Evaluation of using distal part of endotracheal tube samples for SARS-COV-2 diagnosis by RT-PCR

Objective: The reverse transcription-polymerase chain reaction (RT-PCR) analyses method is the most important diagnostic method in the diagnosis of SARS-CoV-2 virus infection. In this research, we aimed to investigate the positivity of SARS-CoV-2 by RT-PCR from distal part of the endotracheal tube (DPET) samples, which have not been investigated in any study yet. Materials and Methods: A total of 48 patients with a diagnosis of COVID-19 hospitalized in the intensive care unit receiving mechanical ventilation and whose conditions resulted in death or extubation were included in the study. The distal 6 cm part of the orotracheal intubation tube was removed from the patient (including the cuff). DPET samples were mixed with viral transport medium and vortexed; then, it was centrifuged at 4500g for 4 minutes. RNA isolation was performed by taking 400 µl from the supernatant and then SARS-CoV-2 RT-PCR was studied. Results: In 15 patients (31.25 %) the swab samples were PCR positive, 42 patients (87.5 %) had positive computed tomography finding and 48 patients (100 %) had positive clinical findings. Among the patients whose oropharynx (OP)/nasopharynx (NP) combined swab sample was positive for RT-PCR, the rate of RT-PCR positivity detected in DPET samples was 26.7%. While OP/NP combined swab sample was negative, DPET RT-PCR positivity rate was found to be 9.09%. Conclusions: Patients with positive DPET RT-PCR are detected when the swab is negative. These findings suggest that DPET can be used as a good lower respiratory sample without the risk of particle spread and transmission to healthcare personnel.

Conjunctival polymerase chain reactions in COVID-19 patients and its correlation with clinical and paraclinical indexes

Background and Objectives: This study aimed to detect SARS-CoV-2 in conjunctival samples of COVID-19 patients to investigate the transmission route of COVID-19 and its correlation with laboratory indexes. Materials and Methods: In this cross-sectional study, 44 COVID-19 patients were tested for conjunctival PCR in Ayatollah Rouhani hospital of Babol, Iran, in January and February 2021. The conjunctival samples were collected using a conjunctival swab and suspended in a viral transport medium. After RNA extraction and cDNA synthesis, real-time PCR was performed to investigate the SARS-CoV-2 genome in samples. The ocular manifestations and laboratory indexes were evaluated for all patients. Results: Among 44 COVID-19 patients, 6 samples (13.63%) were positive in terms of conjunctival PCR. The mean ± SD age of conjunctival PCR-positive patients was 76.17 ± 16.61-year-old, while conjunctival PCR-negative COVID-19 pa- tients were aged 57.54 ± 13.61-year-old (p <0.05). D-dimer serum level is significantly higher in conjunctival PCR-positive COVID-19 patients (4001.00 ± 3043.36 µg/ml) compared to normal individuals (496.80 ± 805.92 µg/ml, p <0.01). Conclusion: Our study showed that the conjunctiva and tear contain the SARS-CoV-2 in COVID-19 patients as a possible transmission route.

Ecological surveillance of bat coronaviruses in Sarawak, Malaysian Borneo

Objective Coronaviruses (CoVs) are natural commensals of bats. Two subgenera, namely Sarbecoviruses and Merbecoviruses have a high zoonotic potential and have been associated with three separate spillover events in the past 2 decades, making surveillance of bat-CoVs crucial for the prevention of the next epidemic. The study was aimed to elucidate the presence of coronavirus in fresh bat guano sampled from Wind Cave Nature Reserve (WCNR) in Sarawak, Malaysian Borneo. Samples collected were placed into viral transport medium, transported on ice within the collection day, and preserved at − 80 °C. Nucleic acid was extracted using the column method and screened using consensus PCR primers targeting the RNA-dependent RNA polymerase (RdRp) gene. Amplicons were sequenced bidirectionally using the Sanger method. Phylogenetic tree with maximum-likelihood bootstrap and Bayesian posterior probability were constructed. Results CoV-RNA was detected in ten specimens (47.6%, n = 21). Six alphacoronavirus and four betacoronaviruses were identified. The bat-CoVs can be phylogenetically grouped into four novel clades which are closely related to Decacovirus-1 and Decacovirus-2, Sarbecovirus, and an unclassified CoV. CoVs lineages unique to the Island of Borneo were discovered in Sarawak, Malaysia, with one of them closely related to Sarbecovirus. All of them are distant from currently known human coronaviruses.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/μL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3–99.5%] sensitivity and 100% (95% CI = 94.5–100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non–SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction–free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

