Study protocol: infectious diseases consortium (I3D) for study on integrated and innovative approaches for management of respiratory infections: respiratory infections research and outcome study (RESPIRO).

BACKGROUND: Community-acquired respiratory infections are a leading cause of illness and death globally. The aetiologies of community-acquired pneumonia remain poorly defined. The RESPIRO study is an ongoing prospective observational cohort study aimed at developing pragmatic logistical and analytic platforms to accurately identify the causes of moderate-to-severe community-acquired pneumonia in adults and understand the factors influencing disease caused by individual pathogens. The study is currently underway in Singapore and has plans for expansion into the broader region. METHODS: RESPIRO is being conducted at three major tertiary hospitals in Singapore. Adults hospitalised with acute community-acquired pneumonia or lower respiratory tract infections, based on established clinical, laboratory and radiological criteria, will be recruited. Over the course of the illness, clinical data and biological samples will be collected longitudinally and stored in a biorepository for future analysis. DISCUSSION: The RESPIRO study is designed to be hypothesis generating, complementary to and easily integrated with other research projects and clinical trials. The detailed clinical database and biorepository will yield insights into the epidemiology and outcomes of community-acquired lower respiratory tract infections in Singapore and the surrounding region and offers the opportunity to deeply characterise the microbiology and immunopathology of community-acquired pneumonia.

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.

Update on Non-Culture-Based Diagnostics for Invasive Fungal Disease.

Diagnostic tests for fungi provide the mycological evidence to strengthen diagnosis of invasive fungal disease. Conventional microbiology and histopathology have their limitations. Recognizing this, there have been attempts at developing new methods to improve yield of diagnosing invasive fungal disease (IFD). The recent focus has been on non-culture-based antigen detection and molecular methods. The use of antigen detection of IFD through 1,3-ß-D-glucan and galactomannan assay have been expanded, followed by development of lateral flow assays, and in combination with other diagnostic modalities to further increase diagnostic yield. The molecular diagnostic front has seen initiatives to standardize polymerase chain reaction methodologies to detect fungi and anti-fungal resistance, new platforms such as the T2Candida Biosystems and foray into fungal metagenomics. As these newer assays undergo stringent validation before incorporation into the diagnostic algorithm, the clinician needs to be mindful of their bedside utility as well as their limitation.

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.

Responding to COVID-19: how an academic infectious diseases division mobilized in Singapore.

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.

Distinguishing Zika and Dengue Viruses through Simple Clinical Assessment, Singapore.

Dengue virus and Zika virus coexist in tropical regions in Asia where healthcare resources are limited; differentiating the 2 viruses is challenging. We showed in a case-control discovery cohort, and replicated in a validation cohort, that the diagnostic indices of conjunctivitis, platelet count, and monocyte count reliably distinguished between these viruses.

Performance evaluation of Cepheid Xpert Norovirus kit with a user-modified protocol.

The Cepheid Xpert® Norovirus kit automates sample processing, nucleic acid extraction, and real-time reverse transcription polymerase chain reactions (RT-PCRs) to detect norovirus genogroups I (GI) and II (GII). Eighty-five stool samples collected between February 2015 and May 2017 were used to compare the performance of a user-modified Xpert assay against a clinically validated laboratory-developed test (LDT). Of the 85 samples, 54 were previously archived in -80°C freezer. The remaining 31 were fresh samples tested concurrently with the LDT. The results of all samples tested using the Xpert kit and LDT were found to be concordant, including 12 GI- and 42 GII-positive samples, 1 GI and GII coinfection, and 30 negative samples. Comparison of the assays showed perfect concordance with a kappa coefficient score of 1.00 (95%CI from 1.00 to 1.00). Of the 30 negative stool samples tested, three samples were positive for rotavirus detected using an immunochromatographic assay, with no cross-reactivity shown in both LDT and Xpert assays. In-run sample processing control of the Xpert assay for all negative samples tested showed no/minor inhibition. Compared to the LDT, the Xpert assay produced similar or better Ct values for detection. It also showed better mitigation of PCR inhibition in stool sample testing.

Singapore tuberculosis (TB) clinical management guidelines 2024: A modified Delphi adaptation of international guidelines for drug-susceptible TB infection and pulmonary disease.

Introduction: Tuberculosis (TB) remains endemic in Singapore. Singapore's clinical practice guidelines for the management of tuberculosis were first published in 2016. Since then, there have been major new advances in the clinical management of TB, ranging from diagnostics to new drugs and treatment regimens. The National TB Programme convened a multidisciplinary panel to update guidelines for the clinical management of drug-susceptible TB infection and disease in Singapore, contextualising current evidence for local practice. Method: Following the ADAPTE framework, the panel systematically reviewed, scored and synthesised English-language national and international TB clinical guidelines published from 2016, adapting recommendations for a prioritised list of clinical decisions. For questions related to more recent advances, an additional primary literature review was conducted via a targeted search approach. A 2-round modified Delphi process was implemented to achieve consensus for each recommendation, with a final round of edits after consultation with external stakeholders. Results: Recommendations for 25 clinical questions spanning screening, diagnosis, selection of drug regimen, monitoring and follow-up of TB infection and disease were formulated. The availability of results from recent clinical trials led to the inclusion of shorter treatment regimens for TB infection and disease, as well as consensus positions on the role of newer technologies, such as computer-aided detection-artificial intelligence products for radiological screening of TB disease, next-generation sequencing for drug-susceptibility testing, and video observation of treatment. Conclusion: The panel updated recommendations on the management of drug-susceptible TB infection and disease in Singapore.

