Which Clinical Decisions Benefit from Automation? A Task Complexity Approach

Studies in Health Technology and Informatics. 2002  |  Pubmed ID: 15460772

This paper describes a model for analysing medical decision making tasks and evaluation of their suitability for automation. The overall approach focuses on the assessment of decision complexity and possible reduction of human effort by automated decision support. The approach consists of five subsequent steps: (1) selection of the domain and relevant tasks; (2) evaluation of the knowledge complexity for tasks selected; (3) selection of potentially most cognitively demanding task; (4) assessment of unaided and aided effort requirements for this task accomplishment; and (5) selection of computational tools to achieve this complexity reduction. The model described allows for task automation without lowering of decision quality.

Which Clinical Decisions Benefit from Automation? A Task Complexity Approach

International Journal of Medical Informatics. Jul, 2003  |  Pubmed ID: 12909183

To describe a model for analysing complex medical decision making tasks and for evaluating their suitability for automation.

Comparative Impact of Guidelines, Clinical Data, and Decision Support on Prescribing Decisions: an Interactive Web Experiment with Simulated Cases

Journal of the American Medical Informatics Association : JAMIA. Jan-Feb, 2004  |  Pubmed ID: 14527970

The aim of this study was to compare the clinical impact of computerized decision support with and without electronic access to clinical guidelines and laboratory data on antibiotic prescribing decisions.

Handheld Computer-based Decision Support Reduces Patient Length of Stay and Antibiotic Prescribing in Critical Care

Journal of the American Medical Informatics Association : JAMIA. Jul-Aug, 2005  |  Pubmed ID: 15802478

This study assessed the effect of a handheld computer-based decision support system (DSS) on antibiotic use and patient outcomes in a critical care unit.

Screening for Antibiotic Resistant Gram-negative Bacteria

The Lancet Infectious Diseases. Jun, 2006  |  Pubmed ID: 16728313

A Case of Urinary Tuberculosis Due to Mycobacterium Bovis Subspecies Caprae

Pathology. Aug, 2006  |  Pubmed ID: 16916737

Genotyping of Mycobacterium Tuberculosis in New South Wales: Results from 18 Months of a Statewide Trial

New South Wales Public Health Bulletin. May-Jun, 2006  |  Pubmed ID: 17036086

Decision Complexity Affects the Extent and Type of Decision Support Use

AMIA ... Annual Symposium Proceedings / AMIA Symposium. AMIA Symposium. 2006  |  Pubmed ID: 17238436

The increasing complexity of decision-making has emerged as a risk factor in clinical medicine. The impact that decision task complexity has on the uptake and use of clinical decision support systems (DSS) is also not well understood. Antibiotic prescribing in critical care is a complex, cognitively demanding task, made under time pressure. A web-based experiment was conducted to explore the impact of decision complexity on DSS utilization, comparing utilization of antibiotic guidelines and an interactive probability calculator for ventilator associated pneumonia (VAP) plus laboratory data. Decision support was found to be used more often for less complex decisions. Prescribing decisions of higher complexity were associated with a lower frequency of DSS use, but required the use of the more cognitively demanding situation assessment tool for infection risk along with pathology data. Decision complexity thus seems to impact on the extent and type of information support used by individuals when decision-making.

An Outbreak of Pulmonary Tuberculosis in Young Australians

The Medical Journal of Australia. Mar, 2007  |  Pubmed ID: 17391086

To characterise a pulmonary tuberculosis (TB) cluster in the Hunter Area of New South Wales using a combination of traditional epidemiological methods and molecular typing.

Pathogen Profiling for Disease Management and Surveillance

Nature Reviews. Microbiology. Jun, 2007  |  Pubmed ID: 17487146

The usefulness of rapid pathogen genotyping is widely recognized, but its effective interpretation and application requires integration into clinical and public health decision-making. How can pathogen genotyping data best be translated to inform disease management and surveillance? Pathogen profiling integrates microbial genomics data into communicable disease control by consolidating phenotypic identity-based methods with DNA microarrays, proteomics, metabolomics and sequence-based typing. Sharing data on pathogen profiles should facilitate our understanding of transmission patterns and the dynamics of epidemics.

Point-of-care Testing for Community-acquired Pneumonia: Do We Have All the Answers?

The Medical Journal of Australia. Jul, 2007  |  Pubmed ID: 17605702

Point-of-care tests (POCTs) are available for rapid, "bedside" diagnosis of some causes of community-acquired pneumonia. POCTs complement other laboratory investigations for pneumonia. Although their sensitivity and specificity are improving, they are generally less sensitive than nucleic acid amplification and culture techniques. Questions remain as to the most cost-effective use of POCTs in clinical practice. To ensure their maximum value for both individual patients and the public health system, POCTs are probably best used as part of laboratory-designed algorithms for investigating pneumonia. POCTs are a valuable tool for surveillance, for rapid investigation of outbreaks, and for use in laboratories with limited diagnostic facilities.

