Pyrazinamide susceptibility testing: proposed new standard with the BACTECTM MGITTM 960 system.

The susceptibility of 253 Mycobacterium tuberculosis complex isolates to pyrazinamide (PZA) was assessed using the BACTECTM MGITTM 960 (M960) system. Resistant strains underwent paired repeat testing using 1) a critical concentration of 200 g/ml (PZA-200), and 2) a reduced inoculum of 0.25 ml. They were also examined using the BACTEC 460 (B460) reference method and investigated for pncA mutations. On M960, 37 isolates were resistant. In the PZA-200 assay, 20 of these were resistant and 17 susceptible, while 18 were resistant and 19 susceptible with reduced inoculum. The B460 assay and pncA sequencing confirmed results with reduced inoculum.

Mycobacterial infections in solid organ transplant recipients.

Mycobacterial infections represent a growing challenge for solid organ transplant recipients (SOT). The adverse effects of tuberculosis (TB) therapy present a major difficulty, due to the interactions with immunosuppressive drugs and direct drug toxicity. While TB may be donor-transmitted or community-acquired, it usually develops at a latent infection site in the recipient. Pre-transplant prevention efforts will improve transplant outcomes and avoid the complications associated with post-transplant diagnosis and treatment. The present review and consensus manuscript is based on the updated published information and expert recommendations. The current data about epidemiology, diagnosis, new regimens for the treatment of latent TB infection (LTBI), the experience with rifamycins for the treatment of active TB in the post-transplant period and the experience with isoniazid for LTBI in the liver transplant population, are also reviewed. We attempt to provide useful recommendations for each transplant period and problem concerning mycobacterial infections in SOT recipients.

Performance of a commercial nucleic acid amplification test with extrapulmonary specimens for the diagnosis of tuberculosis.

The laboratory diagnosis of tuberculosis (TB) on extrapulmonary specimens is particularly challenging. A number of commercial nucleic acid amplification tests able to detect and identify Mycobacterium tuberculosis (MTB) complex directly from respiratory secretions have been developed, but their use on extrapulmonary samples still calls for validation. The BDProbeTec ET Mycobacterium tuberculosis Complex Direct Detection Assay (DTB) was applied to 918 consecutive extrapulmonary specimens (collected from 863 patients), including 84 gastric aspirates, 145 urine, 136 sterile body fluids, 83 cerebrospinal (CSF) fluids, 237 fine-needle aspirates, 175 pus, 56 biopsies, and two stool specimens. The results were compared with those of acid-fast staining and culture (solid plus liquid media), setting the combination of culture and clinical diagnosis as the gold standard. Ninety-two specimens yielded culture positive for MTB and 24 (smear- and culture-negative) were from patients with TB clinical diagnosis. Of these, 96 were DTB-positive, including all of those from culture-negative TB cases. From 26 specimens, nontuberculous mycobacteria were grown. Two of these specimens were positive by the DTB assay. Finally, of the 776 samples that were smear- and culture-negative for acid-fast bacilli (AFB), collected from patients for whom the diagnosis of TB was excluded, six were DTB-positive. The overall sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of extrapulmonary samples were 82.7, 99.0, 92.3, and 97.8%, respectively. Although, at present, amplification assays cannot replace culture techniques, DTB proved to be rapid and specific for the detection of MTB in extrapulmonary samples.

Nontuberculous mycobacteria infection in solid organ transplant recipients.

Although advances in surgical technique, drug-induced immunosuppression, and supportive medical therapy have led to improved survival and quality of live after solid organ transplantations, infections still represent a major threat for transplant recipients.Infections with non tuberculous mycobacteria (NTM) are infrequent, but can be associated with relevant morbidity and mortality. With the exception of few comprehensive studies, the available literature consists predominantly of case reports and institutional experiences. This article reviews NTM infection in the setting of solid organ transplant (SOT) recipients dealing with clinical features, diagnosis, treatment and outcome. Published studies have documented a protean presentation of NTM infection in SOT recipients which may include skin and soft tissue lesions, osteoarticular infections and pleuropulmonary disease. An aggressive approach including histopathologic examination and acid-fast bacilli culture of aspirates or biopsy specimens from involved sites are essential for diagnosis. A combined treatment of debridement surgery, reduction of immunosuppressive medications and/or antimycobacterial drugs is frequently associated with a favorable outcome. It is strongly emphasized that a high index of suspicion for NTM infection should be maintained when managing SOT recipients with unusual clinical manifestations. Early diagnosis and initiation of therapy are essential to prevent morbidity and mortality in this vulnerable population.

