Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 425
Filter
1.
Indian J Pathol Microbiol ; 65(4): 907-910, 2022.
Article in English | MEDLINE | ID: covidwho-2100023

ABSTRACT

Context: COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging pandemic that is rapidly spreading with more than 114 million confirmed cases and 2.5 million deaths by far. Nasopharyngeal swab (NPS) in VTM has been used as the gold standard respiratory specimen for SARS-CoV-2 reverse-transcriptase real-time PCR (rRT-PCR) tests. But now the virus can also be detected in other clinical specimens like bronchoalveolar lavage, sputum, saliva, throat swab, blood, and stool specimens. Aims: The aim of this study was to determine the diagnostic potential of saliva as a sample in comparison to NPS for detection of SARS-CoV-2 by rRT-PCR. Settings and Design: A cross-sectional study was conducted among 256 paired samples (NPS and Saliva) received in the Department of Microbiology, SMS Medical College, Jaipur over a period of 2 months. Methods and Material: NPS from individuals were collected in a sterile tube containing Viral Transport Medium™. Before swab collection, whole saliva was collected by spitting from the suspected patient into a sterile container. Both were stored at room temperature and transferred to the diagnostic laboratory within four hours of collection where extraction was done using Perkin Elmer chemagic extractor and rRT- PCR was performed using NIV, Pune mastermix. Results: Sensitivity, specificity, PPV, and NPV of RT-PCR for the diagnosis of COVID-19 in saliva were 84.26%, 100%, 100%, and 54.05%, respectively. The accuracy of detection of COVID-19 by saliva samples compared to the routinely used NPS samples (considered as the standard reference) for RT PCR was 86.72%. Conclusions: Our results show that saliva as a reliable sample type for SARS-CoV-2 detection.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Reverse Transcriptase Polymerase Chain Reaction , COVID-19/diagnosis , Saliva , Cross-Sectional Studies , Nasopharynx , India , Specimen Handling/methods
5.
PLoS One ; 17(9): e0275201, 2022.
Article in English | MEDLINE | ID: covidwho-2054361

ABSTRACT

Molecular diagnostic testing has played a critical role in the global response to the novel Coronavirus disease (COVID-19) pandemic, since its first outbreak in late 2019. At the inception of the COVID-19 pandemic, nasopharyngeal swab sample analysis for COVID-19 diagnosis using the real-time polymerase chain reaction (RT-PCR) technique was the most widely used. However, due to the high cost and difficulty of sample collection, the number of available sample types for COVID-19 diagnosis is rapidly increasing, as is the COVID-19 diagnostic literature. The use of nasal swabs, saliva, and oral fluids as viable sample options for the effective detection of SARS-CoV-2 has been implemented successfully in different settings since 2020. These alternative sample type provides a plethora of advantages including decreasing the high exposure risk to frontline workers, enhancing the chances of home self-sampling, reducing the cost, and significantly increasing testing capacity. This study sought to ascertain the effectiveness of Saliva samples as an alternative for COVID-19 diagnosis in Nigeria. Demographic data, paired samples of Nasopharyngeal Swab and Drooling Saliva were obtained from 309 consenting individuals aged 8-83 years presenting for COVID-19 testing. All samples were simultaneously assayed for the detection of SARS-CoV-2 RdRp, N, and E genes using the GeneFinder™ COVID-19 Plus RT-PCR test kit. Out of 309 participants, only 299 with valid RT-PCR results comprising 159 (53.2%) males and 140 (46.8%) females were analyzed in this study using the R Statistical package. Among the 299 samples analyzed, 39 (13.0%) had SARS-CoV-2 detected in at least one specimen type. Both swabs and saliva were positive in 20 (51.3%) participants. Ten participants (25.6%) had swab positive/saliva-negative results and 9 participants (23.1%) had saliva positive/swab-negative results. The percentage of positive and negative agreement of the saliva samples with the nasopharyngeal swab were 67% and 97% respectively with positive and negative predictive values as 69% and 96% respectively. The findings indicate that drooling saliva samples have good and comparable diagnostic accuracy to the nasopharyngeal swabs with moderate sensitivities and high specificities.


