Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles.

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.

Association Between Minimum Inhibitory Concentration, Beta-lactamase Genes and Mortality for Patients Treated With Piperacillin/Tazobactam or Meropenem From the MERINO Study.

INTRODUCTION: This study aims to assess the association of piperacillin/tazobactam and meropenem minimum inhibitory concentration (MIC) and beta-lactam resistance genes with mortality in the MERINO trial. METHODS: Blood culture isolates from enrolled patients were tested by broth microdilution and whole genome sequencing at a central laboratory. Multivariate logistic regression was performed to account for confounders. Absolute risk increase for 30-day mortality between treatment groups was calculated for the primary analysis (PA) and the microbiologic assessable (MA) populations. RESULTS: In total, 320 isolates from 379 enrolled patients were available with susceptibility to piperacillin/tazobactam 94% and meropenem 100%. The piperacillin/tazobactam nonsusceptible breakpoint (MIC >16 mg/L) best predicted 30-day mortality after accounting for confounders (odds ratio 14.9, 95% confidence interval [CI] 2.8-87.2). The absolute risk increase for 30-day mortality for patients treated with piperacillin/tazobactam compared with meropenem was 9% (95% CI 3%-15%) and 8% (95% CI 2%-15%) for the original PA population and the post hoc MA populations, which reduced to 5% (95% CI -1% to 10%) after excluding strains with piperacillin/tazobactam MIC values >16 mg/L. Isolates coharboring extended spectrum ß-lactamase (ESBL) and OXA-1 genes were associated with elevated piperacillin/tazobactam MICs and the highest risk increase in 30-day mortality of 14% (95% CI 2%-28%). CONCLUSIONS: After excluding nonsusceptible strains, the 30-day mortality difference from the MERINO trial was less pronounced for piperacillin/tazobactam. Poor reliability in susceptibility testing performance for piperacillin/tazobactam and the high prevalence of OXA coharboring ESBLs suggests that meropenem remains the preferred choice for definitive treatment of ceftriaxone nonsusceptible Escherichia coli and Klebsiella.

Donor-Derived Candida dubliniensis Resulting in Perigraft Abscesses in a Liver Transplant Recipient Proven by Whole Genome Sequencing: A Case Report.

BACKGROUND: The transmission of fungi via transplant, although well-known, has not often been molecularly proven. We describe a case of donor-derived candidiasis verified by whole genome sequencing. CASE DESCRIPTION: The multiorgan donor was a 42-year-old woman with subdural hemorrhage. Procurement of the thoracic organs was performed followed by the abdominal organs. Tissue from the left bronchus grew Candida dubliniensis. The liver recipient was a 63-year-old woman with cryptogenic liver cirrhosis. She was noted to have worsening leukocytosis on postoperative day (POD) 9. Computed tomography of the abdomen and pelvis showed multiple rim-enhancing collections around the graft. Percutaneous drainage was performed. Fluid cultures grew C dubliniensis. C dubliniensis isolated from the donor's left bronchus and the liver recipient's abscesses were verified to be related by whole genome sequencing. We postulate that C dubliniensis colonizing the donor's transected trachea could have contaminated the inferior vena cava when the former was left open after explant of the donor's lungs. A portion of the donor's contaminated inferior vena cava was transplanted along with the liver graft, resulting in the infected collections in the recipient. CONCLUSIONS: Our case report highlights the importance of maintaining a sterile field during organ procurement, especially in a multiorgan donor whose organs are explanted in succession.

Sustained meticillin-resistant Staphylococcus aureus control in a hyper-endemic tertiary acute care hospital with infrastructure challenges in Singapore.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) has been entrenched in Singapore hospitals since the 1980s, with an excess of 600 non-duplicate cases of infections (120 bacteraemia episodes) each year in our 995-bed university hospital. Approximately 5% of our hospital beds are used as isolation facilities. AIM: To study the impact of an MRSA control bundle that was implemented via gradual geographic extension across hospital wards. METHODS: The bundle included active surveillance on admission and transfer/discharge to identify ward-based acquisition of MRSA, isolation and cohorting of MRSA-infected patients, enhanced hand hygiene initiatives, and publicly displayed feedback of MRSA acquisition and hand hygiene compliance rates. Implementation was between October 2006 and June 2010 in order to provide lead-time for the incremental development of infrastructural capacity, and to develop an ethic of infection prevention among staff. Results were analysed via interrupted time-series analysis. FINDINGS: MRSA infections fell midway through the implementation, with MRSA bacteraemia declining from 0.26 [95% confidence interval (CI): 0.18-0.34] cases per 1000 inpatient-days in the first quarter of 2004 to 0.11 (95% CI: 0.07-0.19) cases per 1000 inpatient-days in the first quarter of 2012. MRSA acquisition rates fell a year after the programme had been fully implemented, whereas hand hygiene compliance rose significantly from 47% (95% CI: 44-49) in the first quarter of 2009 to 69% (95% CI: 68-71) in the first quarter of 2012. CONCLUSION: Successful staged implementation of an MRSA bundle in a hyper-endemic setting is sustainable and represents a model that may be adapted for similar settings.

