Clinical evaluation of the automated COBAS AMPLICOR MTB assay for testing respiratory and nonrespiratory specimens

We evaluated the COBAS AMPLICOR PCR system (Roche Diagnostics) for the routine detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. Diagnostic culture, considered as the reference method, was performed with BACTEC, L?wenstein-Jensen, Stonebrink, and Kirchner media. Occasionally MB-Redox, ESP, or MGIT medium was also used. A total of 643 respiratory and 506 nonrespiratory specimens collected from 807 patients were investigated. Of the 95 culture-positive specimens, 80 were COBAS AMPLICOR MTB positive, and of the 1,054 culture-negative specimens, 1,044 were COBAS AMPLICOR MTB negative. After resolving discrepancies by review of the medical history, the overall sensitivity, specificity, and positive and negative predictive values for the COBAS AMPLICOR MTB assay, respectively, were 83.5, 98.8, 86.7, and 98.6% compared to those of diagnostic culture. In smear-positive specimens, the sensitivity of the COBAS AMPLICOR MTB assay was 96%, versus 48% for smear-negative specimens. No significant differences in the test performance between respiratory and nonrespiratory specimens were observed. The overall inhibition rate was less than 2%, excluding stool specimens. The clear advantages of the COBAS AMPLICOR PCR system are standardized procedures and reagents for specimen processing as well as an internal control for reliable monitoring of PCR inhibitors. By simplifying the work flow through a completely automated amplification and amplicon detection procedure, the COBAS AMPLICOR PCR system proved itself as a very useful component for routine diagnostic procedures.     

An algorithm for the rational choice of sodium profile during hemodialysis

Two systems, the newly developed Mycobacteria Growth Indicator Tube (MGIT) and biphasic Septi-Chek AFB based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. The difference in the rates of isolation of bacteria between the two groups of media was more remarkable with smear-negative specimens. The isolation of the Mycobacterium tuberculosis complex by MGIT occurred 8 days previous to the isolation by the conventional Ogawa method. The mean time for detecting M. tuberculosis complex by Septi-Chek AFB was similar to those of the Ogawa method. A greater difference in isolation time was observed for mycobacteria other than M. tuberculosis (MOTT) isolates. These results indicate that the MGIT and Septi-Chek AFB systems based on liquid media are efficient for the recovery of mycobacteria. PCR and other nucleic acid amplification methods are widely used for the detection of M. tuberculosis in clinical specimens. Although the sensitivities of the Gen-Probe Amplified Mycobacteria Direct Test (MTD) and Amplicor Mycobacteria for the detection of the M. tuberculosis complex appear to be similar to the sensitivity of the culture method using the Septi-Chek AFB, the two methods should be quite useful for rapid detection of M. tuberculosis infections. On the other hand, two cooperative blind studies conducted between 6 to 9 laboratories to estimate the reliability and reproducibility of these two commercially available kits revealed the necessity of good laboratory practice and development of reference reagents to monitor the performance of the whole assay, including pretreatment of clinical specimens. Considerable progress has been made in recent years toward understanding the molecular basis of the resistance to antituberculosis drugs, isoniazid (katG, inhA, ahpC), rifampin (rpoB), pyrazinamide (pncA), streptomycin (rpsL, rrs), ethambutol (embB), and fluoroquinolones (gyrA). Most cases of resistance are related usually to simple nucleotide substitutions rather than to acquisition of new genetic elements. Multidrug-resistant isolates of M. tuberculosis arise as a consequence of sequential accumulation of mutations conferring resistance to single therapeutic agents. The basis of resistance is not able to be explained yet in a substantial percentage of strains (> 90%) for other antituberculosis drugs than rifampin. Further studies are required to fully understand the molecular mechanisms of resistance.     

Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by ligase chain reaction

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a ligase chain reaction (LCR)-based assay and acid-fast stain and culture techniques. Results from the LCR assay (Abbott LCx Mycobacterium tuberculosis [MTB] Assay) were compared to results from standard culture techniques held for 6 weeks. Four hundred ninety-three specimens from 205 patients suspected of pulmonary tuberculosis were included in the prospective study. Thirty-four (6.9%) of the specimens were culture positive for M. tuberculosis, and 13 (38%) of these were also fluorochrome stain positive. LCR sensitivities and specificities compared to culture were 74 and 98%, respectively. LCR sensitivity was 100% for fluorochrome stain-positive specimens and 57% for fluorochrome stain-negative specimens. Nine LCR-negative, culture-positive specimens were the result of low concentrations of M. tuberculosis. No inhibitors were detected in any of these specimens. Of the eight LCR-positive, culture-negative specimens, five were from patients with active tuberculosis. With these considered culture misses, final LCR sensitivity, specificity, positive predictive value, and negative predictive value were 77, 99, 91, and 98%, respectively. The same performance values for the fluorochrome acid-fast bacillus smear were 33, 98, 62, and 94%, respectively. After normal laboratory sputum processing, the Abbott LCx MTB Assay can be completed in 6 h. Thus, it is possible to have results available within 8 h of specimen submission.     

Limits of commercial molecular tests for diagnosis of pulmonary tuberculosis

Several studies report high specificity, but variable sensitivity, of Amplified Mycobacterium tuberculosis Direct Test (AMTDT, Gen-Probe) based on ribosomal ribonucleic acid (rRNA) amplification and Amplicor Mycobacterium tuberculosis test (Amplicor, Roche) based on deoxyribonucleic acid (DNA) amplification for diagnosis of pulmonary tuberculosis. We have retrospectively evaluated these assays on selected acid-fast bacilli (AFB)-positive and -negative smear specimens and compared the results obtained from nucleic acid amplification with those of AFB staining of semi-quantitative cultures as determined by radiometric Bactec and L?wenstein-Jensen cultures. In comparison to cultures, Amplicor and AMTDT assays exhibited identical overall sensitivities of 80%, while the staining had a lower sensitivity of 62%. The sensitivities of Amplicor and AMTDT were 98% and 100%, respectively, for the AFB-positive specimens, and 50 and 46%, respectively for the AFB-negative specimens in comparison to cultures. The sensitivities of both assays appeared similar, and were directly related to the number of bacilli in the specimens studied. The low sensitivity (50%) for smear-negative specimens showed that current amplification assays may be unsuitable to replace cultures for diagnosis of tuberculosis. The decision to perform these molecular techniques should result from close co-operation between clinicians and microbiologists, taking into account the sensitivity results reported here, as well as the expense of the assays.     

Hydraulic analog for simultaneous representation of pharmacokinetics and pharmacodynamics: application to vecuronium

A variety of different media for the cultivation of mycobacteria have been described but a few of them are in use today. Those currently used can be characterized by three basic types. The first is egg-based media represented by Ogawa and L?wenstein-Jensen. The second type is agar-based media; the most common one are Middlebrook 7H10 and 7H11. The third type is liquid media such as Middlebrook 7H9. Several weeks of incubation may be required for the isolation of M. tuberculosis on solid media. Substantial improvement in the time to detection and the recovery rate was realized by using broth-based culture system such as the BACTEC 460TB, Septi-Chek AFB, MGIT and BACTEC 9000. In the BACTEC 460TB system, the mycobacteria is detected radiometrically. The processed specimen is added to a modified 7H9 medium (BACTEC 12B) containing 14C-labeled palmitic acid and an antibiotic complex, PANTA. Mycobacterial growth can be ascertained by the liberation of 14CO2 and detected by BACTEC 460TB instrument. The Septi-Chek AFB is a biphasic medium which combines broth and solid media. The liquid medium is a modified Middlebrook 7H9 in a carbon-dioxide-enriched culture bottle. After inoculation of the sample, the bottle is capped with a slide consisting of three solid media; a non-selective Middlebrook 7H11 agar, an egg-based medium, and chocolate agar. A novel system is the MGIT, which is a nonradiometric broth method for the detection of mycobacteria from clinical specimens. The MGIT consists of a modified Middlebrook 7H9 broth and a sensor embedded in silicone on the bottom of a tube. The appearance of orange-colored fluorescence in the sensor when excited indicates the growth of mycobacteria. MB Redox is a modified, serum-supplemented Kirchner medium containing p-indonitrotetrazolium violet (INT) as an indicator of microbial growth. The INT is reduced by the redox system of the mycobacteria to deep violet-colored formazan. This substance is water insoluble and is reduced to the cell surface, by which bacterial clamps can be easily detected by their violet color. At present, the egg-based media are the first choice for the culture of clinical samples. However, there are advantages to each type of medium and not all strains of mycobacteria can be recovered on a single medium. Therefore, it is recommended that one representative of each type of medium be used for primary isolation; one example in Japan may be Ogawa egg medium in combination with Middlebrook 7H11 and MGIT.     