Diagnostic accuracy of in-house PCR for pulmonary tuberculosis in smear-positive patients: meta-analysis and metaregression.

In-house PCR (hPCR) could speed differential diagnosis between tuberculosis (TB) and nontuberculous mycobacterial disease in patients with positive smears and pulmonary infiltrates, but its reported accuracy fluctuates across studies. We conducted a systematic review and meta-analysis of hPCR sensitivity and specificity for smear-positive TB diagnosis, using culture as the reference standard. After searching English language studies in MEDLINE and EMBASE, we estimated cumulative accuracy by means of summary receiver operating characteristic analysis. The possible influence of hPCR procedures and study methodological features on accuracy was explored by univariate metaregression, followed by multivariate adjustment of items selected as significant. Thirty-five articles (1991 to 2006) met the inclusion criteria. The pooled estimates of the diagnostic odds ratio, sensitivity, and specificity (random-effect model) were, respectively, 60 (confidence interval [CI], 29 to 123), 0.96 (CI, 0.95 to 0.97), and 0.81 (CI, 0.78 to 0.84), but significant variations (mainly in specificity) limit their clinical applicability. The quality of the reference test, the detection method, and real-time PCR use explained some of the observed heterogeneity. Probably due to the limited study power of our meta-analysis and to the wide differences in both laboratory techniques and methodological quality, only real-time PCR also displayed a positive impact on accuracy in the multivariate model. Currently, hPCR can be confidently used to exclude TB in smear-positive patients, but its low specificity could lead to erroneous initiation of therapy, isolation, and contact investigation. As the inclusion of samples from treated patients could have artificially reduced specificity, future studies should report mycobacterial-culture results for each TB and non-TB sample analyzed.

Comparison of tuberculin skin test and Quantiferon immunological assay for latent tuberculosis infection.

BACKGROUND: Correct identification of individuals with latent tuberculosis infection (LTBI) is a crucial element of the elimination strategy, allowing their adequate treatment. In addition to tuberculin skin test (TST), the Quantiferon test (QFT, based on whole blood gamma-interferon release) had been recently proposed. Aim of the study is to compare this test to TST for identification of LTBI in a non-selected population, in order to verify their value in identifying truly infected individuals (entitled to receive preventive chemotherapy), and to exclude from treatment those having a positive TST for other reasons (e.g. after BCG vaccination). METHODS: 136 consecutive persons (78 males, mean age 34 +/- 9 years) referred to the clinic for TST were recruited (78 born in low--or middle--income countries). Based on their history, the cases were divided into 4 groups: 1) recently traced contacts of whom 18 TST negative and 28 TST positive; 2) 22 screening subjects, all TST negative; 3) BCG vaccinated subjects (14); and 4) 54 subjects already undergoing treatment of LTBI for exposure to TB. RESULTS: The overall agreement between TST and QFT was 72% (64% in TST positive and 88.4% in TST negative subjects). The proportion of TST positive/QFT negative BCG vaccinated individuals was 23.1%. The K coefficient was 0.474 in recently traced contacts, 0.366 in BCG vaccinated individuals and 0.451 overall. CONCLUSIONS: The study results suggest that agreement between TST and QFT is lower in TST positive than in negative subjects, being lower in individuals treated for LTBI. Quantiferon does not seem to have brought significant improvement in the diagnosis of LTBI.

Mycobacterial testing in hospital laboratories: results from a questionnaire survey in Italy.

Between 1999 and 2001, 355 hospital laboratories in Italy were asked to complete a questionnaire addressing mycobacterial test methods, 1-year workloads and laboratory safety features. Analysis of the data showed that rapid methods for mycobacterial testing were being used by most larger laboratories; however, sub-optimal methods were still in use in small and medium-size laboratories. In a country such as Italy, which has a low prevalence of tuberculosis cases, implementation of rapid technologies, combined with regionalisation of mycobacterial diagnostic services, seems to be the most reasonable and cost-effective strategy.

Monitoring the quality of laboratories and the prevalence of resistance to antituberculosis drugs: Italy, 1998-2000.

