Routine use of Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test for detection of Mycobacterium tuberculosis with smear-positive and smear-negative specimens.

SETTING: Nucleic acid amplification tests, such as the Amplified Mycobacterium Tuberculosis Direct (MTD) Test, may improve early diagnosis of tuberculosis when used in combination with acid-fast bacilli smear examination with a similar turnaround time. OBJECTIVE: To evaluate the routine use of MTD in respiratory and non-respiratory samples; to investigate the improvement of MTD specificity and positive predictive value by defining an equivocal zone for result interpretation. DESIGN: MTD was performed according to the instructions supplied by the manufacturer. An equivocal zone was included for interpretation of results. Discordant results with culture were resolved by incorporating clinical data and multiple specimen analysis. RESULTS: The overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens (n = 3308) were 90.8, 99.9, 99.1, and 99.2%, respectively. With extra-pulmonary specimens (n = 1350) those values were 67.4, 99.9, 98.2, and 97.9%, respectively. By implementing an equivocal zone, the specificity and positive predictive value of MTD were improved (from 99.1% and 88.6% to 99.9% and 98.9% respectively) without significantly altering other performance characteristics. CONCLUSIONS: Amplification assays cannot yet replace the conventional diagnostic techniques. Nevertheless, MTD is a reliable method for the direct detection of M. tuberculosis in clinical specimens. The number of false-positive results can be limited by defining an equivocal zone.

Acquired drug resistance in Mycobacterium tuberculosis isolates recovered from compliant patients with human immunodeficiency virus-associated tuberculosis.

We describe five compliant patients with human immunodeficiency virus (HIV)-associated tuberculosis (TB) that relapsed, with acquisition of resistance by the original Mycobacterium tuberculosis strains. Both the first and second isolates from each patient had the same IS (insertion sequence) 6110-based DNA fingerprint patterns. Three of the five patients developed TB that was resistant to rifampin alone; no mutation in the region of the rpoB gene was detected by a line probe assay in two of the isolates from these patients. We discuss several factors presumably associated with acquired drug resistance in HIV-infected patients, including exogenous reinfection, drug interactions, malabsorption of drugs, and the presence of a large organism burden.

[Disseminated Mycobacterium genavense infection in patients with HIV infection. Description of 5 cases and review of the literature]. / Infección diseminada por Mycobacterium genavense en pacientes con infección por HIV. Descripción de 5 casos y revisión de la literatura.

BACKGROUND: Five cases of disseminated infection by Mycobacterium genavense in patients with HIV infection are reported with a review of the literature. MATERIAL AND METHODS: A description of the clinical, epidemiologic and therapeutic characteristics of five cases are presented. The initial isolation of the microorganism was performed in Bactec 13A from blood and bone marrow aspirates. Subcultures were made in different media and the growth characteristics evaluated. Sensitivity to NAP was determined by radiometric techniques and gas chromatography allowed a possible identification. Definitive identification was based on PCR amplification of the gene which codifies the 65kDa protein and the posterior restriction of the amplified fragments by using BstEII and HaeIII. RESULTS: All five patients were males with HIV infection and a lymphocyte count of less than 25 cells/mm3 with an non-specific clinical picture similar to that produced by M. avium complex (MAC). Empiric antiMAC treatment was administered in four of the patients with good clinical response. All five strains were sensitive to NAP. The greatest growth rate was obtained in the subcultures with acid pH in liquid medium. Gas chromatography was very similar to that of M. simiae. Amplification of the gene which codifies the 65 kDa protein and posterior restriction with BstEII resulted in 2 fragments of 325 and 125 bp, while restriction with HaeIII resulted in two fragments of 140 and 105 bp. CONCLUSIONS: Mycobacterium genavense represents 9% of the disseminated infections by mycobacteria in AIDS patients. The clinical manifestations, empiric treatment and response is similar to that of infection by M. avium complex. Growth is favored by acid pH in liquid medium. Susceptibility to NAP leads to possible identification which should be confirmed by molecular techniques.

[Usefulness of DR, PGRS, and spoligotyping in the typing of Mycobacterium tuberculosis. Comparison with IS6110]. / Utilidad de DR, PGRS y spoligotyping en la tipificación de Mycobacterium tuberculosis. Comparación con IS6110.