Subject(s)
COVID-19 , Sialorrhea , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19 Testing , Female , Humans , Male , Nasopharynx , Pandemics , RNA-Dependent RNA Polymerase , SARS-CoV-2/genetics , Saliva , Specimen Handling/methods
6.
JAMA ; 328(10): 935-940, 2022 09 13.
Article in English | MEDLINE | ID: covidwho-2047345

ABSTRACT

Importance: Despite the expansion of SARS-CoV-2 testing, available tests have not received Emergency Use Authorization for performance with self-collected anterior nares (nasal) swabs from children younger than 14 years because the effect of pediatric self-swabbing on SARS-CoV-2 test sensitivity is unknown. Objective: To characterize the ability of school-aged children to self-collect nasal swabs for SARS-CoV-2 testing compared with collection by health care workers. Design, Setting, and Participants: Cross-sectional study of 197 symptomatic children and adolescents aged 4 to 14 years old. Individuals were recruited based on results of testing in the Children's Healthcare of Atlanta system from July to August 2021. Exposures: Children and adolescents were given instructional material consisting of a short instructional video and a handout with written and visual steps for self-swab collection. Participants first provided a self-collected nasal swab. Health care workers then collected a second specimen. Main Outcomes and Measures: The primary outcome was SARS-CoV-2 detection and relative quantitation by cycle threshold (Ct) in self- vs health care worker-collected nasal swabs when tested with a real-time reverse transcriptase-polymerase chain reaction test with Emergency Use Authorization. Results: Among the study participants, 108 of 194 (55.7%) were male and the median age was 9 years (IQR, 6-11). Of the 196 participants, 87 (44.4%) tested positive for SARS-CoV-2 and 105 (53.6%) tested negative by both self- and health care worker-collected swabs. Two children tested positive by self- or health care worker-collected swab alone; 1 child had an invalid health care worker swab. Compared with health care worker-collected swabs, self-collected swabs had 97.8% (95% CI, 94.7%-100.0%) and 98.1% (95% CI, 95.6%-100.0%) positive and negative percent agreement, respectively, and SARS-CoV-2 Ct values did not differ significantly between groups (mean [SD] Ct, self-swab: 26.7 [5.4] vs health care worker swab: 26.3 [6.0]; P = .65). Conclusions and Relevance: After hearing and seeing simple instructional materials, children and adolescents aged 4 to 14 years self-collected nasal swabs that closely agreed on SARS-CoV-2 detection with swabs collected by health care workers.


Subject(s)
COVID-19 , SARS-CoV-2 , Adolescent , COVID-19/diagnosis , COVID-19 Testing , Child , Child, Preschool , Cross-Sectional Studies , Female , Health Personnel , Humans , Male , Specimen Handling/methods
7.
Int J Mol Sci ; 23(18)2022 Sep 14.
Article in English | MEDLINE | ID: covidwho-2039870

ABSTRACT

Body fluid identification at crime scenes can be crucial in retrieving the appropriate evidence that leads to the perpetrator and, in some cases, the victim. For this purpose, immunochromatographic tests are simple, fast and suitable for crime scenes. The potential sample is retrieved with a swab, normally a cotton swab, moistened in a specific buffer. Nonetheless, there are other swab types available, which have been proven to be efficient for DNA isolation and analysis. The aim of this study is to evaluate the efficiency of different swab types for body fluid identification as well as DNA isolation and characterization. Fifty microliters of human saliva were deposited in three different types of fabric (denim, cotton, and polyester). After 24 h at room temperature, samples were recovered by applying three different swab types, and the tests were performed. Subsequently, total DNA was recovered from the sample buffer. Cotton swabs performed worse in denim and cotton fabrics in both immunochromatography tests and DNA yield. No differences were observed for polyester. In contrast, and except for two replicates, it was possible to obtain a full DNA profile per fabric and swab type, and to identify the mtDNA haplogroup. In this paper, the impact of swab types on body fluid identification through the application of immunochromatographic tests is analyzed for the first time. This work corroborates previous research related to the influence of swab types in nuclear DNA isolation and characterization.


