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1.  Bacteraemia during Transurethral Resection of the Prostate: What Are the Risk Factors and Is It More Common than We Think? 
PLoS ONE  2016;11(7):e0157864.
The aim of this work was to investigate the microbial causes, incidence, duration, risk factors and clinical implications of bacteraemia occurring during transurethral resection of the prostate (TURP) surgery to better inform prophylaxis strategies. An ethically approved, prospective, cohort study of patients undergoing TURP was conducted. Clinical information and follow-up details were collected using standardized data collection sheets. Blood was obtained for culture at 6 different time points peri-procedure. Standard of care antibiotic prophylaxis was given prior to surgery. Bacteriuria was assessed in a pre-procedure urine sample. Histopathology from all prostate chips was assessed for inflammation and malignancy. 73 patients were consented and 276 blood samples obtained. No patients developed symptomatic bacteraemia during the procedure, 17 patients developed asymptomatic bacteraemia (23.2%). Enterococcus faecalis and Pseudomonas aeruginosa were the most common organisms cultured. 10 minutes after the start of the TURP, the odds ratio (OR) of developing bacteraemia was 5.38 (CI 0.97–29.87 p = 0.05), and 20 minutes after the start of the procedure, the OR was 6.46 (CI 1.12–37.24, p = 0.03), compared to before the procedure. We also found an association between the development of intra-operative bacteraemia and recent antibiotic use (OR 4.34, CI 1.14–16.62, p = 0.032), the presence of a urinary catheter (OR 4.92, CI 1.13–21.51, p = 0.034) and a malignant histology (OR 4.90, CI 1.30–18.46, p = 0.019). There was no statistical relationship between pre-operative urine culture results and blood culture results. This study shows that asymptomatic bacteraemia is commonly caused by TURP and occurs in spite of antibiotic prophylaxis. Our findings challenge the commonly held view that urine is the primary source of bacteraemia in TURP-associated sepsis and raise the possibility of occult prostatic infection as a cause of bacteraemia. More work will be needed to determine the significance of transient bacteraemia in relation to more serious complications like infective endocarditis and malignancy.
PMCID: PMC4938130  PMID: 27391962
2.  Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study 
Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows.
We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics.
Compared with routine results, WGS predicted species with 93% (95% CI 90–96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91–95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10–26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6–10), a median of 21 days (IQR 14–32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21–44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows.
We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis.
National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.
PMCID: PMC4698465  PMID: 26669893
3.  Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures 
Journal of Clinical Microbiology  2015;53(4):1137-1143.
We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.
PMCID: PMC4365189  PMID: 25631807
4.  Genome analysis shows a common evolutionary origin for the dominant strains of Mycobacterium tuberculosis in a UK South Asian community  
We have investigated the Mycobacterium tuberculosis strain types present in the South Asian population of the UK, in which tuberculosis is particularly prevalent. In contrast to the widespread Beijing strains which have the variable number tandem repeats (VNTR) profile 42435, isolates with the VNTR profile 42235, jointly with 02335 or 42234 profiles, appear more frequently in tuberculosis patients of South Asian ethnic origin (SA-strains) in the UK than in any other ethnic group. Using microarray-based comparative genomics to distinguish total or partially deleted genes, we found that three of the common deleted regions in the SA-strains were identical to some deleted genes in the strain CH, which caused an outbreak among South Asian patients in Leicester in 2001 but were different from genomic deletions found in Beijing/W strains. Analysis of some of the deleted regions revealed differences in comparison to the strain CH including the polymorphism in some of the PE/PPE and Esat-6 genes, which may be responsible for the diversity of antigenic variation or differences in the activation of the host immune response. Interrupted genes or the replacement by insertion elements was confirmed in some of the deleted genomic regions. Our results are consistent with the hypothesis that the SA-strains may present common features, implying a common origin for this group of strains.
PMCID: PMC2963927  PMID: 17719277
Mycobacterium tuberculosis; PE/PPE; Polymorphism; VNTR 42235; South Asian community
5.  Buruli Ulcer in United Kingdom Tourist Returning from Latin America 
Emerging Infectious Diseases  2009;15(11):1827-1829.
