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1.
Am J Clin Pathol ; 156(1): 24-33, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1211384

ABSTRACT

OBJECTIVES: We conducted an analytic and clinical comparison of a novel high-definition polymerase chain reaction PCR (HDPCR) assay to traditional real-time PCR (RT-PCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper respiratory specimens. METHODS: Analytic performance of RT-PCR, HDPCR, and extraction-free HDPCR was established through replicate testing of a serially diluted clinical specimen containing SARS-CoV-2. A clinical comparison of all 3 assays was conducted using 351 prospectively collected upper respiratory swab specimens obtained from symptomatic and asymptomatic individuals collected in various transport media. RESULTS: RT-PCR and HDPCR assays using extracted nucleic acid demonstrated similar analytic limits of detection (LoD) and clinical performance, with 100% positive and negative agreement. Extraction-free HDPCR demonstrated a 1.5 to 2.0 log10 increase in LoD based on cycle threshold values. However, clinical performance of extraction-free HDPCR remained high, demonstrating 97.8% positive and 99.6% negative agreement with RT-PCR. An overall increase in "invalid" and "presumptive" results was observed when using the extraction-free method, but this was highly variable based on transport medium used. CONCLUSIONS: HDPCR performs similar to RT-PCR for the detection of SARS-CoV-2. The use of an extraction-free HDPCR protocol maintained high clinical performance despite reduced analytic LoD, with the benefit of reduced hands-on time and cost of reagents associated with nucleic acid extraction.


Subject(s)
COVID-19/genetics , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19 Testing/methods , Humans , Limit of Detection , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity , Sensitivity and Specificity , Specimen Handling/methods
2.
J Infect ; 82(6): 253-259, 2021 06.
Article in English | MEDLINE | ID: covidwho-1152506

ABSTRACT

BACKGROUND: Human to human transmission of SARS-CoV-2 is driven by the respiratory route but little is known about the pattern and quantity of virus output from exhaled breath. We have previously shown that face-mask sampling (FMS) can detect exhaled tubercle bacilli and have adapted its use to quantify exhaled SARS-CoV-2 RNA in patients admitted to hospital with Coronavirus Disease-2019 (COVID-19). METHODS: Between May and December 2020, we took two concomitant FMS and nasopharyngeal samples (NPS) over two days, starting within 24 h of a routine virus positive NPS in patients hospitalised with COVID-19, at University Hospitals of Leicester NHS Trust, UK. Participants were asked to wear a modified duckbilled facemask for 30 min, followed by a nasopharyngeal swab. Demographic, clinical, and radiological data, as well as International Severe Acute Respiratory and emerging Infections Consortium (ISARIC) mortality and deterioration scores were obtained. Exposed masks were processed by removal, dissolution and analysis of sampling matrix strips fixed within the mask by RT-qPCR. Viral genome copy numbers were determined and results classified as Negative; Low: ≤999 copies; Medium: 1000-99,999 copies and High ≥ 100,000 copies per strip for FMS or per 100 µl for NPS. RESULTS: 102 FMS and NPS were collected from 66 routinely positive patients; median age: 61 (IQR 49 - 77), of which FMS was positive in 38% of individuals and concomitant NPS was positive in 50%. Positive FMS viral loads varied over five orders of magnitude (<10-3.3 x 106 genome copies/strip); 21 (32%) patients were asymptomatic at the time of sampling. High FMS viral load was associated with respiratory symptoms at time of sampling and shorter interval between sampling and symptom onset (FMS High: median (IQR) 2 days (2-3) vs FMS Negative: 7 days (7-10), p = 0.002). On multivariable linear regression analysis, higher FMS viral loads were associated with higher ISARIC mortality (Medium FMS vs Negative FMS gave an adjusted coefficient of 15.7, 95% CI 3.7-27.7, p = 0.01) and deterioration scores (High FMS vs Negative FMS gave an adjusted coefficient of 37.6, 95% CI 14.0 to 61.3, p = 0.002), while NPS viral loads showed no significant association. CONCLUSION: We demonstrate a simple and effective method for detecting and quantifying exhaled SARS-CoV-2 in hospitalised patients with COVID-19. Higher FMS viral loads were more likely to be associated with developing severe disease compared to NPS viral loads. Similar to NPS, FMS viral load was highest in early disease and in those with active respiratory symptoms, highlighting the potential role of FMS in understanding infectivity.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Masks , Middle Aged , RNA, Viral , Viral Load
3.
J Assist Reprod Genet ; 38(4): 785-789, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1116946

