A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

Distinguishing SARS-CoV-2 Infection and Non-SARS-CoV-2 Viral Infections in Adult Patients through Clinical Score Tools.

This study aimed to determine distinguishing predictors and develop a clinical score to differentiate COVID-19 and common viral infections (influenza, respiratory syncytial virus (RSV), dengue, chikungunya (CKV), and zika (ZKV)). This retrospective study enrolled 549 adults (100 COVID-19, 100 dengue, 100 influenza, 100 RSV, 100 CKV, and 49 ZKV) during the period 2017−2020. CKV and ZKV infections had specific clinical features (i.e., arthralgia and rash); therefore, these diseases were excluded. Multiple binary logistic regression models were fitted to identify significant predictors, and two scores were developed differentiating influenza/RSV from COVID-19 (Flu-RSV/COVID) and dengue from COVID-19 (Dengue/COVID). The five independent predictors of influenza/RSV were age > 50 years, the presence of underlying disease, rhinorrhea, productive sputum, and lymphocyte count < 1000 cell/mm3. Likewise, the five independent predictors of dengue were headache, myalgia, no cough, platelet count < 150,000/mm3, and lymphocyte count < 1000 cell/mm3. The Flu-RSV/COVID score (cut-off value of 4) demonstrated 88% sensitivity and specificity for predicting influenza/RSV (AUROC = 0.94). The Dengue/COVID score (cut-off value of 4) achieved 91% sensitivity and 94% specificity for differentiating dengue and COVID-19 (AUROC = 0.98). The Flu-RSV/COVID and Dengue/COVID scores had a high discriminative ability for differentiating influenza/RSV or dengue infection and COVID-19. The further validation of these scores is needed to ensure their utility in clinical practice.

Prevalence and factors associated with COVID-19 among healthcare workers at a university hospital in Thailand.

Globally, healthcare workers (HCWs) have a high risk of SARS-CoV-2 infection, but less is known about healthcare workers in Thailand. We estimated the prevalence and risk factors for COVID-19 among HCWs in Bangkok, Thailand. A retrospective cohort study was conducted at a large tertiary care academic hospital in Thailand from May 2020 to May 2021. HCWs that presented with fever and/or acute respiratory tract symptoms who tested with RT-PCR were identified, and their clinical data were collected. There were 1432 HCWs with fever and/or acute respiratory tract symptoms during May 2020 and May 2021. A total of 167 patients were front-line HCWs and 1265 were non-front-line HCWs. Sixty HCWs (4.2%) developed COVID-19; 2 were front-line and 58 were non-front-line HCWs. The prevalence of COVID-19 in front-line HCWs was 1.7% (2/167), and 4.6% (58/1265) in non-front-line HCWs (P = .04). In addition, non-front-line HCWs, non-medical staffs, history of contact with a confirmed COVID-19 case at home/family, unvaccinated status, fair compliance to personal protective equipment (PPE) standard, and initial presentation with pneumonia were significantly more common in HCWs with COVID-19 than those without COVID-19 (P < .05). Front-line HCWs, history of contact with a confirmed COVID-19 case at the clinical care areas in the hospital, vaccinated status, good compliance to PPE standards, and initial presentation with upper respiratory infection were significantly more common in HCWs without COVID-19 than those with COVID-19 (P < .05). Multivariate analysis revealed history of exposure with confirmed COVID-19 case at home or in family, unvaccinated status, non-frontline-HCWs, non-medical staffs, and fair compliance to PPE standard to be independent factors associated with COVID-19 in HCWs. COVID-19 was more common in non-front-line HCWs at this tertiary hospital. Thai guidelines on infection prevention and control for COVID-19 seem to be effective in preventing SARS-CoV-2 transmission. Therefore, the adherence to these recommendations should be encouraged.

Epidemiology, clinical characteristics, and treatment outcomes of patients with COVID-19 at Thailand's university-based referral hospital.

BACKGROUND: The epidemiology and outcomes of COVID-19 patients in Thailand are scarce. METHODS: This retrospective cohort study included adult hospitalized patients who were diagnosed with COVID-19 at Siriraj Hospital during February 2020 to April 2020. RESULTS: The prevalence of COVID-19 was 7.5% (107 COVID-19 patients) among 1409 patients who underwent RT-PCR for SARS-CoV-2 detection at our hospital during the outbreak period. Patients with COVID-19 presented with symptoms in 94.4%. Among the 104 patients who were treated with antiviral medications, 78 (75%) received 2-drug regimen (lopinavir/ritonavir or darunavir/ritonavir plus chloroquine or hydroxychloroquine), and 26 (25%) received a 3-drug regimen with favipiravir added to the 2-drug regimen. Disease progression was observed in 18 patients (16.8%). All patients with COVID-19 were discharged alive. CONCLUSIONS: The prevalence of COVID-19 was 7.5% among patients who underwent RT-PCR testing, and 10% among those having risk factors for COVID-19 acquisition. Combination antiviral therapies for COVID-19 patients were well-tolerated and produced a favorable outcome.

Rapid SARS-CoV-2 antigen detection assay in comparison with real-time RT-PCR assay for laboratory diagnosis of COVID-19 in Thailand.

BACKGROUND: The Coronavirus disease 2019 (COVID-19) pandemic continues to spread across the world. Hence, there is an urgent need for rapid, simple, and accurate tests to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Performance characteristics of the rapid SARS-CoV-2 antigen detection test should be evaluated and compared with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) test for diagnosis of COVID-19 cases. METHODS: The rapid SARS-CoV-2 antigen detection test, Standard™ Q COVID-19 Ag kit (SD Biosensor®, Republic of Korea), was compared with the real-time RT-PCR test, Allplex™ 2019-nCoV Assay (Seegene®, Korea) for detection of SARS-CoV-2 in respiratory specimens. Four hundred fifty-four respiratory samples (mainly nasopharyngeal and throat swabs) were obtained from COVID-19 suspected cases and contact individuals, including pre-operative patients at Siriraj Hospital, Bangkok, Thailand during March-May 2020. RESULTS: Of 454 respiratory samples, 60 (13.2%) were positive, and 394 (86.8%) were negative for SARS-CoV-2 RNA by real-time RT-PCR assay. The duration from onset to laboratory test in COVID-19 suspected cases and contact individuals ranged from 0 to 14 days with a median of 3 days. The rapid SARS-CoV-2 antigen detection test's sensitivity and specificity were 98.33% (95% CI, 91.06-99.96%) and 98.73% (95% CI, 97.06-99.59%), respectively. One false negative test result was from a sample with a high real-time RT-PCR cycle threshold (Ct), while five false positive test results were from specimens of pre-operative patients. CONCLUSIONS: The rapid assay for SARS-CoV-2 antigen detection showed comparable sensitivity and specificity with the real-time RT-PCR assay. Thus, there is a potential use of this rapid and simple SARS-CoV-2 antigen detection test as a screening assay.

Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.