Successful Confirmation of Dual Genital Herpes Co-Infection with Herpes Simplex Virus 1 and Herpes Simplex Virus 2 Using Unbiased Metagenomic Next-Generation Sequencing.

Dual co-infection with both HSV-1 and HSV-2 is rare, with few cases reported in the literature. In this case report, we describe the successful use of unbiased metagenomic next-generation sequencing (mNGS) as a rapid and alternative method for confirming dual genital herpes co-infection. Our case involves a 74-year-old woman who presented with genital lesions and initially tested positive for both HSV-1 and HSV-2 via the Luminex ARIES HSV 1&2 assay. The entire mNGS process, from nucleic acid extraction to result analysis, was completed in less than 48 h. Using mNGS, we identified mapped reads specific to either HSV-1 or HSV-2 and screened the sequences to rule out mis-genotyping by the Luminex ARIES assay. Notably, the generated sequences can reveal sequence variations within multiple gene regions, demonstrating the potential of mNGS for identifying novel HSV-1 and HSV-2 variants. Our findings suggest that mNGS can serve as a rapid and reliable alternative confirmatory method for dual genital herpes infections, providing valuable information to guide appropriate treatment options for patients. By eliminating the need for prior knowledge of causative agents, mNGS offers an unbiased approach for detecting and characterizing viral co-infections.

Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms.

Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.

Sub genomic analysis of SARS-CoV-2 using short read amplicon-based sequencing.

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.

Towards Next-Generation Sequencing for HIV-1 Drug Resistance Testing in a Clinical Setting.

The HIV genotypic resistance test (GRT) is a standard of care for the clinical management of HIV/AIDS patients. In recent decades, population or Sanger sequencing has been the foundation for drug resistance monitoring in clinical settings. However, the advent of high-throughput or next-generation sequencing has caused a paradigm shift towards the detection and characterization of low-abundance covert mutations that would otherwise be missed by population sequencing. This is clinically significant, as these mutations can potentially compromise the efficacy of antiretroviral therapy, causing poor virologic suppression. Therefore, it is important to develop a more sensitive method so as to reliably detect clinically actionable drug-resistant mutations (DRMs). Here, we evaluated the diagnostic performance of a laboratory-developed, high-throughput, sequencing-based GRT using 103 archived clinical samples that were previously tested for drug resistance using population sequencing. As expected, high-throughput sequencing found all the DRMs that were detectable by population sequencing. Significantly, 78 additional DRMs were identified only by high-throughput sequencing, which is statistically significant based on McNemar's test. Overall, our results complement previous studies, supporting the notion that the two methods are well correlated, and the high-throughput sequencing method appears to be an excellent alternative for drug resistance testing in a clinical setting.

Strategy for Safe Bronchoscopy During the COVID-19 Pandemic.

Aerosol-generating procedures are avoided for patients with coronavirus disease 2019 (COVID-19) to lower the risk of transmission to health care providers. However, when bronchoscopy is indicated, it remains unclear whether the procedure performed while the patient is under general anesthesia leads to contamination of the surroundings and whether standard endoscopy reprocessing methods are effective in eradicating severe acute respiratory syndrome coronavirus 2. This report describes a case of bronchoscopic retrieval of a foreign body in the airway of a patient under general anesthesia who tested positive for COVID-19. The report focuses on anesthesia techniques to minimize aerosolization.

Effects of acute severe acute respiratory syndrome coronavirus 2 infection on male hormone profile, ACE2 and TMPRSS2 expression, and potential for transmission of severe acute respiratory syndrome coronavirus 2 in semen of Asian men.