In 1998 a network of 20 regional tuberculosis (TB) laboratories (the Italian Multicentre Study on Resistance to Antituberculosis drugs (SMIRA) network) was established in Italy to implement proficiency testing and to monitor the prevalence of drug resistance nationwide. The network managed 30% of all TB cases reported in Italy each year. The aim of the present report is to describe: 1) the accuracy of drug-susceptibility testing in the network; 2) the prevalence of drug resistance for the period 1998-2000. Data were collected from the network laboratories. Sensitivity to streptomycin and ethambutol increased from the first survey (1998-1999) to the second survey (2000) from 87.7 to 91.9%. Specificity, predictive values for resistance and susceptibility, efficiency and reproducibility were consistent in both surveys. In previously untreated cases, the prevalence of multidrug-resistance was the same in both surveys (1.2%), while a slight decrease from the first to the second survey was observed for monoresistance to rifampicin (from 0.8 to 0.4%) and isoniazid (from 2.9 to 2%). The significant association found between isoniazid resistance and immigration is a useful indicator for both clinicians managing individual tuberculosis cases and public health services planning control strategies.

Evaluation of the Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium bovis in bovine tissue samples.

The commercial LCx amplification assay, usually employed to detect the Mycobacterium tuberculosis complex in respiratory specimens, was evaluated by comparing the results it gave with those obtained using Lowenstein-Jensen solid medium and pathological findings in 55 lymph nodes from cattle with positive and 10 lymph nodes from cattle with negative skin tests for tuberculosis. Fifty-three cultures (51 and 2, respectively) were positive for M. bovis, while the results for the LCx assay and the histological method were positive in 48 (45, 3) and 24 (20, 4) samples, respectively. None of the samples from cattle from certified tuberculosis-free herds were positive by any of the procedures. The results obtained with the LCx assay, compared with the culture procedure, regarded as the gold standard among the diagnostic techniques, gave a specificity of 91.6% and sensitivity of 90.5%. Although the sensitivity of LCx was suboptimal, DNA of M. bovis was detected in 81.8% of the skin test-positive animals. Amplification techniques could provide a rapid and reasonably reliable tool for detecting bovine tuberculosis.

Direct identification of mycobacteria from MB/BacT alert 3D bottles: comparative evaluation of two commercial probe assays.

The new INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium), a reverse-hybridization-based line probe assay, and the AccuProbe assay (Gen-Probe Inc., San Diego, Calif.) were applied to MB/BacT Alert 3D (MB/BacT) system (Organon Teknika, Boxtel, The Netherlands) culture bottles and evaluated for mycobacterial identification. From 2,532 respiratory and extrapulmonary specimens submitted for culture, 168 were flagged positive by the MB/BacT system and promptly evaluated for identification (within 24 h). Each of 163 vials grew one mycobacterial isolate, including Mycobacterium tuberculosis complex (n = 73), M. avium complex (n = 3), M. avium (n = 8), M. intracellulare (n = 5), M. kansasii (n = 15), M. gordonae (n = 8), M. malmoense (n = 3), M. chelonae (n = 13), M. abscessus (n = 2), M. xenopi (n = 11), M. scrofulaceum (n = 2), M. fortuitum (n = 7), M. terrae (n = 3), M. simiae (n = 2), M. celatum (n = 3), M. flavescens (n = 1), M. interjectum (n = 1), M. bohemicum (n = 1), and M. pulveris (n = 2). Five cultures yielded mixed growth of two mycobacterial species: M. tuberculosis complex plus M. gordonae (n = 2), M. tuberculosis complex plus M. chelonae (n = 1), M. tuberculosis complex plus M. xenopi (n = 1), and M. avium plus M. chelonae (n = 1). In testing of one-isolate vials, both systems showed excellent sensitivity and specificity for all species and complexes for which they are licensed (nine for INNO-LiPA Mycobacteria versus six for AccuProbe). There were minor discrepancies in results for two isolates identified by INNO-LiPA Mycobacteria as M. avium - M. intracellulare - M. scrofulaceum (MAIS) complex and by AccuProbe as M. intracellulare. In testing of two-isolate vials, INNO-LiPA Mycobacteria correctly identified all isolates, while the AccuProbe assay failed to identify three M. tuberculosis complex isolates and one M. avium isolate. The AccuProbe assay was completed within 2 h, while INNO-LiPA Mycobacteria required a 6-h period. In our opinion, INNO-LiPA Mycobacteria offers the following advantages: (i) it contains a genus-specific probe that, in addition to being used in genus identification, may be used as an internal control for both the amplification and hybridization steps; (ii) it simultaneously identifies M. tuberculosis complex, MAIS complex, and seven other mycobacterial species, even from mixed cultures; (iii) its mycobacterial DNA amplification ensures reliable results independent from the concentration of viable microorganisms; and (iv) it genotypically identifies M. kansasii and M. chelonae. In conclusion, even though INNO-LiPA Mycobacteria is considerably less easy to use than AccuProbe, requiring personnel skilled in molecular biology techniques, it represents an excellent approach for routine identification of frequently encountered mycobacteria.

