Detection and characterisation of a sixth Candida auris clade in Singapore: a genomic and phenotypic study.

BACKGROUND: The emerging fungal pathogen Candida auris poses a serious threat to global public health due to its worldwide distribution, multidrug resistance, high transmissibility, propensity to cause outbreaks, and high mortality. We aimed to characterise three unusual C auris isolates detected in Singapore, and to determine whether they constitute a novel clade distinct from all previously known C auris clades (I-V). METHODS: In this genotypic and phenotypic study, we characterised three C auris clinical isolates, which were cultured from epidemiologically unlinked inpatients at a large tertiary hospital in Singapore. The index isolate was detected in April, 2023. We performed whole-genome sequencing (WGS) and obtained hybrid assemblies of these C auris isolates. The complete genomes were compared with representative genomes of all known C auris clades. To provide a global context, 3651 international WGS data from the National Center for Biotechnology Information (NCBI) database were included in a high-resolution single nucleotide polymorphism (SNP) analysis. Antifungal susceptibility testing was done and antifungal resistance genes, mating-type locus, and chromosomal rearrangements were characterised from the WGS data of the three investigated isolates. We further implemented Bayesian logistic regression models to classify isolates into known clades and simulate the automatic detection of isolates belonging to novel clades as their WGS data became available. FINDINGS: The three investigated isolates were separated by at least 37 000 SNPs (range 37 000-236 900) from all existing C auris clades. These isolates had opposite mating-type allele and different chromosomal rearrangements when compared with their closest clade IV relatives. The isolates were susceptible to all tested antifungals. Therefore, we propose that these isolates represent a new clade of C auris, clade VI. Furthermore, an independent WGS dataset from Bangladesh, accessed via the NCBI Sequence Read Archive, was found to belong to this new clade. As a proof-of-concept, our Bayesian logistic regression model was able to flag these outlier genomes as a potential new clade. INTERPRETATION: The discovery of a new C auris clade in Singapore and Bangladesh in the Indomalayan zone, showing a close relationship to clade IV members most commonly found in South America, highlights the unknown genetic diversity and origin of C auris, particularly in under-resourced regions. Active surveillance in clinical settings, along with effective sequencing strategies and downstream analysis, will be essential in the identification of novel strains, tracking of transmission, and containment of adverse clinical effects of C auris infections. FUNDING: Duke-NUS Academic Medical Center Nurturing Clinician Researcher Scheme, and the Genedant-GIS Innovation Program.

Rapid management of Serratia marcescens outbreak in neonatology unit in Singapore: Risk factors and infection control measures.

BACKGROUND: We describe the investigations for control of two consecutive Serratia marcescens outbreaks in neonatology unit of Singapore General Hospital. METHODS: Epidemiological investigations, environmental sampling and risk-factors analysis were performed to guide infection control measures. Active surveillance sampling of nasopharyngeal aspirate and/or stool from neonates was conducted during both outbreaks. Whole-genome-sequencing was done to determine clonal links. Retrospective case-control study was conducted for second outbreak to identify risk factors for S marcescens acquisition. RESULTS: In 2022, two genetically unrelated S marcescens outbreaks were managed involving five neonates in March 2022 (outbreak 1) and eight neonates in November 2022 (outbreak 2). A link to positive isolates from sinks in intensive care units and milk preparation room was identified during outbreak 1. Neonatal jaundice (aOR, 16.46; p-value= 0.023) and non-formula milk feeding (aOR, 13.88; p-value= 0.02) were identified as risk factors during second outbreak. Multiple interventions adopted were cohorting of positive cases, carriage-screening, enhanced environmental cleaning, and emphasis on alcohol-based handrubs for hand-hygiene. CONCLUSION: The two outbreaks were likely due to infection prevention practices lapses and favourable environmental conditions. Nosocomial S marcescens outbreaks in neonatology units are difficult to control and require multidisciplinary approach with strict infection prevention measures to mitigate risk factors.

Genomic Identification of Two Phytobacter diazotrophicus Isolates from a Neonatal Intensive Care Unit in Singapore.

We report the draft genome sequences of two Phytobacter diazotrophicus isolates recovered from a swab specimen from the water faucet located in the Neonatal Intensive Care Unit (ICU), National University Hospital, Singapore. The isolates were misidentified as Cronobacter sakazakii and Klebsiella oxytoca using biochemical methods. Whole-genome sequencing (WGS) was performed to determine their identity.

