The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTD) is a direct specimen assay for the identification ofMycobacterium tuberculosis from respiratory samples. rRNA is amplified, and the product is detected with a specific chemiluminescent probe. We performed a retrospective evaluation of three separate respiratory specimens from each of 250 patients by using the AMTD and compared the results with those of microscopy, culturing, and a patient chart review. From the latter results, 198 patients (594 specimens) were found negative for M. tuberculosis by culturing and clinical criteria. The overall specificity of the AMTD after discrepancy resolution was 98.5% (585 of 594). There were 52 patients with culture-proven and/or clinically diagnosed tuberculosis. Of these 156 specimens, the organism was cultured from 142 (91%), and acid-fast microscopy was positive for 105 (67.3%). The AMTD was positive for 142 (91%) specimens from these patients. Tuberculosis patient samples were tested by a PCR assay which uses primers for amplification of the IS6110 insertion sequence of the M. tuberculosis complex. The PCR assay detected 144 of the 156 (92.3%) specimens. Overall, when three specimens per patient were examined, the AMTD found all 52 patients positive for tuberculosis, while the PCR assay found 51 patients positive by agarose gel analysis and all 52 patients positive by Southern blot hybridization.

A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.Mycobacterium tuberculosis remains a serious public health issue due to its high risk of person-to-person transmission, morbidity, and mortality (15,20). Currently, approximately 8 million new infections and 3 million deaths are attributed to tuberculosis (TB) each year (L. B. Reichman, Letter, Chest 112:855, 1997). Progressive increases in TB infections are expected, and a worldwide annual incidence of 12 million cases by 2005 is predicted by the World Health Organization (20). The resurgence of TB in industrialized countries since the mid-1980s, primarily due to the increased incidence of immunocompromised patients with AIDS, and the emergence of multidrug-resistant strains of M. tuberculosis has accented the need for rapid diagnosis of this disease (15,20). Rapid detection of active TB infection is critical for effective patient management and implementation of infection control measures.Conventional detection of mycobacteria is based on a number of protocols, including microscopic examination of smears stained with the Ziehl-Neelsen stain or auramine fluorescent dye and selective culture techniques (8,17,26,39). The key aspect of TB control is rapid diagnosis, which for many years has been based on the staining of smears for the presence of acid-fast bacilli (AFB). The AFB smear test lacks specificity, so there is a need for a laboratory test for specific detection of the M. tuberculosis complex (MTB) that can be performe...

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A procedure using the Smart Cycler instrument and a fluorescence quencher (FQ) probe for the specific identification of Mycobacterium tuberculosis complex (MTB) was used to detect organisms in 366 acid-fast bacillus smear-positive respiratory specimens. It was compared to culture and the AMPLICOR M. tuberculosis PCR test. MTB was isolated from 198 of these samples. The FQ PCR assay was sensitive (197 of 198, 99.5%) and specific (165 of 168, 98.2%); no significant difference was observed between the two PCR protocols. After DNA extraction, a final result was available within 1.5 h with the real-time PCR protocol.Because of the high risk of person-to-person transmission, rapid detection of Mycobacterium tuberculosis directly in patient specimens is recognized as an important step in the control of infection due to this organism (9, 12). The detection of acid-fast bacilli (AFB) in smears prepared from respiratory secretions has been the mainstay of rapid identification of potentially infectious individuals. Direct staining of prepared smears is generally performed with Ziehl-Neelsen stain or auramine fluorescent dye (8). Because the AFB smear lacks specificity, there is a need for a laboratory test for specific detection of the M. tuberculosis complex (MTB) that can be performed within a short period of time. Currently, nucleic acid amplification tests offer a rapid, specific, and sensitive approach to the detection and characterization of MTB (2,3,6,14,16,20,21). However, many of these assays require multiple user-dependent steps for amplification and detection and have the potential for error and sample contamination. Real-time PCR techniques (22), involving fluorescent dyes or fluorophores with a spectrofluorometric thermal cycler, have been developed and evaluated for the detection of mycobacteria and for the detection of drug resistance (5,11,18,19). The ABI 7700 TaqMan system (Applied Biosystems, Foster City, Calif.) has been used to quantitate MTB DNA in sputum during the treatment of tuberculosis patients (4). The probe used in this assay was specific for the IS6110 gene region (7).In addition, we recently reported a rapid and sensitive method for the identification of MTB by amplification of the internal transcribed spacer and the use of a specific fluorogenic probes for MTB in the LightCycler system (10, 15).In this study, we compared the fluorescence quencher (FQ) PCR assay to the AMPLICOR PCR test and conventional culture techniques. We examined AFB smear-positive respiratory specimens that were submitted for culture from January 2001 through December 2002. All specimens were liquefied and decontaminated with N-acetyl cysteine-2.5% NaOH and concentrated by centrifugation (17). The sediment was used to inoculate a selective 7H11 agar plate and a supplemented BacT/ALERT MP (MP; Organon Teknika, Durham, N.C.) culture bottle. Also, two smears were prepared and stained with an auramine fluorochrome dye (8). Isolates of mycobacteria growing on solid media were identified by DNA probes (Accuprobe; Gen-Probe, I...

Evidence is presented that supports the contention that a small subset of patients who experience viral load elevations after influenza vaccination can develop mutational changes in the RT region of the viral genome either acutely or after a failure of the viral load to return to undetectable levels.