Are We Measuring the Right End-points? Variables That Affect the Impact of Computerised Decision Support on Patient Outcomes: a Systematic Review

Medical Informatics and the Internet in Medicine. Sep, 2007  |  Pubmed ID: 17701828

Previous reviews of electronic decision-support systems (EDSS) have often treated them as a single category, and factors that may modify their effectiveness of EDSS have not been examined. The objective was to summarise the evidence associating the use of computerised decision support and improved patient outcomes. PubMed/Medline and the Database of Abstracts were searched for randomised controlled trials (RCT) of EDSS from 1 January 1994 to 31 January 2006. Twenty-four RCT studies from 97 reviewed were selected, eight of them examined systems supporting decisions for patients who presented with an acute illness, and 16 studies enrolled patients with chronic conditions. Overall, 13 (54%) of the studies showed a positive result, and 11 (46%) were with no impact. Critiquing and consultative systems showed the impact in 71% and 47% of studies, respectively. All systems targeting decisions related to acute disease improved patient outcomes compared with 38% of systems focused on the management of chronic conditions (P = 0.005). Provision of EDSS improves prescribing practices and treatment outcomes of patients with acute illnesses; however, EDSS were less effective in primary care. Complex interventions as clinical EDSS may require new metrics of assessment to describe the impact on patient outcomes.

Comparison of Single- and Multilocus Sequence Typing and Toxin Gene Profiling for Characterization of Methicillin-resistant Staphylococcus Aureus

Journal of Clinical Microbiology. Oct, 2007  |  Pubmed ID: 17715374

We compared three novel methicillin-resistant Staphylococcus aureus (MRSA) genotyping methods with multilocus sequence typing (MLST) and spa typing to assess their utility for routine strain typing. The new methods were femA and nuc sequence typing and toxin gene profiling (TGP), using a multiplex-PCR-based reverse line blot assay to detect 13 pyrogenic superantigen and exfoliative toxin genes. Forty-two well-characterized MRSA strains, representing 15 MLSTs or 9 clonal clusters (CCs), were genotyped by all methods. Twenty-two spa, nine femA, and seven nuc sequence types were identified. The femA sequence types correlated exactly with CCs; nuc sequences types were less discriminatory but generally correlated well with femA types and CCs. Ten isolates contained none of 13 toxin genes; TGPs of the remainder comprised 1 to 5 toxin genes. The combination of spa typing and TGPs identified 26 genotypes among the 42 strains studied. A combination of two or three rapid, inexpensive genotyping methods could potentially provide rapid MRSA strain typing as well as useful information about clonal origin and virulence.

Is Bordetella Pertussis Susceptibility to Erythromycin Changing? MIC Trends Among Australian Isolates 1971-2006

The Journal of Antimicrobial Chemotherapy. Nov, 2007  |  Pubmed ID: 17827145

Are Clinicians' Information Needs and Decision Support Affected by Different Models of Care? Experimental Study

Studies in Health Technology and Informatics. 2007  |  Pubmed ID: 17911845

This study explores task- and healthcare model-specific differences in clinicians' information needs which can affect the uptake of decision support. Results of a web experiment involving 104 general practitioners are presented. Respondents indicated that guidelines were the most important source of information with almost equal weighting for acute, chronic and preventive care. A patient's quality of life was identified as the most important determinant of decision-making in all three models of care. Risk assessment tools and information about outcomes were more valuable (P<0.05) for chronic and preventive care than for acute cases. The participants accessed electronic risk assessment tools in 54%, 45% and 81% of acute, chronic and preventive care scenarios, respectively. Participants estimated that the electronic decision support would have a significantly higher impact in preventive care than in chronic or acute care settings (P=0.01). The differences in the information needs of clinicians related to different care models have to be considered in the design of clinical decision support systems. Systems that target preventive model decisions may have higher adoption and impact.

Utility of Genotyping of Mycobacterium Tuberculosis in the Contact Investigation: a Decision Analysis

