The dissemination of antimicrobial resistance (AMR) is an escalating problem and a threat to public health. Comparative metagenomics was used to investigate the occurrence of antibiotic resistant ...genes (ARGs) in wastewater and urban surface water environments in Singapore. Hospital and municipal wastewater (
= 6) were found to have higher diversity and average abundance of ARGs (303 ARG subtypes, 197,816 x/Gb) compared to treated wastewater effluent (
= 2, 58 ARG subtypes, 2,692 x/Gb) and surface water (
= 5, 35 subtypes, 7,985 x/Gb). A cluster analysis showed that the taxonomic composition of wastewaters was highly similar and had a bacterial community composition enriched in gut bacteria (
), the
group (
) and opportunistic pathogens (
). Wastewater, treated effluents and surface waters had a shared resistome of 21 ARGs encoding multidrug resistant efflux pumps or resistance to aminoglycoside, macrolide-lincosamide-streptogramins (MLS), quinolones, sulfonamide, and tetracycline resistance which suggests that these genes are wide spread across different environments. Wastewater had a distinctively higher average abundance of clinically relevant, class A beta-lactamase resistant genes (i.e.,
,
,
,
). The wastewaters from clinical isolation wards, in particular, had a exceedingly high levels of
genes (142,200 x/Gb), encoding for carbapenem resistance. Assembled scaffolds (16 and 30 kbp) from isolation ward wastewater samples indicated this gene was located on a Tn3-based transposon (Tn
), a mobilization element found in
plasmids. In the longer scaffold, transposable elements were flanked by a toxin-antitoxin (TA) system and other metal resistant genes that likely increase the persistence, fitness and propagation of the plasmid in the bacterial host under conditions of stress. A few bacterial species (
) that were cultured from the isolation ward wastewaters on CHROMagar media harbored the
gene. This suggests that hospital wastewaters derived from clinical specialty wards are hotspots for the spread of AMR. Assembled scaffolds of other mobile genetic elements such as IncQ and IncF plasmids bearing quinolone resistance genes (
) and the class A beta-lactamase gene (
) were recovered in wastewater samples which may aid the transfer of AMR.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and ...other countries. We investigated the effect of this deletion on the clinical features of infection.
We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study—a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed.
Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 0% of 29 patients) than in the wild-type only group (26 28% of 92; absolute difference 28% 95% CI 14–28). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 95% CI 0·00–0·48) compared with infection with wild-type virus only.
The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines.
National Medical Research Council Singapore.
Testing for SARS-CoV-2: Can We Stop at 2? Lee, Tau Hong; Junhao Lin, Ray; Lin, Raymond T P ...
Clinical infectious diseases,
11/2020, Letnik:
71, Številka:
16
Journal Article
Recenzirano
Odprti dostop
Abstract
The COVID-19 epidemic requires accurate identification and isolation of confirmed cases for effective control. This report describes the effectiveness of our testing strategy and highlights ...the importance of repeat testing in suspected cases in our cohort.
Abstract
Background
Rapid identification of COVID-19 cases, which is crucial to outbreak containment efforts, is challenging due to the lack of pathognomonic symptoms and in settings with limited ...capacity for specialized nucleic acid–based reverse transcription polymerase chain reaction (PCR) testing.
Methods
This retrospective case-control study involves subjects (7–98 years) presenting at the designated national outbreak screening center and tertiary care hospital in Singapore for SARS-CoV-2 testing from 26 January to 16 February 2020. COVID-19 status was confirmed by PCR testing of sputum, nasopharyngeal swabs, or throat swabs. Demographic, clinical, laboratory, and exposure-risk variables ascertainable at presentation were analyzed to develop an algorithm for estimating the risk of COVID-19. Model development used Akaike’s information criterion in a stepwise fashion to build logistic regression models, which were then translated into prediction scores. Performance was measured using receiver operating characteristic curves, adjusting for overconfidence using leave-one-out cross-validation.
Results
The study population included 788 subjects, of whom 54 (6.9%) were SARS-CoV-2 positive and 734 (93.1%) were SARS-CoV-2 negative. The median age was 34 years, and 407 (51.7%) were female. Using leave-one-out cross-validation, all the models incorporating clinical tests (models 1, 2, and 3) performed well with areas under the receiver operating characteristic curve (AUCs) of 0.91, 0.88, and 0.88, respectively. In comparison, model 4 had an AUC of 0.65.
Conclusions
Rapidly ascertainable clinical and laboratory data could identify individuals at high risk of COVID-19 and enable prioritization of PCR testing and containment efforts. Basic laboratory test results were crucial to prediction models.
A risk score incorporating easily ascertainable demographic, clinical evaluation, and clinical testing covariates to identify patients at high risk of COVID-19 can help prioritize subjects for testing and public health measures to prevent onward transmission, especially in resource-limited settings.
There is currently no available test for monitoring the effect of treatment of latent tuberculosis infection (LTBI) to indicate cure or predict risk of subsequent progression to disease.
We used the ...T-SPOT.TB assay, which measures T-cell interferon-gamma responses to the Mycobacterium tuberculosis-specific peptides early secretory antigenic target 6-kD protein (ESAT-6) and culture filtrate protein 10 (CFP-10), to determine the effect of LTBI treatment on these responses.
