Direct identification of major Gram-negative pathogens in respiratory specimens by respiFISH® HAP Gram (-) Panel, a beacon-based FISH methodology.

Rapid detection of microorganisms in respiratory specimens is of paramount importance to drive the proper antibiotic regimen to prevent complications and transmission of infections. In the present study, the respiFISH® HAP Gram (-) Panel (miacom diagnostics GmbH, Duesseldorf, Germany) for the etiological diagnosis of hospital-acquired pneumonia was compared with the traditional culture method for the detection of major Gram-negative pathogens in respiratory specimens. respiFISH® combined the classical fluorescence in situ hybridization (FISH) technology with fluorescence-labeled DNA molecular beacons as probes. From September 2011 to January 2012, 165 samples were analyzed: the sensitivity and specificity were 94.39 and 87.93%, respectively. Only six pathogens (3.6%) were not identified with respiFISH®, while seven specimens (3%) provided false-positive results. This beacon-based identification shortens the time to result by at least one work day, providing species-level identification within half an hour. Considering the high sensitivity and specificity and the significant time saving, the introduction of bbFISH® assays could effectively complement traditional systems in microbiology laboratories.

Interference of confounding factors on the use of (1,3)-beta-D-glucan in the diagnosis of invasive candidiasis in the intensive care unit.

Invasive fungal infections (IFIs) are an increasing problem in intensive care units (ICUs), and conventional diagnostic methods are not always reliable or timely enough to deliver appropriate antimicrobial therapy. The dosage of fungal antigens in serum is a promising diagnostic technique, but several confounding factors, such as treatment with immunoglobulins (Ig), albumin, or antifungals, could interfere with the correct interpretation of the (1,3)-beta-D-glucan (BG) assay. This study assessed the reliability of the BG assay and the influence of timing and dosage of major confounding factors on circulating levels of IFI biomarkers. 267 ICU patients who underwent a BG assay were retrospectively studied. The timing and dosage of albumin, use of azole treatment, and infusions of intravenous IgG, red blood cells, concentrated platelets, and frozen plasma were analyzed to find possible correlations with the BG results. The sensitivity and specificity of the BG assay were calculated. The BG test in serum showed high sensitivity (82.9 %) but low specificity (56.7 %). The optimal cut-off for the test was 95.9 pg/mL. The mean BG level in proven invasive candidiasis was around 400 pg/mL. The only factor that was found to significantly confound (p < 0.05) the diagnostic performance of the BG assay was the administration of more than 30 g of albumin within 2 days prior to BG testing. The BG assay remains a useful diagnostic test in ICU patients and the levels of BG are useful in evaluating the positive predictive value of this biomarker. The only confounding factor in our study was the use of albumin.

A case of Beauveria bassiana keratitis confirmed by internal transcribed spacer and LSU rDNA D1-D2 sequencing.

We describe a case of fungal keratitis due to Beauveria bassiana in a farmer with Fuchs' dystrophy, treated with amphotericin B. Surgery with penetrating keratoplasty was necessary to resolve the lesions. Susceptibility testing and molecular sequencing permitted the identification and treatment of this rare aetiological agent of invasive fungal disease.

Prevalence of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae strains isolated in North-East Italy.

We investigated the prevalence of plasmid-mediated quinolone resistance genes in 756 clinical isolates of Enterobacteriaceae originating from Microbiology Diagnostic Laboratories of North-East Italy. Five point zero two percent of isolates carried a qnr determinant while the aac(6')-Ib-cr determinant was detected in 9·25% of isolates. We also investigated the association between the plasmid-mediated quinolone resistance and the beta-lactamase genes, and characterized the plasmids carrying these determinants of resistance.

Description and plasmid characterization of qnrD determinants in Proteus mirabilis and Morganella morganii.

We investigated the presence of qnrC and qnrD among 756 non-replicate Enterobacteriaceae isolated in Italy, selected for being non-susceptible to fluoroquinolones and/or resistant to third-generation cephalosporins. Four Proteus mirabilis and one Morganella morganii (0.66% of the total) presented a qnrD gene, located in a 2687-base-pair plasmid that was entirely sequenced. The plasmid is un-typable, and contains no known coding region other than qnrD. That the qnrD gene was found in four unrelated P. mirabilis and in one M. morganii isolate might suggest a frequent association of this gene with the tribe Proteeae.

