COVID-19: Test, Test and Test.

A new virus was identified in late December 2019 when China reported the first cases of pneumonia in Wuhan, and a global COVID-19 pandemic followed. The world was not late to respond, with a number of sweeping measures ranging from social distancing protocols, stringent hygienic practices, and nation-wide lockdowns, as well as COVID-19 testing campaigns in an attempt to prevent the transmission of the disease and contain the pandemic. Currently, different types of diagnostic testing have been adopted globally, such as nucleic acid detection tests, immunological tests and imaging approaches; however, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) remains the "gold standard" for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pre-analytical factors, such as specimen selection and collection, are crucial for RT-PCR, and any suboptimal collection may contribute to false-negative results. Herein, we address some of the specimen types that have been used in molecular detection methods for COVID-19. However, the pandemic is still evolving, and information might change as more studies are conducted.

[Rt or RDt, that is the question!] / Rt or RDt, that is the question!

The article compares two of the most followed indices in the monitoring of COVID-19 epidemic cases: the Rt and the RDt indices. The first was disseminated by the Italian National Institute of Health (ISS) and the second, which is more usable due to the lower difficulty of calculation and the availability of data, was adopted by various regional and local institutions.The rationale for the Rt index refers to that for the R0 index, the basic reproduction number, which is used by infectivologists as a measure of contagiousness of a given infectious agent in a completely susceptible population. The RDt index, on the other hand, is borrowed from the techniques of time series analysis for the trend of an event measurement that develops as a function of time. The RDt index does not take into account the time of infection, but the date of the diagnosis of positivity and for this reason it is defined as diagnostic replication index, as it aims to describe the intensity of the development of frequency for cases recognized as positive in the population.The comparison between different possible applications of the methods and the use of different types of monitoring data was limited to four areas for which complete individual data were available in March and April 2020. The main problems in the use of Rt, which is based on the date of symptoms onset, arise from the lack of completeness of this information due both to the difficulty in the recording and to the absence in asymptomatic subjects.The general trend of RDt, at least at an intermediate lag of 6 or 7 days, is very similar to that of Rt, as confirmed by the very high value of the correlation index between the two indices. The maximum correlation between Rt and RDt is reached at lag 7 with a value of R exceeding 0.97 (R2=0.944).The two indices, albeit formally distinct, are both valid; they show specific aspects of the phenomenon, but provide basically similar information to the public health decision-maker. Their distinction lies not so much in the method of calculation, rather in the use of different information, i.e., the beginning of symptoms and the swabs outcome.Therefore, it is not appropriate to make a judgment of preference for one of the two indices, but only to invite people to understand their different potentials so that they can choose the one they consider the most appropriate for the purpose they want to use it for.

Prevalence of SARS-CoV-2 (Covid-19) in Italians and in immigrants in an area of Northern Italy (Reggio Emilia).

It has been hypothesized that bacille Calmette-Guerin (BCG), the anti-tuberculosis vaccine, can be protective against Covid-19. Using data of performed swabs and RT-PCR results for SARS-CoV-2 in the Reggio Emilia province (Emilia-Romagna Region, Northern Italy) from March 6th to March 26th, 2020, we computed age, gender, and place of birth (Italy or abroad) specific risk of being tested, prevalence of positive tests, and probability of testing positive given that a swab has been taken during the epidemic peak. We report that immigrants resident in Reggio Emilia province, mostly coming from Countries with high BCG vaccination coverage, and Italians had a similar prevalence of infection (odds ratio - OR 0.99; 95%CI 0.82-1.20) and similar probability of being tested (OR 0.93; 95%CI 0.81-1.10). Our data do not support the hypothesis that immigrants from Countries where BCG vaccination is recommended have a lower risk of Covid-19 infection.

Comparison between Nasal and Nasopharyngeal Swabs for SARS-CoV-2 Rapid Antigen Detection in an Asymptomatic Population, and Direct Confirmation by RT-PCR from the Residual Buffer.

