Molecular typing and epidemiological investigation of clinical populations of Pseudomonas aeruginosa using an oligonucleotide-microarray.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen which has the potential to become extremely harmful in the nosocomial environment, especially for cystic fibrosis (CF) patients, who are easily affected by chronic lung infections. For epidemiological purposes, discriminating P.aeruginosa isolates is a critical step, to define distribution of clones among hospital departments, to predict occurring microevolution events and to correlate clones to their source. A collection of 182 P. aeruginosa clinical strains isolated within Italian hospitals from patients with chronic infections, i.e. cystic fibrosis (CF) patients, and with acute infections were genotyped. Molecular typing was performed with the ArrayTube (AT) multimarker microarray (Alere Technologies GmbH, Jena, Germany), a cost-effective, time-saving and standardized method, which addresses genes from both the core and accessory P.aeruginosa genome. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were employed as reference genotyping techniques to estimate the ArrayTube resolution power. RESULTS: 41 AT-genotypes were identified within our collection, among which 14 were novel and 27 had been previously described in publicly available AT-databases. Almost 30% of the genotypes belonged to a main cluster of clones. 4B9A, EC2A, 3C2A were mostly associated to CF-patients whereas F469, 2C1A, 6C22 to non CF. An investigation on co-infections events revealed that almost 40% of CF patients were colonized by more than one genotype, whereas less than 4% were observed in non CF patients. The presence of the exoU gene correlated with non-CF patients within the intensive care unit (ICU) whereas the pKLC102-like island appeared to be prevalent in the CF centre. The congruence between the ArrayTube typing and PFGE or MLST was 0.077 and 0.559 (Adjusted Rand coefficient), respectively.AT typing of this Italian collection could be easily integrated with the global P. aeruginosa AT-typed population, uncovering that most AT-genotypes identified (> 80%) belonged to two large clonal clusters, and included 12 among the most abundant clones of the global population. CONCLUSIONS: The ArrayTube (AT) multimarker array represented a robust and portable alternative to reference techniques for performing P. aeruginosa molecular typing, and allowed us to draw conclusions especially suitable for epidemiologists on an Italian clinical collection from chronic and acute infections.

Comparative evaluation of an automated repetitive-sequence-based PCR instrument versus pulsed-field gel electrophoresis in the setting of a Serratia marcescens nosocomial infection outbreak.

A semiautomated, repetitive-sequence-based PCR (rep-PCR) instrument (DiversiLab system) was evaluated in comparison with pulsed-field gel electrophoresis (PFGE) to investigate an outbreak of Serratia marcescens infections in a neonatal intensive care unit (NICU). A selection of 36 epidemiologically related and 8 epidemiologically unrelated isolates was analyzed. Among the epidemiologically related isolates, PFGE identified five genetically unrelated patterns. Thirty-two isolates from patients and wet nurses showed the same PFGE profile (pattern A). Genetically unrelated PFGE patterns were found in one patient (pattern B), in two wet nurses (patterns C and D), and in an environmental isolate from the NICU (pattern G). Rep-PCR identified seven different patterns, three of which included the 32 isolates of PFGE type A. One or two band differences in isolates of these three types allowed isolates to be categorized as similar and included in a unique cluster. Isolates of different PFGE types were also of unrelated rep-PCR types. All of the epidemiologically unrelated isolates were of different PFGE and rep-PCR types. The level of discrimination exhibited by rep-PCR with the DiversiLab system allowed us to conclude that this method was able to identify genetic similarity in a spatio-temporal cluster of S. marcescens isolates.

A two-year prospective study of clinical criteria and polymerase chain reaction assay of cerebrospinal fluid for the diagnosis of viral infections of the central nervous system.

Cerebrospinal fluid specimens from 226 patients with suspected viral infections of the central nervous system (CNS) were tested by polymerase chain reaction (PCR) to identify the most frequent viruses involved in these infections. A positive PCR result was obtained in 18 patients (7 cases positive for herpes simplex viruses, 5 for enterovirus, 3 for JC virus, 2 for varicella-zoster virus, and 1 for Epstein-Barr virus). All patients with positive PCR results had a definite diagnosis of CNS viral infection. However, a negative result did not rule out the possibility of viral infection of the CNS.