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1.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-318389

ABSTRACT

This protocol presents the Variant Nucleotide Guard (VaNGuard) assay, which is robust towards viral mutations and can be performed on purified RNA or directly on nasopharyngeal (NP) swab samples. The procedure typically comprises three parts, namely sample preparation, RT-LAMP reaction, and Cas12a-based detection via fluorescence or lateral flow assay. Sample preparation from NP swabs involves Proteinase K digestion followed by heat inactivation. Purified RNA or digested NP swab samples are then added as templates into RT-LAMP reactions and incubated at 65ºC for 22 minutes. Next, enAsCas12a and ssDNA-probes are added and the reactions are incubated at 60ºC for another 5 minutes. End-point fluorescence can be detected by a plate reader or a real-time PCR machine. Alternatively, a lateral flow strip can be inserted into each reaction tube for equipment-free read-out. The VaNGuard assay is a rapid and convenient point-of-care test for SARS-CoV-2 and is applicable to resource poor settings.

2.
Front Immunol ; 12: 710217, 2021.
Article in English | MEDLINE | ID: covidwho-1555700

ABSTRACT

Severe SARS-CoV-2 infection can trigger uncontrolled innate and adaptive immune responses, which are commonly associated with lymphopenia and increased neutrophil counts. However, whether the immune abnormalities observed in mild to severely infected patients persist into convalescence remains unclear. Herein, comparisons were drawn between the immune responses of COVID-19 infected and convalescent adults. Strikingly, survivors of severe COVID-19 had decreased proportions of NKT and Vδ2 T cells, and increased proportions of low-density neutrophils, IgA+/CD86+/CD123+ non-classical monocytes and hyperactivated HLADR+CD38+ CD8+ T cells, and elevated levels of pro-inflammatory cytokines such as hepatocyte growth factor and vascular endothelial growth factor A, long after virus clearance. Our study suggests potential immune correlates of "long COVID-19", and defines key cells and cytokines that delineate true and quasi-convalescent states.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , COVID-19/complications , Cohort Studies , Convalescence , Female , Humans , Male , Middle Aged
3.
Infect Chemother ; 53(2): 391-394, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1295981

ABSTRACT

There have been recent descriptions of the novel coronavirus disease 2019 (COVID-19) presenting as 'varicella-like exanthem'. We report three cases of patients with Varicella-Zoster Virus (VZV) and COVID-19 co-infections, presenting in three varied ways. These cases highlight the need for heightened alertness to how such co-infections can present, to pick up overlapping 'dual pathologies' during this current pandemic given that infection control measures including airborne precautions are crucial for both COVID-19 and VZV.

4.
J Med Virol ; 93(7): 4603-4607, 2021 07.
Article in English | MEDLINE | ID: covidwho-1263098

ABSTRACT

We compared the performance of five assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on nasopharyngeal swab samples: Roche "cobas," Luminex "ARIES," MiRXES "Fortitude," Altona "RealStar," and Thermo Fisher Scientific "TaqPath." A total of 94 nasopharyngeal swab samples were obtained from 80 confirmed coronavirus disease 2019 cases in the first 2 weeks of illness (median, 7 days; range, 2-14 days) and 14 healthy controls. After collection, all samples were transported to the hospital clinical laboratory within 24 h. These samples were tested on all five assays within 3 days of sample receipt. Of the 94 samples, 69 yielded the same result on all platforms, resulting in an agreement of 73.4% (69 of 94). Of these, 14 were the healthy control swabs which all tested negative, demonstrating good specificity across all platforms. The ARIES assay had the lowest detection rate (68.8%), followed by Fortitude (85.0%), RealStar (86.3%), cobas (95.0%), and TaqPath (100%). Statistically significant differences were observed for ARIES, Fortitude, and RealStar when compared against the best performing TaqPath using McNemar's χ2 test. A consensus result was established based on the results obtained by the cobas, Fortitude, RealStar, and TaqPath. Six discrepancies had failed to reach a consensus and were adjudicated using the Cepheid Xpert Xpress SARS-CoV-2. Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS-CoV-2 gene targets. On the other hand, the ARIES assay was the least sensitive, thus warranting the need for assay re-optimization before go-live at the testing laboratory.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Genes, Viral/genetics , Humans , Nasopharynx/virology , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
6.
Nat Commun ; 12(1): 1739, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1142438

ABSTRACT

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Guide , SARS-CoV-2/genetics , Bacterial Proteins/genetics , COVID-19/virology , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Endodeoxyribonucleases/genetics , Humans , Molecular Diagnostic Techniques/methods , Mutation , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Sensitivity and Specificity
7.
J Med Virol ; 93(7): 4603-4607, 2021 07.
Article in English | MEDLINE | ID: covidwho-1130631

ABSTRACT

We compared the performance of five assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on nasopharyngeal swab samples: Roche "cobas," Luminex "ARIES," MiRXES "Fortitude," Altona "RealStar," and Thermo Fisher Scientific "TaqPath." A total of 94 nasopharyngeal swab samples were obtained from 80 confirmed coronavirus disease 2019 cases in the first 2 weeks of illness (median, 7 days; range, 2-14 days) and 14 healthy controls. After collection, all samples were transported to the hospital clinical laboratory within 24 h. These samples were tested on all five assays within 3 days of sample receipt. Of the 94 samples, 69 yielded the same result on all platforms, resulting in an agreement of 73.4% (69 of 94). Of these, 14 were the healthy control swabs which all tested negative, demonstrating good specificity across all platforms. The ARIES assay had the lowest detection rate (68.8%), followed by Fortitude (85.0%), RealStar (86.3%), cobas (95.0%), and TaqPath (100%). Statistically significant differences were observed for ARIES, Fortitude, and RealStar when compared against the best performing TaqPath using McNemar's χ2 test. A consensus result was established based on the results obtained by the cobas, Fortitude, RealStar, and TaqPath. Six discrepancies had failed to reach a consensus and were adjudicated using the Cepheid Xpert Xpress SARS-CoV-2. Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS-CoV-2 gene targets. On the other hand, the ARIES assay was the least sensitive, thus warranting the need for assay re-optimization before go-live at the testing laboratory.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Genes, Viral/genetics , Humans , Nasopharynx/virology , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
10.
mBio ; 11(4)2020 07 21.
Article in English | MEDLINE | ID: covidwho-660818

