Search

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for the generation of accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, the impact on SARS-CoV-2 detectability by reverse transcriptase PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2–positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on reverse transcriptase PCR detection. SARS-CoV-2 RNA was still reliably detected by reverse transcriptase PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The change of Ct value per day was 0.023 to 0.046 (±0.018 to 0.019). In addition, viral RNA was still detected after 15 freeze-thaw cycles, with a change of Ct value of 0.106 to 0.197 (±0.009 to 0.061) per freeze-thaw cycle. However, compared with storage at 4°C, RNA was significantly less detectable when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels, as measured by reverse transcriptase PCR assays, are significantly, but not substantially, altered by prolonged unrefrigerated storage of up to 21 days at temperatures ranging up to 35°C or after repeated freeze-thaw cycles.

Introduction The sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained. Methods VTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR. Results The best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4 °C, and tested within 48 h. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature. Discussion The manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS-CoV-2, and for the testing of vaccine escape mutants.

Context: In the absence of effective treatment or vaccine, the current strategy for the prevention of further transmission of severe acute respiratory syndrome (SARS) CoV-2 (COVID-19) infection is early diagnosis and isolation of cases. The diagnosis of SARS-CoV-2 is done by detecting viral RNA in the nasopharyngeal and throat swabs by real-time polymerase chain reaction (PCR). Many commercial assays are now available for performing the PCR assay. Aims: The aim was to evaluate the performance of the SD Biosensor nCoV real-time detection kit with the real-time PCR kit provided by the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune (NIV Protocol). Subjects and Methods: A total of 253 pairs of nasopharyngeal-oropharyngeal swabs combined in a single viral transport medium were tested for viral RNA by both the protocols. The sensitivity and specificity of the SD Biosensor were calculated considering the ICMR-NIV kit as the gold standard. Matched pairs of recorded cycle threshold values (Ct values) were compared by Pearson’s correlation coefficient. Results: Concordant COVID-19 negative and positive PCR results were reported for 113 and 77 samples, respectively. The SD Biosensor kit additionally detected 62 cases, which were found negative by the NIV protocol. In all discordant positive results by the SD Biosensor kit, the average Ct values were higher than the concordant positive results. A total of forty samples tested positive for E gene by SD Biosensor and having Ct values 32 were all found negative by the NIV protocol. Conclusions: The results highlight the need for careful evaluation of commercial kits before being deployed for screening of COVID-19 infections.

Current conventional detection of SARS-CoV-2 involves collection of a patient sample with a nasopharyngeal swab, storage of the swab during transport in a viral transport medium, extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). We developed a simplified preparation method using a chelating resin, Chelex, that obviates RNA extraction during viral testing. Direct detection RT-qPCR and digital-droplet PCR was compared to the current conventional method with RNA extraction for simulated samples and patient specimens. The heat-treatment in the presence of Chelex markedly improved detection sensitivity as compared to heat alone, and lack of RNA extraction shortens the overall diagnostic workflow. Furthermore, the initial sample heating step inactivates SARS-CoV-2 infectivity, thus improving workflow safety. This fast RNA preparation and detection method is versatile for a variety of samples, safe for testing personnel, and suitable for standard clinical collection and testing on high throughput platforms., Graphical Abstract

Introduction: Influenza testing is carried out all over the world but in developing countries including India, there is paucity in timely identification of new strains/type of influenza viruses and their active surveillance. Aim: This study aims to determine the record based analysis for the prevalence of influenza infection and circulating subtype, in and around Kangra District of Himachal Pradesh from January 2016 to December 2017. Materials and Methods: Nasopharyngeal and throat swabs were collected in Viral Transport Medium and RNA were extracted by using commercially available kit (Qiagen). Real Time polymerase chain reaction (RTPCR) was carried out to amplify the influenza type A, H1N1/Pdm 2009, H3N2 and influenza B. Results: Total of 281 cases for influenza infection in the year of 2016 and 2017, 133 (47.3%) cases were positives for influenza A, 43 (15.3%) positives for influenza H1N1/Pdm2009, 8 (2.84%) positives for H3N2 and 3 (1.06%) positives for influenza B. Nine deaths were reported in the year of 2017 due to H1N1/Pdm 2009 infection while no mortality was observed in year 2016 at DRPGMC Tanda. Discussion and Conclusion: Influenza infection is more common in age group of 18 to 50 years followed by age of > 50 years. Active testing and data interpretation at all influenza testing laboratories throughout India will help the health authorities to expedite the need of influenza vaccination. Further, aggregating the data would help in understanding the circulation of influenza virus subtype at particular geographical areas. Keywords: Influenza infection, Real time polymerase chain reaction, Data analysis.

