Sointu Mero

Accurate and rapid identification of bacterial species from positive blood cultures with a DNA-based microarray platform: an observational study

BACKGROUND New DNA-based microarray platforms enable rapid detection and species identification of many pathogens, including bacteria. We assessed the sensitivity, specificity, and turnaround time of a new molecular sepsis assay. METHODS 2107 positive blood-culture samples of 3318 blood samples from patients with clinically suspected sepsis were investigated for bacterial species by both conventional culture and Prove-it sepsis assay (Mobidiag, Helsinki, Finland) in two centres (UK and Finland). The assay is a novel PCR and microarray method that is based on amplification and detection of gyrB, parE, and mecA genes of 50 bacterial species. Operators of the test assay were not aware of culture results. We calculated sensitivity, specificity, and turnaround time according to Clinical and Laboratory Standards Institute recommendations. FINDINGS 1807 of 2107 (86%) positive blood-culture samples included a pathogen covered by the assay. The assay had a clinical sensitivity of 94.7% (95% CI 93.6-95.7) and a specificity of 98.8% (98.1-99.2), and 100% for both measures for meticillin-resistant Staphylococcus aureus bacteraemia. The assay was a mean 18 h faster than was the conventional culture-based method, which takes an additional 1-2 working days. 34 of 3284 (1.0%) samples were excluded because of technical and operator errors. INTERPRETATION Definitive identification of bacterial species with this microarray platform was highly sensitive, specific, and faster than was the gold-standard culture-based method. This assay could enable fast and earlier evidence-based management for clinical sepsis.

Antimicrobials Increase Travelers' Risk of Colonization by Extended-Spectrum Betalactamase-Producing Enterobacteriaceae

Colonized travelers contribute to the pandemic spread of resistant intestinal bacteria. This study is the first to show that antimicrobial use during travel predisposes to colonization by intestinal extended-spectrum beta-lactamase-producing Enterobacteriaceae. Travelers refrain from taking unnecessary antibiotics.

Increased Risk for ESBL-Producing Bacteria from Co-administration of Loperamide and Antimicrobial Drugs for Travelers' Diarrhea.

Antimicrobial drug treatment of travelers’ diarrhea is known to increase the risk for colonization with extended-spectrum β-lactamase-producing Enterobacteriaceae. Among 288 travelers with travelers’ diarrhea, the colonization rate without medications was 21%. For treatment with loperamide only, the rate was 20%; with antimicrobial drugs alone, 40%; and with loperamide and antimicrobial drugs, 71%.

Comparison of repetitive extragenic palindromic sequence-based PCR with PCR ribotyping and pulsed-field gel electrophoresis in studying the clonality of Clostridium difficile.

Clostridium difficile infection is most often induced by antibiotic treatment. Recently, morbidity and mortality resulting especially from C. difficile PCR ribotype 027 have increased significantly. In addition, more severe disease has been associated with C. difficile PCR ribotype 078 strains. Thus, reliable typing methods for epidemic control are needed. In the present study, we compared an automated repetitive extragenic palindromic sequence-based PCR (rep-PCR) method (DiversiLab; Bacterial Barcodes, Inc., Athens, GA, USA) to PCR ribotyping and pulsed-field gel electrophoresis (PFGE) typing using 205 isolates of C. difficile (including 24 previously characterized isolates). Among the 181 clinical isolates, a total of 31 different PCR ribotypes, 38 different PFGE types and subtypes and 28 different rep-PCR types were found. Six major rep-PCR groups (DL1-DL6) harboured 86% of the clinical isolates. All isolates belonging to PCR ribotypes 027 and 001 clustered in their own rep-PCR groups, enabling us to screen out the hypervirulent ribotype 027 strain. Within the PCR ribotype 001, four subgroups were found using rep-PCR. Overall, in 75% (135/181) of the isolates, the classification attributed following rep-PCR and PCR ribotyping was comparable. In conclusion, the automated rep-PCR-based typing method represents an option for first-line molecular typing in local clinical microbiology laboratories. The method was easy to use as well as rapid, requiring less hands-on time than PCR ribotyping or PFGE typing. The conventional PCR ribotyping or PFGE, however, are needed for confirmatory molecular epidemiology. In addition, more epidemiology-oriented studies are needed to examine the discriminatory power of automated rep-PCR with isolates collected from a larger geographical area and during a longer period of time.