Subject(s)
DNA Fingerprinting , Specimen Handling , DNA Fingerprinting/methods , DNA, Mitochondrial/analysis , Humans , Polyesters , Saliva/chemistry , Specimen Handling/methods
8.
Microbiol Spectr ; 10(5): e0142222, 2022 Oct 26.
Article in English | MEDLINE | ID: covidwho-2038249

ABSTRACT

We examined the detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using reverse transcription-PCR (RT-PCR) of side-by-side saliva and oropharyngeal swab (OPS) samples from 639 symptomatic and asymptomatic subjects, of which 47 subjects were found to be positive for SARS-CoV-2 in the OPS or saliva sample or both. It was found that the detection rate (93.6% for both OPS and saliva) as well as the sensitivity and specificity were comparable between the two sampling methods in this cohort. The sensitivity was 0.932 (95% confidence interval [CI], 0.818 to 0.977) and the specificity was 0.995 (95% CI, 0.985 to 0.998), both for saliva when OPS sampling was used as the reference and for OPS when saliva was used as the reference. Furthermore, the Cohen's kappa value was 0.926 (95% CI, 0.868 to 0.985), indicating strong agreement between the two sampling methods. In addition, the viral RNA stability in pure saliva and saliva mixed with preservation buffers was examined following storage at room temperature and at 4°C. It was found that pure saliva kept the viral RNA stable for 9 days at both temperatures and that the type of preservation buffer can either enhance or reduce the stability of the RNA. We conclude that self-administered saliva sampling is an attractive alternative to oropharyngeal swabbing for SARS-CoV-2 detection, and it might be useful in large-scale testing. IMPORTANCE It is not inconceivable that we will witness recurring surges of COVID-19 before the pandemic finally recedes. It is therefore still relevant to look for feasible, simple, and flexible screening methods so that schools, workplaces, and communities in general can avoid lockdowns. In this work, we analyzed two different sampling methods: oropharyngeal swabs and saliva collection. Oropharyngeal swabs must be collected by trained health personnel at clinics, whereas self-assisted saliva collection can be performed at any given location. It was found that the two sampling methods were comparable. Saliva sampling is a simple method that allows easy mass testing using minimal resources from the existing health care system, and this method may therefore prove to be an effective tool for containing the COVID-19 pandemic.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Pandemics , COVID-19/diagnosis , COVID-19 Testing , Saliva , RNA, Viral , Communicable Disease Control , Specimen Handling/methods
9.
Obstet Gynecol ; 140(3): 470-476, 2022 Sep 01.
Article in English | MEDLINE | ID: covidwho-2032193

ABSTRACT

OBJECTIVE: To assess concordance and acceptability of a modified menstrual pad compared with a clinician-collected high-risk human papillomavirus (HPV) sample. METHODS: This was a prospective observational study. Women presenting for either cervical cancer screening or with a history of high-risk HPV positivity were eligible. Three samples were requested from participants: 1) clinician-collected cervical specimens; 2) self-collected vaginal swabs; and 3) a modified menstrual pad, which was taken home for use during the next menstruation. All samples were processed using the Cobas HPV test. Menstrual pad dried blood spots were eluted, then similarly processed. RESULTS: Of 153 women enrolled in the study, 106 provided menstrual pad samples and clinician-collected cervical specimens for high-risk HPV analysis. For samples in which the interval between the clinician-collected specimen and the menstrual pad sample was less than 2 months, the concordance was 94% (95% CI 83-98). For women who tested positive for high-risk HPV who presented for general screening and those with more than cervical intraepithelial neoplasia 2, menstrual pad and clinician-collected specimen agreement was 100% (95% CI 32.5-100). Among participants, 22.9% expressed discomfort with the self-collected vaginal swabs and opted out of collection. Overall, 94.0% of participants preferred the menstrual pad over clinician-collected sampling. Twelve patients were found to be positive for HPV on the menstrual pad sample but negative on the clinician-collected specimen. CONCLUSION: Among women who tested positive for HPV, the menstrual pad showed highly concordant results compared with clinician-collected sampling. This collection approach shows promise for integration into cervical cancer prevention programs.