Background For infection control measures, rapid accurate diagnostics on admission of patients with suspected seasonal influenza is crucial. Objective Prospective comparison of three rapid molecular tests for detection of influenza A/B RNA. Study design Outpatients presenting at the Medical emergency department of Graz University Hospital with influenza-like illness and a requirement for hospitalization (n = 312) were studied. Nasopharyngeal swabs were collected with the 3 mL-version of the UTM™ Viral Transport Medium (Copan). Specimens were tested for influenza A and B RNA using the Alere™ i Influenza A & B (Abbott), the cobas® Influenza A/B (Roche), and the Xpert® Xpress Flu/RSV (Cepheid) tests. Results were compared to those obtained from the same specimen by the Influenza A/B R-GENE® (bioMerieux) test based on real-time PCR as reference method. Results Overall sensitivities of the Abbott, Roche, and Cepheid tests were 90.5%, 96.0%, and 97.0%, overall specificities 99.4%, 97.6%, and 98.2% respectively. With the Abbott and the Cepheid tests, all specimens gave valid results, while the Roche test showed invalid results in 37 (12.1%) specimens. Total time to result for the Abbott, Roche, and Cepheid tests was 18 min, 22 min, and 32 min respectively. Conclusions The Abbott test lacked sensitivity, the Roche test was impaired by a high number of invalid results. Overall, despite the longest total time to result, the Cepheid test showed the best performance to detect influenza virus RNA in symptomatic patients presenting at an emergency unit in this study.

Highlights It is best to store COVID‐19 swab samples in VTM to get the most reliable PCR results.Positive COVID‐19 swab samples can be stored for at least 5 days at both room temperature and 4°C without loss of positivity.Samples with high viral loads with CTs below 25 can be stored at both room temperature and 4°C without loss of positivity for up to 12 days., Reliable and rapid detection of severe acute respiratory syndrome coronavirus 2 in laboratory setting is critical to control the pandemic. We aimed to an evaluated polymerase chain reaction (PCR) efficiency of nasopharyngeal swabs stored in viral transport medium (VTM) in different temperatures. Ninety swabs taken into VTM were analyzed at the first hour, then divided into two groups with similar numbers of positive and negative samples. Positive samples of each group were also subgrouped according to Fam CT values as low CT (

Phi 6 (Φ6) bacteriophage is a proposed surrogate to study pathogenic enveloped viruses including SARS-CoV-2-the causative agent of COVID-19-based on structural similarities, BSL-1 status, and ease of use. To determine the role of virus-contaminated hands in disease transmission, an enhanced understanding of buffer and method performance for Φ6 recovery needs to be determined. Four buffer types and three methodologies were investigated for the recovery of Φ6 from human fingerpads over a 30 min duration. Phosphate buffered saline (PBS), PBS + 0.1 % Tween, 0.1 M glycine + 3% beef extract, and viral transport medium were evaluated as buffers for recovery of Φ6 via a dish, modified glove juice, and vigorous swabbing method. Φ6 concentrations on fingerpads were determined at 0-, 5-, 10-, and 30-min post-inoculation. While there were observed differences in virus recovery across buffer and method types depending on the time point, log PFU recovery based on buffer type or methodology was not significantly different at any time point (P > 0.05). The results presented in this study will allow for future work on Φ6 persistence, transfer between hands and surfaces, and efficacy of hand hygiene methods to be performed using a well-characterized and validated recovery method.

Aims The objective of the present study was to investigate the presence of SARS-CoV-2 in aerosol and COVID-19 contamination distance asssociated with ultrasonic scaling and tooth preparation. Methods Twenty-four patients with COVID-19 were included in this study. Removal of supragingival plaque with ultrasonic instruments for 10 minutes and high-speed air-turbine using for the simulation of cutting the maxillary right canine tooth with a round diamond bur for 5 minutes were performed. Patients were randomly assigned to 2 groups: In group A, medium-volume suction was used during treatment. In group B, high-volume suction with an aerosol cannula was added to medium-volume suction. Prior to treatment, 5 glass petri dishes containing viral transport medium were placed in the operating room. After treatment, petri dishes were immediately delivered to a microbiology laboratory for real-time polymerase chain reaction (RT-PCR) analysis. Results RT-PCR test results were negative for all specimens in group B. However, 5 positive test results for COVID-19 were detected in group A specimens. Conclusions Suction with an aerosol cannula is very important to prevent COVID-19 viral contamination via aerosol. In addition, a high-volume suction capacity (air volume) of 150 mm Hg or 325 L/min is sufficient for elimination of viral contamination. Thus, high-volume suction should be used during dental treatments in COVID-19 patients.