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.

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 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.

Functional Dentition, Chronic Periodontal Disease and Frailty in Older Adults-A Narrative Review.

BACKGROUND: The likelihood of experiencing the impact of chronic disease rises with age, and cumulative functional deficits over time increase the risk of frailty in older adults. The exact causes of frailty are not clear, and research is needed to identify appropriate intervention measures to reduce risk of developing frailty in old age. OBJECTIVE: To review the evidence on the relationship between frailty, dental status and chronic periodontitis and to determine if improvements in oral health of older adults can contribute to reversal of frailty. RESULTS: The oral cavity is the entry point to the gastro-intestinal tract, and natural teeth facilitate efficient mastication of food prior to swallowing and subsequent digestion. The loss of natural teeth, which is gradual and cumulative over the life course, is associated with diminished nutritional intake, especially in older adults. Furthermore, chronic periodontitis has been postulated as a risk factor for frailty. The evidence supporting a strong relationship between oral health status and frailty is not clearcut. Cross sectional studies suggest an association with missing teeth and chronic periodontal inflammation. However, there are very few longitudinal studies and accordingly, it is not currently possible to claim a causal relationship. As yet, there is no evidence to suggest that improvements in oral health contribute to reversal of frailty. CONCLUSION: Longitudinal studies with robust designs are required to better inform the relationship across functional dentition, chronic periodontitis and frailty in older adults.

The relationship between self-efficacy and oral health status of older adults.

OBJECTIVES: The objectives of this study were to determine the relationship between reported self-efficacy and dental status in older adults, identify factors which might influence self-efficacy and, their willingness to pay (WTP) for preventive care. METHODS: Participants aged 60-90 years of age living in Singapore were recruited. All participants completed an oral health questionnaire and a clinical examination. Details of participants' socio-economic status and educational attainment were collected, and participants also answered a range of questions related to self-efficacy, oral health attitudes, beliefs and dental attendance patterns. Participants were asked to indicate their willingness to pay for preventive care using contingent valuation. The clinical examination recorded decayed, missing and filled teeth [DMFT], root caries, periodontal attachment loss, bleeding on probing index, occlusal status and, denture wearing status. Associations between self-efficacy, self-report and clinical variables were assessed using Kendall's Tau B coefficient. RESULTS: 614 participants [mean age 68.07 (5.99) years] were recruited. There was a high level of dental awareness and nearly 70% of the participants reported visiting a dentist once or twice a year. Self-efficacy was associated with levels of bleeding on probing and self-reported satisfaction with oral health. Nearly 60% of participants were not willing to pay for preventive advice from an oral healthcare professional. CONCLUSION: Older adults with a high level of self-efficacy had good gingival health, with low reported levels of bleeding on probing. Participants with low reported self-efficacy had higher disease levels and were less satisfied with their oral health. CLINICAL SIGNIFICANCE: Self-efficacy in relation to oral hygiene practices is variable, and participants with low self-efficacy had higher gingival bleeding scores. However, willingness to pay for preventive advice is low, and further work is required to increase the value proposition of preventive care to older adults.

Willingness to Pay for Preventive Dental Care Amongst Older Adults.

OBJECTIVE: The aims of this study were (1) to investigate willingness to pay (WTP) for preventive and curative dental care procedures and (2) to determine the factors that influence older adults' WTP for dental care. METHODOLOGY: Older, independently living adults from Singapore aged 60 years and older and eligible for government-subsidised dental care were nonrandomly recruited for this study. Data were collected using questionnaires and a clinical examination which recorded details of caries experience, number and distribution of posterior occluding contacts, prosthodontic status, and periodontal status. Using a contingent valuation method, participants were asked to rate WTP in Singapore dollars [SGD$] for 4 aspects of care: dental fillings, dental scaling, dental extraction, and disease prevention advice. Negative binomial regression was used to assess the relationship between the predictor variables associated with WTP for dental fillings, scaling, extraction, and preventive advice. RESULTS: The mean value of WTP for a dental filling was SGD$30.23 (SGD$31.05), for scaling was SGD$30.28 (SGD$29.46), for dental extraction was SGD$35.08 (SGD$58.54). In a multivariate model, factors associated with higher WTPfees were as follows: (1) dental filling: age (younger), level of education (higher), and frequency of dental visits (regular); (2) scaling: level of education (higher), agree that dental problems affect overall health, and frequency of dental visits (regular); (3) dental extractions: age (younger), level of education (higher), frequency of dental visits (regular), and prosthodontic status (not wearing); (4) preventive advice: age (younger), gender (male), ethnicity (Chinese), level of education (higher), marital status (married), self-perceived oral health (good), and dental visits (regular). CONCLUSIONS: The findings of our study suggest that older adults are willing to pay most for extraction and least for preventive advice.