Subject(s)
Alphapapillomavirus , Papillomavirus Infections , Uterine Cervical Neoplasms , Humans , Female , Papillomaviridae , Papillomavirus Infections/diagnosis , Early Detection of Cancer/methods , Mass Screening/methods , Specimen Handling/methods , Vaginal Smears/methods , Sensitivity and Specificity
10.
J Infect Dev Ctries ; 16(8): 1278-1284, 2022 08 30.
Article in English | MEDLINE | ID: covidwho-2030101

ABSTRACT

INTRODUCTION: Mass testing is essential in the surveillance strategy for fighting the COVID-19 pandemic. It allows early detection of suspected cases and subsequently early isolation to mitigate spread. However, the high cost and limited consumables and reagents hinder the mass testing strategy in developing countries such as Indonesia. The specimen pooling strategy is an option to perform mass screening with limited resources. This study aims to determine the positivity rate cut-off and to evaluate the efficiency of pooling strategy for the laboratory diagnosis of COVID-19. METHODOLOGY: Between August 4th, 2020, and November 11th, 2020, a four-sample pooling strategy testing to detect SARS-CoV-2 was carried out at the Microbiology Diagnostic Laboratory of Diponegoro National Hospital, Semarang, Indonesia. Pools with positive results were subjected to individual specimen retesting. Spearman's correlation and linear regression analysis were used to determine the best positivity rate cut-off to apply pooling strategy. RESULTS: A total of 15,216 individual specimens were pooled into 3,804 four-sample pools. Among these pools, 1,007 (26.47%) were positive. Five hundred and ten (50.64%) were 1/4 positive. A maximum positivity rate of 22% is needed to save at least 50% extraction and qRT-PCR reactions in a four-sample pooling strategy. CT values between individual specimens and pools showed a good interval agreement. CONCLUSIONS: Pooling strategy could reduce personnel workload and reagent cost, and increase laboratory capacity by up to 50% when the positivity rate is less than 22%.


Subject(s)
COVID-19 , COVID-19/diagnosis , COVID-19 Testing , Humans , Pandemics , SARS-CoV-2 , Sensitivity and Specificity , Specimen Handling/methods
11.
Arch Pathol Lab Med ; 146(9): 1056-1061, 2022 09 01.
Article in English | MEDLINE | ID: covidwho-2025231

ABSTRACT

CONTEXT.­: Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains integral to care, particularly for supporting return to and attendance in schools. The concordance of SARS-CoV-2 detection in children, using various specimen types, has not been widely studied. OBJECTIVE.­: To compare 3 sample types for SARS-CoV-2 polymerase chain reaction (PCR) testing in children, collected and tested at a single facility. DESIGN.­: We prospectively recruited 142 symptomatic and asymptomatic children/young adults into a sample comparison study performed in a single health care system. Each child provided self-collected saliva, and a trained health care provider collected a mid-turbinate nasal swab and nasopharyngeal (NP) swab. Specimens were assayed within 24 hours of collection by using reverse transcription-polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 on a single testing platform. RESULTS.­: Concurrently collected saliva and mid-turbinate swabs had greater than 95% positive agreement with NP swabs when obtained within 10 days of symptom onset. Positive agreement of saliva and mid-turbinate samples collected from children with symptom onset >10 days prior, or without symptoms, was 82% compared to NP swab samples. Cycle threshold (Ct) values for mid-turbinate nasal samples more closely correlated with Ct values from NP samples than from saliva samples. CONCLUSIONS.­: These findings suggest that all 3 sample types from children are useful for SARS-CoV-2 diagnostic testing by RT-PCR, and that concordance is greatest when the child has had symptoms of COVID-19 within the past 10 days. This study provides scientific justification for using sample types other than the NP swab for SARS-CoV-2 testing in pediatric populations.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Child , Humans , Nasopharynx , Outpatients , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Reverse Transcription , SARS-CoV-2/genetics , Saliva , Specimen Handling/methods , Turbinates , Young Adult
12.
PLoS One ; 17(7): e0270929, 2022.
Article in English | MEDLINE | ID: covidwho-2021860