A novel VIM-type metallo-beta-lactamase (VIM-14) in a Pseudomonas aeruginosa clinical isolate from a neonatal intensive care unit.

A Pseudomonas aeruginosa highly resistant to carbapenems was isolated in a neonatal intensive care unit in Palermo, Italy. The strain was found to carry a novel VIM-type enzyme, classified as VIM-14. The novel enzyme differs from VIM-4 in a G31S mutation. VIM-14 was harboured in a class 1 integron with a new organization. The integron carried the genes aac7, blaVIM-14, blaOXA-20 and aac4 in that order.

Susceptibilities of Streptococcus pyogenes and Streptococcus pneumoniae to macrolides and telithromycin: data from an Italian multicenter study.

687 isolates of Streptococcus pyogenes and 600 isolates of Streptococcus pneumoniae , isolated over the period 2002-2003 from specimens of different human origin obtained in 16 different Italian centres, were assayed for their susceptibilities to different macrolides and to telithromycin, and were investigated by PCR to detect their different erythromycin resistance genes. 25.5% of the S. pyogenes isolates proved resistant to erythromycin, as well as to clarithromycin and azithromycin. 6.6% of the isolates proved non-susceptible to clindamycin. 4.9% of the isolates were non-susceptible to telithromycin. 22.3% of all erythromycin-resistant isolates exhibited cMLS B resistance, 50.3% iMLS B resistance, and 27.4% Mtype resistance. All cMLS B strains had the erm(B) gene, all M strains had the mef (A) gene, and no resistance genes were found in the erythromycin-susceptible strains. Roughly one quarter of the iMLS(B) strains had erm(A) and roughly three quarters erm(B). 35.2% of the S. pneumoniae isolates proved resistant to erythromycin, and virtually all of them also proved resistant to clarithromycin and azithromycin, too. Only 6.0% of the pneumococcal isolates were resistant to penicillin and a further 11.0% were intermediate. Only 0.2% of the isolates were nonsusceptible to telithromycin. 65.9% of all erythromycin-resistant S. pneumoniae isolates had cMLS B resistance, 18.0% had iMLS B resistance, and 16.1% had M-type resistance. All the MLS B-resistant isolates had an erm(B) gene, and all the M-type isolates had a mef gene. We conclude that macrolide resistance of streptococci still persists in Italy with incidences as high as 40%, more often than not being characterised by the MLS B phenotype. The ketolide telithromycin, structurally related to macrolides and most likely to substitute for them in a number of clinical uses, is confirmed as being extremely active even against recent clinical streptococcal isolates.

Detection of a new SHV-type extended-spectrum beta-lactamase, SHV-31, in a Klebsiella pneumoniae strain causing a large nosocomial outbreak in The Netherlands.

A Klebsiella pneumoniae strain resistant to third-generation cephalosporins was isolated in the eastern Netherlands. The strain was found to carry a novel extended-spectrum beta-lactamase, namely, SHV-31. The combination of the two mutations by which SHV-31 differs from SHV-1, namely, L35Q and E240K, had previously only been described in association with one or more additional mutations.

A strain of Salmonella enterica serovar Virchow isolated in Turkey and carrying a CTX-M-3 extended-spectrum beta-lactamase.

A multiply resistant strain of Salmonella enterica subsp. enterica serovar Virchow was isolated in November 2002 from a catheterized patient admitted to the SSK Training Hospital in Ankara, Turkey. This isolate showed an antimicrobial susceptibility pattern compatible with the presence of a CTX-M-type ESBL, namely resistance to cefotaxime, aztreonam and cefepime, and intermediate susceptibility to ceftazidime. On checking for the presence of the bla(TEM), bla(SHV), and bla(CTX-M )resistance genes by PCR, negative results were obtained with the primers specific for SHV and TEM genes, while positive results were obtained with those specific for CTX-M-type genes. After sequencing, the beta-lactamase was identified as CTX-M-3. This is the first report of this enzyme in Salmonella Virchow and represents a further disquieting threat to the therapy of infections caused by Salmonella isolates.