J. Lonca

Evaluation of a Commercial Probe Assay for Detection of Rifampin Resistance in Mycobacterium tuberculosis Directly from Respiratory and Nonrespiratory Clinical Samples

A commercial assay (Inno-Line Probe Assay; Innogenetics, Belgium) was evaluated to determine its ability to detect rifampin resistance inMycobacterium tuberculosis directly from clinical specimens. Fifty-nine selected specimens (42 respiratory and 17 nonrespiratory) culture positive forMycobacterium tuberculosis were tested along with their corresponding isolates in culture. The results were compared with those obtained by in vitro susceptibility testing. The results of the line probe assay to detect rifampin resistance inMycobacterium tuberculosis present in clinical specimens and in cultured isolates were concordant for 58 of 59 (98.3%) isolates (95% confidence limits = 90.9–99.9%). The line probe assay failed only once, when a fecal specimen was tested; no amplification was observed due to the presence of inhibitory compounds. The most frequently observed mutation was His526→Asp (58.7%), followed by the His526→Tyr (23.9%); together, they represented 82.6% of rifampin-resistant samples. In conclusion, the Inno-Line Probe Assay is a rapid, useful method for detecting the presence ofMycobacterium tuberculosis complex and its resistance to rifampin directly from clinical specimens and culture. Moreover, since rifampin resistance is a potential marker for multidrug resistance inMycobacterium tuberculosis, this assay may constitute an important tool for the control of tuberculosis.

Evaluation of a Fluorescence Hybridisation Assay Using Peptide Nucleic Acid Probes for Identification and Differentiation of Tuberculous and Non-Tuberculous Mycobacteria in Liquid Cultures

Abstract The performance was evaluated of a fluorescence in situ hybridisation assay using peptide nucleic acid probes (Dako Probe MTB Culture Confirmation Test; Dako, Denmark) for identification of Mycobacterium tuberculosis complex (MTC) organisms and differentiation between tuberculous and non-tuberculous mycobacteria (NTM) in material taken directly from Bactec 12B (Becton Dickinson, USA) and MB/BacT (Organon Teknika, USA) bottles. The test was applied to 129 smear-positive (Ziehl-Neelsen stain) clinical specimens, 48 previously identified clinical strains of mycobacteria (12 MTC and 36 NTM), and 51 reference strains (7 MTC and 44 NTM) which were all previously inoculated into Bactec 12B and MB/BacT bottles. The sensitivity and specificity of the assay for MTC-positive cultures was 87.6% and 100%, respectively, for Bactec 12B, and 100%, respectively, for MB/BacT. The sensitivity and specificity of the assay for NTM-positive cultures was 100% for both media.

Comparative evaluation of two commercial assays for direct detection of Mycobacterium tuberculosis in respiratory specimens.

Two commercial systems for the amplification and detection ofMycobacterium tuberculosis directly from respiratory samples were compared. The Roche Cobas Amplicor MTB Test and the Roche manual Amplicor MTB Test (Roche Diagnostic Systems, USA) were applied to 755 decontaminated respiratory specimens collected from 470 patients. Results were compared with those of acid-fast staining and culture. A total of 251 specimens were collected from 156 patients diagnosed with pulmonary tuberculosis, including 28 specimens corresponding to 13 patients that were receiving antituberculous treatment. Given the overall positivity rate of 33.2% (251/755), the sensitivity, specificity, and positive and negative predictive values were 92.4, 100, 100, and 96.5%, respectively, for the Cobas Amplicor MTB Test and 90.8, 100, 100, and 95.8%, respectively, for the Amplicor MTB Test. For 204 (81.3%) smear positive specimens and 47 (19.7%) smear negative specimens, the sensitivity values were 100 and 59.6%, respectively, for the Cobas Amplicor MTB Test and 100 and 51%, respectively, for the Amplicor MTB Test. There were no statistically significant differences in sensitivity or specificity between the two assays and culture (p>0.05). The overall results of both assays were concordant for 99.5% of the samples. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection ofMycobacterium tuberculosis complex in respiratory specimens, the Cobas Amplicor MTB Test appears to be slightly more sensitive than the Amplicor MTB Test when smear negative specimens are investigated.