Recent advance in the isolation of mycobacteria]

Two systems, the newly developed Mycobacteria Growth Indicator Tube (MGIT) and biphasic Septi-Chek AFB based on liquid media, proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. The isolation of Mycobacterium tuberculosis by MGIT occurred 8 days previous to the isolation by the conventional Ogawa method. These results indicate that the MGIT system is efficient for the recovery of mycobacteria.     

Clinical evaluation of a new non-radiometric automatic system for the rapid diagnosis of tuberculosis

BACKGROUND: Tuberculosis (TB) is again a public health problem un many countries and is considered a re-emerging disease. The fastest possible diagnosis in our patients is essential for TB control programs. ESP is a non-radioactive, totally automated, continuously monitored system designed to detect mycobacteria. METHODS: Clinical evaluation of this new system for the rapid diagnosis of tuberculosis. During 1997 a total of 1,022 clinical sputum specimens were investigated. Specimens were processed in triplicate for ESP, BACTEC 460 TB and L?wenstein-Jensen systems. The validity, isolates of Mycobacterium tuberculosis and time required for detecting M. tuberculosis by the three systems were determined. RESULTS: The sensitivity, specificity, positive predictive and negative predictive values of the new systems were 98%, 99.8%, 98% and 99.8%, respectively. No significant differences were found between the recovery rates by the three systems. The mean time for detection was 10 days (range: 7-13 days) for specimens with positive bacilloscopy and 14 days (range: 10-28 days) for specimens with negative bacilloscopy. The difference was statistically significant between ESP and L?wenstein-Jensen, but not between ESP and BACTEC. CONCLUSIONS: The new system proved to have an excellent sensitivity and specificity, which along with its total automation renders it a system of great clinical interest for the rapid diagnosis of TB and an alternative method for radiometric systems.     

Diagnosis of extrapulmonary tuberculosis by a commercial polymerase chain reaction kit

The Roche Amplicor Mycobacterium tuberculosis PCR test was compared with mycobacterial culture for direct detection of M. tuberculosis in extrapulmonary specimens. From January 1995 to October 1996, 124 clinical specimens from 112 patients were assessed, including 47 body fluids, 61 tissue specimens and 16 abscesses. The sensitivity and specificity of Amplicor PCR compared to culture were 63.6% and 93.1% respectively. Analysis of 7 PCR-positive, culture-negative specimens confirmed that all were from patients with recently diagnosed tuberculosis under treatment. Eight specimens were PCR negative-culture positive, including a pleural fluid containing inhibitory substances. On acid-fast bacilli (AFB) smear-negative specimens, sensitivity and specificity were 53 and 100% respectively. The best results for Amplicor PCR were obtained with abscesses and biopsies. It is concluded that this test, highly specific for the diagnosis of tuberculosis, is at least as sensitive on extrapulmonary specimens as on smear-negative respiratory specimens.     

Genus level identification of mycobacteria from clinical specimens by using an easy-to-handle Mycobacterium-specific PCR assay

An easy-to-handle Mycobacterium-specific PCR assay for detection of the presence of a wide range of mycobacterial species in clinical samples was evaluated. The performance of the genus probe was compared with the performance of probes specific for Mycobacterium tuberculosis and Mycobacterium avium and with that of standard culture. In addition, the utility of an internal control in monitoring amplification inhibitors was studied. Of 545 respiratory and 325 nonrespiratory specimens (a total of 870 specimens), 58 (6.7%) showed the presence of amplification inhibitors, as determined by a negative result for the internal control. Of these 58 specimens, 31 (53%) were stool specimens; other material, even citrate blood after lysis of erythrocytes, did not pose a problem with regard to inhibition of PCR amplification. Eighty-one of the remaining 812 specimens had a positive Mycobacterium culture result. Of these culture-positive specimens, 58 (71.6%) showed a positive result with the Mycobacterium genus-specific probe. Seventy-two samples had a positive result with the Mycobacterium-specific probe but a negative culture result. Of these 72 samples, 26 samples were regarded as true positive, either because the M. tuberculosis- or M. avium-specific probe was also positive at the same time or because other specimens from the same patient taken at the same time were culture positive. The sensitivity of the Mycobacterium-specific probe was 78.5% and the specificity was 93.5%. This study showed that pretesting of clinical specimens for mycobacteria to the genus level with a Mycobacterium-specific probe offers the routine clinical laboratory the possibility of detecting tuberculous and nontuberculous mycobacteria with one test. Furthermore, specimens testing positive with the genus-specific probe can be immediately identified with species-specific probes.     