Back groundEarlier studies suggested the use of dry swab method for SARS-CoV-2 detection as it does not need VTM and subsequent RNA extraction step making the process cheaper, safer and faster. In this study we explore whether the virus in the dry swab is viable and can be cultured and propagated.MethodSwabs were spiked with SARS-CoV-2 and stored in three different conditions: a) as dry swab (SD, eluted in 1 mL DMEM), b) in 1 mL of Viral Transport Medium (SVTM), and c) in 1 mL of Tris-EDTA buffer (STE). The sample groups were stored either at room temperature (RT, 25°C±1°C) or at 4°C for 1, 4, 8, 12, 24, 48 and 72 hours before being used as viral inoculums for the propagation studies in Vero cells.ResultsThe RT-qPCR data suggests that SD incubated both at RT and 4°C harbors viral particles that are viable and culturable at par with SVTM and STE.ConclusionThe dry swab method, in addition to its advantages in detection of the virus, also renders viable viral particles that can be cultured and propagated.

Current conventional detection of SARS-CoV-2 involves collection of a patient sample with a nasopharyngeal swab, storage of the swab during transport in a viral transport medium, extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). We developed a simplified and novel preparation method using a Chelex resin that obviates RNA extraction during viral testing. Direct detection RT-qPCR and digital-droplet PCR was compared to the current conventional method with RNA extraction for simulated samples and patient specimens. The heat-treatment in the presence of Chelex markedly improved detection sensitivity as compared to heat alone, and lack of RNA extraction shortens the overall diagnostic workflow. Furthermore, the initial sample heating step inactivates SARS-CoV-2 infectivity, thus improving workflow safety. This fast RNA preparation and detection method is versatile for a variety of samples, safe for testing personnel, and suitable for standard clinical collection and testing on high throughput platforms.

Roles of environmental factors in transmission of COVID-19 have been highlighted. In this study, we sampled the high-touch environmental surfaces in the quarantine room, aiming to detect the distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the environmental surfaces during the incubation period of coronavirus disease 2019 (COVID-19) patients. Fifteen sites were sampled from the quarantine room, distributing in the functional areas such as bedroom, bathroom and living room. All environmental surface samples were collected with sterile polyester-tipped applicator pre-moistened in viral transport medium and tested for SARS-CoV-2. Overall, 34.1% of samples were detected positively for SARS-CoV-2. The positive rates of Patient A, B and C, were 46.2%, 0 and 61.5%, respectively. SARS-CoV-2 was detected positively in bedroom and bathroom, with the positive rate of 50.0% and 46.7%, respectively. In contrast, living room had no positive sample detected. Environmental contamination of SARS-CoV-2 distributes widely during the incubation period of CCOVID-19, and the positive rates of SARS-CoV-2 on environmental surfaces are relatively high in bathroom and bedroom., Highlights • This study reports detection of SARS-CoV-2 on the high-touch surfaces of dwelling environment. • Environmental contamination of SARS-CoV-2 distributes widely during the incubation period. • The positive rates of SARS-CoV-2 on environmental surfaces are relatively high in bathroom and bedroom.

Conducting numerous, rapid, and reliable PCR tests for SARS-CoV-2 is essential for our ability to monitor and control the current COVID-19 pandemic.Here, we tested the sensitivity and efficiency of SARS-CoV-2 detection in clinical samples collected directly into a mix of lysis buffer and RNA preservative, thus inactivating the virus immediately after sampling.We tested 79 COVID-19 patients and 20 healthy controls. We collected two samples (nasopharyngeal swabs) from each participant: one swab was inserted into a test tube with Viral Transport Medium (VTM), following the standard guideline used as the recommended method for sample collection; the other swab was inserted into a lysis buffer supplemented with nucleic acid stabilization mix (coined NSLB).We found that RT-qPCR tests of patients were significantly more sensitive with NSLB sampling, reaching detection threshold 2.1±0.6 (Mean±SE) PCR cycles earlier then VTM samples from the same patient. We show that this improvement is most likely since NSLB samples are not diluted in lysis buffer before RNA extraction. Re-extracting RNA from NSLB samples after 72 hours at room temperature did not affect the sensitivity of detection, demonstrating that NSLB allows for long periods of sample preservation without special cooling equipment. We also show that swirling the swab in NSLB and discarding it did not reduce sensitivity compared to retaining the swab in the tube, thus allowing improved automation of COVID-19 tests. Overall, we show that using NSLB instead of VTM can improve the sensitivity, safety, and rapidity of COVID-19 tests at a time most needed.

