-sectional, diagnostic accuracy study, we recruited adults presenting with clinical symptoms or suggestive history of tuberculosis or COVID-19, or both. Participants were recruited from a total of 35 primary health facilities in Lima, Peru. Participants provided one nasopharyngeal swab and one sputum sample. For COVID-19, we tested nasopharyngeal swabs and sputum using Xpert Xpress; for tuberculosis, we tested sputum using culture and Xpert Ultra. We compared diagnostic accuracy of sputum testing using Xpert Xpress with nasopharyngeal swab testing using Xpert Xpress. Individuals with positive Xpert Xpress nasopharyngeal swab results were considered COVID-19 positive, and a positive culture indicated tuberculosis. To assess testing integration, the proportion of cases identified in sputum by Xpert Xpress was compared with Xpert Xpress on nasopharyngeal swabs, and sputum by Xpert Ultra was compared with culture.
Findings:
Between Jan 11, 2021, and April 26, 2022, we recruited 600 participants (312 [52%] women and 288 [48%] men). In-study prevalence of tuberculosis was 13% (80 participants, 95% CI 11-16) and of SARS-CoV-2 was 35% (212 participants, 32-39). Among tuberculosis cases, 13 (2·2%, 1·2-3·7) participants were concurrently positive for SARS-CoV-2. Regarding the diagnostic yield of integrated testing, Xpert Ultra detected 96% (89-99) of culture-confirmed tuberculosis cases (n=77), and Xpert Xpress-sputum detected 67% (60-73) of COVID-19 cases (n=134). All five study staff reported that integrated molecular testing was easy and acceptable.
Interpretation:
The diagnostic yield of Xpert Xpress on sputum was moderate, but integrated testing for tuberculosis and COVID-19 with GeneXpert was feasible. However, systematic testing for both diseases might not be the ideal approach for everyone presenting with presumptive tuberculosis or COVID-19, as concurrent positive cases were rare during the study period. Further research might help to identify when integrated testing is most worthwhile and its optimal implementation.
Funding:
Canadian Institutes of Health Research and International Development Research Centre.
Translation:
For the Spanish translation of the abstract see Supplementary Materials section.
... SARS-CoV-2 can independently colonize the oral cavity and/or salivary glands via several pathways [10,11] with transmission through saliva droplets perpetuated by activities such as speaking and sneezing [12]. PCR testing of saliva has been shown to have comparable [13][14][15] or higher sensitivity [16] and stability [13] than PCR testing for COVID-19 using nasal or nasopharyngeal (NP) swabs. We posited that SARS-CoV-2 virus might be detectable in saliva for longer periods than nasal swabs, including during the late post-symptomatic period. ...
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Saliva has been a COVID-19 diagnostic specimen of interest due to its simple collection, scalability, and yield. Yet COVID-19 testing and estimates of the infectious period remain largely based on nasopharyngeal and nasal swabs. We sought to evaluate whether saliva testing captured prolonged presence of SARS-CoV-2 and potential infectiousness later in the disease course. We conducted an observational study of symptomatic COVID-19 patients at University Hospital in Newark, NJ. Paired saliva and nasal specimens from 96 patients were analyzed, including longitudinal analysis of paired observations from 28 of these patients who had multiple time-points. Saliva detected significantly more cases of COVID-19 beyond 5 days (86.1% [99/115] saliva vs 48.7% [56/115] nasal, p-value < 0.001), 9 days (79.4% [50/63] saliva vs 36.5% [23/63] nasal, p-value < 0.001) and 14 days (71.4% [20/28] saliva vs 32.1% [9/28] nasal, p-value = 0.010) of symptoms. Additionally, saliva yielded lower cycle thresholds across all time periods, indicative of higher viral loads in saliva. In the longitudinal analysis, a log-rank analysis indicated that the survival curve for saliva was significantly different from the curve for nasal swabs (p<0.001) with a median survival time for saliva of 18 days compared to 13 days for nasal swabs. We additionally performed saliva viral cultures among a similar COVID-19 patient cohort and noted patients with positive saliva viral cultures between 7 to 28 days of symptoms. Findings from this study suggest that SARS-CoV-2 RNA persists longer and in higher abundance in saliva compared to nasal swabs, with potential of prolonged propagating virus. Testing saliva may thus increase yield for detecting potentially infectious virus even beyond the first five days of symptomatic COVID-19.
