Evaluation of using distal part of endotracheal tube samples for SARS-COV-2 diagnosis by RT-PCR (original) (raw)
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Diagnosis of SARS-CoV-2 by RT-PCR Using Different Sample Sources: Review of the Literature
Ear, Nose & Throat Journal, 2020
Objective: The most widely used diagnostic technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is real-time reverse transcriptase-polymerase chain reaction (RT-PCR). It can be done on different samples: nasopharyngeal swabs (NPS) or oropharyngeal swabs (OPS), and self-collected saliva. However, negative findings do not rule out infection. Methods: A review was conceived to discuss advantages and limitations of the available diagnostic modalities for nonserologic diagnosis of SARS-CoV-2 based on RT-PCR; the article also proposes some practical suggestions to improve diagnostic reliability. Results: A total of 16 papers (corresponding to 452 patients) of the 56 initially identified were included. Most of the papers describe findings from different samples obtained in limited case series; comparative studies are missing. Conclusions: Diagnostic accuracy of NPS and OPS is suboptimal and the risk of contaminated aerosol dispersal is not negligible. The SA...
A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples
Healthcare
Introduction: the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of acute respiratory disease (COVID-19). SARS-CoV-2 is a positive-strand RNA virus and its genomic characterization has played a vital role in the design of appropriate diagnostics tests. The current RT-PCR protocol for SARS-CoV-2 detects two regions of the viral genome, requiring RNA extraction and several hours. There is a need for fast, simple, and cost-effective detection strategies. Methods: we optimized a protocol for direct RT-PCR detection of SARS-CoV-2 without the need for nucleic acid extraction. Nasopharyngeal samples were diluted to 1:3 using diethyl pyrocarbonate (DEPC)-treated water. The diluted samples were incubated at 95 °C for 5 min in a thermal cycler, followed by a cooling step at 4 °C for 5 min. Samples then underwent reverse transcription real-time RT-PCR in the E and RdRp genes. Results: our direct detection protocol showed 100% concordance ...
Travel Medicine and Infectious Disease
Background: It has been found that patients recovered from COVID 19 may still test Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) positive without being infectious; the reasons are unclear. The occurrence of false-negative results of RT-PCR interferes with a proper diagnosis. The objectives of that work were to determine factors associated with persistently detectable SARS-CoV-2 RNA among recovered hospitalized patients and to determine the incidence of false-negative RT-PCR results and associated factors. Methods: Relevant data were collected from 482 COVID 19 patients hospitalized in six referral centers from four countries. Results: The median duration of RT-PCR conversion to negative was 20 days. Out of 482 studied patients, 8.7% tested positive after more than four weeks and were considered prolonged convertors. Binary logistic regression analysis revealed headache as an independent risk factor for short conversion time while fever, hypertension, chronic obstructive pulmonary disease, lymphopenia, elevated erythrocyte sedimentation rate, and the number of lobes affected, and bilateralism were found to be independent risk factors for prolonged positivity. Eighteen patients had initial negative results then turned positive after 24-48 h. Associated factors and outcomes were identified. Conclusion: Identifying patients with a high likelihood of COVID-19 despite a negative RT-PCR is critical for effective clinical care. However, patient isolation resumption depending on positive RT-PCR despite clinical and radiological recovery is an overrating that greatly burdens the health sector.
Open Forum Infectious Diseases
Background The performance of real-time reverse transcription polymerase chain reaction (rRT-PCR) for SARS-CoV-2 varies with sampling site(s), illness stage, and infection site. Methods Unilateral nasopharyngeal, nasal midturbinate, throat swabs, and saliva were simultaneously sampled for SARS-CoV-2 rRT-PCR from suspected or confirmed cases of COVID-19. True positives were defined as patients with at least 1 SARS-CoV-2 detected by rRT-PCR from any site on the evaluation day or at any time point thereafter, until discharge. Diagnostic performance was assessed and extrapolated for site combinations. Results We evaluated 105 patients; 73 had active SARS-CoV-2 infection. Overall, nasopharyngeal specimens had the highest clinical sensitivity at 85%, followed by throat, 80%, midturbinate, 62%, and saliva, 38%–52%. Clinical sensitivity for nasopharyngeal, throat, midturbinate, and saliva was 95%, 88%, 72%, and 44%–56%, respectively, if taken ≤7 days from onset of illness, and 70%, 67%, 47%...
Allergologia et Immunopathologia, 2021
Coronavirus disease 2019 (COVID-19) is a disease caused by a new strain of coronavirus named as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Globally, since the outbreak, more than seven million confirmed cases of COVID-19 have been reported. The rapid spread and increase in the number of new cases is due to person-to-person transmission. To further control its transmission, early laboratory diagnosis of both asymptomatic and symptomatic patients is crucial. Presently, the COVID-19 diagnosis of infected individuals is dependent on computed tomography scanning and real-time polymerase chain reaction (PCR). The latter is considered more sensitive and efficient for early diagnosis. In this review, general comparisons are made (cases, fatality rate, incubation period, clinical features, and reservoirs) and diag-nostic laboratory procedures (specimens, extraction methods, and positive rates by real-time PCR) are compared between SARS, Middle East Respiratory Syndrome, an...