With increasing demand for large numbers of testing during the coronavirus disease 2019 pandemic, alternative protocols were developed with shortened turn-around time. We evaluated the performance of such a protocol 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 a blinded fashion. Prior to conducting real-time polymerase chain reaction, swabs collected in viral transport medium (VTM) were processed for RNA extraction (reference method) and swabs collected in a dry tube without VTM were incubated in Tris–EDTA–proteinase K buffer for 30 min and heat-inactivated at 98 °C for 6 min (index method). Overall sensitivity and specificity of the index method were 78.9% (95% confidence interval (CI) 71–86) and 99% (95% CI 98–99.6), respectively. Agreement between the index and reference method was 96.8% (k= 0.83,s.e.= 0.03). The reference method exhibited an enhanced detection of viral genes (E, N and RNA-dependent RNA polymerase) with lower Ct values compared to the index method. The index method can be used for detecting severe acute respiratory syndrome corona virus-2 infection with an appropriately chosen primer–probe set and heat treatment approach in pressing time; low sensitivity constrains its potential wider use.

Introduction: Coronavirus Disease 2019 (COVID-19) has been haunting the world since December 2019 and has grown to pandemic proportions from March 2020. Even after a full year of research and study, the most effective way to control the spread of this infection is early diagnosis and isolation of the cases. Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) is considered the standard test all over the world for the diagnosis of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection. All the sample collection guidelines have recommended stringent maintenance of the cold chain for the sample transport. However, it is not possible for the resource constrained developing countries with inadequate infrastructure to comply with these guidelines all the time. Aim: To determine necessity of stringent transport criteria and the effect of temperature on the clinical sensitivity of a RT-PCR assay for diagnosis of SARS-CoV-2 infection. Materials and Methods: In this prospective experimental study conducted in November 2020, 49 positive samples were kept at ambient room temperature and were tested everyday with RT- PCR for the detection of SARS-CoV-2 Ribonucleic Acid (RNA). The samples were also kept under refrigeration at 4°C and were also tested by RT-PCR and the results were compared with their respective counterparts kept at room temperature till nine days. Python Jupiter notebook SciPy and Anaconda software was used for statistical analysis. Results: It was observed that the positivity of the RT-PCR results were not deteriorated till five days and there was no significant deterioration even after nine days of samples being stored at room temperature suggesting that even if the viral RNA itself is not stable outside strict temperature control but small fragment or target genetic sequences are enough for detection of virus by RT-PCR. Conclusion: It is possible to keep samples at this ambient temperature for five days without any loss of positivity in RT-PCR.

IntroductionThe sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained.MethodsVTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR (qPCR).ResultsThe best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4oC, and tested within 48 hours. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature (RT).DiscussionThe manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS--CoV-2, and for the testing of vaccine escape mutants.

The current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being deployed to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N) gene, we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65°C for 30 minutes, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N=51), and all positives with Ct less than 20 (N=24), 65% of positives with Ct between 20-30 (N=17), and no positives with Ct greater than 30 (N=9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources and gold standard tests for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results – “glass half empty” – in exchange for reliable detection of those with high levels of virus within an hour, using

INTRODUCTION: We describe cytologic and immunohistologic findings in virus transport medium on cases under investigation of SARS-CoV-2 infection. METHODS: Cytologic findings in cases under investigation of SARS-CoV-2 infection from one hundred consecutive nasopharyngeal swab were reviewed. Immunohistochemistry and SARSCoV-2 RT-PCR determination were performed to detect virus. RESULTS: No viral inclusions were noted in squamous cells obtained from virus transport medium. Immunohistochemical study with monoclonal antibody against SARS-CoV-2 viral nucleoprotein showed positivity in squamous cells. No positivity was present in others cellular components. CONCLUSIONS: SARS-CoV-2 predominantly localizes squamous cells in cytology samples of patients with RT-PCR positive determination of SARSCoV-2. The results of the current study support the notion that the nasopharyngeal region is the anatomical station that SARS-CoV-2 infects first, and the infection can lead to the migration of the virus into the lower airways.