We report a case of Buruli ulcer in a tourist from the United Kingdom. The disease was almost certainly acquired in Brazil, where only 1 case had previously been reported. The delay in diagnosis highlights the need for physicians to be aware of the disease and its epidemiology.
PMCID: PMC2857232  PMID: 19891876
Buruli ulcer; Mycobacterium ulcerans; tuberculosis and other mycobacteria; bacteria; Latin America; Brazil; United Kingdom; travel; dispatch
6.  Snapshot of Moving and Expanding Clones of Mycobacterium tuberculosis and Their Global Distribution Assessed by Spoligotyping in an International Study†  
Journal of Clinical Microbiology  2003;41(5):1963-1970.
The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.
PMCID: PMC154710  PMID: 12734235
7.  Global Distribution of Mycobacterium tuberculosis Spoligotypes 
Emerging Infectious Diseases  2002;8(11):1347-1349.
We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.
PMCID: PMC2738532  PMID: 12453368
Mycobacterium tuberculosis; spoligotyping
8.  Comparison of Variable Number Tandem Repeat and IS6110-Restriction Fragment Length Polymorphism Analyses for Discrimination of High- and Low-Copy-Number IS6110 Mycobacterium tuberculosis Isolates 
Journal of Clinical Microbiology  2001;39(7):2453-2457.
The present study was designed to evaluate the use of variable number tandem repeat (VNTR) and IS6110-restriction fragment length polymorphism (RFLP) analyses in combination as a two-step strategy for discrimination (as measured by the Hunter-Gaston Discrimination Index [HGDI]) of both high- and low-copy-number IS6110 Mycobacterium tuberculosis isolates compared to IS6110-RFLP alone with an unselected collection of isolates. Individually, IS6110-RFLP fingerprinting produced six clusters that accounted for 69% of the low-copy-number IS6110 isolates (five clusters) and 5% of the high-copy-number IS6110 isolates (one cluster). A total of 39% of all the isolates were clustered (HGDI = 0.97). VNTR analysis generated a total of 35 different VNTR allele profile sets from 93 isolates (HGDI = 0.938). Combining IS6110-RFLP analysis with VNTR analysis reduced the overall percentage of clustered isolates to 29% (HGDI = 0.988) and discriminated a further 27% of low-copy-number isolates that would have been clustered by IS6110-RFLP alone. The use of VNTR analysis as an initial typing strategy facilitates further analysis by IS6110-RFLP, and more importantly, VNTR analysis subdivides some IS6110-RFLP-defined clusters containing low- and single-copy IS6110 isolates.
PMCID: PMC88169  PMID: 11427553
9.  Rapid Identification of Laboratory Contamination with Mycobacterium tuberculosis Using Variable Number Tandem Repeat Analysis 
Compared with solid media, broth-based mycobacterial culture systems have increased sensitivity but also have higher false-positive rates due to cross-contamination. Systematic strain typing is rarely undertaken because the techniques are technically demanding and the data are difficult to organize. Variable number tandem repeat (VNTR) analysis by PCR is rapid and reproducible. The digital profile is easily manipulated in a database. We undertook a retrospective study of Mycobacterium tuberculosis isolates collected over an 18-month period following the introduction of the BACTEC MGIT 960 system. VNTR allele profiles were determined with early positive broth cultures and entered into a database with the specimen processing date and other specimen data. We found 36 distinct VNTR profiles in cultures from 144 patients. Three common VNTR profiles accounted for 45% of true-positive cases. By combining VNTR results with specimen data, we identified nine cross-contamination incidents, six of which were previously unsuspected. These nine incidents resulted in 34 false-positive cultures for 29 patients. False-positive cultures were identified for three patients who had previously been culture positive for tuberculosis and were receiving treatment. Identification of cross-contamination incidents requires careful documentation of specimen data and good communication between clinical and laboratory staff. Automated broth culture systems should be supplemented with molecular analysis to identify cross-contamination events. VNTR analysis is reproducible and provides timely results when applied to early positive broth cultures. This method should ensure that patients are not placed on unnecessary tuberculosis therapy or that cases are not falsely identified as treatment failures. In addition, areas where existing procedures may be improved can be identified.
PMCID: PMC87682  PMID: 11136751

Results 1-9 (9)