ABSTRACT

BACKGROUND: Saliva sampling is a promising alternative to nasopharyngeal swabs for SARS-CoV-2 testing, but acceptability data is lacking. We characterize the acceptability of saliva sampling and nasopharyngeal swabs for primary decision makers and their children after experiencing both testing modalities. METHODS: We administered a cross-sectional survey to participants aged 6-to-17 years and their primary decision makers at an Ottawa community COVID-19 testing centre in March 2021. Included were participants meeting local guidelines for testing. Excluded were those identified prior to participation as having inability to complete the consent, sampling, or survey process. Acceptability in multiple hypothetical scenarios was rated using a 5-point Likert scale. Pain was measured using the Faces Pain Scale-Revised (FPS-R). Preference for testing was assessed with direct binary questions. RESULTS: 48 participants and 48 primary decision makers completed the survey. Nasopharyngeal swab acceptability differed between scenarios, ranging 79% [95%CI: 66, 88] to 100% [95%CI: 95, 100]; saliva sampling acceptability was similar across scenarios, ranging 92% [95%CI: 82, 97] to 98% [95%CI: 89, 99]. 58% of youth described significant pain with nasopharyngeal swabbing, versus none with saliva sampling. 90% of children prefer saliva sampling. 66% of primary decision makers would prefer nasopharyngeal swabbing if it were 10% more sensitive. CONCLUSION: Though youth prefer saliva sampling over nasopharyngeal swabs, primary decision makers present for testing remain highly accepting of both. Acceptance of nasopharyngeal swabs, however, varies with the testing indication and is influenced by perceived test accuracy. Understanding factors that influence sampling acceptance will inform more successful testing strategies.


Subject(s)
COVID-19 , SARS-CoV-2 , Adolescent , COVID-19/diagnosis , COVID-19 Testing , Caregivers , Child , Cross-Sectional Studies , Humans , Nasopharynx , Pain/diagnosis , Saliva , Specimen Handling/methods
13.
Int J Infect Dis ; 124: 11-13, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2007752

ABSTRACT

OBJECTIVES: To describe the trend of SARS-CoV-2 RNA in saliva samples from children attending nine schools in Rome in the local surveillance unit RM3 during the period of September 2021-March 2022, in parallel with the trend of SARS-CoV-2 RNA observed in nasopharyngeal swabs (NPSs) from the population in the same catchment area that was routinely tested at our laboratory in the same period. METHODS: Saliva samples were collected using the Copan LolliSpongeTM device and analyzed by Aptima® SARS-CoV-2 Assay on the Panther® System. NPSs were tested using either Aptima® SARS-CoV-2 Assay or Alinity m SARS-CoV-2 Assay. RESULTS: The percentage of positivity in the two populations was different; of the 2222 saliva samples from students, 0.99% had positive results, whereas the percentage was higher (33.43%) in the 8994 NPSs representing the population from local surveillance unit RM3. Interestingly, the trend of SARS-CoV-2 RNA in saliva samples from students was consistent with that observed in NPSs from the population in same catchment area, although with peaks slightly anticipated. CONCLUSION: Overall, screening of saliva in the schools represents a good system to monitor SARS-CoV-2 circulation in the population, allowing early detection and quick isolation of students who are asymptomatic with positive test results and thus prevention of virus transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , Child , Humans , SARS-CoV-2/genetics , Saliva , RNA, Viral/genetics , Rome/epidemiology , COVID-19/diagnosis , COVID-19/epidemiology , Specimen Handling/methods , Nasopharynx
14.
Viruses ; 14(9)2022 08 30.
Article in English | MEDLINE | ID: covidwho-2006232

