Multiplex PCR-ligation detection reaction assay for simultaneous detection of drug resistance and toxin genes from Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium.

A multiplex PCR-ligation detection reaction (PCR-LDR) assay was developed for rapid detection of methicillin, tetracycline, and vancomycin resistance, as well as toxic shock toxin and Panton-Valentine leukocidin. The assay was tested on 470 positive blood culture bottles containing Staphylococcus aureus or enterococci. PCR-LDR exhibited a sensitivity and specificity of > or = 98% for all components except tetracycline resistance, which had a sensitivity of 94.7%. Rapid and sensitive detection of antimicrobial resistance and virulence genes could help guide therapy and appropriate infection control measures.

Development of multiplex PCR-ligase detection reaction assay for detection of West Nile virus.

We have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.

Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay.

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

Osteomyelitis caused by Mycobacterium haemophilum: successful therapy in two patients with AIDS.

OBJECTIVE: Difficulties involved in diagnosis and response to antimicrobial therapy are described in detail for two cases of biopsy-proven osteomyelitis caused by Mycobacterium haemophilum in AIDS patients. SETTING: Two large, private teaching hospitals in New York City, New York, USA. PATIENTS, PARTICIPANTS: A 31-year-old woman with previous diagnoses of candida esophagitis and peripheral neuropathy (patient 1), and a 37-year-old man with Kaposi's sarcoma (patient 2). INTERVENTIONS: One patient was treated with a combination of rifampin, ethambutol, clofazimine, and ciprofloxacin, while the other received rifampin, ciprofloxacin and doxycycline. Both patients also received a short course of intravenous amikacin. MAIN OUTCOME MEASURES: Disease activity was monitored clinically by observing resolution of skin ulcers, lymphadenopathy, and pain and swelling in areas affected by osteomyelitis. RESULTS: Both patients experienced complete resolution of signs and symptoms of M. haemophilum infection. Patient 1 was treated for 17 months and remains well after 10 months without therapy. Patient 2 shows no evidence of infection after 14 months of therapy. CONCLUSIONS: M. haemophilum infection must be considered in the differential diagnosis of osteomyelitis in AIDS patients, although specialized culture techniques are required to isolate and identify this pathogen. Excellent clinical response can be achieved with oral antimicrobial therapy.

Comparison of two commercial systems (Vitek and MicroScan-WalkAway) for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Antimicrobial susceptibility testing of cystic fibrosis (CF) isolates of Pseudomonas aeruginosa is difficult because the organisms are often mucoid and slow-growing. This study of 498 CF strains examined the correlation of results derived from two commonly used commercial systems (Vitek, MicroScan-WalkAway) with a reference method for 10 antimicrobials. Correlation to reference results was unacceptably low for all agents and both commercial systems had a high rate of very major (false-susceptible) errors. Although mucoid strains produced a 4.8% greater intermethod error, it was not markedly different than non-mucoid strains for the Vitek System. Overall, these tested commercial systems performed poorly for CF isolates in contrast to earlier reported, high correlations with the reference methods (broth microdilution frozen panels and agar dilution) of the National Committee for Clinical Laboratory Standards, the standardized disk diffusion test, and the Etest (AB BIODISK, Solna, Sweden).

Comparative study of the new colorimetric VITEK 2 yeast identification card versus the older fluorometric card and of CHROMagar Candida as a source medium with the new card.

The new VITEK 2 colorimetric card was compared to the previous fluorometric card for identification of yeast. API 20C was considered the "gold standard." The new card consistently performed better than the older card. Isolates from CHROMagar Candida plates were identified equally as well as those from Sabouraud dextrose agar.

Comparison of the Oxi/Ferm and N/F systems for identification of infrequently encountered nonfermentative and oxidase-positive fermentative bacilli.

The N/F system (Flow Laboratories, Inc. Rockville, Md) and the Oxi/Ferm Tube, (Roche Diagnostics, Div. Hoffman-La Roche, Inc. Nutley, N.J.) were evaluated in parallel for identification of infrequently encountered nonfermentative gram-negative bacilli and oxidase-positive, fermentative gram-negative bacilli selected from fresh clinical isolates and stock cultures. When compared with conventional media, the Flow N/F system correctly identified 97.7% (86 or 88) of all strains tested. No organisms were misidentified, but this system failed to identify two strains. Six supplemental tests were needed for the complete identification of 16 strains (18%); however, correct results were obtained within 48 h for 85% of the isolates. The Oxi/Ferm method correctly identified 87.5% of the isolates; 7% were incorrectly identified, and the method failed to identify five strains. Seventeen supplemental tests were required to identify 64 strains (73%). Complete identification within 48 h was obtained for 60% of the organisms tested.

Comparison of agar diffusion methodologies for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of patients with cystic fibrosis (CF). Improved antimicrobial chemotherapy has significantly increased the life expectancy of these patients. However, accurate susceptibility testing of P. aeruginosa isolates from CF sputum may be difficult because the organisms are often mucoid and slow growing. This study of 597 CF isolates of P. aeruginosa examined the correlation of disk diffusion and Etest (AB BIODISK, Solna, Sweden) results with a reference broth microdilution method. The rates of interpretive errors for 12 commonly used antipseudomonal antimicrobials were determined. The disk diffusion method correlated well (zone diameter versus MIC) for all of the agents tested. However, for mucoid isolates, correlation coefficients (r values) for piperacillin, piperacillin-tazobactam, and meropenem were <0.80. The Etest correlation with reference broth microdilution results (MIC versus MIC) was acceptable for all of the agents tested, for both mucoid and nonmucoid isolates. Category interpretation errors were similar for the disk diffusion and Etest methods with 0.4 and 0.1%, respectively, very major errors (false susceptibility) and 1.1 and 2.2% major errors (false resistance). Overall, both agar diffusion methods appear to be broadly acceptable for routine clinical use in susceptibility testing of CF isolates of P. aeruginosa.