Willem B. van Leeuwen

Comparison of restriction fragment length polymorphism, microsatellite length polymorphism, and random amplification of polymorphic DNA analyses for fingerprinting Aspergillus fumigatus isolates.

ABSTRACT Aspergillus fumigatus fingerprints generated by random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) upon hybridization with repeated DNA sequences, and PCR detection of microsatellite length polymorphism (MLP) were compared among 67 isolates. In contrast to RAPD, RFLP and MLP gave discriminating and significantly concordant genotyping results.

Prevention of catheter-related bacteremia with a daily ethanol lock in patients with tunnelled catheters: A randomized, placebo-controlled trial

Background Catheter-related bloodstream infection (CRBSI) results in significant attributable morbidity and mortality. In this randomized, double-blind, placebo-controlled trial, we studied the efficacy and safety of a daily ethanol lock for the prevention of CRBSI in patients with a tunnelled central venous catheter (CVC). Methodology From 2005 through 2008, each lumen of the CVC of adult hematology patients was locked for 15 minutes per day with either 70%-ethanol or placebo, where after the lock solution was flushed through. As a primary endpoint, the incidence rates of endoluminal CRBSI were compared. Principal Findings The intent-to-treat analysis was based on 376 patients, accounting for 448 CVCs and 27,745 catheter days. For ethanol locks, the incidence of endoluminal CRBSI per 1000 CVC-days was 0.70 (95%-CI, 0.4–1.3), compared to 1.19 (95% confidence interval, 0.7–1.9) for placebo (incidence rate-ratio, 0.59; 95% confidence interval, 0.27–1.30; P = .19). For endoluminal CRBSI according to the strictest definition (positive hub culture and identical bacterial strain in blood), a 3.6-fold, non-significant, reduction was observed for patients receiving ethanol (2 of 226 versus 7 of 222; P = .103). No life-threatening adverse events were observed. More patients receiving ethanol discontinued lock-therapy (11 of 226 versus 1 of 222; P = .006) or continued with decreased lock-frequency (10 of 226 versus 0 of 222; P = .002), due to non-severe adverse events. Conclusions In this study, the reduction in the incidence of endoluminal CRBSI using preventive ethanol locks was non-significant, although the low incidence of endoluminal CRBSI precludes definite conclusions. Therefore, the lack of statistical significance may partially reflect a lack of power. Significantly more patients treated with ethanol locks discontinued their prophylactic treatment due to adverse effects, which were non-severe but reasonably ethanol related. Additional studies should be performed in populations with higher incidence of (endoluminal) CRBSI. Alternative sources of bacteremia, like exoluminal CRBSI or microbial translocation during chemotherapy-induced mucositis may have been more important in our patients. Trial Registration ClinicalTrials.gov NCT00122642

Natural population dynamics and expansion of pathogenic clones of Staphylococcus aureus

The population structure of Staphylococcus aureus carried by healthy humans was determined using a large strain collection of nonclinical origin (n = 829). High-throughput amplified fragment length polymorphism (AFLP) analysis revealed 3 major and 2 minor genetic clusters of S. aureus, which were corroborated by multilocus sequence typing. Major AFLP cluster I comprised 44.4% of the carriage isolates and showed additional heterogeneity whereas major AFLP groups II and III presented 2 homogeneous clusters, including 47.3% of all carriage isolates. Coanalysis of invasive S. aureus strains and epidemic methicillin-resistant S. aureus (MRSA) revealed that all major clusters contained invasive and multiresistant isolates. However, clusters and subclusters with overrepresentation of invasive isolates were also identified. Bacteremia in elderly adults, for instance, was caused by a IVa cluster-derived strain significantly more often than by strains from other AFLP clusters. Furthermore, expansion of multiresistant clones or clones associated with skin disease (impetigo) was detected, which suggests that epidemic potential is present in pathogenic strains of S. aureus. In addition, the virulence gene encoding Panton-Valentine leukocidin was significantly enriched in S. aureus strains causing abscesses and arthritis in comparison with the carriage group. We provide evidence that essentially any S. aureus genotype carried by humans can transform into a life-threatening human pathogen but that certain clones are more virulent than others.

Evaluation of Molecular Typing Methods in Characterizing a European Collection of Epidemic Methicillin-Resistant Staphylococcus aureus Strains: the HARMONY Collection

ABSTRACT We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCCmec typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.

Mycetoma caused by Madurella mycetomatis: a neglected infectious burden

Tropical eumycetoma is frequently caused by the fungus Madurella mycetomatis. The disease is characterised by extensive subcutaneous masses, usually with sinuses draining pus, blood, and fungal grains. The disease affects individuals of all ages, although disability is most severe in adults who work outdoors. Compared with major diseases such as tuberculosis, malaria, and HIV, disease from M mycetomatis is underestimated but socioeconomically important. Many scientific case reports on mycetoma exist, but fundamental research was lacking until recently. We present a review on developments in the clinical, epidemiological, and diagnostic management of M mycetomatis eumycetoma. We describe newly developed molecular diagnostic and gene typing procedures, and their application for management of patients and environmental research. Fungal susceptibility tests have been developed as well as a mouse model of infection. These advances should greatly further our understanding of the molecular basis of eumycetoma.