Detection of SARS-CoV-2 infection by RT-PCR test: factors influencing interpretation of results
VirusDisease
In this current pandemic of coronavirus disease 2019 (COVID-19), prompt interventions in terms of early detection and clinical management along with isolation of positive cases is of utmost importance. This helps to limit not only the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections but also the morbidity and mortality associated with it. Different strategies for screening of COVID-19 in containment zones and noncontainment areas include testing of symptomatic patients and their contacts in fever clinics, hospital-based testing, testing on demand and population-based screening. The choice of tests like reverse-transcription polymerase chain reaction (RT-PCR), rapid antigen testing (RAT) or antibody test depends upon these strategies and also the turnaround time. Currently, RT-PCR is considered the gold standard for COVID-19 detection. This commentary provides the insights and experiences on COVID-19 diagnosis by RT-PCR. The utility of this test is limited by several false positive, false negative and inconclusive results at early stages of infection, scarcity of reagents and lack of well-equipped labs including trained staff. Moreover, appropriate sample collection and transport, standard laboratory protocols, stringent quality control norms, good quality RNA extraction kits, PCR kits with suitable primers can help in improving accuracy of the test results. A careful assessment of clinical, radiological and molecular findings is required for identifying potential cases of COVID-19.
Diagnostics
Nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the most widely used upper respiratory tract specimens for diagnosis of SARS-CoV-2 using RT-qPCR. In contrast, nasal swab (NS) and saliva (SS), recently recommended by the WHO, are rarely used, and their test accuracy is limited. The method for direct RT-PCR detection of SARS-CoV-2 does not require an RNA extraction and is faster and easier than standard RT-PCR tests with RNA extraction. This study aimed to compare the diagnostic performance of upper respiratory tract samples for SARS-CoV-2 detection using the direct RT-PCR without preliminary heat inactivation. Here we report the application and validation of direct RT-PCR SARS-CoV-2 RNA on 165 clinical specimens of NPS/OP, and 36 samples of NS/NPS and 37 saliva samples (for the latter with prior deproteinization). The overall sensitivity estimates were 95.9%, 94.2%, 88.9%, and 94.6% for NPS/OPS/NS/SS samples, respectively, and the specificity was 100% against standard RT-P...
Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n ؍ 170) and stool (n ؍ 44) were reversetranscribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.
Infection
Molecular diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction (RT-PCR) in respiratory specimens is considered the gold standard method. This method is highly sensitive and specific but it has some limitations such as being expensive and requiring special laboratory equipment and skilled personnel. RapidFor™ Antigen Rapid Test Kit is a commercially available Ag-RDT which is produced in Turkey and designed to detect the nucleocapsid antigen of SARS-CoV-2 in nasopharyngeal swab samples. The aim of this study was to evaluate the performance of this novel SARS-CoV-2 antigen detection considering the RT-PCR method as the gold standard. Four hundred forty-four nasopharyngeal swab samples which were collected from the patients who met clinical criteria of COVID-19 from ten centers in Turkey between September 2020 and February 2021 were included in the study. All the nasopharyngeal swab samples were tested for SARS-CoV-2 RNA using commercial RT-PCR kits (Bioeksen and A1 Lifesciences, İstanbul, Turkey) according to the manufacturer's instructions. Viral loads were assessed according to the cycle threshold (Ct) values. RapidFor™ SARS-CoV-2 antigen test (Vitrosens Biotechnology, Istanbul, Turkey) was used to investigate the presence of SARS-CoV-2 antigen in all samples following the manufacturer's instructions. Out of 444 nasopharyngeal swab samples tested, 346 (77.9%) were positive and 98 (22.1%) were negative for SARS-CoV-2 RNA by RTPCR. Overall sensitivity of the RapidFor™. Antigen Rapid Test Kit was 80.3% whereas specificity was found to be 87.8%. Positivity rate of rapid antigen test in samples with Ct values over 25 and below 30 was 82.7%, while it increased to 95.7% in samples 20 ≤ Ct < 25 and reached 100% in samples with Ct values below 20. RapidFor™ SARS-CoV-2 Ag test might be a good choice in the screening of symptomatic and asymptomatic patients and their contacts for taking isolation measures early, with advantages over RT-PCR as being rapid, easy and being applicable in every laboratory and even at point of care.
Frontiers in Pediatrics, 2022
Reliable testing methods for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in children are essential to allow normal activities. Diagnosis of SARS-CoV-2 infection is currently based on real-time reverse transcriptase-polymerase chain reaction (RT-PCR) performed on nasopharyngeal (NP) swabs; concerns have been raised regarding NP swab accuracy in children to detect the virus because of potential lack of cooperation of the patients or due to general uncertainties about concordance between high and low respiratory tract specimens in children. The aim of the study (IRB approval: ST/2020/405) is to prospectively compare RT-PCR results on NP and tracheo-bronchial aspirate (TA) in children admitted to the hospital for surgery or admitted to the Pediatric Intensive Care Unit (PICU) of a tertiary children hospital in Milano, Italy, during a peak of COVID-19 infections in the city. A total of 385 patients were enrolled in the study: 364 from surgical theater and 21 from PICU. Two patients (0.5%) tested positive on TA and were negative on NP; both cases occurred in November 2020, during a peak of infection in the city. Specificity of NP swab was.995 (95% CI: 0.980-0.999). Two patients with positive NP swabs tested negative on TA. Conclusion: Our study shows that the specificity of SARS-CoV-2 RT-PCR on TA swab, compared to results of SARS-CoV-2 RT-PCR on NP, was very high for negative cases in our pediatric cohort during a period of high epidemiological pressure.