Containment measures employed during the COVID-19 pandemic included prompt recognition of cases, isolation, and contact tracing. Bilateral nasal (NA) swabs applied to a commercial antigen-based rapid diagnostic test (Ag-RDT) offer a simpler and more comfortable alternative to nasopharyngeal (NP) collection; however, little is known about the sensitivity of this method in an asymptomatic population. Participants in community-based asymptomatic testing sites were screened for SARS-CoV-2 using an Ag-RDT with NP sampling. Positive individuals returned for confirmatory molecular testing and consented to repeating the Ag-RDT using a bilateral NA swab for comparison. Residual test buffer (RTB) from Ag-RDTs was subjected to real-time reverse transcription-PCR (RT-PCR). Of 123,617 asymptomatic individuals, 197 NP Ag-RDT-positive participants were included, with 175 confirmed positive by RT-PCR. Of these cases, 154 were identified from the NA swab collection with Ag-RDT, with a sensitivity of 88.0% compared to the NP swab collection. Stratifying results by RT-PCR cycle threshold demonstrated that sensitivity of the nasal collection method varied based on the cycle threshold (CT) value of the paired RT-PCR sample. RT-PCR testing on the RTB from the Ag-RDT using NP and NA swab collections resulted in 100.0% and 98.7% sensitivity, respectively. NA swabs provide an adequate alternative to NP swab collection for use with Ag-RDT, with the recognition that the test is most sensitive in specimens with high viral loads. With the high sensitivity of RT-PCR testing on RTB from Ag-RDT, a more streamlined approach to confirmatory testing is possible without recollection or use of paired collections strategies. IMPORTANCE Nasal swabbing for SARS-CoV-2 (COVID-19) comes with many benefits but is slightly less sensitive than traditional nasopharyngeal swabbing; however, confirmatory lab-based testing could be performed directly from the residual buffer from either sample type.

YouTube Videos Demonstrating the Nasopharyngeal Swab Technique for SARS-CoV-2 Specimen Collection: Content Analysis.

BACKGROUND: Real-time polymerase chain reaction using nasopharyngeal swabs is currently the most widely used diagnostic test for SARS-CoV-2 detection. However, false negatives and the sensitivity of this mode of testing have posed challenges in the accurate estimation of the prevalence of SARS-CoV-2 infection rates. OBJECTIVE: The purpose of this study was to evaluate whether technical and, therefore, correctable errors were being made with regard to nasopharyngeal swab procedures. METHODS: We searched a web-based video database (YouTube) for videos demonstrating SARS-CoV-2 nasopharyngeal swab tests, posted from January 1 to May 15, 2020. Videos were rated by 3 blinded rhinologists for accuracy of swab angle and depth. The overall score for swab angle and swab depth for each nasopharyngeal swab demonstration video was determined based on the majority score with agreement between at least 2 of the 3 reviewers. We then comparatively evaluated video data collected from YouTube videos demonstrating the correct nasopharyngeal swab technique with data from videos demonstrating an incorrect nasopharyngeal swab technique. Multiple linear regression analysis with statistical significance set at P=.05 was performed to determine video data variables associated with the correct nasopharyngeal swab technique. RESULTS: In all, 126 videos met the study inclusion and exclusion criteria. Of these, 52.3% (66/126) of all videos demonstrated the correct swab angle, and 46% (58/126) of the videos demonstrated an appropriate swab depth. Moreover, 45.2% (57/126) of the videos demonstrated both correct nasopharyngeal swab angle and appropriate depth, whereas 46.8% (59/126) of the videos demonstrated both incorrect nasopharyngeal swab angle and inappropriate depth. Videos with correct nasopharyngeal swab technique were associated with the swab operators identifying themselves as a medical professional or as an Ear, Nose, Throat-related medical professional. We also found an association between correct nasopharyngeal swab techniques and recency of video publication date (relative to May 15, 2020). CONCLUSIONS: Our findings show that over half of the videos documenting the nasopharyngeal swab test showed an incorrect technique, which could elevate false-negative test rates. Therefore, greater attention needs to be provided toward educating frontline health care workers who routinely perform nasopharyngeal swab procedures.

