Jeanette Teo

Molecular characterization of newly emerged blaKPC-2-producing Klebsiella pneumoniae in Singapore

ABSTRACT In Asia, bla KPC detection has been limited to East Asia and not yet seen in Southeast Asia. We report four bla KPC-2-containing Klebsiella pneumoniae isolates from two different hospitals in Singapore. All isolates belonged to strain type 11 (ST11) and were indistinguishable by pulsed-field gel electrophoresis (PFGE). bla KPC-2 was located on nonconjugative plasmids and flanked by mobile genetic structures composed of a partial Tn4401 transposon and a Tn3-based transposon which previously have been described only in Chinese isolates.

Comparison of Two Nucleic Acid Amplification Assays, the Xpert MTB/RIF Assay and the Amplified Mycobacterium Tuberculosis Direct Assay, for Detection of Mycobacterium tuberculosis in Respiratory and Nonrespiratory Specimens

ABSTRACT We compared the performance of the Xpert MTB/RIF assay, a new real-time tuberculosis (TB) PCR test, with that of the Amplified Mycobacterium Tuberculosis Direct (MTD) assay using 162 respiratory and nonrespiratory specimens. Based on culture as the gold standard, the overall sensitivity and specificity for all sample types for the Xpert MTB/RIF assay were 90.9 and 89%, respectively, while for the MTD assay, the overall sensitivity and specificity were 97.3 and 87.1%, respectively. A higher proportion of total equivocal results were obtained for the MTD assay, at 10.5% (17/162), while the Xpert MTB/RIF assay generated 5.5% (9/162) of invalid reads.

VereFlu™: an integrated multiplex RT-PCR and microarray assay for rapid detection and identification of human influenza A and B viruses using lab-on-chip technology

Threatening sporadic outbreaks of avian influenza and the H1N1 pandemic of 2009 highlight the need for rapid and accurate detection and typing of influenza viruses. In this paper, we describe the validation of the VereFlu™ Lab-on-Chip Influenza Assay, which is based on the integration of two technologies: multiplex reverse transcription (RT)-PCR followed by microarray amplicon detection. This assay simultaneously detects five influenza virus subtypes, including the 2009 pandemic influenza A (H1N1), seasonal H1N1, H3N2, H5N1 and influenza B virus. The VereFlu™ assay was clinically validated in Singapore and compared against reference methods of real-time PCR, virus detection by immunofluorescence of cell cultures and sequencing. A sensitivity and specificity of 96.8% and 92.8%, respectively, was demonstrated for pandemic H1N1; 95.7% and 100%, respectively, for seasonal H1N1; 91.2% and 97.6%, respectively, for seasonal H3N2; 95.2% and 100%, respectively, for influenza B. Additional evaluations carried out at the World Health Organization (WHO) Collaborating Centre, Melbourne, Australia, confirmed that the test was able to reliably detect H5N1. This portable, fast time-to-answer (3 hours) device is particularly suited for diagnostic applications of detection, differentiation and identification of human influenza virus subtypes.

OXA-181-producing Klebsiella pneumoniae establishing in Singapore

BackgroundCarbapenemase producing Enterobacteriaceae are becoming a major public health concern globally, however, relatively little is known about the molecular and clinical epidemiology of these organisms in many parts of the world.MethodsAs part of a laboratory surveillance programme, 96 carbapenem non-susceptible Enterobacteriaceae isolates from clinical samples from patients in seven hospitals were referred for investigation for carbapenemases. Using polymerase chain reaction (PCR) to screen for a collection of genes encoding carbapenemases, 33 of 96 (34.5%) isolates were confirmed as carbapenemase producers. NDM-1 producers were the most prevalent at 64% (21/33) whilst OXA-181 was the second most common carbapenemase constituting 24.5% (8/33) of the carbapenemase producing isolates. Seven of these eight OXA-181 positive isolates underwent further characterisation with screening for other transmissible antimicrobial resistance determinants using PCR. Clonal relatedness was explored using Multilocus sequence typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Plasmid characterisation was performed including restriction analysis and transfer by conjugation or transformation.ResultsIn addition to the OXA-181 gene, all contained other transmissible resistance determinants including extended spectrum β-lactamases, oxacillinases or 16S rRNA methylase genes, but none contained metallo-β-lactamases or serine carbapenemases. All isolates had a multidrug resistant phenotype with two isolates being resistant to every antibiotic tested including colistin. Multilocus sequence typing confirmed five isolates belonged to ST17 and two to ST14, with those belonging to the same sequence type having identical PFGE profiles. The OXA-181 gene was typically carried on large plasmids which were mostly non-conjugative.ConclusionsOXA-181 carbapenemase appears to be an important and probably under-recognised cause of carbapenem resistance in Enterobacteriaceae in Singapore. Further coordinated research into clinical and molecular epidemiology of carbapenemases is urgently required in Singapore and throughout Asia.