RNA-extraction-free diagnostic method to detect SARS-CoV-2: an assessment from two States, India

With increasing demand for large numbers of testing during COVID-19 pandemic, came alternative protocols with shortened turn-around time. We evaluated the performance of such an approach wherein 1138 consecutive clinic attendees were enrolled; 584 and 554 respectively from two independent study sites in the cities of Pune and Kolkata. Paired nasopharyngeal and oropharyngeal swabs were tested by using both reference and index methods in blinded fashion. Prior to conducting RT-PCR, swabs collected in viral transport medium (VTM) were processed for RNA extraction (reference method) and swabs collected in dry tube without VTM were incubated in Tris-EDTA-Proteinase K buffer for 30 minutes and heat inactivated at 98oC for 6 minutes (index method). Overall sensitivity and specificity of the index method were 78.9% (95% CI 71% to 86%) and 99 % (95% CI 98% to 99.6%) respectively. Agreement between the index and reference method was 96.8 % (k = 0.83, SE=0.030). The reference method exhibited enhanced detection of viral genes (E, N and RdRP) with lower Ct values compared to the index method. The index method can be used for detecting SARS-CoV-2 infection with appropriately chosen primer-probe set and heat treatment approach in pressing time; low sensitivity constrains its potential wider use.

Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

Assessing Quality Indicators for the COVID-19 RTPCR in a Molecular Laboratory

A quality indicator is a tool that enables the user to quantify the quality of a selected aspect of care by comparing it with a set benchmark. The objective of this study was to review quality indicators for COVID-19 molecular testing at S.S. Institute of Medical Sciences and Research Centre and to compare with the predefined quality indicators in order to improve the performance of the molecular laboratory and to initiate the corrective and preventive measures. Over the period of one year we assessed different quality indicators collected from the molecular laboratory of a tertiary care hospital in Central Karnataka which has processed 36000 throat swabs for the diagnosis of COVID-19. Twelve quality indicators under pre-analytical, analytical and post analytical stage were assessed for the quality by referring it with the select criteria. Missing test request form / specimen (1.36/1000) was the most common inconsistency observed during the assessment of pre-analytical indicators followed by specimen inadequacy (0.194/1000), duplicate specimen referral forms (SRF) generated in ICMR portal (0.277/1000) and color change in the viral transport medium. In analytical phase, non-conformity with QC was seen in 2.83/1000 samples. In post analytical phase, excessive turnaround time was seen in 0.75/1000 samples followed by revised reports due to transcription error (0.38/1000) and duplicate reports (0.13/1000). The results of assessment of quality indicators in the molecular laboratory explicitly supports that laboratory could keep the incidence of errors to the minimum level by following proper corrective and preventive measures. Thus, catering quality laboratory services during devastated COVID pandemic year.

Sensitivity of Rapid Antigen Testing and RT-PCR Performed on Nasopharyngeal Swabs versus Saliva Samples in COVID-19 Hospitalized Patients: Results of a Prospective Comparative Trial (RESTART)