ABSTRACT

The use of saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sparks debate due to presumed lower sensitivity and lack of standardization. Our aim was to evaluate the performance characteristics of (i) saliva collected by the ORAcollectTM device as a matrix for SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR), and (ii) 2 saliva rapid antigen tests (AgRDT). From 342 ambulatory individuals, both a nasopharyngeal swab and saliva sample via ORAcollectTM were obtained for a SARS-CoV-2 RT-PCR test. Furthermore, 54 and 123 additionally performed the V-ChekTM or WhistlingTM saliva AgRDT. In total, 35% of individuals screened positive for SARS-CoV-2 via nasopharyngeal swab. Saliva, as a matrix for the RT-PCR, had a specificity of 96.5% and a negative predictive value (NPV) of 91.3%. Interestingly, 6 out of 8 patients thought to be false positive in saliva re-tested positive by nasopharyngeal sampling after 2 to 9 days. Both V-ChekTM and WhistlingTM AgRDT had a lack of sensitivity, resulting in an NPV of 66.9 and 67.3%, respectively. Saliva proved to be a sensitive and specific matrix for SARS-CoV-2 detection by the RT-PCR. In this setting, saliva might have an earlier window of detection than the nasopharyngeal swab. By contrast, both AgRDT showed an unacceptably low sensitivity and NPV.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Clinical Laboratory Techniques/methods , Humans , Nasopharynx , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Saliva , Sensitivity and Specificity , Specimen Handling/methods
15.
Am J Emerg Med ; 61: 111-116, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2003796

ABSTRACT

BACKGROUND: The nasopharyngeal swab is the gold standard collection method for COVID-19, but is invasive and painful, subsequently resulting in poor patient acceptance. This investigation explores the process of developing and validating an alternative respiratory pathogen collection device that relies on a nasopharyngeal irrigation mechanic. The primary objective was to determine if sufficient pathological sampling can be achieved by mechanism of nasopharyngeal irrigation that is proportionate to the nasopharyngeal swab method. METHODS: The study device was designed using Shapr3D modeling software and fabricated on a fused deposition modeling printer. Fifteen participants were enrolled with each receiving a saline nasopharyngeal washing using the study device. Specimen adequacy was evaluated by two real-time reverse transcriptase polymerase chain reaction (PCR) testing methods to identify the presence of the human RNase P gene. Results were evaluated quantitatively through interpretation of the PCR cycle threshold (Ct). RESULTS: All 15 specimens tested positive for the presence of RNaseP, demonstrating specimen cellularity, adequate extraction of nucleic acids, and the absence of inhibitors to amplification. The mean Ct value was 29.5 (Applied Biosystems TaqPath RT-qPCR) and 30.7 (NECoV19). All participants felt the study device irrigation procedure was faster than the nasopharyngeal swab, with none experiencing any discomfort from the irrigation mechanism. CONCLUSION: The importance of early diagnostic testing and its role in countermeasures for communicable diseases such as COVID-19 is well established in the literature. Innovation to bolster our testing infrastructure is more important now than ever. This study was successful in developing and validating an alternative nasopharyngeal respiratory pathogen collection device that utilizes fluid debridement as its core mechanic. Data from this pilot study demonstrated the study device was successful in producing high-quality specimens for PCR testing. Feedback from the study participants was also in favor of the study device when compared to the nasopharyngeal swab.