Tuberculosis (Edinburgh, Scotland). May, 2007  |  Pubmed ID: 17161653

The objective of this study was to compare the traditional tuberculosis contact-tracing strategy with a two-stage strategy, in low prevalence countries. We compared the utility of contact tracing of pulmonary tuberculosis patients using a single interview (Strategy I) with that of two-stage strategies, namely traditional 'stone-in the pond' contact tracing (strategy II) and a strategy involving second interviews of patients whose Mycobacterium tuberculosis isolates are genotypically clustered (Strategy III). Factors affecting the utility and impact of each were explored using sensitivity analysis of probabilistic decision trees and a quantitative Markov simulation. Contact tracing using Strategy III demonstrated a higher utility and a 12% lower probability of secondary infection being missed compared with Strategy II. The threshold level, at which a change, from a traditional to a two-stage contact tracing strategy is indicated, is when the rate of clustering is 4% or more. The utility of Strategy III is optimal when the probability of detecting new epidemiological links is more than 10%. Strategy III allows detection of 58% of infected patients within 2 years after exposure compared with Strategy II and Strategy I which will detect 47% and 32% of infected contacts within 2 years, respectively. Strategy III allows detection of 58% of infected patients within 2 years after exposure, compared with 32% and 47% for Strategies I and II, respectively. There is a linear relationship between the rate of clustering of isolates and the probability of secondary cases being prevented by the use of Strategy III. A two-stage tuberculosis contact tracing strategy, based on clustering of genetically related M. tuberculosis isolates, should improve identification of epidemiologic links and prevent more cases of secondary infections in low prevalence settings and so augment traditional contact tracing. The main factors affecting utilities were the likelihood of new epidemiological links being identified after the second interview and the local rate of clustering of M. tuberculosis isolates.

Developing Decision Support Systems in Clinical Bioinformatics

Methods in Molecular Medicine. 2008  |  Pubmed ID: 18453098

There is a growing demand for tools to support clinicians utilize genomic results generated by molecular diagnostic and cytogenetic methods in support of their decision-making. This chapter reviews existing experience and methods for the design, implementation and evaluation of clinical bioinformatics electronic decision support systems (EDSS). It provides a roadmap for identifying decision tasks for automation and selecting optimal tools for building task-specific systems. Key success factors for EDSS implementation and evaluation are also outlined.

Genome-wide Analysis of Single Nucleotide Polymorphisms in Bordetella Pertussis Using Comparative Genomic Sequencing

Research in Microbiology. Nov-Dec, 2008  |  Pubmed ID: 18790049

Bordetella pertussis is known to be a genotypically homogeneous pathogen but the extent of homogeneity at the genomic level is unknown. A currently circulating B. pertussis isolate from Australia was compared with the genome-sequenced Tohama I strain isolated in Japan in the 1950s from a distantly related lineage. Microarray-based comparative genome sequencing (CGS) was used to detect single nucleotide polymorphisms (SNPs) in a total of 1.4 Mb of the 4.09 Mb genome, including 1012 coding-regions, 217 pseudogenes and 268 intergenic regions. The CGS analysis, followed by validation using real-time PCR and DNA sequencing, identified 70 SNPs and five 1-3 bp indels, giving an overall frequency of base changes of 1 per 20 kb. Thirty-two of the 56 SNPs in coding regions were non-synonymous, including five located in virulence-associated genes. The data also allowed us to compare genomic diversity with other "clonal" human pathogens such as Mycobacterium tuberculosis and Yersinia pestis, showing that B. pertussis may be one of the least variable pathogenic bacterial species.

Decision Support Systems for Antibiotic Prescribing

Current Opinion in Infectious Diseases. Dec, 2008  |  Pubmed ID: 18988320

To explore recent developments in computerized evidence-based guidelines and decision support systems that have been designed to improve the effectiveness and efficiency of antibiotic prescribing.

The Re-emergence of Pertussis: Implications for Diagnosis and Surveillance

New South Wales Public Health Bulletin. Jul-Aug, 2008  |  Pubmed ID: 19007547

Pertussis, or whooping cough, a highly contagious disease caused by Bordetella pertussis, is making a comeback globally and nationally in spite of reasonable vaccination coverage. This paper provides an update on laboratory testing methods that assist the confirmation of clinical disease and investigation of outbreaks. Laboratory confirmation of the diagnosis by polymerase chain reaction or serology should be attempted, especially when atypical pertussis is suspected clinically. Genetic and antigenic variations in virulence factors of strains circulating in the population should also be monitored.

Towards Bioinformatics Assisted Infectious Disease Control

BMC Bioinformatics. 2009  |  Pubmed ID: 19208185

This paper proposes a novel framework for bioinformatics assisted biosurveillance and early warning to address the inefficiencies in traditional surveillance as well as the need for more timely and comprehensive infection monitoring and control. It leverages on breakthroughs in rapid, high-throughput molecular profiling of microorganisms and text mining.

In Silico Prioritisation of Candidate Genes for Prokaryotic Gene Function Discovery: an Application of Phylogenetic Profiles

BMC Bioinformatics. 2009  |  Pubmed ID: 19292914

In silico candidate gene prioritisation (CGP) aids the discovery of gene functions by ranking genes according to an objective relevance score. While several CGP methods have been described for identifying human disease genes, corresponding methods for prokaryotic gene function discovery are lacking. Here we present two prokaryotic CGP methods, based on phylogenetic profiles, to assist with this task.