Performance assessment of new multiplex probe assay for identification of mycobacteria.

A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of Mycobacterium tuberculosis complex, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium gordonae, the species of the Mycobacterium avium complex (MAC), Mycobacterium scrofulaceum, and Mycobacterium chelonae was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus Mycobacterium, correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as Mycobacterium intracellulare by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species M. kansasii and M. chelonae, while the declared overlapping reactivity of probe 4 with some M. kansasii and Mycobacterium gastri organisms and of probe 9 with MAC, Mycobacterium haemophilum, and Mycobacterium malmoense, may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.

Comparison of MB/Bact alert 3D system with radiometric BACTEC system and Löwenstein-Jensen medium for recovery and identification of mycobacteria from clinical specimens: a multicenter study.

The MB/BacT ALERT 3D System (MB/BacT) (Organon Teknika, Boxtel, The Netherlands) is a fully automated, nonradiometric system with a revised antibiotic supplement kit designed for the recovery of mycobacteria from clinical specimens. In a multicenter study, the recovery rate of acid-fast bacilli (AFB) and the mean time to their detection from clinical specimens was determined by using the MB/BacT system. Data were compared to those assessed by the radiometric BACTEC 460 system (B460) and by culture on Löwenstein-Jensen (L-J) solid medium. A total of 2,859 respiratory and extrapulmonary specimens were processed by the N-acetyl-L-cysteine (NALC)-NaOH method using two different concentrations of sodium hydroxide; 1.5% was adopted in study design A (1,766 specimens), and 1.0% was used in study design B (1,093 specimens). The contamination rates for MB/BacT were 4.6% (study design A) and 7.1% (study design B). One hundred seventy-nine mycobacterial isolates were detected by study design A, with 148 Mycobacterium tuberculosis complex (MTB) isolates and 31 nontuberculous mycobacteria (NTM) isolates. Overall recovery rates were 78.8% for MB/BacT (P = 0.0049), 64.2% for L-J (P < 0.0001), and 87.1% for B460, whereas they were 84.5, 70.9, and 91.2%, respectively, for MTB alone. A total of 125 mycobacteria were detected by study design B, with 46 MTB and 79 NTM. Overall recovery rates by the individual systems were 57.6% (P = 0.0002), 56.8% (P = 0.0001), and 80% for MB/BacT, L-J, and B460, respectively, whereas the rates were 91.3, 78.3, and 97.8% for MTB alone. By study design A, the mean times to detection of smear-positive MTB, smear-negative MTB, and NTM were 11.5, 19.9, and 19.6 days, respectively, with the MB/BacT; 8.3, 16.8, and 16.6 days, respectively, with the B460; and 20.6, 32.1, and 27.8 days, respectively, with L-J medium. By study design B, the mean times were 15.1, 26.7, and 26 days with the MB/BacT; 11.7, 21.3, and 24.8 days with the B460; and 20.4, 28.7, and 28.4 days with L-J medium. Identification was attempted by probing (Accuprobe) MB/BacT-positive bottles within the first working day following instrument positive flag. Results were compared to those obtained in the B460 positive vials by the p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test (study design A) or by the Accuprobe assay (study design B). About 90% of MTB and 100% of NTM could be identified, showing turnaround times closely related to those obtained by combining B460 and the NAP test or the Accuprobe assay. In conclusion, even though recovery rates were shown to be lower than B460, especially for NTM, and contaminants were somewhat higher, MB/BacT represents a valuable alternative to the radiometric system, especially in those laboratories where disposal of radioactive waste is restricted. Finally, when AFB are cultured in nonradiometric liquid media, our data (detection times and bacterial overgrowth rates) suggest that decontamination with 1.5% NaOH may be more suitable than the standard NALC-NaOH.

Mycobacterium doricum sp. nov.

A novel mycobacterial species is described in this study. The strain was isolated from the cerebrospinal fluid of a severely immunocompromised AIDS patient. It was scotochromogenic and slow-growing. Characteristic features for its differentiation from other mycobacteria are its lipid pattern and the unique gene sequences within the hypervariable regions of the 16S rDNA. The strain shows susceptibility to current antimycobacterial drugs. The pathogenicity of the novel mycobacterium and its clinical significance are not certain, as the neurological symptoms of the patient could also be due to concomitant infection with Cryptococcus neoformans. The name Mycobacterium doricum sp. nov. is proposed for the novel mycobacterium; the type strain is strain FI-13295T (= DSM 44339T = CIP 106867T).