BACKGROUND: To evaluate 4 markers (IS6110, DR, PGRS and spoligotyping) to differentiate the strains of Mycobacterium tuberculosis isolated in our surroundings, most specially in those which contain a reduced number of IS6110 copies. In addition, to confirm the identity of the strains that share the same IS6110 restriction-hybridization pattern. METHODS: We selected 37 strains from a previous study: 25 had a unique IS6110 pattern and 12 grouped in 3 clusters (8 strains with 11 bands, 2 with 17 bands and 2 which shared the same six-band pattern). The PGRS and DR-RFLPs were obtained by AluI restriction and synthetic oligonucleotides specific to these sequences. The polymorphism of the DR region spacers was analyzed by spoloigotyping. For the amplification of the spacers we used the DRa and DRb primers. Detection was done hybridizing the PCR products on a membrane in which 43 specific spacers had been previously immobilized. RESULTS: Twenty-three different PGRS patterns and 18 spolygotyping patterns were obtained from 25 strains with unique IS6110 pattern. Eight patterns resulted from the 10 strains studied by DR. The 8 strains which shared an 11-band pattern, as well as the 2 strains which shared a 17-band pattern, resulted identical by the other markers. However, 2 strains which shared a 6-band pattern were different by both PGRS and DR or spoligotyping. CONCLUSIONS: 1) IS6110 resulted the most discriminative marker of all. 2) The clonality of clusters with a low number of bands has to be confirmed with alternative markers.

[Fiberoptic bronchoscope disinfection with glutaraldehyde phenolate in 1:8 solution]. / Desinfección del broncofibroscopio con glutaraldehído fenolato a la dilución 1:8.

We evaluated the efficacy of phenolated glutaraldehyde in a 1:8 solution for the disinfection of bronchofibroscopes that were highly contaminated with Serratia marcescens and Pseudomonas aeruginosa. An Olympus BF-P10 bronchofibroscope was contaminated with artificial samples containing one of the aforementioned microorganisms in concentrations nearing 10(8) colony forming units per milliliter (cfu/ml). The instruments were then washed with soap and water and submerged in a 1:8 solution of phenolated glutaraldehyde. Samples were taken for culturing after contamination, after washing, and after 10, 15 and 30 min in the disinfectant solution. The level of cfu/ml in the cultures was measured and the definition of failure-to-disinfect was a finding of > or = 1 cfu/ml after each experimental procedure. Twenty procedures, 10 for each microorganism, were carried out. Washing of the bronchofibroscope afforded significant elimination of microorganisms and no colony growth was observed in cultures after 10 min submersion in phenolated glutaraldehyde. We conclude that immersion in a 1:8 solution of phenolated glutaraldehyde after careful washing is a valid way to disinfect bronchofibroscopes that are highly contaminated with S. marcescens and P. aeruginosa.

[Utility of the direct culture examination (Ziehl-Neelsen) using the Bactec system for the presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium xenopi, and Mycobacterium kansasii]. / Valor del examen directo (Ziehl-Neelsen) del cultivo en sistema Bactec para la identificación presuntiva de: Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium xenopi y Mycobacterium kansasii.

BACKGROUND: The usefulness of the microscopic examination of Bactec 12B and 13A growth medium as a method for the possible identification of M. tuberculosis complex, M. avium complex, M. xenopi, and M. kansasii was performed out to guide the selection of different genetic identification probes and, in the case of M. xenopi, the selection of the temperature of subcultures incubation. METHODS: Upon detection of an index of growth greater than 100 in Bactec tubes, staining was performed by the Ziehl-Neelsen technique. On the basis of the morphology observed, the possible identification was performed by genetic probes. Subcultures were used for definitive identification. RESULTS: Three hundred forty-four positive samples were studied by radiometric technique. A total of 190 strains were identified as M. tuberculosis, 88 strains as M. avium-intracellulare (MAI), 33 strains as M. xenopi, 14 strains as M. kansasii and 19 strains were identified as: M. gordonae (10), unpigmented rapid growth microbacteria (7), and M. simiae (2). Sensitivity, specificity, positive predictive value, and negative predictive value were 97.9%, 95.4%, 96.4%, and 97.3%, respectively for M. tuberculosis complex, 84.0%, 99.2%, 97.3% 94.7% for M. avium complex; 63.6%, 98.3%, 80.7%, 96.2% for M. xenopi; 35.7%, 98.1%, 45.5% 97.2% for M. kansasii. CONCLUSIONS: The morphology of M. tuberculosis complex examined in the radiometric system in useful to differentiate this species from other microbacteria (MOTT), allowing the selection of specific probe used. Within the MOTT, M. avium complex also has morphological characteristics which are useful for its differentiation, the morphology usually described for the remaining species was frequently not observed.