Rapid and Accurate Typing of Bordetella Pertussis Targeting Genes Encoding Acellular Vaccine Antigens Using Real Time PCR and High Resolution Melt Analysis

Journal of Microbiological Methods. Jun, 2009  |  Pubmed ID: 19341769

Real Time-PCR (RT-PCR) and high resolution melt (HRM) analyses were used for rapid typing of genes encoding components of the pertussis acellular vaccine, namely prn, ptxA, fhaB, fim2 and fim3. The length polymorphisms in prn were detected by RT-PCR followed by HRM; single nucleotide polymorphisms in prn and other genes were detected by hairpin primer RT-PCR. These rapid methods are suitable for large-scale studies of vaccine-driven evolution of Bordetella pertussis.

Laboratory-guided Detection of Disease Outbreaks: Three Generations of Surveillance Systems

Archives of Pathology & Laboratory Medicine. Jun, 2009  |  Pubmed ID: 19492884

Traditional biothreat surveillance systems are vulnerable to incomplete and delayed reporting of public health threats.

Identification of Non-tuberculous Mycobacteria: Utility of the GenoType Mycobacterium CM/AS Assay Compared with HPLC and 16S RRNA Gene Sequencing

Journal of Medical Microbiology. Jul, 2009  |  Pubmed ID: 19502366

Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. A new reverse line probe assay, GenoType Mycobacterium CM/AS (Hain Lifescience), was evaluated for identification of a broad range of NTM. It was compared with phenotypic (HPLC) and molecular (DNA probes, in-house real-time multiplex species-specific PCR, 16S rRNA gene PCR and sequencing) identification techniques, which together provided the reference 'gold standard'. A total of 131 clinical isolates belonging to 31 Mycobacterium species and 19 controls, including 5 non-Mycobacterium species, was used. Concordant results between the GenoType Mycobacterium assay and the reference identification were obtained in 119/131 clinical isolates (90.8 %). Identification of Mycobacterium abscessus and Mycobacterium lentiflavum by the assay was problematic. The GenoType Mycobacterium assay enables rapid identification of a broad range of potentially clinically significant Mycobacterium species, but some species require further testing to differentiate or confirm ambiguous results.

A New Multiplex PCR-based Reverse Line-blot Hybridization (mPCR/RLB) Assay for Rapid Staphylococcal Cassette Chromosome Mec (SCCmec) Typing

Journal of Medical Microbiology. Aug, 2009  |  Pubmed ID: 19528184

The aim of this study was to develop a new discriminatory method for MRSA SCCmec typing based on multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay to enable rapid identification and classification of MRSA SCCmec types in a clinical laboratory. Forty-five primer sets and 49 probes were designed and tested in uniplex PCR (uPCR) and mPCR/RLB. Probes were compared in silico to 14 whole-genome sequences and 18 partial SCCmec gene sequences of Staphylococcus aureus and complete genome and partial SCCmec genes of seven non-MRSA strains, including meticillin-susceptible S. aureus and meticillin-resistant coagulase-negative staphylococci. The method was tested on a set of 42 well-characterized reference MRSA strains. It identified all five epidemiologically relevant SCCmec types and 26 subtypes, including established and new subtypes of SCCmec III, IV (eight subtypes each) and V (three subtypes). The discriminatory power of mPCR/RLB SCCmec typing was similar to that of MLST and spa typing (Simpson indices of diversity of 0.916, 0.926 and 0.882, respectively; differences not statistically significant). The application of mPCR/RLB hybridization assay to MRSA SCCmec typing can improve the specificity, discriminatory power and throughput of the typing procedure. The detection of up to 43 mPCR products in a single hybridization assay transforms MRSA SCCmec typing from passive epidemiological library typing into a potential tool for near-real-time infection control surveillance and tracking of MRSA transmission in hospitals.

Rolling Circle Amplification and Multiplex Allele-specific PCR for Rapid Detection of KatG and InhA Gene Mutations in Mycobacterium Tuberculosis

International Journal of Medical Microbiology : IJMM. Dec, 2009  |  Pubmed ID: 19604720

The aim of the study was to compare a novel, rolling circle amplification (RCA) assay for detection of common isoniazid (INH) resistance mutations in Mycobacterium tuberculosis with a multiplex allele-specific PCR (MAS-PCR) and sequencing of katG and the fabG1-inhA promoter region. One or more mutations were identified by RCA, MAS-PCR, and sequencing in 21 (68%), 22 (71%), and 23 (74%), respectively, of 31 epidemiologically unrelated INH-resistant isolates, and in none of 8 INH-susceptible isolates. The RCA assay is a rapid, inexpensive, and practical screening method for INH resistance in M. tuberculosis in countries with high prevalence of INH resistance.