Bad design, bad practices, bad bugs: frustrations in controlling an outbreak of Elizabethkingia meningoseptica in intensive care units.

BACKGROUND: Elizabethkingia meningoseptica is a nosocomial-adapted Gram-negative bacillus intrinsically resistant to antibiotics commonly used in the intensive care setting. An outbreak investigation commenced when five patients developed E. meningoseptica infection in two intensive care units (ICUs). METHODS: Analysis of laboratory data, case reviews, ICU workflows and extensive environmental sampling were undertaken. Molecular typing was performed using repetitive element palindromic polymerase chain reaction. Follow-up studies after interventions included environmental monitoring and a survey of staff compliance with interventions. FINDINGS: Laboratory data revealed increasing incidence of E. meningoseptica colonization or infection in ICU patients compared with preceding years. E. meningoseptica was cultured from 44% (35/79) of taps, but not from other sources. Hand hygiene sinks were used for disposal of patient secretions and rinsing re-usable patient care items. Sinks misused in this way were contaminated more often than sinks that were not misused (odds ratio 4.38, 95% confidence interval 1.68-11.39; P = 0.004). Molecular typing revealed that patient isolates had identical patterns to several isolates from hand hygiene taps. An urgent education programme was instituted to change these practices. Taps were cleaned systematically and aerators were changed. A temporary reduction in case numbers was achieved. Recolonization of taps was demonstrated on follow-up environmental screening, and cases recurred after two months. A survey revealed that 77.3% (163/213) of nursing staff still misused sinks due to time constraints or other problems adhering to the interventions. CONCLUSION: Introduction of non-sanctioned practices due to suboptimal unit design may have unintentional consequences for ICU patients. Room design and staff workflows must be optimized for patient safety as lapses in procedure can inadvertently put patients at risk.

Emergence of Klebsiella pneumoniae co-producing NDM-type and OXA-181 carbapenemases.

The emergence of carbapenemase-producing Enterobacteriaceae is a rapidly evolving threat worldwide. Here, we report the molecular characterization of two Klebsiella pneumoniae isolates carrying both bla(OXA -181) and bla(NDM -1) or bla(NDM -5) isolated from epidemiologically unrelated patients in Singapore. The bla(OXA -181) genes were found existing in different genetic environments.

Emergence of clinical Klebsiella pneumoniae producing OXA-232 carbapenemase in Singapore.

We report the emergence of OXA-232, a newly described OXA-48-like carbapenemase variant, in Southeast Asia. Molecular characterization of eight Klebsiella pneumoniae obtained from local and foreign patients reveals clonality of the isolates. bla OXA-232 was located on a non-conjugative plasmid of 6141 base pairs (GenBank accession number JX423831.1).

Hot and steamy: outbreak of Bacillus cereus in Singapore associated with construction work and laundry practices.

BACKGROUND: A sudden increase in invasive infections caused by Bacillus cereus group organisms prompted an investigation at the National University Hospital in Singapore. AIM: To describe the investigation and management and subsequent difficulties controlling the outbreak. METHODS: Clinical case reviews were performed on all patients with B. cereus group recovered from clinical samples. Widespread environmental sampling was performed followed by review of hospital ventilation systems, domestic cleaning and laundry practices. FINDINGS: B. cereus was recovered from 171 patients during a six-month period coinciding with large-scale construction work beside the hospital. Most patients presented with bacteraemia (146/171; 85.4%) with 46/171 (26.9%) requiring extended treatment courses with vancomycin or other interventions. Sampling confirmed extensive airborne dispersal inside the hospital, including isolation rooms and air-conditioned wards. Hospital linen was heavily contaminated [7403 cfu/cm(2); 95% confidence interval (CI): 6349-8457; for 30 towels sampled], encouraged by inappropriate storage in airtight plastic bags (4437 cfu/cm(2); CI: 3125-5750) compared with storage in porous canvas bags (166 cfu/cm(2); CI: 76-256; P < 0.001). Interventions introduced included revision of laundry practices, transport and storage of hospital linen and towels; bleach-based environmental cleaning; and upgrading of ventilation systems throughout the hospital. Clinical case numbers returned to baseline levels within three months, only to rise again following relaxation of laundry practices. CONCLUSIONS: Construction work beside this Singapore hospital encouraged heavy contamination of air and environment with Bacillus spp., assumed to be responsible for the outbreak described. Failure to maintain revised laundry practices allowed resurgence of clinical cases, particularly among immunocompromised patients.

Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria.

AIMS: To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S-23S rRNA internal transcribed spacer (ITS) region for timely patient management. METHODS AND RESULTS: Two mPCR assays were developed: Slow-Growers (SG) mPCR was used for the detection of slow-growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast-Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species-specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S-23S ITS region and when further confirmation was needed, hsp65 sequencing was performed. CONCLUSIONS: The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005-2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.

