Repeated Reverse Transcription Polymerase Chain Reaction Research Articles

Comparison of RT-PCR cycle threshold values between individual and pooled SARS-CoV-2 infected nasopharyngeal swab specimens.

The molecular reverse transcription-polymerase chain reaction (RT-PCR) testing of respiratory tract swabs has become mandatory to confirm the diagnosis of coronavirus disease 2019 (COVID-19). However, RT-PCR tests are expensive, require standardized equipment, and relatively long testing times, and the sample pooling method has been introduced to solve this issue. The aim of this study was to compare the cycle threshold (Ct) values of the individual sample and pooled sample methods to assess how accurate the pooling method was. Repeat RT-PCR examinations were initially performed to confirm the Ct values for each sample before running the pooled test procedure. Sample extraction and amplification were performed in both assays to detect ORF1ab, N, and E genes with a cut-off point value of Ct <38. Overall, there was no difference in Ct values between individual sample and pooled sample groups at all concentrations (p=0.259) and for all pooled sizes. Only pooled size of five could detect the Ct value in the pooled samples for all concentration samples, including low-concentration sample (Ct values 36 to 38). This study highlighted that pooled RT-PCR testing strategy did not reduce the quality of individually measured RT-PCR Ct values. A pool size of five could provide a practical technique to expand the screening capacity of RT-PCR.

PP56 The Use of Computed Tomography for Detecting COVID-19 Pneumonia: Rapid Evidence Review

IntroductionThe reverse transcription polymerase chain reaction (RT-PCR) test is the gold standard for detecting coronavirus disease 2019 (COVID-19), but recent studies have reported false negative results for RT-PCR. Consequently, several countries are looking at chest computed tomography (CT) for the diagnostic workup of patients with suspected or probable COVID-19 to inform clinical management. Therefore, this rapid review provides the best available evidence on the use of chest CT for detecting COVID-19 pneumonia.MethodsLiterature searches were conducted in the PubMed, Cochrane Library, JBI Library, Epistemonikos, and Evidence Aid databases for relevant papers published from 1 December 2019 to 12 June 2020. The search strategy was based on the identified population, concept, and context with respect to the objective of the review. The methodological quality of the seven systematic reviews identified was appraised using the AMSTAR checklist.ResultsThe use of CT as a first-line screening tool for COVID-19 is not beneficial. Nevertheless, for patients with suspected COVID-19 pneumonia who had a false-negative RT-PCR results, cross-checking with CT and a repeated RT-PCR test is essential to avoid misdiagnosis. Chest CT offered high sensitivity for detecting COVID-19 pneumonia among symptomatic individuals, especially in those with severe disease. It was also useful for evaluating the potential complications, disease severity, and progression of COVID-19 pneumonia. The features of CT images differed at the early, intermediate, and late stages of COVID-19 pneumonia, but the most common imaging findings were patchy and ground glass opacities. It was also found that diagnosing COVID-19 in children using CT is challenging.ConclusionsEven though chest CT is not useful for primary screening of COVID-19, it is useful for detecting later stage disease and for evaluating patients with COVID-19 pneumonia. For patients with suspected COVID-19 pneumonia and a false negative RT-PCR result, cross-checking with a chest CT and a repeat RT-PCR test is essential to avoid misdiagnosis.

Optimisation of COVID-19 diagnostic pathways in acute hospital admissions to prevent nosocomial transmission.

IntroductionIn the management of acute hospital admissions during the COVID‐19 pandemic, safe patient cohorting depends on robust admission diagnostic strategies. It is essential that screening strategies are sensitive and rapid, to prevent nosocomial transmission of COVID‐19 and maintain patient flow.MethodsWe retrospectively identified all COVID‐19 positive and suspected cases at our institution screened by reverse transcription polymerase chain reaction (RT‐PCR) between 4 April and 28 June 2020. Using RT‐PCR positivity within 7 days as our reference standard, we assessed sensitivity and net‐benefit of three admission screening strategies: single admission RT‐PCR, composite admission RT‐PCR and CXR and repeat RT‐PCR with 48 h.ResultsRT‐PCR single‐test sensitivity was 91.5% (87.8%–94.4%) versus 97.7% (95.4%–99.1%) (p = 0.025) for RT‐PCR/CXR composite testing and 95.1% (92.1%–97.2%) (p = 0.03) for repeated RT‐PCR. Net‐benefit was 0.83 for single RT‐PCR versus 0.89 for RT‐PCR/CXR and 0.87 for repeated RT‐PCR at 0.02% threshold probability.ConclusionThe RT‐PCR/CXR composite testing strategy was highly sensitive when screening patients at the point of hospital admission. Real‐world sensitivity of this approach was comparable to repeat RT‐PCR testing within 48 h; however, faster facilitating improved patient flow.