Comparison of a Nonradiometric System with Bactec 12B and Culture on Egg-Based Media for Recovery of Mycobacteria from Clinical Specimens

Abstract The MB/BacT (Organon-Teknika, USA) is a fully automated, rapid, nonradiometric system for the culture of mycobacteria from clinical samples. The rate of recovery of mycobacteria and the time to detection obtained with the MB/BacT were compared with those obtained with Löwenstein-Jensen and Coletsos solid media and Bactec 7H12 (12B) (Becton-Dickinson, USA) broth when 600 processed specimens were inoculated into all media in parallel. Specimens included 383 respiratory samples, 20 urine samples, 23 purulent exudates, 13 stool samples, 103 blood samples, 14 bone marrow aspirates, and 44 body fluid samples or aspirates. Overall, 106 mycobacterial isolates comprising six species were recovered, of which 100 (94.3%) were detected with MB/BacT, 98 (92.5%) with egg-based media, and 96 (90.2%) with Bactec 12B. The recovery rates of Mycobacteriumtuberculosis complex with MB/BacT, egg-based media, and Bactec 12B were 98.7%, 93.7, and 89.9%, respectively. The average number of days to detection of single mycobacterial isolates was 14.2 days for MB/BacT, 26.1 days for egg-based media, and 11.7 days for Bactec 12B. The contamination rates were higher in MB/BacT (5%) than in Bactec 12B (1.8%) or egg-based media (1.5%). MB/BacT is a reliable, nonradiometric, less labor-intensive alternative to Bactec 12B for recovery of mycobacteria, but, as with other liquid culture methods, MB/BacT should be used in combination with a solid medium, not on its own.

Catalase-peroxidase activity has no influence on virulence in a murine model of tuberculosis.

The capacity to generate a chronic and persistent infection in the experimental murine model of tuberculosis induced aerogenically by a low-dose inoculum was determined in eight isoniazid-resistant clinical strains of Mycobacterium tuberculosis showing different catalase-peroxidase (C-P) activities. Determination of bacillary concentration in lung and spleen and the percentage of pulmonary parenchyma occupied by granulomas were monitored. Data showed no relation between the lack of C-P activity and the ability to develop a persistent infection, highlighting the potential of C-P negative strains to spread through the community.

Comparison of the sodium dodecyl sulfate-sodium hydroxide specimen processing method with the C18-carboxypropylbetaine specimen processing method using the MB/BacT liquid culture system.

The ability of physicians to diagnose tuberculosis is impacted by the use of smear and culture techniques combined with specimen processing methods. The objective of this study was to evaluate the effects of specimen processing on smear and culture sensitivity by comparing the specimen processing method that uses C18-carboxypropylbetaine with the method that combines sodium dodecyl sulfate and sodium hydroxide. A total of 1,201 specimens were entered into this study. Specimens were split approximately equally such that one-half of each specimen was processed with sodium dodecyl sulfate-sodium hydroxide, while the other half was processed with C18-carboxypropylbetaine. All sediments were subjected to acid-fast staining and then analyzed using the MB/BacT liquid culture system (bioMérieux, France) and solid media. The sensitivity of smear following processing with sodium dodecyl sulfate-sodium hydroxide and C18-carboxypropylbetaine was 61.2% and 58.6% (P>0.05), respectively, while the specificities were identical (99.7%). The sensitivity of culture was 84.2% and 96.1% (P<0.05), respectively. The time to detection in the MB/BacT liquid culture system was 13.2±5.6 and 15.0±8.8 days (P>0.05), respectively, and 20.0±7.6 and 15.7±8.9 days (P<0.05), respectively, on solid media. The contamination rates in the MB/BacT system were 0.8% and 8.7%, respectively, whereas the contamination rates on solid media were 2.6% and 4.3%, respectively. C18-carboxypropylbetaine specimen processing was less labor-intensive than sodium dodecyl sulfate-sodium hydroxide processing and improved the ability of laboratory staff to detect the presence of mycobacteria by culture.