Assignment of Reference 5'-end 16S RDNA Sequences and Species-Specific Sequence Polymorphisms Improves Species Identification of Nocardia

The Open Microbiology Journal. 2009  |  Pubmed ID: 19639036

16S rDNA sequence analysis is the most accurate method for definitive species identification of nocardiae. However, conflicting results can be found due to sequence errors in gene databases. This study tested the feasibility of species identification of Nocardia by partial (5'-end 606-bp) 16S rDNA sequencing, based on sequence comparison with "reference" sequences of well-annotated strains. This new approach was evaluated using 96 American Type Culture Collection (n=6), and clinical (n=90) Nocardia isolates. Nucleotide sequence-based polymorphisms within species were indicative of "sequence types" for that species. Sequences were compared with those in the GenBank, Bioinformatics Bacteria Identification and Ribosomal Database Project databases. Compared with the reference sequence set, all 96 isolates were correctly identified using the criterion of >/=99% sequence similarity. Seventy-eight (81.3%) were speciated by database comparison; alignment with reference sequences resolved the identity of 14 (15%) isolates whose sequences yielded 100% similarity to sequences in GenBank under >1 species designation. Of 90 clinical isolates, the commonest species was Nocardia nova (33.3%) followed by Nocardia cyriacigeorgica (26.7%). Recently-described or uncommon species included Nocardia veterana (4.4%), Nocarida bejingensis (2.2%) and, Nocardia abscessus and Nocardia arthriditis (each n=1). Nocardia asteroides sensu stricto was rare (n=1). There were nine sequence types of N. nova, three of Nocardia brasiliensis with two each of N. cyriacigeorgica and Nocardia farcinica. Thirteen novel sequences were identified. Alignment of sequences with reference sequences facilitated species identification of Nocardia and allowed delineation of sequence types within species, suggesting that such a barcoding approach can be clinically useful for identification of bacteria.

Influenza Outbreak During Sydney World Youth Day 2008: the Utility of Laboratory Testing and Case Definitions on Mass Gathering Outbreak Containment

PloS One. 2009  |  Pubmed ID: 19727401

Influenza causes annual epidemics and often results in extensive outbreaks in closed communities. To minimize transmission, a range of interventions have been suggested. For these to be effective, an accurate and timely diagnosis of influenza is required. This is confirmed by a positive laboratory test result in an individual whose symptoms are consistent with a predefined clinical case definition. However, the utility of these clinical case definitions and laboratory testing in mass gathering outbreaks remains unknown.

Commonly Used Molecular Epidemiology Markers of Streptococcus Agalactiae Do Not Appear to Predict Virulence

Pathology. 2009  |  Pubmed ID: 19900108

Several virulent clones of group B streptococcus (GBS) are known to be associated with certain serotypes and molecular epidemiological markers. It is unclear, however, whether the clinical significance of GBS can be predicted based solely on such molecular markers. The aim of this study was to test the hypothesis that GBS virulence can be predicted by using the molecular epidemiology markers.

Biosurveillance of Emerging Biothreats Using Scalable Genotype Clustering

Journal of Biomedical Informatics. Feb, 2009  |  Pubmed ID: 18723122

Developments in molecular fingerprinting of pathogens with epidemic potential have offered new opportunities for improving detection and monitoring of biothreats. However, the lack of scalable definitions for infectious disease clustering presents a barrier for effective use and evaluation of new data types for early warning systems. A novel working definition of an outbreak based on temporal and spatial clustering of molecular genotypes is introduced in this paper. It provides an unambiguous way of clustering of causative pathogens and is adjustable to local disease prevalence and availability of public health resources. The performance of this definition in prospective surveillance is assessed in the context of community outbreaks of food-borne salmonellosis. Molecular fingerprinting augmented with the scalable clustering allows the detection of more than 50% of the potential outbreaks before they reach the midpoint of the cluster duration. Clustering in time by imposing restrictions on intervals between collection dates results in a smaller number of outbreaks but does not significantly affect the timeliness of detection. Clustering in space and time by imposing restrictions on the spatial and temporal distance between cases results in a further reduction in the number of outbreaks and decreases the overall efficiency of prospective detection. Innovative bacterial genotyping technologies can enhance early warning systems for public health by aiding the detection of moderate and small epidemics.

Bug Breakfast in the Bulletin: Biosecurity and Infectious Diseases

New South Wales Public Health Bulletin. May-Jun, 2010  |  Pubmed ID: 21604605

Identification of Pathogenic Nocardia Species by Reverse Line Blot Hybridization Targeting the 16S RRNA and 16S-23S RRNA Gene Spacer Regions

Journal of Clinical Microbiology. Feb, 2010  |  Pubmed ID: 19955277

Although 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardia species, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined Nocardia PCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardia isolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 species-specific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivorans strains (n = 10 strains) and a Nocardia asteroides isolate with a novel 16S rRNA gene sequence. Nocardia farcinica and Nocardia cyriacigeorgica strains were identified by the sequential use of an N. farcinica-"negative" probe and a combined N. farcinica/N. cyriacigeorgica probe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensis probe with the Nocardia thailandica DNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivorans strains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacer-directed probes were required to identify all N. farcinica stains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardia species and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended.