Comparative Activities of Novel Therapeutic Agents against Molecularly Characterized Clinical Carbapenem-Resistant Enterobacterales Isolates.

Limited treatment options exist for the treatment of carbapenem-resistant Enterobacterales (CRE) bacteria. Fortunately, there are several recently approved antibiotics indicated for CRE infections. Here, we examine the in vitro activity of various novel agents (eravacycline, plazomicin, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam) and comparators (tigecycline, amikacin, levofloxacin, fosfomycin, polymyxin B) against 365 well-characterized CRE clinical isolates with various genotypes. Nonduplicate isolates collected from the largest public health hospital in Singapore between 2007 and 2020 were subjected to antimicrobial susceptibility testing (broth microdilution or antibiotic gradient test strips). Susceptibilities were defined using Clinical and Laboratory Standards Institute (CLSI) or Food and Drug Administration (FDA) interpretative criteria. Sequence types and resistance mechanisms were characterized using short-read whole-genome sequencing. Overall, tigecycline and plazomicin exhibited the highest susceptibility rates (89.6% and 80.8%, respectively). However, the tigecycline susceptibility breakpoint utilized here may be outdated in view of prevailing pharmacokinetic-pharmacodynamic (PK/PD) data. Susceptibility varied by carbapenemase genotype; the ß-lactam/ß-lactamase inhibitor combinations were equally active (92.3 to 99.2% susceptible) against KPC producers, but only ceftazidime-avibactam retained high susceptibility (98.7%) against OXA-48-like producers. Against metallo-ß-lactamase producers, only plazomicin exhibited moderate activity (77.0% susceptible). Aminoglycoside activity was also influenced by carbapenemase genotypes. This work provides an insight into the comparative activity and presumptive utility of novel agents in this geographic region. IMPORTANCE This study determined the susceptibilities of carbapenem-resistant Enterobacterales isolates to various novel antimicrobial agents (ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, eravacycline, and plazomicin). Whole-genome sequencing was performed for all strains. Our study findings provide insights into the comparative activities of novel agents in this geographic region. Plazomicin and ceftazidime-avibactam exhibited the lowest nonsusceptibility rates and may be considered promising agents in the management of carbapenem-resistant Enterobacterales infections. We note also that antibiotic activity is influenced by genotypes and that understanding the geographic region's molecular epidemiology could aid in the definition of the presumptive utility of novel agents and contribute to antibiotic decision-making.

Environmental contamination and evaluation of healthcare-associated SARS-CoV-2 transmission risk in temporary isolation wards during the COVID-19 pandemic.

BACKGROUND: Temporary isolation wards have been introduced to meet demands for airborne-infection-isolation-rooms (AIIRs) during the COVID-19 pandemic. Environmental sampling and outbreak investigation was conducted in temporary isolation wards converted from general wards and/or prefabricated containers, in order to evaluate the ability of such temporary isolation wards to safely manage COVID-19 cases over a period of sustained use. METHODS: Environmental sampling for SARS-CoV-2 RNA was conducted in temporary isolation ward rooms constructed from pre-fabricated containers (N = 20) or converted from normal-pressure general wards (N = 47). Whole genome sequencing (WGS) was utilized to ascertain health care-associated transmission when clusters were reported amongst HCWs working in isolation areas from July 2020 to December 2021. RESULTS: A total of 355 environmental swabs were collected; 22.4% (15/67) of patients had at least one positive environmental sample. Patients housed in temporary isolation ward rooms constructed from pre-fabricated containers (adjusted-odds-ratio, aOR = 10.46, 95% CI = 3.89-58.91, P = .008) had greater odds of detectable environmental contamination, with positive environmental samples obtained from the toilet area (60.0%, 12/20) and patient equipment, including electronic devices used for patient communication (8/20, 40.0%). A single HCW cluster was reported amongst staff working in the temporary isolation ward constructed from pre-fabricated containers; however, health care-associated transmission was deemed unlikely based on WGS and/or epidemiological investigations. CONCLUSION: Environmental contamination with SARS-CoV-2 RNA was observed in temporary isolation wards, particularly from the toilet area and smartphones used for patient communication. However, despite intensive surveillance, no healthcare-associated transmission was detected in temporary isolation wards over 18 months of prolonged usage, demonstrating their capacity for sustained use during succeeding pandemic waves.