Molecular characterization of NDM-1 producing Enterobacteriaceae isolates in Singapore hospitals

OBJECTIVE In this study, we molecularly characterized 12 NDM-1 producing clinical Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae) isolates that were part of a collection of non-carbapenem susceptible isolates obtained during a one-year period. These isolates were obtained from four local general hospitals in Singapore. METHODS Polymerase chain reaction (PCR) assays and sequencing was used to determine the presence of β-lactamase encoding genes (bla) including bla NDM-1 and plasmid-mediated quinolone and aminoglycoside resistance determinants. Conjugation experiments were performed to determine the transferability of bla NDM-1. Isolate relatedness was determined by multilocus sequence typing (MLST). RESULTS The isolates were completely resistant to the second- and third-generation cephalosporins tested as well as carbapenems. Susceptibility profiling of the isolates indicated that 100% retained susceptibility to tigecycline while 11/12 (91.7%) were susceptible to colistin. The bla NDM-1 gene was encoded on plasmids that were easily transferable. None of the patients had a travel history to countries where NDM-1 has been reported. The isolates appear clonally unrelated with MLST, revealing a diversity of clonal types among the Klebsiella pneumoniae and Escherichia coli isolates. CONCLUSION The ease of NDM-1 plasmid transmissibility may help their dissemination among the Enterobacteriaceae. Although it appears that the isolates are clonally unrelated, epidemiological links cannot be fully excluded without further research.

First case of E anophelis outbreak in an intensive-care unit

www.thelancet.com Vol 382 September 7, 2013 855 2 Grant RM, Lama JR, Anderson PL, et al, and the iPrEx Study Team. Pre-exposure chemoprophylaxis for HIV prevention in men who have sex with men. N Engl J Med 2010; 363: 2587–99. 3 Thigpen MC, Kebaabetswe PM, Paxton LA, et al, and the TDF2 Study Group. Antiretroviral preexposure prophylaxis for heterosexual HIV transmission in Botswana. N Engl J Med 2012; 367: 423–34. 4 Baeten JM, Donnell D, Ndase P, et al, and the Partners PrEP Study Team. Antiretroviral prophylaxis for HIV prevention in heterosexual men and women. N Engl J Med 2012; 367: 399–410. 5 Vanichseni S, Martin M, Suntharasamai P, et al. HIV-associated risk behavior among injecting drug users participating in an HIV preexposure prophylaxis trial in Bangkok, Thailand. IAS Conference on HIV Pathogenesis, Treatment and Prevention, Kuala Lumpur, Malaysia, July 2013 (abstract: MOLBPE27). among participants who became infected during follow-up. We agree with Andrew Craig and colleagues that individuals and policy makers will need to consider cost when deciding which HIV prevention method or combination of methods to use. We note that the incremental cost of providing preexposure prophylaxis in settings where HIV-prevention services are available would be relatively small, and targeting individuals at highest risk of HIV infection would decrease the cost per infection averted. Data from trials suggest that when pre-exposure prophylaxis is provided with a package of HIV prevention services, HIVassociated risk behavior will decrease rather than increase. Nonetheless, it will be important to monitor for risk compensation in future pre-exposure prophylaxis projects. Additional work is needed to establish how best to implement preexposure prophylaxis in conjunction with other proven prevention measures among people who inject drugs, including: how to support adherence, assessing the cost of pre-exposure prophylaxis, and determining appropriate venues for pre-exposure prophylaxis delivery. Nonetheless, for people who will not or cannot stop injecting drugs, preexposure prophylaxis might provide an important new HIV prevention method to complement other available prevention strategies.

Development of a novel multiplex PCR for the detection and differentiation of Salmonella enterica serovars Typhi and Paratyphi A.

In this study, we developed a multiplex polymerase chain reaction (mPCR) assay for pan-Salmonella detection as well as for specific detection of serovars Typhi and Paratyphi A. The assay detects members of the Salmonella genus by amplifying the outer membrane protein C (ompC). The presence of either Salmonella serovar Typhi or Paratyphi A is indicated by amplification of the putative regulatory protein gene STY4220, which is common to both serovars. Further differentiation of the serovars was achieved by targeting the intergenic region (SSPAI) between SSPA1723a and SSPA1724 in serovar Paratyphi A, and stgA, a fimbrial subunit protein, in serovar Typhi. mPCR was evaluated using 124 clinical and reference Salmonella serovars. S. enterica serovars Typhi and Paratyphi A were detected at 100% specificity and sensitivity. Each PCR reaction detected approximately 1 pg of Salmonella genomic DNA. Sensitivity of the PCR when tested on 8-h-enriched spiked blood samples of serovars Typhi and Paratyphi A was estimated at 4.5 x 10(4)-5.5 x 10(4)CFU/ml, with similar detection levels observed for spiked fecal samples. This mPCR could prove to be a useful diagnostic tool for the detection and differentiation of serovars Typhi and Paratyphi A.