OBJECTIVE: To confirm if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen of men with acute coronavirus disease 2019 and if their male hormone profile (testosterone, follicle-stimulating hormone, luteinizing hormone, sex hormone binding globulin, and free androgen index) is adversely affected during the acute phase of infection and any relation to the ACE2 and/or TMPRSS2 expression in human semen. DESIGN: Clinical study. SETTING: National University Hospital, Singapore. PATIENTS: Asian men aged 21-55 years who were admitted to National University Hospital, Singapore, with a laboratory-confirmed diagnosis of SARS-CoV-2 infection via nasopharyngeal swab in the acute phase of the infection, within 2-14 days of the development of symptoms or contact history, were recruited for the study. INTERVENTIONS: Blood was collected in the morning to assess the male hormone profile. Human semen were obtained by masturbation and sent to the molecular diagnostic laboratories to detect the presence of SARS-CoV-2 RNA and assess the ACE2 and TMPRSS2 expression. MAIN OUTCOME MEASURES: Male hormone profile level and expression of SARS-CoV-2 RNA, ACE2, and TMPRSS2 in human semen. RESULTS: A total of 63 men of Asian ethnicities agreed to participate in the study. Subsequently, 65% of recruited men had completely normal levels of male hormone profile. Moreover, 27% were noted to have higher luteinizing hormone levels between 6.6 and 16.1 IU/L (normal range, 0.8-6.1 IU/L), and 10% had higher follicle-stimulating hormone levels between 13.6 and 41.6 IU/L (normal range, 1.5-12.4 IU/L); all had normal testosterone levels. No SARS-CoV-2 RNAs were detected in all human semen. The ACE2 and TMPRSS2 expression was undetectable in 26 samples, whereas 23 samples only had a detectable TMPRSS2 expression and 4 only had an ACE2 expression. The remaining 3 expressed both ACE2 and TMPRSS2. CONCLUSIONS: Severe acute respiratory syndrome coronavirus 2 could not be found in the semen of a cohort of young to middle-aged Asian men with mild acute SARS-CoV-2 infection. However, there was a detectable expression of ACE2 and TMPRSS2 in semen, although not causal, and it may be correlated to changes in male hormone profiles and male age.

Validation of a multi-gene qPCR-based pharmacogenomics panel across major ethnic groups in Singapore and Indonesia.

Aim: The clinical utility of pharmacogenomics (PGx) has been gaining traction alongside growing evidence that adverse drug reactions (ADRs) have significant genetic associations. Nala PGx Core® is a multi-gene qPCR-based panel of 20 allele variants, comprising 18 SNPs and two CYP2D6 copy number markers across four pharmacogenes - CYP2C9, CYP2C19, CYP2D6 and SLCO1B1. Methods: In this study, we validated the performance of Nala PGx Core® against benchmark methods, on the Singaporean and Indonesian populations. Results & conclusion: Nala PGx Core® demonstrated robust and accurate genotyping when compared with other established benchmarks. Furthermore, the panel successfully characterized alleles of clinical relevance, such as CYP2D6*10 and CYP2D6*36, across major ethnic groups present of Singapore and Indonesia, suggesting its potential for adoption in clinical workflows regionally.

Range of Varicella Zoster Co-Infections with COVID-19, Singapore.

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.

Correlating indoor and outdoor temperature and humidity in a sample of buildings in tropical climates.

The incidence of several respiratory viral infections has been shown to be related to climate. Because humans spend most of their time indoors, measures of indoor climate, rather than outdoor climate, may be better predictors of disease incidence and transmission. Therefore, understanding the relationship between indoor and outdoor climate will help illuminate their influence on the seasonality of diseases caused by respiratory viruses. Indoor-outdoor relationships between temperature and humidity have been documented in temperate regions, but little information is available for tropical regions, where seasonal patterns of respiratory viral diseases differ. We have examined indoor-outdoor correlations of temperature, relative humidity (RH), and absolute humidity (AH) over a 1-year period in each of seven tropical cities. Across all cities, the average monthly indoor temperature was 25 ± 3°C (mean ± standard deviation) with a range of 20-30°C. The average monthly indoor RH was 66 ± 9% with a range of 50-78%, and the average monthly indoor AH was 15 ± 3 g/m3 with a range of 10-23 g/m3 . Indoor AH and RH were linearly correlated with outdoor AH when the air conditioning (AC) was off, suggesting that outdoor AH may be a good proxy of indoor humidity in the absence of AC. All indoor measurements were more strongly correlated with outdoor measurements as distance from the equator increased. Such correlations were weaker during the wet season, especially when AC was in operation. These correlations will provide insight for assessing the seasonality of respiratory viral infections using outdoor climate data, which is more widely available than indoor data, even though transmission of these diseases mainly occurs indoors.

Clinical performance of Roche cobas 6800, Luminex ARIES, MiRXES Fortitude Kit 2.1, Altona RealStar, and Applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.

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.

An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing.

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.

Design and Multicenter Clinical Validation of a 3-Dimensionally Printed Nasopharyngeal Swab for SARS-CoV-2 Testing.