Virucidal Effect of Povidone Iodine on SARS-CoV-2 in Nasopharynx: An Open-label Randomized Clinical Trial.

To assess the virucidal effect of povidone iodine (PVP-I) on severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) located in the nasopharynx and suitable dose-formulation for nasal application were the purpose of this clinical trial. This single-center, open-label randomized clinical trial with a 7-arm parallel-group design was conducted in Dhaka Medical College (DMC) Hospital. A total of 189 reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS CoV-2 positive cases aged 12–90 years with symptoms was sequentially enrolled following randomization. Nasopharyngeal clearance of SARS-CoV-2 was tested against PVP-I nasal irrigation (NI) at diluted concentrations of 0.4%, 0.5% and 0.6%, and PVP-I nasal spray (NS) at diluted concentrations of 0.5% and 0.6%. All groups were compared to the corresponding controls (distilled water). Written informed consent was ensured before participation. All procedures were conducted in after ethical clearance from the Ethical Review Board and in accordance with the Declaration of Helsinki. Viral clearance in a repeat RT-PCR (qualitative) was the primary outcome, and occurrence of any adverse event following administration of testing drug was considered as the secondary outcome. Analysis was performed using SPSS (Version 26). All cases were randomized into seven groups and each group consists of 27-patient. Mean age of the cases 43.98 ± 12.67 years (SD). All strength of NI were effective in nasopharyngeal clearance compared to the control (0.4%, p = 0.006; 0.5%, p < 0.001; and 0.6%, p = 0.018). Similarly, all strength of the NS is also effective than control (0.5%, p = < 0.001; and 0.6%, p ≤ 0.001). Highest nasopharyngeal clearance was observed in patients using 0.5% NI (n = 25, 92.6%, p = 0.018). Nasal irritation was the single most adverse event recorded in this trial and found in two patients using 0.4%, and 0.6% PVP-I NI, respectively. Both PVP-I NS and NI are effective for nasopharyngeal clearance in-vivo. However, further community trials are needed to repurpose these solutions as preventive agents against SARS-CoV2. Ethical clearance memo no ERC-DMC/ECC/2020/93.Trial registration NCT Identifier number NCT04549376.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12070-022-03106-0.

Gargling with 7.5% Sodium Bicarbonate Solution for SARS-CoV-2 Viremia Clearance: Our Institutional Clinical Experience.

BackgroundGargling had been reported to have some roles in the prevention and treatment of respiratory tract infections. The purpose of this study was to assess the ability of regular gargling using 7.5% sodium bicarbonate to eliminate SARS-CoV-2 in the oropharynx and nasopharynx.Materials and methodsThis pilot, open-labeled, nonrandomized, parallel single-center study. The effect of 30 seconds, three times per day gargling using 7.5% sodium bicarbonate solution—25 mL on SARS-CoV-2 viral clearance among coronavirus disease-2019 (COVID-19) patients in a dedicated COVID hospital at All India Institute of Medical Sciences, Patna, Bihar, India. We monitored the progress on by days 0, 1, 2, 3, 4, 5, 6, and 7 by observing variables like clinical category, P/F ratio, neutrophil/lymphocyte ratio (NLR) ratio, platelet count, ferritin, lactate dehydrogenase (LDH), CRP, procalcitonin, d-dimer, INR, APTT, and sequential organ function assessment (SOFA) score. We have also done repeat reverse transcription-polymerase chain reaction (RT-PCR) testing on day 5 and day 7.ResultsA total of 10 patients (7 males and 3 females) were included in our study after confirmed COVID positivity. The age range was from 30 to 61 years. Based on clinical severity and P/F ratio, 7 patients were included in the milder group as their ratio was more than 200 and the rest 3 patients were included in the moderate group as P/F ratio was less than 200. Two respondents had comorbidities, which were non-Hodgkin's lymphoma and ovarian carcinoma. Viral clearance was achieved at day 7 in 3 of 10 patients. However, the analysis of using 7.5% sodium bicarbonate 25 mL gargle statistically showed nonsignificant p-value for all of our studied variables. However, the PCR results were negative on 24 hours apart, i.e., on day 5 and day 7.ConclusionsThis is only a preliminary study which showed that gargling with 7.5% sodium bicarbonate may not be effective in achieving early SARS-CoV-2 viral clearance among mild COVID-19 patients. However, still larger studies are required to ascertain the benefit of gargling for different stages of COVID-19 patients with keeping in mind the important variables suggestive of viremia clearance.How to cite this articleKumar N, Kumar A, Mahto M, Singh PK. Gargling with 7.5% Sodium Bicarbonate Solution for SARS-CoV-2 Viremia Clearance: Our Institutional Clinical Experience. Indian J Crit Care Med 2021;25(7):791–794.