Nosocomial SARS-CoV-2 transmission in multi-bedded hospital cubicles over successive pandemic waves: Lower mortality but wider spread with Omicron despite enhanced infection-prevention measures.

BACKGROUND: Increased transmissibility of severe-acute-respiratory-syndrome-coronavirus-2(SARS-CoV-2) variants, such as the Omicron-variant, presents an infection-control challenge. We contrasted nosocomial transmission amongst hospitalized inpatients across successive pandemic waves attributed to the Delta- and Omicron variants, over a 9-month period in which enhanced-infection-prevention-measures were constantly maintained. METHODS: Enhanced-infection-prevention-measures in-place at a large tertiary hospital included universal N95-usage, routine-rostered-testing (RRT) for all inpatient/healthcare-workers (HCWs), rapid-antigen-testing (RAT) for visitors, and outbreak-investigation coupled with enhanced-surveillance (daily-testing) of exposed patients. The study-period lasted from 21st June 2021-21st March 2022. Chi-square test and multivariate-logistic-regression was utilized to identify factors associated with onward transmission and 28d-mortality amongst inpatient cases of hospital-onset COVID-19. RESULTS: During the Delta-wave, hospital-onset cases formed 2.7% (47/1727) of all COVID-19 cases requiring hospitalisation; in contrast, hospital onset-cases formed a greater proportion (17.7%, 265/1483; odds-ratio, OR = 7.78, 95%CI = 5.65-10.70) during the Omicron-wave, despite universal N95-usage and other enhanced infection-prevention measures that remained unchanged. The odds of 28d-mortality were higher during the Delta-wave compared to the Omicron-wave (27.7%, 13/47, vs. 10.6%, 28/265, adjusted-odds-ratio, aOR = 2.78, 95%CI = 1.02-7.69). Onward-transmission occurred in 21.2% (66/312) of hospital-onset cases; being on enhanced-surveillance (daily-testing) was independently associated with lower odds of onward-transmission (aOR = 0.18, 95%CI = 0.09-0.38). Costs amounted to $USD7141 per-hospital-onset COVID-19 case. CONCLUSION: A surge of hospital-onset COVID-19 cases was encountered during the Omicron-wave, despite continuation of enhanced infection-prevention measures; mortality amongst hospital-onset cases was reduced. The Omicron variant poses an infection-control challenge in contrast to Delta; surveillance is important especially in settings where infrastructural limitations make room-sharing unavoidable, despite the high risk of transmission.

No evidence for a common blood microbiome based on a population study of 9,770 healthy humans.

Human blood is conventionally considered sterile but recent studies suggest the presence of a blood microbiome in healthy individuals. Here we characterized the DNA signatures of microbes in the blood of 9,770 healthy individuals using sequencing data from multiple cohorts. After filtering for contaminants, we identified 117 microbial species in blood, some of which had DNA signatures of microbial replication. They were primarily commensals associated with the gut (n = 40), mouth (n = 32) and genitourinary tract (n = 18), and were distinct from pathogens detected in hospital blood cultures. No species were detected in 84% of individuals, while the remainder only had a median of one species. Less than 5% of individuals shared the same species, no co-occurrence patterns between different species were observed and no associations between host phenotypes and microbes were found. Overall, these results do not support the hypothesis of a consistent core microbiome endogenous to human blood. Rather, our findings support the transient and sporadic translocation of commensal microbes from other body sites into the bloodstream.

Relapsing COVID-19 infection as a manifestation of Good syndrome: a case report and literature review.

Good syndrome (GS) is a rare acquired immunodeficiency disease characterized by the presence of thymoma with combined B and T cell immunodeficiency in adults. Recurrent bacterial infections, particularly sinopulmonary infections caused by encapsulated bacteria, remain the most common infective presentation of GS; however, relapsing viral infections have also been reported, likely due to impaired T cell-mediated immunity. Relapsing COVID-19 infection, however, has not been previously reported as a manifestation of GS. We present two cases of relapsing COVID-19 infection in patients with GS; in one case, relapsing COVID-19 was the first manifestation of newly diagnosed GS.

Healthcare-associated multispecies outbreaks of OXA-48-positive carbapenemase-producing Enterobacteriaceae in a Singapore tertiary-care hospital.