Subject(s)
COVID-19 , Nucleic Acids , Humans , Pandemics , Pilot Projects , COVID-19/diagnosis , COVID-19/epidemiology , Ribonuclease P/genetics , SARS-CoV-2 , Nasopharynx , Specimen Handling/methods , Real-Time Polymerase Chain Reaction
16.
Microbiol Spectr ; 10(4): e0135822, 2022 08 31.
Article in English | MEDLINE | ID: covidwho-1992948

ABSTRACT

High-throughput diagnostic assays are required for large-scale population testing for severe acute respiratory coronavirus 2 (SARS-CoV-2). The gold standard technique for SARS-CoV-2 detection in nasopharyngeal swab specimens is nucleic acid extraction followed by real-time reverse transcription-PCR. Two high-throughput commercial extraction and detection systems are used routinely in our laboratory: the Roche cobas SARS-CoV-2 assay (cobas) and the Roche MagNA Pure 96 system combined with the SpeeDx PlexPCR SARS-CoV-2 assay (Plex). As an alternative to more costly instrumentation, or tedious sample pooling to increase throughput, we developed a high-throughput extraction-free sample preparation method for naso-oropharyngeal swabs using the PlexPCR SARS-CoV-2 assay (Direct). A collection of SARS-CoV-2-positive (n = 185) and -negative (n = 354) naso-oropharyngeal swabs in transport medium were tested in parallel to compare Plex to Direct. The overall agreement comparing the qualitative outcomes was 99.3%. The mean cycle of quantification (Cq) increase and corresponding mean reduction in viral load for Direct ORF1ab and RdRp compared to Plex was 3.11 Cq (-0.91 log10 IU/mL) and 4.78 Cq (-1.35 log10 IU/mL), respectively. We also compared Direct to a four-sample pool by combining each positive sample (n = 185) with three SARS-CoV-2-negative samples extracted with MagNA Pure 96 and tested with the PlexPCR SARS-CoV-2 assay (Pool). Although less sensitive than Plex or Pool, the Direct method is a sufficiently sensitive and viable approach to increase our throughput by 12,032 results per day. Combining cobas, Plex, and Direct, an overall throughput of 19,364 results can be achieved in a 24-h period. IMPORTANCE Laboratories have experienced extraordinary demand globally for reagents, consumables, and instrumentation, while facing unprecedented testing demand needed for the diagnosis of SARS-CoV-2 infection. A major bottleneck in testing throughput is the purification of viral RNA. Extraction-based methods provide the greatest yield and purity of RNA for downstream PCR. However, these techniques are expensive, time-consuming, and depend on commercial availability of consumables. Extraction-free methods offer an accessible and cost-effective alternative for sample preparation. However, extraction-free methods often lack sensitivity compared to extraction-based methods. We describe a sensitive extraction-free protocol based on a simple purification step using a chelating resin, combined with proteinase K and thermal treatment. We compare the sensitivity qualitatively and quantitatively to a well-known commercial extraction-based system, using a PCR assay calibrated to the 1st WHO international standard for SARS-CoV-2 RNA. This method entails high throughput and is suitable for all laboratories, particularly in jurisdictions where access to instrumentation and reagents is problematic.


Subject(s)
COVID-19 Testing , COVID-19 , COVID-19/diagnosis , Humans , Nasopharynx , RNA, Viral/analysis , SARS-CoV-2/genetics , Specimen Handling/methods
17.
J Virol Methods ; 309: 114607, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-1983588

ABSTRACT

Demand for accurate SARS-CoV-2 diagnostics is high. Most samples in the UK are collected in the community and rely on the postal service for delivery to the laboratories. The current recommendation remains that swabs should be collected in Viral Transport Media (VTM) and transported with a cold chain to the laboratory for RNA extraction and RT-qPCR. This is not always possible. We aimed to test the stability of SARS-CoV-2 RNA subjected to different pre-analytical conditions. Swabs were dipped into PBS containing cultured SARS-CoV-2 and placed in either a dry tube or a tube containing either normal saline or VTM. The tubes were then stored at different temperatures (20-50 °C) for variable periods (8 h to 5 days). Samples were tested by RT-qPCR targeting SARS-CoV-2 E gene. VTM outperformed swabs in saline and dry swabs in all conditions. Samples in VTM were stable, independent of a cold chain, for 5 days, with a maximum increase in cycle threshold (Ct) of 1.34 when held at 40 °C. Using normal saline as the transport media resulted in a loss of sensitivity (increased Ct) over time and with increasing temperature (up to 7.8 cycles compared to VTM). SARS-CoV-2 was not detected in 3/9 samples in normal saline when tested after 120 h incubation. Transportation of samples in VTM provides a high level of confidence in the results despite the potential for considerable, uncontrolled variation in temperature and longer transportation periods. False negative results may be seen after 96 h in saline and viral loads will appear lower.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Clinical Laboratory Techniques/methods , Humans , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Saline Solution , Specimen Handling/methods
18.
Int J Infect Dis ; 121: 166-171, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1983193

