Hock-a-loogie saliva as a diagnostic specimen for SARS-CoV-2 by a PCR-based assay: A diagnostic validity study.

To clarify the effect of different respiratory sample types on SARS-CoV-2 detection, we collected throat swabs, nasal swabs and hock-a-loogie saliva or sputum, and compared their detection rates and viral loads. The detection rates of sputum (95.65%, 22/23) and hock-a-loogie saliva (88.09%, 37/42) were significantly higher than those in throat swabs (41.54%, 27/65) and nasal swabs (72.31%, 47/65) (P < 0.001). The Ct Values of sputum, hock-a-loogie saliva and nasal swabs were significantly higher than that in throat swabs, whereas no significant difference was observed between sputum and saliva samples. Hock-a-loogie saliva are reliable sample types that can be used to detect SARS-CoV-2, and worthy of clinical promotion.

Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics.

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.

S-Variant SARS-CoV-2 Lineage B1.1.7 Is Associated With Significantly Higher Viral Load in Samples Tested by TaqPath Polymerase Chain Reaction.

A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

Quantification of the Tradeoff between Test Sensitivity and Test Frequency in a COVID-19 Epidemic-A Multi-Scale Modeling Approach.

Control strategies that employ real time polymerase chain reaction (RT-PCR) tests for the diagnosis and surveillance of COVID-19 epidemic are inefficient in fighting the epidemic due to high cost, delays in obtaining results, and the need of specialized personnel and equipment for laboratory processing. Cheaper and faster alternatives, such as antigen and paper-strip tests, have been proposed. They return results rapidly, but have lower sensitivity thresholds for detecting virus. To quantify the effects of the tradeoffs between sensitivity, cost, testing frequency, and delay in test return on the overall course of an outbreak, we built a multi-scale immuno-epidemiological model that connects the virus profile of infected individuals with transmission and testing at the population level. We investigated various randomized testing strategies and found that, for fixed testing capacity, lower sensitivity tests with shorter return delays slightly flatten the daily incidence curve and delay the time to the peak daily incidence. However, compared with RT-PCR testing, they do not always reduce the cumulative case count at half a year into the outbreak. When testing frequency is increased to account for the lower cost of less sensitive tests, we observe a large reduction in cumulative case counts, from 55.4% to as low as 1.22% half a year into the outbreak. The improvement is preserved even when the testing budget is reduced by one half or one third. Our results predict that surveillance testing that employs low-sensitivity tests at high frequency is an effective tool for epidemic control.

A SARS-CoV-2 Reference Standard Quantified by Multiple Digital PCR Platforms for Quality Assessment of Molecular Tests.

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 105 ± 6.5 × 104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.

Challenges and Controversies to Testing for COVID-19.

The coronavirus disease (COVID-19) pandemic has placed the clinical laboratory and testing for SARS-CoV-2 front and center in the worldwide discussion of how to end the outbreak. Clinical laboratories have responded by developing, validating, and implementing a variety of molecular and serologic assays to test for SARS-CoV-2 infection. This has played an essential role in identifying cases, informing isolation decisions, and helping to curb the spread of disease. However, as the demand for COVID-19 testing has increased, laboratory professionals have faced a growing list of challenges, uncertainties, and, in some situations, controversy, as they have attempted to balance the need for increasing test capacity with maintaining a high-quality laboratory operation. The emergence of this new viral pathogen has raised unique diagnostic questions for which there have not always been straightforward answers. In this commentary, the author addresses several areas of current debate, including (i) the role of molecular assays in defining the duration of isolation/quarantine, (ii) whether the PCR cycle threshold value should be included on patient reports, (iii) if specimen pooling and testing by research staff represent acceptable solutions to expand screening, and (iv) whether testing a large percentage of the population is feasible and represents a viable strategy to end the pandemic.

Increasing SARS-CoV-2 nucleic acid testing capacity during the COVID-19 epidemic in Beijing: experience from a general hospital.

Under the ongoing COVID-19 prevention and control measures in China, increasing the laboratory severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid testing capacity has become the top priority. Since the COVID-19 outbreak in Xinfadi market in Beijing in June 2020, large-scale screening of key populations has been carried out, challenging the nucleic acid testing capabilities of hospital laboratories. Therefore, within 48 hours, Peking University People's Hospital (PKUPH) transformed a non-nucleic acid testing laboratory into a SARS-CoV-2 nucleic acid testing laboratory. Based on the original structure of the building, we adapted measures to local conditions, sorted out a new testing process, and quickly started testing for COVID-19. The nucleic acid testing process has been optimized, including quality control, personal operating specifications, and the timeliness of the release of LIS results to form closed-loop management. This high-throughput COVID-19 testing optimization process provides a reference model for other countries that are fighting the epidemic.

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing.

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.

Simulation-based training and assessment of mobile pre-hospital SARS-CoV-2 diagnostic teams in Styria, Austria.