OBJECTIVE: To describe OXA-48-like carbapenem-producing Enterobacteriaceae (CPE) outbreaks at Singapore General Hospital between 2018 and 2020 and to determine the risk associated with OXA-48 carriage in the 2020 outbreak. DESIGN: Outbreak report and case-control study. SETTING: Singapore General Hospital (SGH) is a tertiary-care academic medical center in Singapore with 1,750 beds. METHODS: Active surveillance for CPE is conducted for selected high-risk patient cohorts through molecular testing on rectal swabs or stool samples. Patients with CPE are isolated or placed in cohorts under contact precautions. During outbreak investigations, rectal swabs are repeated for culture. For the 2020 outbreak, a retrospective case-control study was conducted in which controls were inpatients who tested negative for OXA-48 and were selected at a 1:3 case-to-control ratio. RESULTS: Hospital wide, the median number of patients with healthcare-associated OXA-48 was 2 per month. In the 3-year period between 2018 and 2020, 3 OXA-48 outbreaks were investigated and managed, involving 4 patients with Klebsiella pneumoniae in 2018, 55 patients with K. pneumoniae or Escherichia coli in 2019, and 49 patients with multispecies Enterobacterales in 2020. During the 2020 outbreak, independent risk factors for OXA-48 carriage on multivariate analysis (49 patients and 147 controls) were diarrhea within the preceding 2 weeks (OR, 3.3; 95% CI, 1.1-10.7; P = .039), contact with an OXA-48-carrying patient (OR, 8.7; 95% CI, 1.9-39.3; P = .005), and exposure to carbapenems (OR, 17.2; 95% CI, 2.2-136; P = .007) or penicillin (OR, 16.6; 95% CI, 3.8-71.0; P < .001). CONCLUSIONS: Multispecies OXA-48 outbreaks in our institution are likely related to a favorable ecological condition and selective pressure exerted by antimicrobial use. The integration of molecular surveillance epidemiology of the healthcare environment is important in understanding the risk of healthcare-associated infection to patients.

Linking sporadic hospital clusters during a community surge of the severe acute respiratory coronavirus virus 2 (SARS-CoV-2) B.1.617.2 delta variant: The utility of whole-genome sequencing.

Sporadic clusters of healthcare-associated coronavirus disease 2019 (COVID-19) occurred despite intense rostered routine surveillance and a highly vaccinated healthcare worker (HCW) population, during a community surge of the severe acute respiratory coronavirus virus 2 (SARS-CoV-2) B.1.617.2 δ (delta) variant. Genomic analysis facilitated timely cluster detection and uncovered additional linkages via HCWs moving between clinical areas and among HCWs sharing a common lunch area, enabling early intervention.

CREAMMIST: an integrative probabilistic database for cancer drug response prediction.

Extensive in vitro cancer drug screening datasets have enabled scientists to identify biomarkers and develop machine learning models for predicting drug sensitivity. While most advancements have focused on omics profiles, cancer drug sensitivity scores precalculated by the original sources are often used as-is, without consideration for variabilities between studies. It is well-known that significant inconsistencies exist between the drug sensitivity scores across datasets due to differences in experimental setups and preprocessing methods used to obtain the sensitivity scores. As a result, many studies opt to focus only on a single dataset, leading to underutilization of available data and a limited interpretation of cancer pharmacogenomics analysis. To overcome these caveats, we have developed CREAMMIST (https://creammist.mtms.dev), an integrative database that enables users to obtain an integrative dose-response curve, to capture uncertainty (or high certainty when multiple datasets well align) across five widely used cancer cell-line drug-response datasets. We utilized the Bayesian framework to systematically integrate all available dose-response values across datasets (>14 millions dose-response data points). CREAMMIST provides easy-to-use statistics derived from the integrative dose-response curves for various downstream analyses such as identifying biomarkers, selecting drug concentrations for experiments, and training robust machine learning models.

Clonal serotype 1c multidrug-resistant Shigella flexneri detected in multiple institutions by sentinel-site sequencing.

Shigella flexneri is a major diarrhoeal pathogen, and the emergence of multidrug-resistant S. flexneri is of public health concern. We report the detection of a clonal cluster of multidrug-resistant serotype 1c (7a) S. flexneri in Singapore in April 2022. Long-read whole-genome sequence analysis found five S. flexneri isolates to be clonal and harboring the extended-spectrum ß-lactamases bla CTX-M-15 and bla TEM-1. The isolates were phenotypically resistant to ceftriaxone and had intermediate susceptibility to ciprofloxacin. The S. flexneri clonal cluster was first detected in a tertiary hospital diagnostic laboratory (sentinel-site), to which the S. flexneri isolates were sent from other hospitals for routine serogrouping. Long-read whole-genome sequence analysis was performed in the sentinel-site near real-time in view of the unusually high number of S. flexneri isolates received within a short time frame. This study demonstrates that near real-time sentinel-site sequence-based surveillance of convenience samples can detect possible clonal outbreak clusters and may provide alerts useful for public health mitigations at the earliest possible opportunity.

