Viral Transport Medium Research Articles

Sequencing SARS-CoV-2 from antigen tests.

Genomic surveillance empowers agile responses to SARS-CoV-2 by enabling scientists and public health analysts to issue recommendations aimed at slowing transmission, prioritizing contact tracing, and building a robust genomic sequencing surveillance strategy. Since the start of the pandemic, real time RT-PCR diagnostic testing from upper respiratory specimens, such as nasopharyngeal (NP) swabs, has been the standard. Moreover, respiratory samples in viral transport media are the ideal specimen for SARS-CoV-2 whole-genome sequencing (WGS). In early 2021, many clinicians transitioned to antigen-based SARS-CoV-2 detection tests, which use anterior nasal swabs for SARS-CoV-2 antigen detection. Despite this shift in testing methods, the need for whole-genome sequence surveillance remains. Thus, we developed a workflow for whole-genome sequencing with antigen test-derived swabs as an input rather than nasopharyngeal swabs. In this study, we use excess clinical specimens processed using the BinaxNOW™ COVID-19 Ag Card. We demonstrate that whole-genome sequencing from antigen tests is feasible and yields similar results from RT-PCR-based assays utilizing a swab in viral transport media.

DirectDetect SARS-CoV-2 Direct Real-Time RT-PCR Study Using Patient Samples.

COVID-19 is an infectious disease that caused a global pandemic affecting people worldwide. As disease detection and vaccine rollout continue to progress, there is still a need for efficient diagnostic tools to satisfy continued testing needs. This preliminary study evaluated a novel SARS-CoV-2 diagnostic test called DirectDetect SARS-CoV-2 Direct Real-time reverse transcriptase polymerase chain reaction (RT-PCR) based on a limited sample size of 24 respiratory samples from 14 SARS-CoV-2-positive patients. The test is advantageous compared to others on the market since it does not require viral transport medium or viral RNA extraction prior to nucleic acid amplification and detection. This capability transforms the hours-long sample preparation time into a minutes-long procedure while also eliminating the need for many costly reagents which may be difficult to obtain during the surge in nucleic acid-based testing during the pandemic. The results show a positive agreement of 94.7, 100, and 94.7% between dry sample swabs, treated samples, and untreated samples tested using the DirectDetect SARS-CoV-2 Direct Real-time RT-PCR compared to tests used in a clinical laboratory, respectively. The findings indicate that DirectDetect can be used for multiple different sample types while reducing the number of reagents and time needed for diagnosis. Although this study shows promising results using the DirectDetect results, further validation of this test using a larger sample set is required to assess the true performance of this test.

SARS-CoV-2 Direct Real Time PCR without RNA Extraction: Appropriate Method or Not?

Real time PCR (RT-PCR) detection method is the widely used for COVID-19 virus detection. This includes sample collection in viral transport medium (VTM), viral RNA extraction followed by detection of virus using fluorescence dye-based system using RT-PCR machine. Several studies have demonstrated a new method which replaces the extraction step by a simple method involving DTT and Proteinase-K and heat treatment. ICMR and few other governing bodies have approved such protocols but are they appropriate in clinical context? In present study, we tried to evaluate one such protocol by using ICMR and WHO approved COVID-19 detection protocol (of CoviPath™ COVID-19 RT-PCR Kit) by replacing RNA extraction step. We used 228 clinical COVID-19 samples for studying method which includes 176 positive (CT values from 14 to 23; 24 to 31 and 32 to 37 were considered as high, moderate and low positive respectively) and 52 negative nasopharyngeal and oropharyngeal swabs samples. We got 100% concordant results with negative samples and 92% concordant and 8% non-concordant results for positive samples. Non-concordant results are with low positive samples. Low level of positivity in the samples could indicate the initial/end stage of COVID-19 disease. If they are at the initial stage, they can be the potential carrier and spread the disease. Authors believe that direct methods can be used for screening bit not for diagnosis of COVID-19 disease.