Omicron Variant Generates a Higher and More Sustained Viral Load in Nasopharynx and Saliva Than the Delta Variant of SARS-CoV-2.

The Omicron variant of SARS-CoV-2 spreads more easily than earlier variants, possibly as a result of a higher viral load in the upper respiratory tract and oral cavity. Hence, we investigated whether the Omicron variant generates a higher viral load than that of the Delta variant in saliva and nasopharynx. Both specimens were collected from 52 Omicron and 17 Delta cases at two time points one week apart and analyzed by qRT-PCR. Viral load was measured as 10 log RNA genome copies per 1000 human cells according to the WHO reference standard. We found that Omicron cases carried a higher viral load and had more sustained viral shedding compared to the Delta cases, especially in the nasopharynx.

Secondary attack rates in primary and secondary school bubbles following a confirmed case: Active, prospective national surveillance, November to December 2020, England.

BACKGROUND: Following the full re-opening of schools in England and emergence of the SARS-CoV-2 Alpha variant, we investigated the risk of SARS-CoV-2 infection in students and staff who were contacts of a confirmed case in a school bubble (school groupings with limited interactions), along with their household members. METHODS: Primary and secondary school bubbles were recruited into sKIDsBUBBLE after being sent home to self-isolate following a confirmed case of COVID-19 in the bubble. Bubble participants and their household members were sent home-testing kits comprising nasal swabs for RT-PCR testing and whole genome sequencing, and oral fluid swabs for SARS-CoV-2 antibodies. RESULTS: During November-December 2020, 14 bubbles were recruited from 7 schools, including 269 bubble contacts (248 students, 21 staff) and 823 household contacts (524 adults, 299 children). The secondary attack rate was 10.0% (6/60) in primary and 3.9% (4/102) in secondary school students, compared to 6.3% (1/16) and 0% (0/1) among staff, respectively. The incidence rate for household contacts of primary school students was 6.6% (12/183) and 3.7% (1/27) for household contacts of primary school staff. In secondary schools, this was 3.5% (11/317) and 0% (0/1), respectively. Household contacts were more likely to test positive if their bubble contact tested positive although there were new infections among household contacts of uninfected bubble contacts. INTERPRETATION: Compared to other institutional settings, the overall risk of secondary infection in school bubbles and their household contacts was low. Our findings are important for developing evidence-based infection prevention guidelines for educational settings.

Saliva for molecular detection of SARS-CoV-2 in pre-school and school-age children.

SARS-CoV-2 diagnosis is a cornerstone for the management of coronavirus disease 2019 (COVID-19). Numerous studies have assessed saliva performance over nasopharyngeal sampling (NPS), but data in young children are still rare. We explored saliva performance for SARS-CoV-2 detection by RT-PCR according to the time interval from initial symptoms or patient serological status. We collected 509 NPS and saliva paired samples at initial diagnosis from 166 children under 12 years of age (including 57 children under 6), 106 between 12 and 17, and 237 adults. In children under 12, overall detection rate for SARS-CoV-2 was comparable in saliva and NPS, with an overall agreement of 89.8%. Saliva sensitivity was significantly lower than that of NPS (77.1% compared to 95.8%) in pre-school and school-age children but regained 96% when considering seronegative children only. This pattern was also observed to a lesser degree in adolescents but not in adults. Sensitivity of saliva was independent of symptoms, in contrary to NPS, whose sensitivity decreased significantly in asymptomatic subjects. Performance of saliva is excellent in children under 12 at early stages of infection. This reinforces saliva as a collection method for early and unbiased SARS-CoV-2 detection and a less invasive alternative for young children.

Clinical testing on SARS-CoV-2 swab samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

BACKGROUND: High cost of commercial RNA extraction kits limits the testing efficiency of SARS-CoV-2. Here, we developed a simple nucleic acid extraction method for the detection of SARS-CoV-2 directly from nasopharyngeal swab samples. METHODS: A pH sensitive dye was used as the end point detection method. The obvious colour changes between positive and negative reactions eliminates the need of other equipment. RESULTS: Clinical testing using 260 samples showed 92.7% sensitivity (95% CI 87.3-96.3%) and 93.6% specificity (95% CI 87.3-97.4%) of RT-LAMP. CONCLUSIONS: The simple RNA extraction method minimizes the need for any extensive laboratory set-up. We suggest combining this simple nucleic acid extraction method and RT-LAMP technology as the point-of care diagnostic tool.