ABSTRACT

OBJECTIVES: There remain challenges in using SARS-CoV-2 RNA diagnostic assays in the respiratory tract in a pandemic. More so certain countries such as Hong Kong have already included saliva as part of their mass-testing protocol. The aim of this study was to conduct a systematic review on the alternate use of saliva as a SARS-CoV-2 RNA testing specimen in the context of mass screening with reverse transcription polymerase chain reaction. METHODS: Our search methodology was modeled after the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist, and the risk of bias of the selected studies was qualitatively assessed. The percentage individual positive and percentage agreement of both index (saliva) and reference (nasopharyngeal swab), in preference to specificity and sensitivity, were estimated using Kappa statistics. RESULTS: A total of 44 studies met the inclusion criteria. The average percentage positive saliva cases was 72.7% (95% confidence interval), which was lower but not substantially different from the percentage positive NPS of 78.7% (95% confidence interval), and there was an average overall agreement of 89.7% (95% confidence interval). CONCLUSION: Although the literature supports nasopharyngeal swab as a superior testing specimen, an alternative clinical specimen in saliva may offer potential benefits such that a potentially reduced accuracy may be tolerated, especially in low socioeconomic regions.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Nasopharynx , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Saliva , Specimen Handling/methods
19.
Sci Rep ; 12(1): 12612, 2022 07 23.
Article in English | MEDLINE | ID: covidwho-1956424

ABSTRACT

Saliva has been demonstrated as feasible alternative to naso-oropharyngeal swab (NOS) for SARS-CoV-2 detection through reverse transcription quantitative/real-time polymerase chain reaction (RT-qPCR). This study compared the diagnostic agreement of conventional NOS, saliva with RNA extraction (SE) and saliva without RNA extraction (SalivaDirect) processing for RT-qPCR in identifying SARS-CoV-2. All techniques were also compared, as separate index tests, to a composite reference standard (CRS) where positive and negative results were defined as SARS-CoV-2 detection in either one or no sample, respectively. Of 517 paired samples, SARS-CoV-2 was detected in 150 (29.01%) NOS and 151 (29.21%) saliva specimens. The saliva-based tests were noted to have a sensitivity, specificity and accuracy (95% confidence interval) of 92.67% (87.26%, 96.28%), 97.55% (95.40%, 98.87%) and 96.13% (94.09%, 97.62%), respectively, for SE RT-qPCR and 91.33% (85.64%, 95.30%), 98.91% (97.23%, 99.70%) and 96.71% (94.79%, 98.07%), respectively, for SalivaDirect RT-qPCR compared to NOS RT-qPCR. Compared to CRS, all platforms demonstrated statistically similar diagnostic performance. These findings suggest that both conventional and streamlined saliva RT-qPCR are at least non-inferior to conventional NOS RT-qPCR in detecting SARS-CoV-2.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Nasopharynx/virology , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Saliva/virology , Clinical Laboratory Techniques/methods , Cross-Sectional Studies , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Saliva/chemistry , Sensitivity and Specificity , Specimen Handling/methods
20.
Sci Rep ; 12(1): 11553, 2022 07 07.
Article in English | MEDLINE | ID: covidwho-1921718

ABSTRACT

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Clinical Laboratory Techniques/methods , Humans , Nasopharynx , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Saliva/chemistry , Sensitivity and Specificity , Specimen Handling/methods
SELECTION OF CITATIONS
SEARCH DETAIL