Effect of not vortexing nasopharyngeal and throat swabs on SARS-CoV-2 nucleic acid detection: A pilot study

The processing of swabs for respiratory virus detection involves vortexing while still in the viral transport medium (VTM). The effect of not vortexing swabs prior to analysis has not been studied extensively for SARS-CoV-2 detection, and presents an opportunity to improve pre-analytic laboratory workflow. We aimed to assess the impact of not vortexing nasopharyngeal/throat swabs submitted in VTM for SARS-CoV-2 testing. To assess the impact of not vortexing swabs, 277 swab samples were tested for SARS-CoV-2 RNA in paired vortexed and non-vortexed aliquots using eight routine nucleic acid amplification assays. We compared the qualitative (positive/negative) and semi-quantitative (cycle threshold, Ct) results. Following discordant analysis, all but one non-vortexed sample had the same qualitative result as the vortexed sample. 27.4 % of samples were SARS-CoV-2 positive. Comparison of Ct values revealed an apparent reduction in human cellular nucleic acid in the non-vortexed samples (mean Ct values of 24.0 and 26.5 for vortexed and non-vortexed samples, respectively, p < 0.0001) and increased Ct values for non-vortexed samples using a laboratory-developed SARS-CoV-2 assay (mean Ct values of 4.1 and 4.2 for vortexed and non-vortexed samples, respectively; p < 0.0001), but this was not observed for a more automated commercial SARS-CoV-2 assay (mean Ct values of 15.2 for both vortexed and non-vortexed samples, respectively; p = 0.68). While vortexing swabs appears to improve the recovery of cellular material, it does not have an appreciable impact on the qualitative sensitivity of SARS-CoV-2 nucleic acid tests, which may support omission of this step and simplification of front-end sample processing.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). “Extraction-less” or “direct” real time–reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Predicting Direct-Specimen SARS-CoV-2 Assay Performance Using Residual Patient Samples.

BackgroundDiagnostic sensitivities of point-of-care SARS-CoV-2 assays depend on specimen type and population-specific viral loads. Evaluation of these assays require ‘direct’ specimens from paired-swab studies rather than more accessible residual specimens in viral transport media (VTM).MethodsResidual VTM and limit-of-detection studies were conducted on Abbott ID NOW™ COVID-19, Quidel Sofia 2™ SARS Antigen FIA, and DiaSorin Simplexa™ COVID-19 Direct assays, with cycle threshold (CT) adjustments to approximate direct-specimen testing based on gene-target doubling each PCR cycle. Logistic regression was used to model assay performance by specimen CT. These models were applied to CT distributions of symptomatic and asymptomatic populations presenting to emergency services to predict the percent of specimens that would be detected by each assay. A 96-sample paired-swab study was conducted to confirm model results.ResultsWhen using direct nasopharyngeal samples and fit with either VTM or limit-of-detection data, percent positivities for ID NOW (symptomatic 94.9%/97.4%; asymptomatic 88.4.0%/89.6%) and Simplexa (symptomatic 97.8%/97.2%; asymptomatic 91.1%/90.8%) were predicted to be similar. Likewise, fit with VTM data, percent positivities for ID NOW with direct nasal specimens (symptomatic 77.8%; asymptomatic 64.5%) and Sofia 2 with direct nasopharyngeal specimens (symptomatic 76.6%, asymptomatic 60.3%) were similar. The paired-swab study comparing direct nasopharyngeal specimens on ID NOW and nasopharyngeal VTM specimens on Simplexa showed 99% concordance.ConclusionsAssay performance can be modeled as dependent on viral load, fit using laboratory bench study results, and adjusted to account for direct-specimen testing. When using nasopharyngeal specimens, direct testing on Abbott ID NOW and VTM testing on DiaSorin Simplexa have similar performance.