To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach towards these ends. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. Presence of the SARS-CoV-2 spike protein elicits a robust, two-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism, and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.Abstract Figure

SummarySevere acute respiratory syndrome - coronavirus 2 (SARS-CoV2) is detected by a highly sensitive molecular method, reverse transcriptase-polymerase chain reaction (RT-PCR) from nasopharyngeal swab (NPS) samples collected in 2-3ml of viral transport medium (VTM). Unlike body fluids, NPS samples are undermined by high variability in the amount of cells that get suspended into the VTM. Hence, the cell density used for RNA extraction becomes an important analytical variable that contributes to the overall sensitivity of the RT-PCR. In this study, we compared the sensitivity of SARS-CoV2 RT-PCR in 50 NPS samples collected from in-patients of the COVID wards using the concentration and direct methods. The concentration method detected the viral RNA in all 50 samples, while the direct method was positive in only 41 (82%) samples (p=0.003). Additionally, the Ct values were lower in the direct method compared to concentration method among the 41 positive samples (p=0.03 for N gene and p=0.04 for RdRp gene). The mean CV% was also ≥10%. Thus, the concentration of the cells prior to RNA extraction drastically improves the sensitivity of detection of SARS-CoV2 in NPS samples.

ObjectiveWith the COVID-19 pandemic, there have been supply challenges necessitating that laboratories must prepare their own viral transport medium (VTM), which provides stability for clinical specimens for diagnostic viral testing.MethodsWithin a veteran affairs medical center clinical laboratory, VTM was prepared with a Hanks Balanced Salt Solution (HBSS) 500 mL bottle with phenol red, sterile heat-inactivated fetal bovine serum (FBS), gentamicin sulfate (50 mg/mL), and amphotericin B (250 μg/mL). An antimicrobial mixture was made of 50 mL each of amphotericin B and gentamicin sulfate. Ten mL of FBS and 2 mL of the antimicrobial mixture were mixed into the HBSS bottle, from which 3 mL aliquots were made. Sterility and efficacy check were assessed. These preparations were conducted at our VAMC’s clinical laboratory to assure adequate VTM supply during the COVID-19 shortage.ResultsThe VTM was successfully prepared in-house, supporting uninterrupted testing for the facility and other affiliated medical facilities/centers and community living centers.ConclusionThis quality assurance/improvement report represents the first published manuscript on feasible VTM preparation exclusively within a clinical microbiology laboratory during the COVID-19 pandemic.

Introduction/Objective With the outbreak of COVID-19 caused by SARS-CoV-2, there have been challenges in the maintenance of adequate supplies both in terms of PPE and for testing. The shortage of commercial VTM for the transport of specimens for PCR testing has created a situation in which laboratories would need to manufacture their own in-house VTM as commonly used commercial VTM is unavailable. However, there is sparse literature on the emergency manufacture of VTM. Here, we describe the VAMC experience in manufacturing/quality control on its own VTM. Results VTM was manufactured by pathology and laboratory medicine using strict aseptic technique with Hanks Balanced Salt Solution (HBSS) 500 ml bottle with phenol red, sterile heat-inactivated fetal bovine serum (FBS) gentamicin sulfate (50 mg/mL) and amphotericin B (250 ug/ml). First, 50 ml of amphotericin B and 50 ml of gentamicin sulfate were mixed. Then 10 ml of FBS was mixed with the HBSS bottle and then 2 ml of the gentamicin/amphotericin B mixture was also mixed into the HBSS bottle. 3 ml aliquots were made from this mixture to constitute individual tubes of VTM for clinical use. Sterility for each batch (after 24-hour incubation at 37o C in the CO2 incubator) was assessed visually and by culture on a blood agar, chocolate agar, and thioglycolate mediums. An efficacy check was performed for each batch by spiking positive and negative controls into the VTM aliquots; RT-PCR for SARS-CoV-2 was executed to verify the medium did not degrade viral RNA and produced expected results for room temperature, refrigerated, and frozen samples. Previously manufactured VTM without phenol red also underwent sterility and efficacy checks. Results VTM was successfully manufactured in-house, allowing testing to continue despite the shortage. Sterility and efficacy checks on all lots and bottles from which the VTM aliquots were made passed with no growth detected and efficacy passing with all expected positives and negatives resulting as expected. Conclusion To the author’s knowledge, this represents the first published abstract on VTM manufacture in this most unprecedented crisis involving COVID-19. In this national emergency with corresponding shortage of testing supplies including commercial VTM, the in-house manufacture of VTM is both feasible and prudent to ensure continuity of testing and quality patient/laboratory care.