Serovars of Mycobacterium avium complex isolated from AIDS and non‐AIDS patients in Spain

Antigen fingerprinting based on surface glycolipid antigens was applied to the epidemiology of clinical isolates of the Mycobacterium avium complex from 128 acquired immunodeficiency syndrome (AIDS) and 31 non‐AIDS patients from several different regions of Spain. The application of thin‐layer chromatography, gas chromatography–mass spectrometry and monoclonal antibodies, combined with ELISA, allowed a facile identification, differentiation and classification of the isolates. The cumulative results demonstrate that, among the clinical isolates, serovar 4 was predominant in both AIDS (33·6%) and non‐AIDS (22·6%) isolates. In general, the results demonstrate geographical as well as disease‐related differences in the distribution of Myco. avium complex serovars of clinical importance.

Rapid Detection of Several Mycobacterial Species Using a Polymerase Chain Reaction Reverse Hybridisation Assay

Abstract. The ability of a polymerase-chain-reaction-based technique combined with a reverse hybridisation line-probe assay to detect and identify eight of the most clinically significant mycobacterial species directly from cultures in liquid medium was evaluated. The line-probe assay allows simultaneous identification of eight different mycobacterial species. Ninety-seven mycobacterial strains belonging to 22 different species were evaluated. All strains were previously inoculated into MB/BacT bottles (Organon Teknika, USA). The sensitivity and specificity of the test when applied on positive MB/BacT liquid cultures containing isolates from mycobacterial species included as specific probes on the line-probe assay strip was 100% and 100%, respectively. These results further support the potential clinical usefulness of this technique in the diagnosis of mycobacterial infections.

Analysis of the contaminant spectrum in the MB/BacT liquid culture system following C18-Carboxypropylbetaine specimen processing

A detailed study comparing specimen processing methods for the isolation of mycobacteria was recently reported [1]. In the follow-up study presented here, 1,201 specimens (95% respiratory) were collected and split approximately equally such that one-half of each specimen was processed with 3% sodium dodecyl sulfate-1% sodium hydroxide (SDS-NaOH) [2] and the other half was processed with C18-carboxypropylbetaine (CB-18) followed by lytic enzyme decontamination [3, 4]. All processed specimens were analyzed by culture using the MB/BacT liquid culture system containing the antibiotic supplement MAS (bioM rieux, France). The SDS-NaOHprocessed specimens were also inoculated onto L wenstein-Jensen slants, while the CB-18-processed specimens were also inoculated onto 7H11-selective plates (50 g/ml carbenicillin, 200 U/ml polymyxin B, 10 g/ml amphotericin B, and 20 g/ml trimethoprim lactate). CB-18/lytic enzyme processing increased overall culture sensitivity by approximately 14.1% (P<0.05); however, the contamination rates in the MB/BacT system following SDS-NaOH and CB-18 processing were 0.8% and 8.7%, respectively, and on solid media the contamination rates were 2.6% and 4.3%, respectively [1]. All specimens that had been processed by the CB-18/ lytic enzyme method, and that were either flagged as positive in MB/BacT cultures or were observed with growth on the 7H11-selective plates, were subjected to acid-fast staining. Those culture-positive specimens that were negative for the presence of acid-fast bacteria, or that suggested the presence of contaminating microorganisms, were further analyzed to determine the nature of the contaminant. Portions of these cultures were first subcultured onto blood agar plates. Any microorganisms that grew were then subjected to Gram staining. Contaminant speciation was performed using conventional methods [5]. A total of 105 MB/BacT cultures were considered contaminated following processing with CB-18. These 105 cultures produced 116 contaminants (Table 1): 62.1%