Multilocus Sequence Typing of Staphylococcus aureus with DNA Array Technology

ABSTRACT A newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically concordant.

Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates

ABSTRACT Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.

RespiFinder: a New Multiparameter Test To Differentially Identify Fifteen Respiratory Viruses

ABSTRACT Broad-spectrum analysis for pathogens in patients with respiratory tract infections is becoming more relevant as the number of potential infectious agents is still increasing. Here we describe the new multiparameter RespiFinder assay, which is based on the multiplex ligation-dependent probe amplification (MLPA) technology. This assay detects 15 respiratory viruses in one reaction. The MLPA reaction is preceded by a preamplification step which ensures the detection of both RNA and DNA viruses with the same specificity and sensitivity as individual monoplex real-time reverse transcription-PCRs. The RespiFinder assay was validated with 144 clinical samples, and the results of the assay were compared to those of cell culture and a respiratory syncytial virus (RSV)-specific immunochromatography assay (ICA). Compared to the cell culture results, the RespiFinder assay showed specificities and sensitivities of 98.2% and 100%, respectively, for adenovirus; 96.4% and 100%, respectively, for human metapneumovirus; 98.2% and 100%, respectively, for influenza A virus (InfA); 99.1% and 100%, respectively, for parainfluenza virus type 1 (PIV-1); 99.1% and 80%, respectively, for PIV-3; 90.1% and 100%, respectively, for rhinovirus; and 94.6% and 100%, respectively, for RSV. Compared to the results of the RSV-specific ICA, the RespiFinder assay gave a specificity and a sensitivity of 82.4% and 80%, respectively. PIV-2, PIV-4, influenza B virus, InfA H5N1, and coronavirus 229E were not detected in the clinical specimens tested. The use of the RespiFinder assay resulted in an increase in the diagnostic yield compared to that obtained by cell culture (diagnostic yields, 60% and 35.5%, respectively). In conclusion, the RespiFinder assay provides a user-friendly and high-throughput tool for the simultaneous detection of 15 respiratory viruses with excellent overall performance statistics.

Host- and Tissue-Specific Pathogenic Traits of Staphylococcus aureus

Comparative genomics were used to assess genetic differences between Staphylococcus aureus strains derived from infected animals versus colonized or infected humans. A total of 77 veterinary isolates were genetically characterized by high-throughput amplified fragment length polymorphism (AFLP). Bacterial genotypes were introduced in a large AFLP database containing similar information for 1,056 human S. aureus strains. All S. aureus strains isolated from animals in close contact with humans (e.g., pet animals) were predominantly classified in one of the five main clusters of the AFLP database (cluster I). In essence, mastitis-associated strains from animals were categorized separately (cluster IVa) and cosegregated with bacteremia-associated strains from humans. Distribution of only 2 out of 10 different virulence genes differed across the clusters. The gene encoding the toxic shock syndrome protein (tst) was more often encountered among veterinary strains (P < 0.0001) and even more in the mastitis-related strains (P<0.0001) compared to human isolate results. The gene encoding the collagen binding protein (cna) was rarely detected among invasive human strains. The virulence potential, as indicated by the number of virulence genes per strain, did not differ significantly between the human- and animal-related strains. Our data show that invasive infections in pets and humans are usually due to S. aureus strains with the same genetic background. Mastitis-associated S. aureus isolated in diverse farm animal species form a distinct genetic cluster, characterized by an overrepresentation of the toxic shock syndrome toxin superantigen-encoding gene.

Optical Fingerprinting in Bacterial Epidemiology: Raman Spectroscopy as a Real-Time Typing Method

Hospital-acquired infections (HAI) increase morbidity and mortality and constitute a high financial burden on health care systems. An effective weapon against HAI is early detection of potential outbreaks and sources of contamination. Such monitoring requires microbial typing with sufficient reproducibility and discriminatory power. Here, a microbial-typing method is presented, based on Raman spectroscopy. This technique provides strain-specific optical fingerprints in a few minutes instead of several hours to days, as is the case with genotyping methods. Although the method is generally applicable, we used 118 Staphylococcus aureus isolates to illustrate that the discriminatory power matches that of established genotyping techniques (numerical index of diversity [D] = 0.989) and that concordance with the gold standard (pulsed-field gel electrophoresis) is high (95%). The Raman clustering of isolates was reproducible to the strain level for five independent cultures, despite the various culture times from 18 h to 24 h. Furthermore, this technique was able to classify stored (−80°C) and recent isolates of a methicillin-resistant Staphylococcus aureus-colonized individual during surveillance studies and did so days earlier than established genotyping techniques did. Its high throughput and ease of use make it suitable for use in routine diagnostic laboratory settings. This will set the stage for continuous, automated, real-time epidemiological monitoring of bacterial infections in a hospital, which can then be followed by timely corrective action by infection prevention teams.