Donor Selection and Deferral Pattern in Convalescent Plasma Donor in Plasmapheresis Unit of a Tertiary Care Hospital

Context: The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in Wuhan, China, in December 2019. The management plan is supportive care with oxygen supplementation and mechanical ventilation. US FDA approved convalescent plasma (CP) for COVID-19 for clinical trials and as an emergency investigational new drug. Although numerous trials are currently investigating the safety and efficacy of CP in COVID-19 patients, there is a paucity of ongoing and published studies evaluating the CP donors' side. This retrospective study reports the CP donors' selection and deferrals. Aim: To evaluate and analyze the donor deferral pattern and its causes among CP donors in a tertiary care hospital blood bank apheresis unit. Settings and Design: Hospital-based retrospective analysis. Subjects and Methods: Donors aged 18–65 years who had recovered from COVID-19 at least 4 weeks previously coming for plasma donation were retrospectively analyzed from July 25, 2020 to January 24, 2021 for a period of 6 months at blood bank apheresis unit, Assam Medical College and Hospital. Results: A total of 396 potential plasma donors were screened during the study period. The donor deferral rate was 39.1%. The permanent deferral was 36.8%, and the temporary deferral was 63.2%. The maximum number of donors deferred because of low antibody (18.7%) followed by low hemoglobin (14.8%). Associated comorbidities, low platelet count, repeat reverse transcription-polymerase chain reaction positivity, transfusion transmitted infection reactivity, intake of medicines, infections, vaccination, and underweight were other causes identified. Conclusion: The pattern of donor deferral is an important tool for blood safety and also provides key areas to focus on a demographic region or policy formulation for donor selection as well as to ensure donor safety.

Re-positive of SARS-CoV-2 test is common in COVID-19 patients after hospital discharge. Data from high standard post-discharge quarantined patients in Macao SAR, China.

During the pandemic of COVID-19, Macau faces tremendous pressure because it is a famous gambling and tourism city with the world’s highest population density. The Macau government implemented decisive public health intervention to control the transmission of COVID-19, and there were only two independent outbreaks in Macau. In the second outbreak, all 35 cases were infected in foreign countries. They were quarantined in airborne infection isolation rooms for at least 14 days with reverse transcription-polymerase chain reaction (RT-PCR) tests after hospital discharge. Twelve (34.3%) of them had re-positive SARS-CoV-2 test results, and none of them presented any COVID-19 signs or symptoms during the entire quarantine period. In this study, the re-positive patients were more likely to be diagnosed in the early stage of the disease with a longer hospital stay. Virus re-infection is impossible in this high standard isolation setting, and reactivation is also unlikely, so that residual virus nucleic acid should be the possible reason for this phenomenon. Due to limited data about the risk of re-positive patients, it is better to quarantine patients after discharge for a prolonged period with repeat RT-PCR tests to minimize the community’s potential risk, particularly in the regions with relative plenty of resources and low community infection rate such as Macau.

Proactive Measures to Combat a SARS-CoV-2 Transmission Among High Risk Patients and Health Care Workers in an Outpatient Dialysis Facility.