Effects of protein kinase inhibitors on heat-induced hsp72 gene expression in a human glioblastoma cell line

This study aims to evaluate the performance of a new diagnostic method (LCx Tuberculosis Assay, Abbott Laboratories) based on Ligase Chain Reaction (LCR) technology, for the detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens and compare it with standard microbiological data and the clinical diagnosis of tuberculosis. Nine hundred specimens were collected from patients with a high suspicion of tuberculosis (740 respiratory samples and 160 non-respiratory specimens). The study was divided into two separate groups: samples washed and distilled water (207 samples) and unwashed samples that were directly resuspended in phosphate buffer (693 samples). The overall sensitivity, specificity, positive and negative predictive values of samples washed with distilled water after decontamination with SDS-NaOH were: 54%, 100%, 100%, and 94%, respectively. If these results were divided according to origin of specimens, the sensitivity, specificity, positive and negative predictive values in respiratory and non-respiratory samples were 54.5%, 100%, 100%, 94% and 50 100%, 100%, 93%, respectively. In contrast, for the non-washed samples, values were 85%, 95%, 80% and 98%, respectively. Respiratory and non-respiratory samples gave values of 84%, 96%, 77%, and 97.5% versus 89%, 99%, 94%, and 98%. The LCx M. tuberculosis assay is a novel, semi-automated assay and a rapid and highly specific technique for screening all forms of tuberculosis, including non-respiratory forms.     

Comparison of a nonradiometric system with Bactec 12B and culture on egg-based media for recovery of mycobacteria from clinical specimens

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 L?wenstein-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 Mycobacterium tuberculosis 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.     

Evaluation of 2-SP transport medium for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by two automated amplification systems and culture for chlamydia

AIMS: To assess the performance of 2-sucrose-phosphate based transport medium (2-SP) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by an automated commercial polymerase chain reaction (PCR) and ligase chain reaction (LCR) compared to centrifugation culture on McCoy cells for C trachomatis. Second, to compare both amplification systems for initial diagnostic testing of a low prevalence population for sexually transmitted diseases. METHODS: Four hundred and eighty one consecutive urogenital and conjunctival specimens were examined. All tests were performed on the same specimen collected with a dacron swab and transported in 2-SP medium. Samples that were positive by culture or by both PCR and LCR were considered to be true positives. RESULTS: The prevalences of C trachomatis and of N gonorrhoeae were 2.7% and 0.4%, respectively. PCR had a resolved sensitivity and specificity of 100% and 99.8%, respectively, for C trachomatis, and 100% and 98.9%, respectively, for N gonorrhoeae. LCR was 100% sensitive and specific for both pathogens. The resolved sensitivity of the shell vial assay was 85%. No culture positive sample would have been missed by PCR or LCR. The inhibition rate for PCR was 4.8%. CONCLUSIONS: 2-SP medium proved to be suitable for both PCR and LCR. It is not limited to any one test manufacturer and allows the performance of amplification techniques and viral and chlamydia culture from the same specimen. The LCR was more reliable than PCR on initial testing. However, hands on time is longer, and no amplification control is available for LCR.     

Bacteriological diagnosis of tuberculosis

Microscopic examination and culture are still today essential elements of the bacteriological diagnosis of tuberculosis. Microscopic examination of a Ziehl fuchsin or auramine stained specimen allows detection of most strains in less than an hour. Culture on L?wenstein-Jensen medium is more sensitive than the microscopic examination and is required for identification and to measure sensitivity to antibiotics. Mycobacterium colonies, generally the causal agent in tuberculosis, usually grow within 28 days and are easily recognized by their "cauliflower" aspect. The niacin test is used for formal identification. Currently, radiometric respirometry allows detection of M. tuberculosis growth and provides antibiotic sensitivity results more rapidly, usually within 10 days. Use of this technique is however limited because the culture medium contains radioactive carbon. Genetic probes are on the other hand quite easy to use and allow identification of cultured bacteria in only a few hours. After polymerization chain reaction gene amplification, M. tuberculosis strains can be detected directly in the specimen within 2 or 3 hours, but in practice, this method has not become a routine laboratory technique, particularly due to lack of sufficient specificity and sensitivity. No other serologic tests are currently reliable enough for the diagnosis of tuberculosis. For cases with low-count specimens, there still is no reliable "on-the-spot" diagnostic test.     