The World Health Organization has declared coronavirus disease 2019 (COVID-19) a pandemic. Polymerase chain reaction testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the diagnostic gold standard of COVID-19. We have developed a simulation-based training program for mobile prehospital diagnostic teams in the province of Styria, Austria, and performed a prospective observational study on its applicability and effectivity.The 1-day curriculum uses theoretical instruction, technical skills training, and simulator-based algorithm training to teach and train prehospital patient identification and communication, donning the personal protective equipment, collection of naso-/oropharyngeal swabs for SARS-CoV-2 polymerase chain reaction testing, doffing the personal protective equipment, and sample logistics. Trainings were conducted at the SIM CAMPUS simulation hospital, Eisenerz, using high-fidelity patient simulation. To ensure achievement of predefined learning outcomes, participants had to undergo a final simulator-based objective structured clinical examination.In March 2020, 45 emergency medical assistants and 1 physician of the Austrian Red Cross participated on a voluntary basis. Forty-five of the 46 participants (97.8%) completed the curriculum successfully, with mean objective structured clinical examination ratings of 98.6%.Using several proven educational concepts, we have successfully drafted and implemented a training program for mobile prehospital SARS-CoV-2 diagnostic teams. Based on simulation-based objective structured examinations, it has prepared participants effectively for preclinical duties.

An infant with coronavirus disease 2019 in China: A case report.

RATIONALE: In December 2019, an outbreak of coronavirus disease 2019 (COVID-19) occurred in Wuhan, China. The initial epidemiological investigations showed that COVID-19 occurred more likely in adults, with patients younger than 10 years old accounting for less than 1% of the total number of confirmed cases, and infant infections were more rare. In our case, we present an infant who was only 35 days old when he was tested positive for COVID-19. PATIENT CONCERNS: In this report, a 35 day-old male infant with atypical symptoms had close contact with 2 confirmed patients of COVID-19 who were his grandmother and mother. DIAGNOSIS: The patient was diagnosed as COVID-19 after his oropharyngeal swab tested positive for severe acute respiratory syndrome coronavirus 2 by reverse transcription-polymerase chain reaction assay. INTERVENTIONS: The therapeutic schedule included aerosol inhalation of recombinant human interferon α-2b and supportive therapy. OUTCOMES: Two consecutive (1 day apart) oropharyngeal swabs tested negative for severe acute respiratory syndrome coronavirus 2; then, the patient was discharged on February 27, 2020. LESSONS: Strengthening infants' virus screening in families with infected kins is important for early diagnosis, isolation, and treatment when symptoms are atypical. The infectivity of infants with mild or asymptomatic COVID-19 should not be ignored because this may be a source of transmission in the community.

Utility of Stool PCR for the Diagnosis of COVID-19: Comparison of Two Commercial Platforms.

The ability to detect SARS-CoV-2 in the upper respiratory tract ceases after 2 to 3 weeks post-symptom-onset in most patients. In contrast, SARS-CoV-2 can be detected in the stool of some patients for greater than 4 weeks, suggesting that stool may hold utility as an additional source for diagnosis. We validated the Cepheid Xpert Xpress SARS-CoV-2 and Hologic Panther Fusion real-time RT-PCR assays for detection of viral RNA in stool specimens and compared performance. We utilized remnant stool specimens (n = 79) from 77 patients with gastrointestinal symptoms. Forty-eight patients had PCR-confirmed COVID-19, and 29 either were nasopharyngeal/oropharyngeal PCR negative or presented for reasons unrelated to COVID-19 and were not tested. Positive percent agreement between the Cepheid and Hologic assays was 93% (95% confidence interval [CI]: 81.1% to 98.2%), and negative percent agreement was 96% (95% CI: 89% to 0.99%). Four discrepant specimens (Cepheid positive only, n = 2; Hologic positive only, n = 2) exhibited average cycle threshold (CT ) values of >37 for the targets detected. Of the 48 patients with PCR-confirmed COVID-19, 23 were positive by both assays (47.9%). For the negative patient group, 2/29 were positive by both assays (6.9%). The two stool PCR-positive, nasopharyngeal/oropharyngeal PCR-negative patients were SARS-CoV-2 IgG positive. Our results demonstrate acceptable agreement between two commercially available molecular assays and support the use of stool PCR to confirm diagnosis when SARS-CoV-2 is undetectable in the upper respiratory tract.

Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses.

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 102 copies/µL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

[Optimization of a fluorescent qPCR detection for RNA of SARS-CoV-2].

We optimized a fluorescent quantitative polymerase chain reaction (qPCR) assay system for rapid and real time detection of SARS-CoV-2 RNA. The results show that the lowest dilution of RNA samples used for the detection of SARS-CoV-2 RNA could reach 1/10 000 (the initial value is set as 10 ng/µL). Moreover, the cycle threshold (Ct) for samples of clinically diagnosed COVID-19 was lower than 35 or 40. The sensitivity of this method was satisfactory. The results were consistent with those of the COVID-19 detection kit on the market under the same conditions, but the number of cycles required was shortened by about 2. Therefore, the optimized assay developed in this study can be used in screening and early clinical diagnosis. Our work provides a tool to facilitate rapid clinical diagnosis of COVID-19.

Significance of serology testing to assist timely diagnosis of SARS-CoV-2 infections: implication from a family cluster.

Confirmative diagnosis of SARS-CoV-2 infections has been challenged due to unsatisfactory positive rate of molecular assays. Here we identified a family cluster of SARS-CoV-2 infections, with five of six family members were SARS-CoV-2-specific immunoglobin serology testing positive, while molecular assays only detected two of this five patients even repeated twice. We comprehensively analyzed this familial cluster of cases based on the clinical characteristics, chest CT images, SARS-CoV-2 molecular detection results, and serology testing results. At last, two patients were diagnosed with COVID-19, two were suspected of COVID-19, and two were considered close contacts. Our results emphasized the significance of serology testing to assist timely diagnosis of SARS-CoV-2 infections, especially for COVID-19 close contacts screening.