Background: End-stage-renal-failure (ESRF) patients attending clustered out-patient dialysis are susceptible to SARS-CoV-2 infection. Comorbidities render them vulnerable to severe COVID-19. Although preventative and mitigation strategies are recommended, the effect of these are unknown. A period of “potential-high-infectivity” results if a health-care-worker (HCWs) or a patient becomes infected. Aim: We describe and analyze early, universal SARS-CoV-2 real time reverse transcription polymerase chain reaction (RT-PCR) tests, biomarker monitoring and SARS-CoV-2 preventative strategies, in a single dialysis center, after a positive patient was identified. Methodology: The setting was a single outpatient dialysis center in Johannesburg, South Africa which had already implemented preventative strategies. We describe the management of 57 patients and 11 HCWs, after one of the patients tested positive for SARS-CoV-2. All individuals were subjected to RT-PCR tests and biomarkers (Neutrophil-Lymphocyte Ratio, C-reactive protein, and D-Dimer) within 72 h (initial-tests). Individuals with initial negative RT-PCR and abnormal biomarkers (one or more) were subjected to repeat RT-PCR and biomarkers (retest subgroup) during the second week. Additional stringent measures (awareness of viral transmission, dialysis distancing and screening) were implemented during the period of “potential high infectivity.” The patient retest subgroup also underwent clustered dialysis until retest results became available. Results: A second positive-patient was identified as a result of early universal RT-PCR tests. In the two positive-patients, biomarker improvement coincided with RT-PCR negative tests. We identified 13 individuals for retesting. None of these retested individuals tested positive for SARS-CoV-2 and there was no deterioration in median biomarker values between initial and retests. Collectively, none of the negative individuals developed COVID-19 symptoms during the period “potential high infectivity.” Conclusion: A SARS-CoV-2 outbreak may necessitate additional proactive steps to counteract spread of infection. This includes early universal RT-PCR testing and creating further awareness of the risk of transmission and modifying preventative strategies. Abnormal biomarkers may be poorly predictive of SARS-CoV-2 infection in ESRF patients due to underlying illnesses. Observing dynamic changes in biomarkers in RT-PCR positive and negative-patients may provide insights into general state of health.

Evaluation of COVID-19 Testing Strategies for Repopulating College and University Campuses: A Decision Tree Analysis

PurposeThe optimal approach to identify SARS-CoV-2 infection among college students returning to campus is unknown. Recommendations vary from no testing to two tests per student. This research determined the strategy that optimizes the number of true positives and negatives detected and reverse transcription polymerase chain reaction (RT-PCR) tests needed. MethodsA decision tree analysis evaluated five strategies: (1) classifying students with symptoms as having COVID-19, (2) RT-PCR testing for symptomatic students, (3) RT-PCR testing for all students, (4) RT-PCR testing for all students and retesting symptomatic students with a negative first test, and (5) RT-PCR testing for all students and retesting all students with a negative first test. The number of true positives, true negatives, RT-PCR tests, and RT-PCR tests per true positive (TTP) was calculated. ResultsStrategy 5 detected the most true positives but also required the most tests. The percentage of correctly identified infections was 40.6%, 29.0%, 53.7%, 72.5%, and 86.9% for Strategies 1–5, respectively. All RT-PCR strategies detected more true negatives than the symptom-only strategy. Analysis of TTP demonstrated that the repeat RT-PCR strategies weakly dominated the single RT-PCR strategy and that the thresholds for more intensive RT-PCR testing decreased as the prevalence of infection increased. ConclusionBased on TTP, the single RT-PCR strategy is never preferred. If the cost of RT-PCR testing is of concern, a staged approach involving initial testing of all returning students followed by a repeat testing decision based on the measured prevalence of infection might be considered.

Detecting SARS-CoV-2 at point of care: preliminary data comparing loop-mediated isothermal amplification (LAMP) to polymerase chain reaction (PCR)

BackgroundA cost effective and efficient diagnostic tool for COVID-19 as near to the point of care (PoC) as possible would be a game changer in the current pandemic. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 min, alongside standard methods in a real-life clinical setting.MethodsThis prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a single swab using RT-LAMP compared with the current standard, RT-PCR, as per Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines.ResultsThe novel method accurately detected 8/10 RT-PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a “gold standard”, the sensitivity and specificity of a single novel test were 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19.ConclusionsRT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the PoC. RT-LAMP could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread.

Fatal COVID-19 in a Patient with End-Stage Liver Disease Wait-Listed for Liver Transplantation: An Evidence-Based Review of COVID-19 Screening Modalities Prior to Transplant.

Fatal COVID-19 in a Patient with End-Stage Liver Disease Wait-Listed for Liver Transplantation: An Evidence-Based Review of COVID-19 Screening Modalities Prior to Transplant.