Saliva as alternative to naso-oropharyngeal swab for SARS-CoV-2 detection by RT-qPCR: a multicenter cross-sectional diagnostic validation study.

Saliva has been demonstrated as feasible alternative to naso-oropharyngeal swab (NOS) for SARS-CoV-2 detection through reverse transcription quantitative/real-time polymerase chain reaction (RT-qPCR). This study compared the diagnostic agreement of conventional NOS, saliva with RNA extraction (SE) and saliva without RNA extraction (SalivaDirect) processing for RT-qPCR in identifying SARS-CoV-2. All techniques were also compared, as separate index tests, to a composite reference standard (CRS) where positive and negative results were defined as SARS-CoV-2 detection in either one or no sample, respectively. Of 517 paired samples, SARS-CoV-2 was detected in 150 (29.01%) NOS and 151 (29.21%) saliva specimens. The saliva-based tests were noted to have a sensitivity, specificity and accuracy (95% confidence interval) of 92.67% (87.26%, 96.28%), 97.55% (95.40%, 98.87%) and 96.13% (94.09%, 97.62%), respectively, for SE RT-qPCR and 91.33% (85.64%, 95.30%), 98.91% (97.23%, 99.70%) and 96.71% (94.79%, 98.07%), respectively, for SalivaDirect RT-qPCR compared to NOS RT-qPCR. Compared to CRS, all platforms demonstrated statistically similar diagnostic performance. These findings suggest that both conventional and streamlined saliva RT-qPCR are at least non-inferior to conventional NOS RT-qPCR in detecting SARS-CoV-2.

Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing.

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69-91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.

Whole genome sequence analysis showing unique SARS-CoV-2 lineages of B.1.524 and AU.2 in Malaysia.

SARS-CoV-2 has spread throughout the world since its discovery in China, and Malaysia is no exception. WGS has been a crucial approach in studying the evolution and genetic diversity of SARS-CoV-2 in the ongoing pandemic. Despite considerable number of SARS-CoV-2 genome sequences have been submitted to GISAID and NCBI databases, there is still scarcity of data from Malaysia. This study aims to report new Malaysian lineages of the virus, responsible for the sustained spikes in COVID-19 cases during the third wave of the pandemic. Patients with nasopharyngeal and/or oropharyngeal swabs confirmed COVID-19 positive by real-time RT-PCR with CT value < 25 were chosen for WGS. The selected SARS-CoV-2 isolates were then sequenced, characterized and analyzed along with 986 sequences of the dominant lineages of D614G variants currently circulating throughout Malaysia. The prevalence of clade GH and G formed strong ground for the presence of two Malaysian lineages of AU.2 and B.1.524 that has caused sustained spikes of cases in the country. Statistical analysis on the association of gender and age group with Malaysian lineages revealed a significant association (p <0.05). Phylogenetic analysis revealed dispersion of 41 lineages, of these, 22 lineages are still active. Mutational analysis showed presence of unique G1223C missense mutation in transmembrane domain of the spike protein. For better understanding of the SARS-CoV-2 evolution in Malaysia especially with reference to the reported lineages, large scale studies based on WGS are warranted.

A deep learning-driven low-power, accurate, and portable platform for rapid detection of COVID-19 using reverse-transcription loop-mediated isothermal amplification.