Pooling of urine samples for screening for Neisseria gonorrhoeae by ligase chain reaction: accuracy and application

The accuracy of detection of genital Neisseria gonorrhoeae infection in pooled urine samples by ligase chain reaction (LCR) was examined in three populations. Firstly, urine specimens from 300 female military recruits (FMR) were tested by LCR individually and in pools of four and six. Secondly, 300 urine specimens from middle-school students (MSS) were tested individually by LCR, and then the processed specimens were stored frozen for subsequent testing in pools of 4 and 10. Thirdly, 600 frozen urine specimens from high-school students (HSS) were tested by using the LCR pooling algorithm, i.e., testing processed specimens in pools of four in one test unit dose, and retesting individual specimens from positive pools. Finally, the pooling algorithm results were compared to culture results for a subset of 344 students from the original 600 HSS from whom cervical or urethral samples were taken at the discretion of the school nurse practitioners. Compared to individual testing of specimens by LCR in the FMR population, the pooling-by-four algorithm was 100% sensitive (5 of 5) and 100% pool specific (70 of 70), and the pool-by-six algorithm was 100% sensitive (5 of 5) and 100% pool specific (45 of 45). In the MSS population, the pool-by-4 algorithm was 95.8% sensitive (23 of 24) and 100% (52 of 52) pool specific, and the pool-by-10 algorithm was 95.8% sensitive (23 of 24) and 100% (17 of 17) pool specific. In the subset of 344 HSS from whom endocervical or urethral specimens were collected for culture, 31 were positive by LCR in urine and 26 were positive by culture. After results discrepant between culture and LCR were adjudicated by a confirmatory LCR test, the pooling algorithm was 93.8% (30 of 32) sensitive and 99.7% (311 of 312) specific. Culture from these 344 HSS was 81.3% (26 of 32) sensitive. The pooling algorithm reduced the cost of the N. gonorrhoeae LCR assay by 60% compared to individual testing of the HSS specimens and was both sensitive and specific.     

Identification of mycobacteria strains isolated in our laboratory 1972-1975]

111 (3,34%) acid-fast bacteria were found by direct, and 128 (3.86%) acid-fast bacteria by homogenisation methods, in 3315 specimens sent to the tuberculosis laboratory of our institute from various clinics of Gülhane Military Medical Academy, between October 11, 1972 and December 31, 1975. It was found that there was no great difference between the direct and the homogenisation methods, provided that these tests are carried out with great care. After the isolation of 128 mycobacteria from 3315 specimens by culturing in L?wenstein-Jensen medium they were typed by niacin, nitrate reduction, catalase, and peroxydase reactions. It was found that 124 out of 128 strains were M. tuberculosis var. bovis, atypical and saprophytic mycobacteria (3.125%).     

Comparative genomic hybridization of cancer of the gastroesophageal junction: deletion of 14Q31-32.1 discriminates between esophageal (Barrett's) and gastric cardia adenocarcinomas