Evaluation of the LumiraDx SARS-CoV-2 antigen assay for large-scale population testing in Senegal

Purpose: Real-time reverse-transcription polymerase chain reaction (RT-PCR)-based testing remains the gold standard for the diagnosis of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the high diagnosis demand of SARS-CoV-2 and the limited resources for RT-PCR testing, especially in Low-Income Countries (LICs), antigen-based methods are being considered as an option. The aim of this study was to assess the performance of LumiraDx SARS-CoV-2 antigen assay for large population screening compared to RT-PCR. Methods: This evaluation was conducted on 4146 participants including travelers and participants under household survey and vaccine evaluation studies before injection of the first dose. Oropharyngeal and nasopharyngeal swaps were collected from each participant into 2 mL of viral transport medium (VTM) and 400 μl of VTM were used to assess the performance of LumiraDx SARS-CoV-2 antigen assay, compared to RT-PCR. Results: The prevalence of SARS-CoV-2 of the cohort was 4.5% with RT-PCR and 4.1% with LumiraDx antigen test. Compared to the RT-PCR, the sensitivity and specificity of the LumiraDx antigen SARS-CoV-2 test were 82,7% [95% CI 74.1-89,7] and 99.9% [95% CI 99.6-99.9] respectively. Given the RT-PCR threshold cycle (Ct) range, the sensitivity was 92.1% [95% CI 84.6-96.3] when the Ct value was below or equal 33 cycles, and 38.1% [95% CI 18.9-61.3] when it was above 33 cycles. The inter-rater reliability showed a kappa coefficient of 0.88 when considering all the patients and 0.94 for Ct values below 33 cycles. Conclusion: Our data have shown that the LumiraDx platform can be considered for large-scale testing of SARS-CoV-2.

Screening for cytomegalovirus shedding in vagina and saliva: Significant differences between biological fluids, swab types and storage durations in DNA recovery.

Cytomegalovirus (CMV) shedding in genital and oral secretions during pregnancy is associated with adverse outcomes. Sample collection methods between studies are not uniform and currently there are limited data on the impact of biological fluids, swab types and storage durations on the detection of CMV DNA. To evaluate the absorption efficiency and CMV DNA recovery of various commercially available swabs in vaginal and saliva fluids. The absorption volume of different swab types was evaluated. The recovery of CMV DNA over time from vaginal and saliva fluids, and vaginal and saliva swabs, was evaluated by PCR measurements of samples spiked with CMV standard. Absorption efficiency of swabs varied significantly. The duration of storage did not affect CMV DNA recovery from vaginal fluid or swabs, but did significantly affect CMV DNA recovery from neat saliva fluid and saliva swabs stored dry or in viral transport media (VTM). Flocked swab/eNAT media recovered the highest amount of CMV DNA from both fluids. In saliva, flocked swab/eNAT media and polyester swab/cobas media demonstrated a higher CMV DNA recovery than foam swab/VTM. 25% of dry saliva foam swabs were falsely negative for CMV DNA. Recovery of CMV DNA is dependent on sample type and swab type used. Flocked swab/eNAT media and polyester swab/cobas media appear acceptable, though flocked swab/eNAT media was superior demonstrating the best recovery of CMV DNA. For saliva, foam swabs stored dry or in VTM was shown to be inferior to flocked or polyester swabs.

Evaluation of Using Direct Viral Transport Medium Samples without Nucleic Acid Isolation for SARS-CoV-2 Diagnosis by RT-PCR.

Diagnosis with reverse transcriptase polymerase chain reaction (RT-PCR) test is a very important step for the control of the COVID-19 pandemic. The aim of this study is to compare the RT-PCR results of the samples taken directly from the viral transport medium (VTM) without extraction with the RT-PCR results of two different extraction methods, one automated and the other manual, in the diagnosis of COVID-19. Among the respiratory tract samples sent to Sakarya Training and Research Hospital Microbiology Laboratory for COVID-PCR study, 20 negative and 43 positive samples with different cycle threshold (CT) values were included in the study. Both manual nucleic acid isolation with the vNAT isolation kit (Bioeksen, Turkey) and automatic nucleic acid isolation with the EZ1 Virus Mini Kit v2.0 in the isolation device were performed simulta-neously from the patient samples included in the study and the results were compared. The mean Ct values of the samples were found to be 21.58 using manual vNAT as the extraction method, 17.63 using the automated magnetic bead method, and 21.45 in PCR from direct VTM without extraction. When the automatic magnetic beads extraction method was taken as the reference method, the sensitivity of direct PCR was 97.3%, the specificity was 95%, the positive predictive value was 97.3%, and the negative predictive value was 95%. Phi coefficients were found to be 0.927 between vNAT and direct PCR, 1 between vNAT and EZ1, and 0.922 between direct PCR and EZ1. Direct PCR has advantages such as eliminating RNA extraction and purification steps, providing a shorter detection time, and using less labor and less consumables without reducing the diagnostic accuracy. It is thought that this method can help as a useful process management for the control of the epidemic in countries with limited resources.