Knowledge that upper respiratory tract infection resolves on its own is associated with more appropriate health-seeking behaviour and antibiotic cognition.

INTRODUCTION: This study aims to study whether knowledge that upper respiratory tract infection (URTI) resolves on its own is associated with more appropriate antibiotic cognition and treatment-seeking behaviour in adult patients seeking consultation for upper respiratory tract symptoms in all nine polyclinics of the National Healthcare Group in Singapore. METHODS: A prospective study of 595 adult patients who attended for URTI symptoms of less than seven days duration was performed. We collected data using an interviewer-administered structured questionnaire eliciting the participant's personal and demographical data, his knowledge about URTI, past experience with URTI and treatment-seeking behaviour for this current episode of URTI. These variables were then regressed against the variable "URTI resolves on its own", adjusting for "antibiotics relieve URTI faster", "antibiotic used unnecessarily for URTI", "recovery faster with antibiotics" for antibiotic cognition; and "number of days with URTI", "first line of action: self-medicate", "reason for attendance: felt unwell", "reason for attendance: get well faster" and "used prescribed medication before current visit" for health-seeking behaviour. RESULTS: More than one-third of patients believed that URTI resolved on its own. Subjects who believed that URTI resolved on its own were not significantly different in terms of gender, ethnic group, educational status, age, knowledge of germs as causal, and number of days sick before attendance. However, such patients were 1.68 times (confidence interval [CI] 1.17, 2.41) more likely to agree that, nowadays, antibiotics are used unnecessarily for "flu", and 2.07 times (CI 1.32, 3.24) more likely to self-medicate. They were 0.51 (CI 0.35, 0.76) times more likely to be dependant on prescribed medication. CONCLUSION: Knowledge that URTI resolves on its own is associated with more appropriate antibiotic cognition and health-seeking behaviour.

An evaluation of antimicrobial prophylaxis in paediatric surgery and its financial implication.

OBJECTIVE: To assess the appropriateness of use of antimicrobial prophylaxis in paediatric surgery in Singapore. METHODS: A prospective evaluation of the use of antimicrobial prophylaxis in paediatric patients undergoing surgery in KK Hospital, Singapore was carried out from September and December 2001. Process measures for the study included adherence to local or international guidelines, choice and dose of antimicrobial agent and timing and duration of prophylaxis. Outcome measures included surgical site and other related infections. A cost analysis was also performed to look at the extra expenditure due to any excessive use of antimicrobials. RESULTS: A total of 171 patients with a mean age of 5.7 years (SD=4.5) were included in the study. Among the 117 (68.4%) elective and 54 (31.6%) emergency procedures, 22 cases (12.8%) were fully compliant with the guidelines. The most frequently encountered non-compliance types were unnecessary prolongation of prophylaxis (54.4%), inappropriate choice of antibiotics (42.7%) and overdose of antibiotics (26.3%). The estimated extra cost to patients for the study period was Sing$13,879.41 (US 8164.36). CONCLUSION: The results showed a significantly high level of inappropriate use of antimicrobial prophylaxis in paediatric surgery in Singapore. However, when the individual factors such as appropriate choice of antibiotics, appropriate timing and duration were considered, the situation was very similar to the results obtained from overseas studies.

A retrospective drug utilization evaluation of vancomycin usage in paediatric patients.

OBJECTIVE: To evaluate the appropriateness of use of vancomycin in paediatric patients at KK Women's and Children's Hospital, the major paediatric hospital in Singapore to identify potential problems in prescribing practices that may necessitate intervention to optimize vancomycin usage. METHODS: A retrospective drug utilization evaluation was performed for paediatric patients who received intravenous vancomycin from 1 June 1998 to 31 June 1999. The outcome measures were consistency of vancomycin indication with recommended guidelines, dosing regimens, microbiological data, monitoring of serum drug levels, renal function, clinical outcomes and adverse drug reactions (ADRs). RESULTS: A total of 96 cases was available for evaluation. Sixty-two (64.6%) courses of vancomycin were consistent with guidelines for indication of therapy. Eighty-six (89.6%) of the dosing regimen were consistent. All infusion times that were recorded (56.3%) were consistent with criteria. Of the patients treated with vancomycin for more than 1 day, peak and/or trough serum vancomycin levels were ordered for 70 cases. Of the 56 cases with paired levels ordered, 46 cases had at least one level that fell outside the therapeutic range. Nineteen (19.8%) cases of ADRs were documented. Fifty-eight (60.4%) cases received concurrent nephrotoxic drugs. However, a substantial portion of vancomycin courses were apparently not prescribed for appropriate indications, and there was poor recording of vancomycin administration information and sampling time. CONCLUSION: The majority of dosing regimens of vancomycin was consistent with guideline criteria. The most evident problem was the sub-optimal use of the monitoring of vancomycin serum levels. The information derived from this study may be used as a for further study and for the development of strategies for optimize vancomycin usage.