ABSTRACT

INTRODUCTION: Whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen and transmitted sexually is a vital question that has, thus far, been inconclusive. Prior studies, with limited numbers, have included men in various stages of infection with most in the recovery phase of the illness. The timing of test results and severity of illness has made recruiting study participants a significant challenge. Our pilot study will examine semen from men with a recent diagnosis of COVID-19 as well as those in the convalescent phase to determine if SARS-CoV-2 can be detected and its relationship, if any, with the severity of the disease. METHODS: Eighteen men with a median age of 32 (range, 24-57) who tested positive for COVID-19 by rt-PCR analysis were enrolled and provided a semen sample. The study group demonstrated symptoms of COVID-19 ranging from asymptomatic to moderate and none required hospitalization. Samples were subjected to viral RNA extraction and then processed by real-time RT-PCR using the US Centers for Disease Control and Prevention (CDC, USA) panel of 2019-Novel Coronavirus (2019-nCoV) primers and probes to detect the presence of SARS-CoV-2 RNA. RESULTS: Length of time from diagnosis to providing a specimen ranged from 1 to 28 days (median, 6 days). Fifteen participants were symptomatic and three were asymptomatic, including recovering men, at the time of semen collection. No SARS-CoV-2 was detected in any of the semen samples. CONCLUSION: Based on these preliminary results and consistent with prior findings, we suggest SARS-CoV-2 is not present in semen during the acute or convalescent phase of COVID-19.


Subject(s)
Body Fluids/virology , COVID-19/virology , SARS-CoV-2/pathogenicity , Semen/virology , Adult , COVID-19/genetics , COVID-19/transmission , Cohort Studies , Female , Humans , Male , Middle Aged , Pilot Projects , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Spermatozoa/virology , Young Adult
4.
Lancet Microbe ; 1(5): e218-e225, 2020 09.
Article in English | MEDLINE | ID: covidwho-1087372

ABSTRACT

BACKGROUND: In December, 2019, a novel zoonotic severe acute respiratory syndrome-related coronavirus emerged in China. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became pandemic within weeks and the number of human infections and severe cases is increasing. We aimed to investigate the susceptibilty of potential animal hosts and the risk of anthropozoonotic spill-over infections. METHODS: We intranasally inoculated nine fruit bats (Rousettus aegyptiacus), ferrets (Mustela putorius), pigs (Sus scrofa domesticus), and 17 chickens (Gallus gallus domesticus) with 105 TCID50 of a SARS-CoV-2 isolate per animal. Direct contact animals (n=3) were included 24 h after inoculation to test viral transmission. Animals were monitored for clinical signs and for virus shedding by nucleic acid extraction from nasal washes and rectal swabs (ferrets), oral swabs and pooled faeces samples (fruit bats), nasal and rectal swabs (pigs), or oropharyngeal and cloacal swabs (chickens) on days 2, 4, 8, 12, 16, and 21 after infection by quantitative RT-PCR (RT-qPCR). On days 4, 8, and 12, two inoculated animals (or three in the case of chickens) of each species were euthanised, and all remaining animals, including the contacts, were euthanised at day 21. All animals were subjected to autopsy and various tissues were collected for virus detection by RT-qPCR, histopathology immunohistochemistry, and in situ hybridisation. Presence of SARS-CoV-2 reactive antibodies was tested by indirect immunofluorescence assay and virus neutralisation test in samples collected before inoculation and at autopsy. FINDINGS: Pigs and chickens were not susceptible to SARS-CoV-2. All swabs, organ samples, and contact animals were negative for viral RNA, and none of the pigs or chickens seroconverted. Seven (78%) of nine fruit bats had a transient infection, with virus detectable by RT-qPCR, immunohistochemistry, and in situ hybridisation in the nasal cavity, associated with rhinitis. Viral RNA was also identified in the trachea, lung, and lung-associated lymphatic tissue in two animals euthanised at day 4. One of three contact bats became infected. More efficient virus replication but no clinical signs were observed in ferrets, with transmission to all three direct contact animals. Mild rhinitis was associated with viral antigen detection in the respiratory and olfactory epithelium. Prominent viral RNA loads of 0-104 viral genome copies per mL were detected in the upper respiratory tract of fruit bats and ferrets, and both species developed SARS-CoV-2-reactive antibodies reaching neutralising titres of up to 1/1024 after 21 days. INTERPRETATION: Pigs and chickens could not be infected intranasally by SARS-CoV-2, whereas fruit bats showed characteristics of a reservoir host. Virus replication in ferrets resembled a subclinical human infection with efficient spread. Ferrets might serve as a useful model for further studies-eg, testing vaccines or antivirals. FUNDING: German Federal Ministry of Food and Agriculture.