A total of 226 tuberculosis contacts with positive T-SPOT.TB results underwent repeat testing on LTBI treatment completion. The majority (96%) received 6 months of isoniazid. The pre- and post-treatment T-SPOT.TB results were analyzed according to the combined and separate responses to ESAT-6 and CFP-10 antigens.
The T-SPOT.TB reverted to negative in 85 (37.6%) contacts at treatment completion. Treatment had a significant effect on the response to CFP-10 (p < 0.001; reversion rate, 48.6%), but not on the response to ESAT-6 (p = 0.081; reversion rate, 21.6%). The median number of spot-forming cells (SFCs)/2.5 x 10(5) peripheral blood mononuclear cells (PBMCs) pre- and post-treatment was 6 versus 4.5 for ESAT-6 (p = 0.116) and 11 versus 4 for CFP-10 (p < 0.001). There was a significant difference between the change (fall) in the pre- and post-treatment responses to CFP-10 (6 SFCs/2.5 x 10(5) PBMCs) and ESAT-6 (0 SFCs/2.5 x 10(5) PBMCs; p < 0.001). Significantly different age-related T-cell responses to the two antigens were found.
LTBI treatment had a differential effect on T-cell responses to ESAT-6 and CFP-10 as measured by the T-SPOT.TB. The quantitative response to CFP-10 may be a useful LTBI treatment-monitoring tool.
Streptococcus agalactiae (group B Streptococcus) sequence type 283 bacteremia, found almost exclusively in Southeast Asia, is associated with consuming raw freshwater fish, but some patients deny ...consumption. We detected fecal carriage in 5/184 (2.7%) persons in northeast Thailand. Human carriers might contribute to transmission or be the original source of this sequence type.
The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive ...test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 IL-2, interleukin-17A IL-17A, interleukin-22 IL-22, granulocyte-macrophage colony-stimulating factor GM-CSF, and tumor necrosis factor alpha TNF-α) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154(+) TNF-α(+) cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic ROC curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154(+) TNF-α(+) IFN-γ(+) IL-2(+) and CD154(+) TNF-α(+) CXCR3(+). Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB.
Clostridium difficile infection (CDI) is an increasingly recognized nosocomial infection in Singapore. Surveillance methods include laboratory reporting of Clostridium difficile toxin assays (CDTA) ...or use of International Classification of Diseases, 9(th) Revision (ICD-9) discharge code 008.45. Previous US studies showed good correlation between CDTA and ICD-9 codes. However, the use of ICD-9 codes for CDI surveillance has not been validated in other healthcare settings.
We compared CDI rates based on CDTA to ICD-9 codes for all discharges in 2007 from our hospital to determine sensitivity and specificity of ICD-9 codes. Demographic and hospitalization data were analyzed to determine predictors for missing ICD-9 codes.
During 2007, there were 56,352 discharges. Of these, 268 tested CDTA-positive but only 133 were assigned the CDI ICD-9 code. A total of 141 discharges had the ICD-9 code; 8 were CDTA-negative, the rest were CDTA-positive. Community-acquired CDI accounted for only 3.2% of cases. The sensitivity and specificity of ICD-9 codes compared to CDTA were 49.6% and 100% respectively. Concordance between CDTA and ICD-9 codes was 0.649 (p<.001). Comparing concordant patients (CDTA+/ICD9+) to discordant patients (CDTA+/ICD9-), concordant patients were more likely to be over 50 years of age (OR 3.49, 95% CI 1.66-7.34, p = .001) and have shorter time from admission to testing (OR 0.98, 95% CI 0.97-0.99, p = .009).
Unlike previous studies in the US, ICD-9 codes substantially underestimate CDI in Singapore compared to microbiological data. Older patients with shorter time to testing were less likely to have missing ICD-9 codes.
Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, ε-polylysine, ...disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with ε-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three ε-lysyl residues. Two modified melittin peptides displayed rapid bactericidal properties against antibiotic-resistant strains, low innate resistance development by pathogenic bacteria, remained nonimmunogenic for T lymphocytes, and increased bioavailability in tear fluids. In proof-of-concept
experiments, one of the peptides was noncytotoxic for ocular surfaces and had comparable antimicrobial efficacy to that of fluoroquinolone antibiotics. The results uncover a simple and potential strategy that can enhance the membrane selectivity of cytolytic peptides by ε-lysylation.
Group B
(GBS;
) is the most common cause of neonatal meningitis and a rising cause of sepsis in adults. Recently, it has also been shown to cause foodborne disease. As with many other bacteria, the ...polysaccharide capsule of GBS is antigenic, enabling its use for strain serotyping. Recent advances in DNA sequencing have made sequence-based typing attractive (as has been implemented for several other bacteria, including
,
species complex,
, and others). For GBS, existing WGS-based serotyping systems do not provide complete coverage of all known GBS serotypes (specifically including subtypes of serotype III), and none are simultaneously compatible with the two most common data types, raw short reads and assembled sequences. Here, we create a serotyping database (GBS-SBG, GBS Serotyping by Genome Sequencing), with associated scripts and running instructions, that can be used to call all currently described GBS serotypes, including subtypes of serotype III, using both direct short-read- and assembly-based typing. We achieved higher concordance using GBS-SBG on a previously reported data set of 790 strains. We further validated GBS-SBG on a new set of 572 strains, achieving 99.8% concordance with PCR-based molecular serotyping using either short-read- or assembly-based typing. The GBS-SBG package is publicly available and will hopefully accelerate and simplify serotyping by sequencing for GBS.
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