We evaluated the BACTEC MGIT 960 system, which is a fully automated, noninvasive system for the growth and detection of mycobacteria with a capacity to incubate and continuously monitor 960 7-ml culture tubes. We studied 3,330 specimens, 2,210 respiratory and 1,120 nonrespiratory specimens, collected from 2,346 patients treated at six sites. Processed specimens were inoculated into the BACTEC MGIT 960 and BACTEC 460 TB systems, as well as onto Lowenstein-Jensen slants and Middlebrook 7H11/7H11 selective plates. From all culture systems, a total of 362 isolates of mycobacteria were recovered; these were recovered from 353 specimens collected from 247 patients. The greatest number of isolates of mycobacteria (289, or 80% of the 362 isolates) was recovered with the BACTEC MGIT 960, followed by the BACTEC 460 TB (271, or 75%) and solid media (250, or 69%). From all culture systems a total of 132 isolates of Mycobacterium tuberculosis complex were recovered. The greatest number of isolates of M. tuberculosis complex was recovered when liquid medium was combined with conventional solid media; the number recovered with BACTEC 460 TB plus solid media was 128 (97%), that recovered with BACTEC MGIT 960 plus solid media was 121 (92%), that recovered with BACTEC 460 TB was 119 (90%) and that recovered with all solid media combined was 105 (79%). The recovery with BACTEC MGIT 960 alone was 102 (77%). The mean times to detection (TTD) for M. tuberculosis complex were 14.4 days for BACTEC MGIT, 15.2 days for BACTEC 460 TB, and 24.1 days for solid media. The numbers of isolates of Mycobacterium avium complex (MAC) recovered were 172 (100%) for all systems, 147 (85%) for BACTEC MGIT 960, 123 (72%) for BACTEC 460 TB, and 106 (62%) for all solid media combined. The TTD for MAC in each system were 10.0 days for BACTEC MGIT 960, 10.4 days for BACTEC 460 TB, and 25.9 days for solid media. Breakthrough contamination rates (percentages of isolates) for each of the systems were 8.1% for BACTEC MGIT 960, 4.9% for BACTEC 460 TB, and 21.1% for all solid media combined.     

SMART: identification and annotation of domains from signalling and extracellular protein sequences

Seven hundred thirty-seven clinical samples from 460 patients were processed for direct detection of Mycobacterium tuberculosis complex by a semiautomated ligase chain reaction commercial assay, the LCx Mycobacterium tuberculosis Assay (LCx assay) from Abbott Laboratories. Results were compared to those of direct microscopy and standard microbiological culture. Of 26 patients (5.7%) with a culture positive for M. tuberculosis, 22 (84.6%) were found positive by the LCx assay. The sensitivity of the LCx assay was 98% for smear-positive samples and 27% for smear-negative samples. With an overall culture positivity rate for M. tuberculosis of 8.3% (61 of 737 samples) and after resolution of discrepant results according to clinical data, the sensitivity, specificity, and positive and negative predictive values of the LCx assay were 78, 100, 95, and 98%, respectively, compared to 85, 100, 100, and 98%, respectively, for culture and 67, 99, 87, and 97%, respectively, for acid-fast staining. In conclusion, the LCx assay proved satisfactory and appears to be an easy-to-use 1-day test which must be used with standard culture methods but can considerably reduce diagnosis time versus culture. However, its clinical interest appears to be limited in our population with low mycobacterial prevalence because of its cost considering the small gain in sensitivity versus direct microscopy.     

Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.     

Clinical evaluation of the BDProbeTec strand displacement amplification assay for rapid diagnosis of tuberculosis

The reliability of the BDProbeTec MTB Test (Becton Dickinson, Sparks, Md.) for direct detection of Mycobacterium tuberculosis in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture, with the BACTEC TB 460 and Middlebrook 7H11 biplates. Patients known to have tuberculosis were excluded from analysis. Of 523 specimens from 277 patients, 53 grew a mycobacterium: 24 specimens of M. tuberculosis and 29 specimens of nontuberculous mycobacteria. After initial testing, 42 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 95.8, 96. 2, 54.8, and 99.8%, respectively. After resolution of discrepancies, 28 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 100, 99.2, 85.7, and 100%, respectively. These same values were 100, 80.8, 93.4, and 100%, respectively, for smear-positive samples and 100, 99.4, 75.0, and 100%, respectively, for smear-negative specimens.     

Evaluation of a rapid air thermal cycler for detection of Mycobacterium tuberculosis

The Air Thermal Cycler (ATC) (Idaho Technology, Idaho Falls, Idaho) utilizes the unique technology of small-volume glass capillary tubes and high-velocity air for the heating and cooling medium for the PCR. Standard heat block thermal cycler (HBTC) and ATC performance characteristics were compared for the detection of Mycobacterium tuberculosis. Sensitivity was 100% for all smear-positive, M. tuberculosis culture-positive specimens for both the HBTC and the ATC. Of smear-negative, M. tuberculosis culture-positive specimens, sensitivity was 42.9% with the HBTC and 22.0% with the ATC. Specificity was 100% for both assay systems. Total assay time was 6.5 and 4 h and the reagent cost was 84 and 32 cents for the HBTC and ATC, respectively. The ATC offered an excellent alternative to the traditional HBTC for diagnosis of M. tuberculosis in smear-positive specimens by PCR.