Reverse Line Blot Hybridization and DNA Sequencing Studies of the 16S-23S RRNA Gene Intergenic Spacer Regions of Five Emerging Pathogenic Nocardia Species

Journal of Medical Microbiology. May, 2010  |  Pubmed ID: 20110385

The objective of this study was to examine DNA sequence polymorphisms in the 16S-23S rRNA gene intergenic spacer (ITS) regions of five emerging pathogenic Nocardia species: Nocardia beijingensis, Nocardia blacklockiae, Nocardia thailandica, Nocardia elegans and Nocardia vinacea. A set of six isolates belonging to the species of interest and 135 isolates belonging to other Nocardia species was studied. A PCR-based reverse line blot (RLB) hybridization assay incorporating species- or intraspecies ITS rRNA gene operon-specific probes was then developed for species identification. Substantial intraspecies sequence variation among different ITS operons was identified. Four sequence types of N. thailandica, eight sequence types of N. beijingensis (four types for each of two strains) and five sequence types of N. blacklockiae, N. elegans and N. vinacea were found. The results represent the first evidence of ITS sequence heterogeneity in emerging species of Nocardia. By incorporating species/operon-specific probes into a RLB assay, unique RLB patterns were identified for each of the species and every sequence type. The PCR/RLB assay demonstrated high specificity and showed promise in both the identification and genotyping of Nocardia species. More detailed studies of the polymorphism within the ITS locus may further advance our capacity to reliably identify and subtype medically important Nocardia species.

Bordetella Pertussis Clones Identified by Multilocus Variable-number Tandem-repeat Analysis

Emerging Infectious Diseases. Feb, 2010  |  Pubmed ID: 20113564

Multilocus variable-number tandem-repeat analysis (MLVA) of 316 Bordetella pertussis isolates collected over 40 years from Australia and 3 other continents identified 66 MLVA types (MTs), including 6 predominant MTs. Typing of genes encoding acellular vaccine antigens showed changes that may be vaccine driven in 2 MTs prevalent in Australia.

A PubMed-wide Associational Study of Infectious Diseases

PloS One. 2010  |  Pubmed ID: 20224767

Computational discovery is playing an ever-greater role in supporting the processes of knowledge synthesis. A significant proportion of the more than 18 million manuscripts indexed in the PubMed database describe infectious disease syndromes and various infectious agents. This study is the first attempt to integrate online repositories of text-based publications and microbial genome databases in order to explore the dynamics of relationships between pathogens and infectious diseases.

Three-year Longitudinal Study of Genotypes of Mycobacterium Tuberculosis in a Low Prevalence Population

Pathology. Apr, 2010  |  Pubmed ID: 20350221

To investigate the molecular epidemiology of tuberculosis, temporal and spatial distribution of Mycobacterium tuberculosis isolates and associations between genotypes and clinical characteristics, in a low prevalence population.

Rapid Identification of Methicillin-resistant Staphylococcus Aureus Transmission in Hospitals by Use of Phage-derived Open Reading Frame Typing Enhanced by Multiplex PCR and Reverse Line Blot Assay

Journal of Clinical Microbiology. Aug, 2010  |  Pubmed ID: 20519463

The relatively high-level clonality of methicillin-resistant Staphylococcus aureus (MRSA) and its frequent high-level endemicity in nosocomial settings complicate the development of methods for rapid subtyping of MRSA strains that are capable of identifying person-to-person transmission in hospitals. Phage-derived open reading frame (PDORF) typing is an MRSA typing method targeting mobile genetic elements that was recently described and evaluated using a geographically restricted set of isolates. The objective of this study was to develop a multiplex PCR-reverse line blot (mPCR/RLB) assay for PDORF typing and to test its applicability on a broad range of isolates and in an environment where MRSA is highly endemic. The 16 targets were identified using a 23-primer-pair mPCR/RLB assay with two probes for each target. The method was evaluated using 42 MRSA reference strains, including those representing major international clones, and 35 isolates from episodes of suspected nosocomial transmission. In vivo stability was explored using 81 isolate pairs. Pulsed-field gel electrophoresis (PFGE) and spa typing were performed for comparison. Among the 42 reference strains, there were 33 PFGE subtypes, 30 PDORF types, and 22 spa types. Simpson's index of diversity was 0.987, 0.971, and 0.926 for PFGE subtyping, PDORF typing, and spa typing, respectively. Typing of clinical isolates by PDORF typing and PFGE demonstrated concordant results. mPCR/RLB-based PDORF typing has similar discriminatory power to that of PFGE, can assist in tracking MRSA transmission events in a setting of high-level endemicity, and has the advantage of being a high-throughput technique.