Colistin and polymyxin B susceptibility testing for carbapenem-resistant and mcr-positive Enterobacteriaceae: Comparison of Sensititre, Microscan, Vitek 2, and Etest with broth microdilution

ABSTRACT Colistin and polymyxin B remain part of the last line of antibiotics for multidrug-resistant Gram-negative bacteria, such as carbapenem-resistant Enterobacteriaceae. Current joint EUCAST-CLSI recommendations are for broth microdilution (BMD) to be performed for MIC testing of colistin. Commercial susceptibility testing methods were evaluated and compared against the reference BMD, using a susceptibility breakpoint of ≤2 mg/liter for both colistin and polymyxin B. Seventy-six Enterobacteriaceae were included, of which 21 were mcr-1 positive (18 Escherichia coli isolates, 2 Klebsiella pneumoniae isolates, and 1 Enterobacter aerogenes isolate). Rates of essential agreement (EA) of colistin test results between BMD and Vitek 2, Sensititre, and Etest were 93.4%, 89.5%, and 75.0%, respectively. Rates of EA of polymyxin B test results between BMD and Vitek 2, Sensititre, and Etest were 96.1%, 96.1%, and 48.7%, respectively. A positive MIC correlation with a categorical agreement of >90% was achieved for Sensititre (colistin Spearmans ρ = 0.863, and polymyxin B Spearmans ρ = 0.877) and Vitek 2 (polymyxin B [only] Spearmans ρ = 0.8917). Although a positive MIC correlation (Spearmans ρ = 0.873) with the reference method was achieved for colistin testing with Vitek 2, categorical agreement was <90%, with very major error rates of 36%. Correlation with the Etest MIC was lower, with very major error rates of 12% (colistin) and 26.1% (polymyxin B). MicroScan (colistin) categorical agreement was 88.2%, with a very major error rate of 4%. Colistin MICs for 15 of the 21 mcr-1-positive isolates were >2 mg/liter, and polymyxin MICs for 17 of them were >2 mg/liter by broth microdilution. The use of a lower breakpoint of ≤1 mg/liter further improves detection of mcr-1 for all testing methods. However, further data on the correlation between MICs and clinical outcome are required to determine the most suitable breakpoint to guide clinical management.

Comparative Genomic Analysis of Malaria Mosquito Vector-Associated Novel Pathogen Elizabethkingia anophelis

Acquisition of Elizabethkingia infections in intensive care units (ICUs) has risen in the past decade. Treatment of Elizabethkingia infections is challenging due to the lack of effective therapeutic regimens, leading to a high mortality rate. Elizabethkingia infections have long been attributed to Elizabethkingia meningoseptica. Recently, we used whole-genome sequencing to reveal that E. anophelis is the pathogenic agent for an Elizabethkingia outbreak at two ICUs. We performed comparative genomic analysis of seven hospital-isolated E. anophelis strains with five available Elizabethkingia spp. genomes deposited in the National Center for Biotechnology Information Database. A pan-genomic approach was applied to identify the core- and pan-genome for the Elizabethkingia genus. We showed that unlike the hospital-isolated pathogen E. meningoseptica ATCC 12535 strain, the hospital-isolated E. anophelis strains have genome content and organization similar to the E. anophelis Ag1 and R26 strains isolated from the midgut microbiota of the malaria mosquito vector Anopheles gambiae. Both the core- and accessory genomes of Elizabethkingia spp. possess genes conferring antibiotic resistance and virulence. Our study highlights that E. anophelis is an emerging bacterial pathogen for hospital environments.

Detection and genetic characterisation of qnrB in hospital isolates of Klebsiella pneumoniae in Singapore

Polymerase chain reaction (PCR) screening of 116 ciprofloxacin-resistant Klebsiella pneumoniae hospital isolates for the presence of qnr genes that mediate plasmid quinolone resistance revealed that none were positive for qnrA or qnrS. However, qnrB was detected in ca. 5.2% of the isolates. Southern hybridisation demonstrated that the qnrB-hybridising plasmids were large (>70kb) and capable of transferring quinolone resistance by conjugation. Sequence analysis of the qnrB genes detected in this study showed that they were identical to previously identified qnrB1, qnrB4 and qnrB6 genes, although a novel variant designated qnrB20 was also identified. Analysis of the genetic environment around the cloned qnrB genes showed that they were present in diverse plasmid backbones, sometimes within novel genetic contexts, but always associated with mobile or transposable elements.