Severe Acute Respiratory Syndrome Coronavirus-2 - A Surge of CoronaVirus Disease-2019: An Epidemiological Study in Coimbatore District.

Context:Coronavirus disease-2019 (COVID-19) is an ongoing pneumonia-like cluster syndrome which originated in Wuhan city of China and is still now on escalation, causing severe outbreaks all over the world. Being a ribonucleic acid (RNA) virus which has the low proofreading RNA-dependent RNA polymerase leads to many mutations and that serves as the major cause for the progress of the disease. As per the recent research works done, 99% of COVID-19 severe acute respiratory syndrome coronavirus (SARS-CoV-2) are due to pangolin-associated coronavirus which causes the super spreading events of coronavirus. SARS-CoV-2 was identified in the nasopharyngeal swabs received in the viral transport medium at optimum temperature.Materials and Methods:The tests were conducted for a time period of 1 year from July 2020 to June 2021. A total of 77,824 samples were tested in the laboratory as per ICMR guidelines using approved RNA extraction kits and polymerase chain reaction kits.Results:In the total of 77,824 samples tested in our laboratory, 14174 positives were identified. In that, about seven positive cases (0.004%) were identified in the month of July 2020 which increased to the maximum in September 2020 to about 865 positive cases (6%) which is the peak of first wave COVID-19 in Coimbatore district, Tamil Nadu. Out of 77,824 samples tested, the actual cumulative laboratory-confirmed positive cases of about 14174 were identified. In that, 7731 (55%) male positive cases were identified, 6171 (43%) female positive cases were identified, and 270 (2%) children who were below 12 years of age also were tested positive.Conclusions:The findings of the study indicated a high predominance of SARS-CoV-2 infection in the male gender population when compared to females and children below 12 years of age in Coimbatore district as of June 2021. The surge of cases was high in September 2020 as well as in May 2021, indicating the first and second wave of COVID-19.

Effects of Non-Pharmacological Interventions on Respiratory Viruses Other Than SARS-CoV-2: Analysis of Laboratory Surveillance and Literature Review From 2018 to 2021.

BackgroundSince the global coronavirus disease 2019 (COVID-19) pandemic, non-pharmacological interventions (NPIs) such as extensive and comprehensive hand hygiene, mask-wearing, and social distancing have been implemented globally. This study aimed to investigate changes in respiratory viruses other than severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that occurred following the implementation of these NPIs.MethodsFrom January 2018 to December 2021, influenza-like illness patient specimens and specimens from the Korea Influenza and Respiratory Viruses Surveillance System were analyzed at the Incheon Metropolitan City Institute of Public Health and Environment. Oropharyngeal or nasopharyngeal swab samples from respiratory infection patients were transferred in a virus transport medium at 4°C. After RNA or DNA extraction, respiratory virus-specific genes for human influenza virus (IFV), adenovirus (ADV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), human rhinovirus (hRV), human coronavirus, human bocavirus, and human metapneumovirus were detected by individual real-time reverse transcription polymerase chain reaction.ResultsA total 3,334 samples were collected. After NPI was implemented, the detection of respiratory viruses other than SARS-CoV-2 decreased overall. The yearly detection rate of respiratory viruses was decreased from 69.5% (399/574) in 2018 and 73.3% (505/689) in 2019 to 19.8% (206/1,043) in 2020 and 34.9% (365/1,028) in 2021. The epidemic was more prominent in respiratory viruses such as IFV and RSV, which were considered dominant viruses, especially those with viral envelopes. Among viruses that were not considered dominant, hRV showed no clear change before and after NPI, while PIV showed a rapid increase compared to the existing dominant viruses between October–December 2021, after the increase in the number of gatherings started at the end of September and the “Relaxing COVID19 and mitigation policy,” which was implemented on November 1.ConclusionNPI seems to have influenced the isolation and transmission of respiratory viruses in South Korea. In the future, additional studies focusing on the isolation and transmission patterns of respiratory viruses following NPI are needed.