Unexpected Diversity of Staphylococcal Cassette Chromosome Mec Type IV in Methicillin-resistant Staphylococcus Aureus Strains

Journal of Clinical Microbiology. Oct, 2010  |  Pubmed ID: 20686095

Staphylococcal cassette chromosome mec (SCCmec) is a large mobile genetic element which is used frequently for subtyping of methicillin-resistant Staphylococcus aureus (MRSA) strains. MRSA SCCmec type IV not only predominates among community-acquired MRSA (CA-MRSA) strains but also is associated with several genetic lineages of hospital-acquired MRSA (HA-MRSA) and with other species. The objective of this study was to investigate the diversity of MRSA strains classified as SCCmec type IV by using a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay as well as spa typing and pulsed-field gel electrophoresis (PFGE). Sixty-two primer pairs and 63 probes were designed to interrogate each open reading frame (ORF) of SCCmec type IV sequences. A set of 131 MRSA SCCmec type IV isolates were classified into 79 subtypes by this method. There was considerable concordance between SCCmec type IV subtyping, spa typing, and PFGE patterns for clinical isolates, and the stability of SCCmec type IV subtyping was comparable to that of the other two methods. Using an in-house computer program, we showed that a subset of 20 genetic markers could achieve the same level of discrimination between isolates as the full set of 62, with a Simpson's index of diversity of 0.975. SCCmec type IV has a much higher level of diversity than previously suggested. The application of the mPCR/RLB hybridization assay to MRSA SCCmec type IV subtyping can improve the discriminatory power and throughput of MRSA typing and has the potential to enhance rapid infection control surveillance and outbreak detection.

SecA1 Gene Sequence Polymorphisms for Species Identification of Nocardia Species and Recognition of Intraspecies Genetic Diversity

Journal of Clinical Microbiology. Nov, 2010  |  Pubmed ID: 20810768

Sequence analysis of the Nocardia essential secretory protein SecA1 gene (secA1) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia (16 species) was studied in comparison with 5'-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1 gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with <99.0% similarity to archived secA1 sequences for the species, including 29 Nocardia cyriacigeorgica (96.6 to 98.9% similarity) and 4 Nocardia veterana (91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia nova strains (identified as various Nocardia species by secA1 sequencing) and 1 isolate each of Nocardia abscessus (identified as Nocardia asiatica), Nocardia elegans (Nocardia africana), and Nocardia transvalensis (Nocardia blacklockiae); both secA1 gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1 locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova (14 secA1 sequence types), followed by Nocardia farcinica and N. veterana (n = 7 each); there was only a single sequence type among eight Nocardia paucivorans strains. The secA1 locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.

Insight into Evolution of Bordetella Pertussis from Comparative Genomic Analysis: Evidence of Vaccine-driven Selection

Molecular Biology and Evolution. Jan, 2011  |  Pubmed ID: 20833694

Despite high vaccine coverage, pertussis incidence has increased substantially in recent years in many countries. A significant factor that may be contributing to this increase is adaptation to the vaccine by Bordetella pertussis, the causative agent of pertussis. In this study, we first assessed the genetic diversity of B. pertussis by microarray-based comparative genome sequencing of 10 isolates representing diverse genotypes and different years of isolation. We discovered 171 single nucleotide polymorphisms (SNPs) in a total of 1.4 Mb genome analyzed. The frequency of base changes was estimated as one per 32 kb per isolate, confirming that B. pertussis is one of the least variable bacterial pathogens. We then analyzed an international collection of 316 B. pertussis isolates using a subset of 65 of the SNPs and identified 42 distinct SNP profiles (SPs). Phylogenetic analysis grouped the SPs into six clusters. The majority of recent isolates belonged to clusters I-IV and were descendants of a single prevaccine lineage. Cluster I appeared to be a major clone with a worldwide distribution. Typing of genes encoding acellular vaccine (ACV) antigens, ptxA, prn, fhaB, fim2, and fim3 revealed the emergence and increasing incidence of non-ACV alleles occurring in clusters I and IV, which may have been driven by ACV immune selection. Our findings suggest that B. pertussis, despite its high population homogeneity, is evolving in response to vaccination pressure with recent expansion of clones carrying variants of genes encoding ACV antigens.

Editor's Choice: Implications of Isoniazid Resistance in Mycobacterium Bovis Bacillus Calmette-Guérin Used for Immunotherapy in Bladder Cancer

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Jan, 2011  |  Pubmed ID: 21148524

We report a case of sepsis from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) with low-level isoniazid resistance following intravesical treatment for bladder cancer. Isoniazid resistance in BCG has therapeutic implications when it causes infections after intravesical instillation. For these circumstances, we propose some modifications to existing treatment guidelines for BCG infection.