Critical to facilitating SARS-CoV-2 point-of-care (POC) testing is assurance that viruses present in specimens are inactivated onsite prior to processing. Here, we conducted experiments to determine the virucidal activity of commercially available Viral Transport Mediums (VTMs) to inactivate SARS-CoV-2. Independent testing methods for viral inactivation testing were applied, including a previously described World Health Organization (WHO) protocol, in addition to a buffer exchange method where the virus is physically separated from the VTM post exposure. The latter method enables sensitive detection of viral viability at higher viral titre when incubated with VTM. We demonstrate that VTM formulations, Primestore&reg, Molecular Transport Medium (MTM) and COPAN eNAT&trade, completely inactivate high-titre SARS-CoV-2 virus (&gt, 1 &times, 107 copies/mL) and are compatible with POC processing. Furthermore, full viral inactivation was rapidly achieved in as little as 2 min of VTM exposure. We conclude that adding certain VTM formulations as a first step post specimen collection will render SARS-CoV-2 non-infectious for transport, or for further in-field POC molecular testing using rapid turnaround GeneXpert platforms or equivalent.

Here, we retrospectively analyzed the comparative results of 182 paired dry nasopharyngeal swabs tested by Abbott ID NOW and nasopharyngeal swabs in viral transport medium by real-time RT-PCR methods. While the overall agreement was 96.2%, we found that of 15 samples that were tested positive with RT-PCR methods, 7 were missed by ID NOW, resulting in a false-negative rate of 47%.

It is unclear if the use of a molecular transport medium (MTM) containing guanidine isothiocyanate (GITC) would be advantageous over the CDC recommended, commonly used viral transport medium (VTM). We retested 70 SARS‐CoV2 cases by RT‐PCR in varying stages of follow‐up using MTM and VTM in parallel and found discrepant results of RNase P, E and N genes. Majority (81%) patients tested positive with MTM as compared with VTM (27.1%). Even patients who were sampled 3 weeks after diagnosis demonstrated a significant discrepancy in the positivity rates between MTM vs VTM raising concerns about the clinical utility of VTM.

The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: 1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes 2) Rapid time to result of 2=0.9259). Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.

The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media (VTM) due to widespread demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription-PCR (RT-PCR) testing. In response to this ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our hospital network. As our diagnostic laboratory was not equipped for reagent production, we took advantage of space and personnel that became available due to closure of the research division of our medical center., The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media (VTM) due to widespread demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription-PCR (RT-PCR) testing. In response to this ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our hospital network. As our diagnostic laboratory was not equipped for reagent production, we took advantage of space and personnel that became available due to closure of the research division of our medical center. We utilized a formulation of VTM described by the CDC that was simple to produce, did not require filtration for sterilization, and used reagents that were available from commercial suppliers. Performance of VTM was evaluated by several quality assurance measures. Based on cycle threshold (CT) values of spiking experiments, we found that our VTM supported highly consistent amplification of the SARS-CoV-2 target (coefficient of variation = 2.95%) using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization (EUA) assay on the Abbott m2000 platform. VTM was also found to be compatible with multiple swab types and, based on accelerated stability studies, able to maintain functionality for at least 4 months at room temperature. We further discuss how we met logistical challenges associated with large-scale VTM production in a crisis setting, including use of a staged assembly line for VTM transport tube production.

BackgroundThe global pandemic of Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV2) has resulted in unprecedented challenges for healthcare systems. One barrier to widespread testing has been a paucity of traditional respiratory viral swab collection kits relative to the demand. Whether other sample collection kits, such as widely available MRSA nasal swabs can be used to detect SARS-CoV-2 is unknown.MethodsWe compared simultaneous nasal MRSA swabs (COPAN ESwabs ® 480C flocked nasal swab in 1mL of liquid Amies medium) and virals wabs (BD H192(07) flexible mini-tip flocked nasopharyngeal swabs in 3mL Universal Transport Medium) for SARS-CoV-2 PCR testing using Simplexa COVID-19 Direct assay on patients over a 4-day period. When the results were discordant, the viral swab sample was run again on the Cepheid Xpert Xpress ® SARS-CoV-2 assay.ResultsOf the 81 included samples, there were 19 positives and 62 negatives in viral media and 18 positives and 63 negative in the MRSA swabs. Amongst all included samples, there was concordance between the COPAN ESwabs ® 480C and the viral swabs in 78 (96.3%).ConclusionWe found a high rate of concordance in test results between COPAN ESwabs ® 480C in Amies solution and BD H192(07) nasopharyngeal swabs in in 3 mL of Universal Viral Transport medium viral media. Clinicians and laboratories should feel better informed and assured using COPAN ESwabs ® 480C to help in the diagnosis of COVID-19.