This paper presents a deep learning-driven portable, accurate, low-cost, and easy-to-use device to perform Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) to facilitate rapid detection of COVID-19. The 3D-printed device-powered using only a 5 Volt AC-DC adapter-can perform 16 simultaneous RT-LAMP reactions and can be used multiple times. Moreover, the experimental protocol is devised to obviate the need for separate, expensive equipment for RNA extraction in addition to eliminating sample evaporation. The entire process from sample preparation to the qualitative assessment of the LAMP amplification takes only 45 min (10 min for pre-heating and 35 min for RT-LAMP reactions). The completion of the amplification reaction yields a fuchsia color for the negative samples and either a yellow or orange color for the positive samples, based on a pH indicator dye. The device is coupled with a novel deep learning system that automatically analyzes the amplification results and pays attention to the pH indicator dye to screen the COVID-19 subjects. The proposed device has been rigorously tested on 250 RT-LAMP clinical samples, where it achieved an overall specificity and sensitivity of 0.9666 and 0.9722, respectively with a recall of 0.9892 for Ct < 30. Also, the proposed system can be widely used as an accurate, sensitive, rapid, and portable tool to detect COVID-19 in settings where access to a lab is difficult, or the results are urgently required.

Self-collected gargle specimen as a patient-friendly sample collection method for COVID-19 diagnosis in a population context.

Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.

SARS-CoV-2 diagnostics in the virology laboratory of a University Hospital in Rome during the lockdown period.

Italy was one of the most affected nations by coronavirus disease 2019 outside China. The infections, initially limited to Northern Italy, spread to all other Italian regions. This study aims to provide a snapshot of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) epidemiology based on a single-center laboratory experience in Rome. The study retrospectively included 6565 subjects tested for SARS-CoV-2 at the Laboratory of Virology of Sapienza University Hospital in Rome from 6 March to 4 May. A total of 9995 clinical specimens were analyzed, including nasopharyngeal swabs, bronchoalveolar lavage fluids, gargle lavages, stools, pleural fluids, and cerebrospinal fluids. Positivity to SARS-CoV-2 was detected in 8% (527/6565) of individuals, increased with age, and was higher in male patients (P < .001). The number of new confirmed cases reached a peak on 18 March and then decreased. The virus was detected in respiratory samples, in stool and in pleural fluids, while none of gargle lavage or cerebrospinal fluid samples gave a positive result. This analysis allowed to gather comprehensive information on SARS-CoV-2 epidemiology in our area, highlighting positivity variations over time and in different sex and age group and the need for a continuous surveillance of the infection, mostly because the pandemic evolution remains unknown.

Analytical performances of the AMPLIQUICK® Respiratory Triplex assay for simultaneous detection and differentiation of SARS-CoV-2, influenza A/B and respiratory syncytial viruses in respiratory specimens.

Although patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B and respiratory syncytial virus (RSV) show comparable or very similar manifestations, the therapeutic approaches of these respiratory viral infections are different, which requires an accurate diagnosis. Recently, the novel multiplex real-time reverse transcription-polymerase chain reaction assay AMPLIQUICK® Respiratory Triplex (BioSynex SA, Illkirch-Graffenstaden, France) allows simultaneous detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory tract samples. We herein evaluated the performance of the AMPLIQUICK® Respiratory Triplex for the detection of the four viruses in respiratory specimens, using Allplex™ Respiratory Panel 1 and 2019-nCoV assays (Seegene, Seoul, Korea) as reference comparator assays. A total of 359 archived predetermined respiratory samples, including 83, 145, 19 and 95 positive specimens for SARS-CoV-2, influenza A, influenza B and RSV respectively, were included. The AMPLIQUICK® Respiratory Triplex showed high concordance with the reference assays, with an overall agreement for SARS-CoV-2, influenza A, influenza B, and RSV at 97.6%, 98.8%, 98.3% and 100.0%, respectively, and high κ values ranging from 0.93 to 1.00, indicating an almost perfect agreement between assays. Furthermore, high correlations of cycle threshold (Ct) values were observed for positive samples of the four viruses between the AMPLIQUICK® Respiratory Triplex and comparator assays, with an overall high agreement between Ct values assessed by Bland-Altman analyses. In conclusion, these observations demonstrate that the multiplex AMPLIQUICK® Respiratory Triplex is a reliable assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory specimens, which may prove useful for streamlining diagnostics during the winter influenza-seasons.