Subject(s)
COVID-19 , Chiroptera , Rhinitis , Animals , Antibodies, Viral , COVID-19/veterinary , Chickens/genetics , Chiroptera/genetics , Ferrets/genetics , RNA, Viral/genetics , SARS-CoV-2
5.
PLoS Biol ; 18(10): e3000896, 2020 10.
Article in English | MEDLINE | ID: covidwho-810274

ABSTRACT

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , Reverse Transcriptase Polymerase Chain Reaction/standards , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/standards , Coronavirus Infections/virology , DNA Primers/standards , Humans , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Sensitivity and Specificity , United States , Viral Load
6.
Indian J Med Res ; 152(1 & 2): 88-94, 2020.
Article in English | MEDLINE | ID: covidwho-745660

ABSTRACT

BACKGROUND & OBJECTIVES: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. METHODS: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed. RESULTS: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ctvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. INTERPRETATION & CONCLUSIONS: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/isolation & purification , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Coronavirus Infections/epidemiology , Coronavirus Infections/genetics , Coronavirus Infections/virology , Diagnostic Tests, Routine/methods , Female , Humans , India/epidemiology , Male , Pandemics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/genetics , Pneumonia, Viral/virology , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Serologic Tests , Specimen Handling , Viral Load/genetics
7.
Acta Biomed ; 91(9-S): 76-78, 2020 07 20.
Article in English | MEDLINE | ID: covidwho-671296

ABSTRACT

The COVID-19 pandemic, affecting 213 countries, with more than 10 million cases and over 500,000 deaths is still causing serious health, social and economic emergency worldwide. Italian Northern regions are among the most badly affected areas. Surfaces represent matrices to which particular attention should be paid for prevention and control of SARS-CoV-2 transmission. A few studies have highlighted virus presence on surfaces. We report the evidence of its presence on hospital surfaces, in a single room hosting a patient whose nose-pharyngeal swab resulted positive for SARS-CoV-2 RNA at the admission. The surfaces sampling was carried out using pre-wetted swabs followed by extraction and amplification of viral RNA by reverse Real-Time Polymerase Chain Reaction (rRT-PCR). A total of 4/15 (26.66%) surfaces were positive for SARS-CoV-2 RNA: the right bed rail, the call button, the bed trapeze bar, the stethoscope; moreover, the patient's inner surgical mask was positive, showing the emission of the virus from the patient. This study is a further confirmation that the surfaces represent a potential vehicle of transmission. This supports the need for strict adherence to hand and environmental hygiene.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/transmission , Pneumonia, Viral/transmission , COVID-19 , Coronavirus Infections/virology , Environmental Microbiology , Hospitals , Humans , Pandemics , Pneumonia, Viral/virology , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , SARS-CoV-2
8.
J Clin Microbiol ; 58(6)2020 05 26.
Article in English | MEDLINE | ID: covidwho-46769

ABSTRACT

Laboratories are currently witnessing extraordinary demand globally for sampling devices, reagents, consumables, and diagnostic instruments needed for timely diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To meet diagnostic needs as the pandemic grows, the U.S. Food and Drug Administration (FDA) recently granted several commercial SARS-CoV-2 tests Emergency Use Authorization (EUA), but manufacturer-independent evaluation data are scarce. We performed the first manufacturer-independent evaluation of the fully automated sample-to-result two-target test cobas 6800 SARS-CoV-2 (cobas) (Roche Molecular Systems, Branchburg, NJ), which received U.S. FDA EUA on 12 March 2020. The comparator was a standardized 3-h SARS-CoV-2 protocol, consisting of RNA extraction using an automated portable instrument, followed by a two-target reverse transcription real-time PCR (RT-PCR), which our laboratory has routinely used since January 2020 [V. M. Corman, O. Landt, M. Kaiser, R. Molenkamp, et al., Euro Surveill 25(3):pii=2000045, 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045]. cobas and the comparator showed overall agreement of 98.1% and a kappa value of 0.95 on an in-house validation panel consisting of 217 well-characterized retrospective samples. Immediate prospective head-to-head comparative evaluation followed on 502 samples, and the diagnostic approaches showed overall agreement of 99.6% and a kappa value of 0.98. A good correlation (r 2 = 0.96) between cycle threshold values for SARS-CoV-2-specific targets obtained by cobas and the comparator was observed. Our results showed that cobas is a reliable assay for qualitative detection of SARS-CoV-2 in nasopharyngeal swab samples collected in the Universal Transport Medium System (UTM-RT) (Copan, Brescia, Italy). Under the extraordinary circumstances that laboratories are facing worldwide, a safe diagnostic platform switch is feasible in only 48 h and in the midst of the COVID-19 pandemic if carefully planned and executed.


Subject(s)
Betacoronavirus , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Humans , Pandemics , SARS-CoV-2 , Time Factors
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