ABSTRACT

To date, limited genetic changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described. Here, we report a 382-nucleotide (nt) deletion in SARS-CoV-2 that truncates open reading frame 7b (ORF7b) and ORF8, removing the ORF8 transcription regulatory sequence (TRS) and eliminating ORF8 transcription. The earliest 382-nt deletion variant was detected in Singapore on 29 January 2020, with the deletion viruses circulating in the country and accounting for 23.6% (45/191) of SARS-CoV-2 samples screened in this study. SARS-CoV-2 with the same deletion has since been detected in Taiwan, and other ORF7b/8 deletions of various lengths, ranging from 62 nt to 345 nt, have been observed in other geographic locations, including Australia, Bangladesh, and Spain. Mutations or deletions in ORF8 of SARS-CoV have been associated with reduced replicative fitness and virus attenuation. In contrast, the SARS-CoV-2 382-nt deletion viruses showed significantly higher replicative fitness in vitro than the wild type, while no difference was observed in patient viral load, indicating that the deletion variant viruses retained their replicative fitness. A robust antibody response to ORF8 has been observed in SARS-CoV-2 infection, suggesting that the emergence of ORF8 deletions may be due to immune-driven selection and that further deletion variants may emerge during the sustained transmission of SARS-CoV-2 in humans.IMPORTANCE During the SARS epidemic in 2003/2004, a number of deletions were observed in ORF8 of SARS-CoV, and eventually deletion variants became predominant, leading to the hypothesis that ORF8 was an evolutionary hot spot for adaptation of SARS-CoV to humans. However, due to the successful control of the SARS epidemic, the importance of these deletions for the epidemiological fitness of SARS-CoV in humans could not be established. The emergence of multiple SARS-CoV-2 strains with ORF8 deletions, combined with evidence of a robust immune response to ORF8, suggests that the lack of ORF8 may assist with host immune evasion. In addition to providing a key insight into the evolutionary behavior of SARS-CoV-2 as the virus adapts to its new human hosts, the emergence of ORF8 deletion variants may also impact vaccination strategies.


Subject(s)
Betacoronavirus/genetics , Genome, Viral , Open Reading Frames , Base Sequence , Betacoronavirus/immunology , Betacoronavirus/physiology , Humans , SARS-CoV-2 , Sequence Deletion , Virus Replication
11.
Emerg Infect Dis ; 26(11): 2764-2766, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-647028

ABSTRACT

Coronavirus disease (COVID-19) and tuberculosis (TB) developed in 4 foreign workers living in dormitories in Singapore during April-May 2020. Clinical manifestations and atypical radiographic features of COVID-19 led to the diagnosis of TB through positive interferon-gamma release assay and culture results. During the COVID-19 pandemic, TB should not be overlooked.


Subject(s)
Betacoronavirus , Coinfection/microbiology , Coronavirus Infections/microbiology , Mycobacterium , Pneumonia, Viral/microbiology , Tuberculosis, Pulmonary/microbiology , Adult , COVID-19 , Female , Humans , Male , Pandemics , SARS-CoV-2 , Singapore , Young Adult
12.
BMC Med ; 18(1): 179, 2020 06 08.
Article in English | MEDLINE | ID: covidwho-549100

ABSTRACT

BACKGROUND: On January 30, COVID-19 was declared a Public Health Emergency of International Concern-a week after Singapore's first imported case and 5 days before local transmission. The National University Hospital (NUH) is Singapore's third largest hospital with 1200 beds, heavy clinical workloads, and major roles in research and teaching. MAIN BODY: With memories of SARS still vivid, there was an urgent requirement for the NUH Division of Infectious Diseases to adapt-undergoing major reorganization to face rapidly changing priorities while ensuring usual essential services and standards. Leveraging on individual strengths, our division mobilized to meet the demands of COVID-19 while engaging in high-level coordination, strategy, and advocacy. We present our experience of the 60 days since the nation's first case. During this time, our hospital has managed 3030 suspect cases, including 1300 inpatients, 37 confirmed cases, and overseen 4384 samples tested for COVID-19. CONCLUSION: Complex hospital adaptations were supported by an unprecedented number of workflows and coordination channels essential to safe and effective operations. The actions we describe, aligned with international recommendations and emerging evidence-based best practices, may serve as a framework for other divisions and institutions facing the spread of COVID-19 globally.


Subject(s)
Coronavirus Infections , Hospitals, University , Organizational Innovation , Pandemics , Pneumonia, Viral , Public Health , Academic Medical Centers , Betacoronavirus , COVID-19 , Communicable Diseases , Coronavirus Infections/epidemiology , Delivery of Health Care , Hospitals, University/organization & administration , Humans , Pneumonia, Viral/epidemiology , SARS-CoV-2 , Singapore/epidemiology , Workload
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