Importance: Three-dimensionally printed nasopharyngeal swabs (3DP swabs) have been used to mitigate swab shortages during the coronavirus disease 2019 (COVID-19) pandemic. Clinical validation for diagnostic accuracy and consistency, as well as patient acceptability, is crucial to evaluate the swab's performance. Objective: To determine the accuracy and acceptability of the 3DP swab for identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Design, Setting, and Participants: A diagnostic study was conducted from May to July 2020 at 2 tertiary care centers in Singapore with different reference swabs (FLOQSwab [COPAN Diagnostics] or Dacron swab [Deltalab]) and swab processing techniques (wet or dry) to evaluate the performance of the 3DP swab compared with traditional, standard-of-care nasopharyngeal swabs used in health care institutions. The participants were patients with COVID-19 in the first 2 weeks of illness and controls with acute respiratory illness with negative test results for SARS-CoV-2. Paired nasopharyngeal swabs were obtained from the same nostril and tested for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction. The sequence of swabs was randomized based on odd and even participant numbers. Main Outcomes and Measures: Primary outcome measures were overall agreement (OA), positive percentage agreement (PPA), and negative percentage agreement of the 3DP swab compared with reference swabs. Secondary outcome measures were the correlation of cycle threshold (Ct) values of both swabs. Results: The mean (SD) age of participants was 45.4 (13.1) years, and most participants were men (87 of 89 [97.8%]), in keeping with the epidemiology of the COVID-19 pandemic in Singapore. A total of 79 patients with COVID-19 and 10 controls were recruited. Among the patients with COVID-19, the overall agreement and PPA of the 3DP swab was 91.1% and 93.5%, respectively, compared with reference swabs. The PPA was 100% for patients with COVID-19 who were tested within the first week of illness. All controls tested negative. The reverse-transcriptase polymerase chain reaction Ct values for the ORF1ab and E-gene targets showed a strong correlation (intraclass correlations coefficient, 0.869-0.920) between the 3DP and reference swab on independent testing at each institution despite differences in sample processing. Discordant results for both gene targets were observed only at high Ct values. Conclusions and Relevance: In this diagnostic study of 79 patients with COVID-19 and 10 controls, the 3DP swab performed accurately and consistently across health care institutions and could help mitigate strained resources in the escalating COVID-19 pandemic.

Clinical Diagnostic Study of a Novel Injection Molded Swab for SARS-Cov-2 Testing.

INTRODUCTION: The gold standard for COVID-19 diagnosis is currently a real-time reverse transcriptase polymerase chain reaction (RT-PCR) to detect SARS-CoV-2. This is most commonly performed on respiratory secretions obtained via a nasopharyngeal swab. Due to supply chain limitations and high demand worldwide because of the COVID-19 pandemic, access to commercial nasopharyngeal swabs has not been assured. 3D printing methods have been used to meet the shortfall. For longer-term considerations, 3D printing may not compare well with injection molding as a production method due to the challenging scalability and greater production costs of 3D printing. METHODS: To secure sufficient nasopharyngeal swab availability for our national healthcare system, we designed a novel injection molded nasopharyngeal swab (the IM2 swab). We performed a clinical diagnostic study comparing the IM2 swab to the Copan FLOQSwab. Forty patients with a known diagnosis of COVID-19 and 10 healthy controls were recruited. Paired nasopharyngeal swabs were obtained from the same nostril of each participant and tested for SARS-CoV-2 by RT-PCR. RESULTS: When compared to the Copan FLOQswab, results from the IM2 swab displayed excellent overall agreement and positive percent agreement of 96.0% and 94.9%, respectively. There was no significant difference in mean RT-PCR cycle threshold values for the ORF1ab (28.05 vs. 28.03, p = 0.97) and E-gene (29.72 vs. 29.37, p = 0.64) targets, respectively. We did not observe any significant adverse events and there was no significant difference in patient-reported pain. CONCLUSION: In summary, the IM2 nasopharyngeal swab is a clinically safe, highly accurate option to commercial nasopharyngeal swabs.

Practical laboratory considerations amidst the COVID-19 outbreak: early experience from Singapore.

The coronavirus disease 2019 (COVID-19) is a zoonotic viral infection originating from Wuhan, China in December 2019. The World Health Organization has classified this pandemic as a global health emergency due to its virulent nature of transmission, which may lead to acute respiratory distress syndrome. Singapore's health ministry has responded with enhanced surveillance of COVID-19 for all suspected pneumonia cases, further increasing the volume of testing via real-time reverse transcription PCR, as well as samples necessitating stringent infectious control. Collectively, this has implications on the total testing process, laboratory operations and its personnel due to biosafety concerns. Turnaround time for routine testing may also be affected. The aim of this article is to present our tertiary institution's early experience with managing this emerging crisis and offer practical considerations for the preanalytical, analytical and postanalytical phases of laboratory testing in this cohort of patients.

Four Patients with COVID-19 and Tuberculosis, Singapore, April-May 2020.

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.