As the coronavirus disease 2019 (COVID-19) pandemic sweeps across the world, the availability of viral transport medium (VTM) has become severely limited, contributing to delays in diagnosis and rationing of diagnostic testing. Given that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA has demonstrated stability, we posited that phosphate-buffered saline (PBS) may be a viable transport medium, as an alternative to VTM, for clinical real-time quantitative PCR (qPCR) testing. The intra-individual reliability and interindividual reliability of SARS-CoV-2 qPCR were assessed in clinical endotracheal secretion samples transported in VTM or PBS to evaluate the stability of the qPCR signal for three viral targets (N gene, ORF1ab, and S gene) when samples were stored in these media at room temperature for up to 18 hours. We report that the use of PBS as a transport medium allows high intra-individual and interindividual reliability, maintains viral stability, and compares with VTM in the detection of the three SARS-CoV-2 genes through 18 hours of storage. This study establishes PBS as a clinically useful medium that can be readily deployed for transporting and short-term preservation of specimens containing SARS-CoV-2. Use of PBS as a transport medium has the potential to increase testing capacity for SARS-CoV-2, aiding more widespread screening and early diagnosis of COVID-19.

The ongoing pandemic of the novel coronavirus, SARS-CoV-2, has led to a global surge in laboratory testing for the virus. The gold standard approach to detecting an active viral infection is the use of RT-qPCR. This approach requires the isolation of viral RNA from respiratory specimens, such as nasopharyngeal swabs.We developed a method using a widely available lysis buffer coupled with solid-phase reverse immobilization (SPRI) beads to extract viral RNA from swabs collected in viral transport medium (VTM) which can be performed manually or on a Hamilton STAR liquid-handling robot. Using a WHO recommended, laboratory-developed RT-qPCR for SARS-CoV-2, we validated this method in a CAP-accredited laboratory, against the IVD-labelled bioMérieux NucliSENS easyMAG automated extraction platform.Our method demonstrates a comparable sensitivity and specificity, making it suitable for large-scale testing and monitoring of suspected COVID-19 cases and health care workers. This is especially important as the world faces critical shortages of viral RNA extraction reagents for the existing commercial extraction systems.

Rapid and accurate detection of influenza virus is critical for proper patient management. The Alere™ i Influenza A&B assay is an isothermal nucleic acid amplification test capable of detecting influenza A and B viruses directly from respiratory specimens. In this multicenter clinical trial conducted in the US, we evaluated the clinical performance of the Alere™ i Influenza A&B assay against that of the Prodesse ProFlu+ assay. A total of 1243 fresh, leftover nasal or nasopharyngeal swabs eluted in viral transport medium were tested by both assays. Sensitivity and specificity of the Alere™ i Influenza A&B assay were 97.8% (95% CI 94.6-99.2) and 96.6% (95% CI 95.2-97.5) for influenza A and 92.9% (95% CI 85.5-96.9) and 98.3% (95% CI 97.4-98.0) for influenza B. The Alere™ i Influenza A&B assay is an ideal molecular assay for influenza virus detection due to its high sensitivity and specificity with minimal hands-on and turn-around-time.