Rapid Molecular Characterization of Acinetobacter baumannii Clones with rep-PCR and Evaluation of Carbapenemase Genes by New Multiplex PCR in Hospital District of Helsinki and Uusimaa

Multidrug-resistant Acinetobacter baumannii (MDRAB) is an increasing problem worldwide. Prevalence of carbapenem resistance in Acinetobacter spp. due to acquired carbapenemase genes is not known in Finland. The purpose of this study was to examine prevalence and clonal spread of multiresistant A. baumannii group species, and their carbapenemase genes. A total of 55 Acinetobacter isolates were evaluated with repetitive PCR (DiversiLab) to analyse clonality of isolates, in conjunction with antimicrobial susceptibility profile for ampicillin/sulbactam, colistin, imipenem, meropenem, rifampicin and tigecycline. In addition, a new real-time PCR assay, detecting most clinically important carbapenemase genes just in two multiplex reactions, was developed. The assay detects genes for KPC, VIM, IMP, GES-1/-10, OXA-48, NDM, GIM-1, SPM-1, IMI/NMC-A, SME, CMY-10, SFC-1, SIM-1, OXA-23-like, OXA-24/40-like, OXA-58 and ISAbaI-OXA-51-like junction, and allows confident detection of isolates harbouring acquired carbapenemase genes. There was a time-dependent, clonal spread of multiresistant A. baumannii strongly correlating with carbapenamase gene profile, at least in this geographically restricted study material. The new carbapenemase screening assay was able to detect all the genes correctly suggesting it might be suitable for epidemiologic screening purposes in clinical laboratories.

A selective broth enrichment combined with real-time nuc-mecA-PCR in the exclusion of MRSA

Pasanen T, Korkeila M, Mero S, Tarkka E, Piiparinen H, Vuopio‐Varkila J, Vaara M, Tissari P. A selective broth enrichment combined with real‐time nuc‐mecA‐PCR in the exclusion of MRSA. APMIS 2010; 118: 74–80.

High number of diarrhoeal co-infections in travellers to Benin, West Africa

BackgroundTravellers’ diarrhoea (TD) is the most frequent health problem among travellers to the tropics. Using routine techniques, the aetiology mostly remains unresolved, whereas modern molecular methods enable reducing the number of equivocal cases considerably. While many studies address the aetiology of TD in Asian, Central American and North African tourist resorts, only few focus on Western Africa.MethodsStool samples from 45 travellers travelling in Benin, West Africa, were analyzed by a new multiplex qPCR assay for Salmonella, Yersinia, Campylobacter, Vibrio cholerae, Shigella or enteroinvasive (EIEC), enterohaemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), and enteropathogenic Escherichia coli (EPEC).ResultsAll 18 pre-travel samples proved negative for bacterial pathogens. Of the 39/45 (87%) travellers having had TD, EPEC was detected in post-travel samples in 30 (77%) cases, EAEC in 23 (59%), ETEC in 22 (56%), Shigella or EIEC in 7 (18%), EHEC in two (5%), and Salmonella in one (3%). In 31(79%) of the TD cases two or more bacterial pathogens were identified. Two (8%) samples remained negative: both patients had taken antimicrobials for TD.ConclusionsEPEC, EAEC and ETEC were the most common findings. 79% of the cases had a co-infection. As modern diagnostics reveals in most patients a multitude of pathogens, the role of each pathogen should be re-evaluated.

Applicability of DiversiLab Repetitive Sequence-Based PCR Method in Epidemiological Typing of Enterohemorrhagic Escherichia coli (EHEC)

Enterohemorrhagic Escherichia coli (EHEC) causes diarrhea, often with severe complications. Rapid and discriminatory typing of EHEC using advanced molecular methods is needed for determination of the genetic relatedness of clones responsible for foodborne outbreaks and for finding out the transmission sources of the outbreaks. This study evaluated the potential of DiversiLab, a semiautomated repetitive sequence-based polymerase chain reaction method for the genotyping of EHEC strains. A set of 52 EHEC strains belonging to 15 O:H serotypes was clustered into 10 DiversiLab groups. All of the O157 strains and one O55 strain were classified into the same group based on a 90% similarity threshold. The other serotypes were classified to their own DiversiLab group, with the exception of one R:H(-) strain that was grouped with O5:H(-) strains. In addition, O26 and O111 strains were grouped together but ultimately subdivided according to their O-serotypes based on a 95% similarity threshold. The O104 strain, which was associated with a major outbreak of hemolytic uremic syndrome in Germany in May 2011, was also classified independently. The DiversiLab performed well in identifying isolates, but the discriminatory power of the repetitive sequence-based polymerase chain reaction method was lower than that of pulsed-field gel electrophoresis. Analysis of 15 enteropathogenic E. coli (EPEC) strains revealed that some EPEC strains clustered together with EHEC strains. Therefore, the DiversiLab system cannot be used to discriminate between these pathogroups. In conclusion, DiversiLab is a rapid and easy system for the primary exclusion of unrelated EHEC strains based on their serotypes, but more discriminatory methods, such as pulsed-field gel electrophoresis, are needed for accurate typing of the EHEC strains.

First isolation of Dietzia cinnamea from a dog bite wound in an adult patient

ABSTRACT We report the first case of deep-wound colonization by Dietzia cinnamea in a patient who had been bitten by a dog.

Evaluation of phenotypic and molecular methods for screening and detection of methicillin-resistant Staphylococcus aureus

An in-house PCR was compared with the GenoType® MRSA and the MRSA EVIGENETM tests, both of which corresponded 100% with the results of in-house PCR. The cefoxitin disk diffusion method was superior to both the oxacillin disk diffusion and minimum inhibitory concentration tests for predicting the mecA status.