Improved Identification of Gordonia, Rhodococcus and Tsukamurella Species by 5'-end 16S RRNA Gene Sequencing

Pathology. Jan, 2011  |  Pubmed ID: 21240067

The identification of fastidious aerobic Actinomycetes such as Gordonia, Rhodococcus, and Tsukamurella has remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5'-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus, and Tsukamurella, and defined potential reference sequences for species from each of these genera.

Computational Bacterial Genome-wide Analysis of Phylogenetic Profiles Reveals Potential Virulence Genes of Streptococcus Agalactiae

PloS One. 2011  |  Pubmed ID: 21483735

The phylogenetic profile of a gene is a reflection of its evolutionary history and can be defined as the differential presence or absence of a gene in a set of reference genomes. It has been employed to facilitate the prediction of gene functions. However, the hypothesis that the application of this concept can also facilitate the discovery of bacterial virulence factors has not been fully examined. In this paper, we test this hypothesis and report a computational pipeline designed to identify previously unknown bacterial virulence genes using group B streptococcus (GBS) as an example. Phylogenetic profiles of all GBS genes across 467 bacterial reference genomes were determined by candidate-against-all BLAST searches,which were then used to identify candidate virulence genes by machine learning models. Evaluation experiments with known GBS virulence genes suggested good functional and model consistency in cross-validation analyses (areas under ROC curve, 0.80 and 0.98 respectively). Inspection of the top-10 genes in each of the 15 virulence functional groups revealed at least 15 (of 119) homologous genes implicated in virulence in other human pathogens but previously unrecognized as potential virulence genes in GBS. Among these highly-ranked genes, many encode hypothetical proteins with possible roles in GBS virulence. Thus, our approach has led to the identification of a set of genes potentially affecting the virulence potential of GBS, which are potential candidates for further in vitro and in vivo investigations. This computational pipeline can also be extended to in silico analysis of virulence determinants of other bacterial pathogens.

Defining Reference Sequences for Nocardia Species by Similarity and Clustering Analyses of 16S RRNA Gene Sequence Data

PloS One. 2011  |  Pubmed ID: 21687706

The intra- and inter-species genetic diversity of bacteria and the absence of 'reference', or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia.

Genetic Relationships of Phage Types and Single Nucleotide Polymorphism Typing of Salmonella Enterica Serovar Typhimurium

Journal of Clinical Microbiology. Jan, 2012  |  Pubmed ID: 22205813

Salmonella enterica serovar Typhimurium is one of the leading causes of gastroenteritis in humans. Phage typing has been used for epidemiological surveillance of Typhimurium for over four decades. However, knowledge of the evolutionary relationships between phage types is very limited. In this study, we used single nucleotide polymorphisms (SNPs) as molecular markers to determine the relationships between common Typhimurium phage types. Forty-four SNPs including 24 identified in a previous study and 20 from 6 available whole genome sequence were used to analyse 215 Typhimurium isolates belonging to 45 phage types. Altogether, 215 isolates and 6 genome strains were differentiated into 33 SNP profiles and four distinctive phylogenetic clusters. Fourteen phage types including DT9, one of the commonest phage types in Australia, were differentiated into multiple SNP profiles. These SNP profiles were distributed in different phylogenetic clusters, indicating that they have arisen independently multiple times. This finding suggests that phage typing may not be useful for long-term epidemiological studies over long periods (years) and diverse localities (different countries or continents). SNP typing provided a discriminative power similar to that of phage typing. However, 12 SNP profiles contained more than one phage type and more SNPs would be needed for further differentiation. SNP typing should be considered as a replacement for phage typing for strain identification of Typhimurium.

Selection and Emergence of Pertussis Toxin Promoter PtxP3 Allele in the Evolution of Bordetella Pertussis

Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases. Jan, 2012  |  Pubmed ID: 22293463

Evolutionary studies using single nucleotide polymorphisms (SNPs) have separated Bordetella pertussis isolates into six major clusters, with recent isolates forming cluster I. The expansion of cluster I isolates was characterised by changes in genes encoding antigenic components in acellular vaccines, including pertactin (Prn). Here, we determined the initial emergence of the pertussis toxin promoter allele, ptxP3, from an evolutionary perspective. This allele was previously shown in a study from the Netherlands to be associated with increased pertussis toxin production as a result of a single base mutation in the ptxP. The ptxP region of 313 worldwide isolates was sequenced, including 208 isolates from Australia collected over a 40year period. Eight alleles were identified, of which only two predominated: ptxP1 and ptxP3. One novel allele was also found. ptxP3 was only found in SNP cluster I of B. pertussis and its emergence is concurrent with the change to the non-vaccine prn2 allele. Our results suggest that the globally distributed cluster I of B. pertussis has the ability to evade vaccine induced selection pressure.