Background Human adenoviruses are common causes of community-acquired respiratory tract and enteric infections. Severe disseminated infections with high mortality rates may be seen in immunocompromised individuals. An accurate and cost-effective quantitative assay is essential not only for laboratory diagnosis of adenoviral infections, but also for monitoring of response to antiviral treatment. The diagnostic performance of an in-house quantitative polymerase chain reaction assay was compared to a commercial system. Methods The analytical sensitivity, specificity, linearity, precision and accuracy of an in-house adenovirus quantitative polymerase chain reaction assay were evaluated against the RealStar® Adenovirus PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany), using 122 clinical specimens and 18 proficiency testing samples. Results Linear regression analysis of the quantitative results by the in-house assay showed the dynamic range from 2.60 to 9 log10 (plasma) and 2.94 to 9 log10 (viral transport medium) copies/mL, with the coefficient of determination (R2) of 0.996 and 0.998, respectively. A dilution series demonstrated the limits of detection and lower limits of quantification for plasma were 2.06 log10 and 2.60 log10 copies/mL and those for viral transport medium were 2.31 log10 and 2.94 log10 copies/mL respectively. The precision of the in-house assay was highly reproducible among runs with coefficients of variance ranging from 0.07 to 3.21% for plasma and 0.17% to 2.11% for viral transport medium. A comparison of 52 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house assay and the RealStar® Adenovirus PCR Kit (R2 = 0.984), with an average bias of − 0.16 log10 copies/mL. Conclusions The in-house adenovirus assay is a sensitive and reliable assay with lower cost for the detection and quantification of adenoviral DNA when compared to the RealStar® Adenovirus PCR Kit. Electronic supplementary material The online version of this article (10.1186/s12985-018-1059-7) contains supplementary material, which is available to authorized users.

BackgroundRapid testing for COVID-19 has been clearly identified as an essential component of the strategy to control the SARS-CoV-2 epidemic, worldwide. The ID NOW COVID-19 assay is a simple, user-friendly, rapid molecular biology test based on nicking and extension amplification reaction (NEAR).ObjectivesThe aim of this study was to evaluate the ID NOW COVID-19 assay when used as a point-of-care test (POCT) in our Emergency Department (ED).Type of studyThis prospective study enrolled 395 consecutive patients; paired nasopharyngeal swabs were collected from each study participant. The first swab was tested with the ID NOW COVID-19 assay at the point-of-care by ED nurses. The second swab was diluted in viral transport medium (VTM) and sent to the clinical microbiology department for analysis by both the RT-PCR Simplexa test COVID-19 Direct assay as the study reference method, and the ID NOW COVID-19 assay performed in the laboratory.ResultsNasopharyngeal swabs directly tested with the ID NOW COVID-19 assay yielded a sensitivity, specificity, PPV and NPV of 98.0%, 97.5%, 96.2% and 98.7%, respectively, in comparison with the RT-PCR study reference assay. When the ID NOW COVID-19 assay was performed in the laboratory using the VTM samples, the sensitivity decreased to 62.5% and the NPV to 79.7%. Three false negative test results were reported with the ID NOW COVID-19 assay when performed using undiluted swabs directly in the ED; these results were obtained from patients with elevated CT values (>30).ConclusionWe demonstrated that the ID NOW COVID-19 assay, performed as a point of care test in the ED using dry swabs, provides a rapid and reliable alternative to laboratory-based RT-PCR methods

Environmental specimens such as faecal droppings are considered important for the detection of avian influenza viruses (AIV). In view of lower rates of AIV isolation from avian faecal droppings, characterization of droppings is imperative to elucidate contributing factors. However, there are no reports on morphological and biochemical characteristics of droppings. The objective of the present study was the characterization of droppings from different avian species and their impact on the AIV detection and isolation. A total of 373 droppings belonging to 61 avian species from 22 families of apparently healthy wild migratory, resident, domestic birds and poultry were studied during five winter migratory bird seasons between 2007 to 2012 and 2016–2017. The colour, morphology and size of the droppings varied from species-to-species. These data could be useful for the identification of avian species. Droppings from 67% of the avian species showed acidic pH (4.5–6.5); nine species showed neutral pH (7.0), and 11 species showed alkaline pH (7.5). The infectious titers of AIV in droppings with acidic pH were significantly lower (p

Alere i RSV is a novel rapid test which applies a nicking enzyme amplification reaction to detect respiratory syncytial virus in point-of-care settings. In this study, we evaluated the Alere i RSV assay by using frozen nasopharyngeal swab samples that were collected in viral transport medium from children hospitalized with acute respiratory tract infection during the 2015-2016 winter season. Alere i RSV assay results were compared to those for Altona RealStar RSV real-time reverse transcription-PCR (RT-PCR). We found that the overall sensitivity and specificity of the Alere i RSV test was 100% (95% confidence intervals [CI], 93% to 100%) and 97% (95% CI, 89% to 100%), respectively. Positive samples were identified within 5 to 7 min from sample collection. Overall, the Alere i RSV test performed well compared to the RT-PCR assay and has the potential to facilitate the detection of RSV in point-of-care settings.