Detection of viable SARS-CoV-2 in deep respiratory specimens despite negative nasopharyngeal SARS-CoV-2 RT-PCR: Occult COVID-19 as an unsuspected cause of pulmonary infiltrates in immunocompromised patients.

Background: Prolonged shedding/relapse of COVID-19 infection has been reported, particularly in patients who received anti-CD20 agents (eg. rituximab). However, cases of occult COVID-19, in which SARS-CoV-2 persistence in lung parenchyma is diagnosed despite clearance from nasopharyngeal (NP) specimens, are uncommon. Case summary: We describe two cases of occult COVID-19 in immunocompromised patients. Both patients had received rituximab previously. Both cases initially presented as ground-glass infiltrates on lung imaging; the diagnosis was originally not suspected due to repeated demonstration of negative SARS-CoV-2 from NP specimens, and alternative etiologies were originally considered. Persistence of SARS-CoV-2 in lung parenchyma, however, was demonstrated on bronchoalveolar lavage (BAL) specimens; additionally, isolation of viable SARS-CoV-2 virus and detection of SARS-CoV-2 nucleocapsid and spike-protein antigen in lung tissue on immunohistochemistry close to 3-months from primary infection strongly suggested ongoing viral persistence and replication as a driver of the lung parenchymal changes, which resolved after antiviral treatment. Discussion: Occult COVID-19 can be a cause of unexplained ground-glass infiltrates on lung imaging; negative NP samples do not rule out SARS-CoV-2 persistence and invasive sampling must be considered. The unsuspected presence of viable virus on BAL, however, highlights that procedurists perfoming aerosol-generating-procedures during an ongoing pandemic wave must also practise appropriate infection-prevention precautions to limit potential exposure.

Emergence of SARS-CoV-2 Spike Mutations during Prolonged Infection in Immunocompromised Hosts.

Immunocompromised hosts with prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been implicated in the emergence of highly mutated SARS-CoV-2 variants. Spike mutations are of particular concern because the spike protein is a key target for vaccines and therapeutics for SARS-CoV-2. Here, we report the emergence of spike mutations in two immunocompromised patients with persistent SARS-CoV-2 reverse transcription (RT)-PCR positivity (>90 days). Whole-genome sequence analysis of samples obtained before and after coronavirus disease 2019 (COVID-19) treatment demonstrated the development of partial therapeutic escape mutations and increased intrahost SARS-CoV-2 genome diversity over time. This case series thus adds to the accumulating evidence that immunocompromised hosts with persistent infections are important sources of SARS-CoV-2 genome diversity and, in particular, clinically important spike protein diversity. IMPORTANCE The emergence of clinically important mutations described in this report highlights the need for sustained vigilance and containment measures when managing immunocompromised patients with persistent COVID-19. Even as jurisdictions across the globe start lifting pandemic control measures, immunocompromised patients with persistent COVID-19 constitute a unique group that requires close genomic monitoring and enhanced infection control measures, to ensure early detection and containment of mutations and variants of therapeutic and public health importance.

Metagenomics-enabled microbial surveillance.

Lessons learnt from the COVID-19 pandemic include increased awareness of the potential for zoonoses and emerging infectious diseases that can adversely affect human health. Although emergent viruses are currently in the spotlight, we must not forget the ongoing toll of morbidity and mortality owing to antimicrobial resistance in bacterial pathogens and to vector-borne, foodborne and waterborne diseases. Population growth, planetary change, international travel and medical tourism all contribute to the increasing frequency of infectious disease outbreaks. Surveillance is therefore of crucial importance, but the diversity of microbial pathogens, coupled with resource-intensive methods, compromises our ability to scale-up such efforts. Innovative technologies that are both easy to use and able to simultaneously identify diverse microorganisms (viral, bacterial or fungal) with precision are necessary to enable informed public health decisions. Metagenomics-enabled surveillance methods offer the opportunity to improve detection of both known and yet-to-emerge pathogens.