Use of a high-resolution melt assay to characterize codon 54 of the cyp51A gene of Aspergillus fumigatus on a Rotor-Gene 6000 instrument.

A high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument was developed to characterize the codon for glycine 54 in the cyp51A genes from 13 reference isolates and 12 clinical isolates of Aspergillus fumigatus. Mutations in this codon confer reduced susceptibility to itraconazole and posaconazole. The assay is simple to perform, and a result of "wild type" or "mutant" is available after approximately 1 h following DNA extraction using commercially available reagents and conventional primers.

Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide.

AIMS: The DNA-intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells by selectively penetrating through the damaged membrane of dead cells and blocking the DNA amplification during the polymerase chain reaction (PCR). We optimized and tested the assay in vitro using Staphylococcus aureus and Staphylococcus epidermidis cultures to distinguish viable from dead bacteria, with the goal of reducing false positive PCR results. METHODS AND RESULTS: Viable and heat-inactivated bacteria were treated with EMA or left untreated before DNA extraction. A real-time PCR assay for the detection of the tuf gene in each DNA extract was used. Our results indicated that EMA influenced viable bacteria as well as dead bacteria, and the effect of EMA depended on the EMA concentration and bacterial number. CONCLUSIONS: EMA is not a suitable indicator of bacterial viability, at least with respect to Staphylococcus species. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the viability of pathogens has a major impact on interpreting the results of molecular tests for bacteria and subsequent clinical management of patients. To this end, several methods are being evaluated. One of these methods--intercalating DNA of dead bacteria by EMA--looked very promising, but our study found it unsatisfactory for S. aureus and coagulase-negative Staphylococci.

Rapid detection of Panton-Valentine leukocidin-positive Staphylococcus aureus by real-time PCR targeting the lukS-PV gene.

In order to assess the speed and accuracy of a real-time PCR assay targeting the lukS-PV gene of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus, 700 S. aureus strains were tested and the results were compared to those achieved with block cycler PCR. Cross-reactivity was tested with 166 other bacterial species. Using this homogeneous real-time PCR assay format, the presence or absence of genetic information for PVL, which is also found in community-associated methicillin-resistant S. aureus, was correctly identified from pure culture and directly in various types of clinical specimens.

Multicenter evaluation of the BDProbeTec ET System for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male urethral swabs.

The performance of the Becton Dickinson BDProbe Tec ET System Chlamydia trachomatis and Neisseria gonorrhoeae Amplified DNA Assays (BD Biosciences, Sparks, Md.) was evaluated in a multicenter study. Specimens were collected from 2,109 men and women, with or without symptoms, attending sexually transmitted disease, family planning, and obstetrics and gynecology clinics. Both swab and urine samples were collected, and the results obtained from 4,131 specimens were compared to those from culture and the LCx nucleic acid amplification test (Abbott Industries, Abbott Park, Ill.). PCR and cytospin of the culture transport medium with chlamydia direct fluorescent antibody staining were used to adjudicate chlamydia culture-negative results. Sensitivity and specificity were calculated both with and without use of the amplification control (AC), with little apparent difference in the results. Without the AC result, sensitivity for C. trachomatis and N. gonorrhoeae were 92.8 and 96.6%, respectively, for cervical swabs and 80.5 and 84.9% for urine from women. C. trachomatis and N. gonorrhoeae sensitivities were 92.5 and 98.5%, respectively, for male urethral swabs and 93.1 and 97.9% for urine from men. This amplified DNA system for simultaneous detection of chlamydial and gonococcal infections demonstrated superior sensitivity compared to chlamydia culture and has performance characteristics comparable to those of other commercially available nucleic acid-based assays for these organisms.

Antimicrobial susceptibility of Abiotrophia adiacens and Abiotrophia defectiva.

The susceptibilities of 27 Abiotrophia adiacens (proposed reclassification Granulicatella adiacens comb.nov., Collins & Lawson, 2000) and 12 Abiotrophia defectiva isolates were tested by microdilution in pyridoxal hydrochloride and lysed horse blood supplemented Mueller-Hinton broth. According to NCCLS interpretative criteria for Streptococcus spp. not Streptococcus pneumoniae, the susceptibilities of A. adiacens and A. defectiva were, respectively: penicillin, 55% and 8%; amoxicillin, 81% and 92%; ceftriaxone, 63% and 83%; meropenem, 96% and 100%; and 100% for both species with clindamycin, rifampin, levofloxacin, ofloxacin, quinupristin/dalfopristin, and vancomycin.