Saliva sampling could serve as an alternative non-invasive sample for SARS-CoV-2 diagnosis while rapid antigen tests (RATs) might help to mitigate the shortage of reagents sporadically encountered with RT-PCR. Thus, in the RESTART study we compared antigen and RT-PCR testing methods on nasopharyngeal (NP) swabs and salivary samples. We conducted a prospective observational study among COVID-19 hospitalized patients between 10 December 2020 and 1 February 2021. Paired saliva and NP samples were investigated by RT-PCR (Cobas 6800, Roche-Switzerland, Basel, Switzerland) and by two rapid antigen tests: One Step Immunoassay Exdia® COVID-19 Ag (Precision Biosensor, Daejeon, Korea) and Standard Q® COVID-19 Rapid Antigen Test (Roche-Switzerland). A total of 58 paired NP-saliva specimens were collected. A total of 32 of 58 (55%) patients were hospitalized in the intensive care unit, and the median duration of symptoms was 11 days (IQR 5-19). NP and salivary RT-PCR exhibited sensitivity of 98% and 69% respectively, whereas the specificity of these RT-PCRs assays was 100%. The NP RATs exhibited much lower diagnostic performance, with sensitivities of 35% and 41% for the Standard Q® and Exdia® assays, respectively, when a wet-swab approach was used (i.e., when the swab was diluted in the viral transport medium (VTM) before testing). The sensitivity of the dry-swab approach was slightly better (47%). These antigen tests exhibited very low sensitivity (4% and 8%) when applied to salivary swabs. Nasopharyngeal RT-PCR is the most accurate test for COVID-19 diagnosis in hospitalized patients. RT-PCR on salivary samples may be used when nasopharyngeal swabs are contraindicated. RATs are not appropriate for hospitalized patients.

Study of vertical transmission of COVID-19 infection in COVID-19 positive obstetrical patients by comparing amniotic fluid and immediate neonatal COVID-19 RT-PCR

Background: The objective of this study was to estimate the incidence of vertical transmission COVID-19 by RT-PCR.Methods: In this hospital based prospective study, all COVID-19 positive pregnant women admitted in COVID ward in Muzaffarnagar medical college and hospital from April 2020 to January 2021 were included. A detailed history and examination was done and all routine investigations were done as per protocol. Samples were taken from amniotic fluid during vaginal delivery or caesarean section and collected in viral transport medium. Sample were also collected as nasal and oropharyngeal swab from neonate immediately after birth and sent for COVID 19 RT-PCR.Results: Out of total 50 cases; 43 (86%) neonates were delivered via LSCS and 7 (14%) by normal delivery, out of these 41 (82%) neonates were normal; 4 (8%) were IUGR, 2 (4%) were IUD, 2 (4%) were pre-term and 1 (2%) neonatal death. All 50 amniotic fluid as well as nasal and oropharyngeal samples of all neonates were negative by RT-PCR.Conclusions: Low vertical transmission may be due to the fact that antibodies are produced by mother that crosses the placenta and saves the fetus or there are highly specific immunological mediators in the placenta that do not allow the infection to pass to the fetus.

SARS COV-2 PANDEMIC: EXPERIENCE AT REFERENCE VIROLOGY LABORATORY SHORT RUNNING TITLE: SARS COV-2 PANDEMIC EXPERIENCE

Objective: To share our large scale SARS CoV-2 PCR test experience in Northern Pakistan. Study Design: Prospective observational study. Place and Duration of Study: Virology Department, Armed Forces Institute of Pathology, Rawalpindi, from Feb to Dec 2020. Methodology: All the patients reporting to COVID-19 desk both indoor and outdoor were included in study. Nasopharyngeal swab specimen was taken from the patients arriving at reception. For hospitalized patient’s samples were received at reception placed in viral transport medium maintaining cold chain. Results: Among 193656 samples tested for SARS CoV-2 RNA by RT-PCR, 24338 (12.6%) were found positive and 169318 (87.4%) were negative. Mean age of patients was 38.25 ± 16.73 (1-110 years). 138781 (71.7%) were males and 54875 (28.3%) were females. 109765 (56.7%) samples were received from in patient department and 83891 (43.3%) samples were received from outpatient department. Highest number of cases (n=6224) seen during month of June followed by 5813 cases during May and 4786 cases during November (p-value <0.001). Most of the positive cases were in age group 21-40 years; 11122 (6%), followed by age group 41-60 years; 8133 (4.2%). More positive samples 14890 (7.7%) were received from in patient department and males 17928 (9.3%) were affected more than females. Conclusion: The two peaks of COVID-19 pandemic in Pakistan were observed during the months of May to July and again during October to December. Most positive patients in our setup were males in age group 21-40 years as this age group is more exposed to external environment.