... The FDA issued emergency use authorizations to commercial RT-PCR tests based on test performance with known positive material from a patient or contrived upper respiratory specimens [4]. Use of either known or contrived samples can often result in overestimation of true clinical test sensitivity, since upper respiratory swabs may miss infected material in practice [5,6]. The decline in SARS-CoV-2 shedding from upper respiratory tract specimens within the 1st week after symptom onset is associated with an increase in RT-PCR false negative rate of 2-29% [7][8][9][10]. ...
Background
COVID-19 is a multi-system infection with emerging evidence-based antiviral and anti-inflammatory therapies to improve disease prognosis. However, a subset of patients with COVID-19 signs and symptoms have repeatedly negative RT-PCR tests, leading to treatment hesitancy. We used comparative serology early in the COVID-19 pandemic when background seroprevalence was low to estimate the likelihood of COVID-19 infection among RT-PCR negative patients with clinical signs and/or symptoms compatible with COVID-19.
Methods
Between April and October 2020, we conducted serologic testing of patients with (i) signs and symptoms of COVID-19 who were repeatedly negative by RT-PCR (‘Probables’; N = 20), (ii) signs and symptoms of COVID-19 but with a potential alternative diagnosis (‘Suspects’; N = 15), (iii) no signs and symptoms of COVID-19 (‘Non-suspects’; N = 43), (iv) RT-PCR confirmed COVID-19 patients (N = 40), and (v) pre-pandemic samples (N = 55).
Results
Probables had similar seropositivity and levels of IgG and IgM antibodies as propensity-score matched RT-PCR confirmed COVID-19 patients (60.0% vs 80.0% for IgG, p-value = 0.13; 50.0% vs 72.5% for IgM, p-value = 0.10), but multi-fold higher seropositivity rates than Suspects and matched Non-suspects (60.0% vs 13.3% and 11.6% for IgG; 50.0% vs 0% and 4.7% for IgM respectively; p-values < 0.01). However, Probables were half as likely to receive COVID-19 treatment than the RT-PCR confirmed COVID-19 patients with similar disease severity.
Conclusions
Findings from this study indicate a high likelihood of acute COVID-19 among RT-PCR negative with typical signs/symptoms, but a common omission of COVID-19 therapies among these patients. Clinically diagnosed COVID-19, independent of RT-PCR positivity, thus has a potential vital role in guiding treatment decisions.
Nasopharyngeal (NP) swabs are considered “gold standard” for diagnosing SARS-CoV-2 infections, but anterior nares or mid-turbinate swabs (nasal swabs) are often used. We performed a meta-analysis comparing the sensitivity of nasal and nasopharyngeal swabs against a composite reference standard for the initial diagnosis of SARS-CoV-2 infection in ambulatory patients. The study is registered on PROSPERO (CRD42020221827). Data sources included studies appearing between January 1, 2020 and March 20, 2021, identified by searches of PubMed, medRxiv and bioRxiv. Studies included at least 20 subjects who simultaneously provided nasal and nasopharyngeal specimens for reverse transcription-polymerase chain reaction testing, and for which confusion matrices could be constructed. Authors individually assessed studies for inclusion and compared assessments. Each author independently extracted all data elements; differences were reconciled by review of initial data sources. Extracted data included specimen site, patient characteristics, collection site, and confusion matrices comparing results for nasal and nasopharyngeal swabs. Assessed against a composite reference standard, anterior nares swabs are less sensitive (82% - 88%) than nasopharyngeal swabs (98%). For populations with 10% specimen positivity, the negative predictive values of all swab types were greater than 98%. Mid-turbinate and anterior nares swabs seem to perform similarly. The lower sensitivity associated with nasal swab SARS-CoV-2 diagnosis is justified by the ability to screen more patients and reduced personal protective equipment requirements. Our conclusions are limited by the small number of studies and the significant heterogeneity of study designs and study outcomes.
Background
Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings.
Methods
We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C.
Results
SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions.
Conclusion
eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.