On-chip integrated graphene aptasensor with portable readout for fast and label-free COVID-19 detection in virus transport medium.

Graphene field-effect transistor (GFET) biosensors exhibit high sensitivity due to a large surface-to-volume ratio and the high sensitivity of the Fermi level to the presence of charged biomolecules near the surface. For most reported GFET biosensors, bulky external reference electrodes are used which prevent their full-scale chip integration and contribute to higher costs per test. In this study, GFET arrays with on-chip integrated liquid electrodes were employed for COVID-19 detection and functionalized with either antibody or aptamer to selectively bind the spike proteins of SARS-CoV-2. In the case of the aptamer-functionalized GFET (aptasensor, Apt-GFET), the limit-of-detection (LOD) achieved was about 103 particles per mL for virus-like particles (VLPs) in clinical transport medium, outperforming the Ab-GFET biosensor counterpart. In addition, the aptasensor achieved a LOD of 160 aM for COVID-19 neutralizing antibodies in serum. The sensors were found to be highly selective, fast (sample-to-result within minutes), and stable (low device-to-device signal variation; relative standard deviations below 0.5%). A home-built portable readout electronic unit was employed for simultaneous real-time measurements of 12 GFETs per chip. Our successful demonstration of a portable GFET biosensing platform has high potential for infectious disease detection and other health-care applications.

Elevated Risk of COVID-19 Infection among People Living with HIV in Mumbai—A Retrospective Study

Introduction: According to Joint United Nations Program on HIV/AIDS 2021, people living with HIV (PLHIV) have higher comorbidities and experience more severe outcomes from COVID-19 as compared to non-PLHIV. Studies from India reporting on the risk of COVID-19 acquisition among PLHIV are lacking. In this study, we attempted to estimate the risk of COVID-19 infection among PLHIV at our high throughput COVID-19 testing facility in Mumbai, India. Methods: A retrospective observational study was undertaken of suspected COVID-19 cases between April and December 2020. The unadjusted and adjusted odds ratios with 95% confidence interval for the risk factors were calculated by logistic regression using the SPSS software (version 19). Results: A total of 58,811 naso/oropharyngeal swabs in viral transport medium samples were tested for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcriptase polymerase chain reaction from April 22, 2020 to December 31, 2020. Out of these, 18,508 (31.4%) samples were found positive for SARS-CoV-2 infection. Of the total samples tested, 65 (0.11%) individuals had reported history of HIV as an underlying medical condition. Out of the total COVID-19 positive patients, 52 (0.28%) had HIV infection. Conclusion: Our study highlights an increased susceptibility of PLHIV to COVID-19 infection, which necessitates active monitoring undertaken of this population for optimal disease management.

DETECTION OF SARS-COV-2 FROM SURFACE OF PATIENTS’ LEFTOVER FOOD PACKAGES AT A COVID-19 QUARANTINE CENTRE

In healthcare facilities, food waste and its packaging are mostly managed as non-infectious general waste. However, waste from SARS-CoV-2 positive patients, are treated as medical waste as they may be contaminated by the virus. We investigated the possibility of SARS-CoV-2 contamination from positive COVID-19 patients to their leftover food packages at a quarantine centre. Food packages surface was swabbed using prewetted cellular foam, placed into viral transport media and analysed using real time reverse transcription polymerase chain reaction. SARS-CoV-2 RNA was detected in two samples (4.5%) from asymptomatic patients who were at day-2 positive SARS-CoV-2 with cycle threshold (Ct) value (RdRp/E), 34.96/35.72 and 37.1/36.48 respectively. Detection of SARS-CoV-2 supports that there is contamination to the waste. These poses risk of exposure as SAR-COV-2 survive on the surfaces, thus, safe handling and disposal of food waste should be maintained. However, further study involving viral culture should be explored to determine the viability of the SARS-CoV-2 from leftover food packages

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.

Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.

Detection of SARS-CoV-2 Virus Amplification Using a Crumpled Graphene Field-Effect Transistor Biosensor.

The rapid and unexpected spread of SARS-CoV-2 worldwide has caused unprecedented disruption to daily life and has brought forward critical challenges for public health. The disease was the largest cause of death in the United States in early 2021. Likewise, the COVID-19 pandemic has highlighted the need for rapid and accurate diagnoses at scales larger than ever before. To improve the availability of current gold standard diagnostic testing methods, the development of point-of-care devices that can maintain gold standard sensitivity while reducing the cost and providing portability is much needed. In this work, we combine the amplification capabilities of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) techniques with high-sensitivity end-point detection of crumpled graphene field-effect transistors (cgFETs) to develop a portable detection cell. This electrical detection method takes advantage of the ability of graphene to adsorb single-stranded DNA due to noncovalent π–π bonds but not double-stranded DNA. These devices have demonstrated the ability to detect the presence of the SARS-CoV-2 virus in a range from 10 to 104 copies/μL in 20 viral transport medium (VTM) clinical samples. As a result, we achieved 100% PPV, NPV, sensitivity, and specificity with 10 positive and 10 negative VTM clinical samples. Further, the cgFET devices can differentiate between positive and negative VTM clinical samples in 35 min based on the Dirac point shift. Likewise, the improved sensing capabilities of the crumpled gFET were compared with those of the traditional flat gFET devices.

431. SARS-CoV-2 Environmental Contamination in Hospitalized COVID-19 Patients’ Rooms

BackgroundThe correlation between SARS-CoV-2 RNA and infectious viral contamination of the hospital environment is poorly understood. Methodshoused in a dedicated COVID-19 unit at an academic medical center. Environmental samples were taken within 24 hours of the first positive SARS-CoV-2 test (day 1) and again on days 3, 6, 10 and 14. Patients were excluded if samples were not obtained on days 1 and 3. Surface samples were obtained with flocked swabs pre-moistened with viral transport media from seven locations inside (bedrail, sink, medical prep area, room computer, exit door handle) and outside the room (nursing station computer). RNA extractions and RT-PCR were completed on all samples. RT-PCR positive samples were used to inoculate Vero E6 cells for 7 days and monitored for cytopathic effect (CPE). If CPE was observed, RT-PCR was used to confirm the presence of SARS-CoV-2.ResultsWe enrolled 14 patients (Table 1, Patient Characteristics) between October 2020 and May 2021. A total of 243 individual samples were obtained – 97 on day 1, 98 on day 3, 34 on day 6, and 14 on day 10. Overall, 18 (7.4%) samples were positive via RT-PCR – 9 from bedrails (12.9%), 4 from sinks (11.4%), 4 from room computers (11.4%) and 1 from the exit door handle (2.9%). Notably, all medical prep and nursing station computer samples were negative (Figure 1). Of the 18 positive samples, 5 were from day 1, 10 on day 3, 1 on day 6 and 2 on day 10. Only one sample, obtained from the bedrails of a symptomatic patient with diarrhea and a fever on day 3, was culture-positive (Figure 2). Table 1. Patient Characteristics Figure 1. Proportion of RT-PCR Positive Samples by Sample Day and Location Figure 2. Cell cultures of negative control (left) and CPE positive sample (right) ConclusionOverall, the amount of environmental contamination of viable SARS-CoV-2 virus in rooms housing COVID-19 infected patients was low. As expected, more samples were considered contaminated via RT-PCR compared to cell culture, supporting the conclusion that the discovery of genetic material in the environment is not an indicator of contamination with live infectious virus. More studies including RT-PCR and viral cell culture assays are needed to determine the significance of discovering SARS-CoV-2 RNA versus infectious virus in the clinical environment.Disclosures David J. Weber, MD, MPH, PDI (Consultant)

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 patients 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.

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.