PC-FACS: COVID-19

PC-FACS (FastArticleCriticalSummaries forClinicians inPalliativeCare) provides hospice and palliative care clinicians with concise summaries of the most important findings from more than 100 medical and scientific journals. If you have colleagues who would benefit from receiving PCFACS, please encourage them to join the AAHPM at aahpm.org. Comments from readers are welcomed at pcfacs@aahpm.org.

Early chest computed tomography to diagnose COVID-19 from suspected patients: A multicenter retrospective study.

ObjectiveThe purpose of this study was to distinguish the imaging features of COVID-19 from those of other infectious pulmonary diseases and evaluate the diagnostic value of chest CT for suspected COVID-19 patients.MethodsAdult patients suspected of COVID-19 aged >18 years who underwent chest CT scans and reverse-transcription polymerase chain reaction (RT-PCR) tests within 14 days of symptom onset were enrolled. The enrolled patients were confirmed and grouped according to the results of the RT-PCR tests. The basic demographics, single chest CT features, and combined chest CT features were analyzed for the confirmed and nonconfirmed groups.ResultsA total of 130 patients were enrolled, with 54 testing positive and 76 testing negative. The typical CT imaging features of the positive group were ground glass opacities (GGOs), the crazy-paving pattern and air bronchogram. The lesions were mostly distributed bilaterally and close to the lower lungs or the pleura. When features were combined, GGOs with bilateral pulmonary distribution and GGOs with pleural distribution were more common among the positive patients, found in 31 (57.4%) and 30 patients (55.6%), respectively. The combinations were almost all statistically significant (P < .05), except for the combination of GGOs with consolidation. Most combinations presented relatively low sensitivity but extremely high specificity. The average specificity of these combinations was approximately 90%.ConclusionsThe combinations with GGOs could be useful in the identification and differential diagnosis of COVID-19, alerting clinicians to isolate patients for prompt treatment and repeat RT-PCR tests until the end of incubation.

Radiological role in the detection, diagnosis and monitoring for the coronavirus disease 2019 (COVID-19).

Coronavirus disease 2019 (COVID-19) has officially been declared a pandemic by the World Health Organization (WHO). Radiological examinations, especially computed tomography (CT), play an important role in the fight against COVID-19. A comprehensive and timely review of radiological role in the fight against COVID-19 remains urgent and mandatory. Hence, the aim of this review is to summarize the radiological role in the fight against COVID-19. This review of current studies on COVID-19 provides insight into the radiological role in the detection, diagnosis, and monitoring for COVID-19. The typical radiological features of COVID-19 include bilateral, multifocal, and multilobar ground glass opacification with patchy consolidation, a peripheral/subpleural or posterior distribution (or both), mainly in the lower lobes. A combination of chest CT and repeat Reverse Transcription-Polymerase Chain Reaction (RT-PCR) testing may be beneficial for the diagnosis of COVID-19 in the setting of strongly clinical suspicion. Chest CT may improve the sensitivity for COVID-19 diagnosis, but patients' exposure to radiation should be kept as low as possible especially for children and pregnant women patients.

Detecting SARS-CoV-2 at Point of Care: Preliminary Data Comparing Loop-Mediated Isothermal Amplification (LAMP) to PCR

Background: The need for a fast and reliable test for COVID-19 is paramount in managing the current pandemic. A cost effective and efficient diagnostic tool as near to the point of care (PoC) as possible would be a game changer in current testing. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 minutes, alongside standard methods in a real-life clinical setting. Methods: This service improvement project piloted a research RT-LAMP method on nasal and pharyngeal swabs on 21 residents in an NHS Category 1 care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We calculated the sensitivity, specificity, positive and negative predictive values of a single RT-LAMP swab compared to RT-PCR, as per STARD guidelines. We also recorded vital signs of patients to correlate clinical and laboratory information. Findings: The novel method accurately detected 8/10 PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a “gold standard”, the sensitivity and specificity of the novel test were 80% and 73% respectively. Positive predictive value (PPV) was 73% and negative predictive value (NPV) was 83%. We also observed hypothermia to be a significant early clinical sign in a number of COVID-19 patients in this setting. Interpretation: RT-LAMP testing for SARS-CoV-2 was found to be promising, fast, easy to use and to work equivalently to RT-PCR methods. Definitive studies to evaluate this method in larger cohorts are underway. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the point of care. This method could be deployed in mobile testing units in the community, care homes and hospitals to detect disease early and prevent spread.