Comparison of enhanced Mycobacterium tuberculosis amplified direct test with COBAS AMPLICOR Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens.

The new Roche COBAS AMPLICOR Mycobacterium tuberculosis Assay was compared to the Gen-Probe enhanced Mycobacterium tuberculosis Amplified Direct Test (AMTDII). A total of 486 specimens (296 respiratory and 190 extrapulmonary) collected from 323 patients were tested in parallel with both assays. Results were compared with those of acid-fast staining and culture, setting the combination of culture and clinical diagnosis as the "gold standard." After resolution of discrepant results, the sensitivity, specificity, and positive and negative predictive values for AMTDII were 85.7, 100, 100, and 90.4% for respiratory specimens and 82.9, 100, 100, and 95. 5% for extrapulmonary specimens, respectively. The corresponding values for AMPLICOR were 94.2, 100, 100, and 96.6% for respiratory specimens and 85, 100, 100, and 96.1% for extrapulmonary specimens, respectively. No significant differences were observed between the results of both assays or, within each one, between respiratory and extrapulmonary specimens. The difference between AMTDII and AMPLICOR sensitivities was related to the presence of inhibitory samples, which the former assay, lacking an internal amplification control (IAC), could not detect. The overall inhibition rate for the AMPLICOR assay was 3.9% (19 specimens). It is concluded that, although both amplification assays proved to be rapid and specific for the detection of M. tuberculosis complex in clinical samples, AMPLICOR, by a completely automated amplification and detection procedure, was shown to be particularly feasible for a routine laboratory setting. Finally, AMTDII is potentially an excellent diagnostic technique for both respiratory and extrapulmonary specimens, provided that an IAC is included with the assay.

Synthesis and in vitro anti-mycobacterium activity of N-alkyl-1, 2-dihydro-2-thioxo-3-pyridinecarbothioamides. Preliminary toxicity and pharmacokinetic evaluation.

Disseminated infections with Mycobacterium tuberculosis (MT) and Mycobacterium avium complex (MAC) are increasingly opportunistic diseases in patients with advanced acquired human immunodeficiency syndrome (AIDS). A series of N-alkyl-1, 2-dihydro-2-thioxo-3-pyridinecarbothioamides has been synthesized, and MICs for MT and MAC strains, either standard or isolated from infected patients, have been determined. Preliminary tests show a good activity and a very low toxicity for some derivatives. Pharmacokinetic studies in the rat show a very rapid elimination from the body after intravenous administration and a poor absorption after oral administration.

Use of BACTEC MGIT 960 for recovery of mycobacteria from clinical specimens: multicenter study.

The BACTEC MGIT 960 instrument is a fully automated system that exploits the fluorescence of an oxygen sensor to detect growth of mycobacteria in culture. Its performance was compared to those of the radiometric BACTEC 460 instrument and egg-based Lowenstein-Jensen medium. An identical volume of sample was inoculated in different media, and incubation was carried out for 6 weeks with the automatic systems and for 8 weeks on solid media. A total of 2,567 specimens obtained from 1,631 patients were cultured in parallel. Mycobacteria belonging to nine different taxa were isolated by at least one of the culture systems, with 75% of them being represented by Mycobacterium tuberculosis complex. The best yield was obtained with the BACTEC 460 system, with 201 isolates, in comparison with 190 isolates with the BACTEC MGIT 960 system and 168 isolates with Lowenstein-Jensen medium. A similar but not significant difference was obtained when the most-represented organisms, the M. tuberculosis complex, Mycobacterium xenopi, and the Mycobacterium avium complex, were analyzed separately and when combinations of a solid medium with the BACTEC MGIT 960 system and with the BACTEC 460 system were considered. The shortest times to detection were obtained with the BACTEC MGIT 960 system (13.3 days); 1.5 days earlier than that with the BACTEC 460 system (14.8 days) and 12 days earlier than that with Lowenstein-Jensen medium (25.6 days). The BACTEC MGIT 960 system had a contamination rate of 10.0%, intermediate between those of the radiometric system (3.7%) and the egg-based medium (17.0%). We conclude, therefore, that the BACTEC MGIT 960 system is a fully automated, nonradiometric instrument that is suitable for the detection of growth of tuberculous and other mycobacterial species and that is characterized by detection times that are even shorter than that of the "gold standard," the BACTEC 460 system. The contamination rate was higher than that for the radiometric BACTEC 460 system and needs to be improved.

Multicenter evaluation of the MB-Redox medium compared with radiometric BACTEC system, mycobacteria growth indicator tube (MGIT), and Löwenstein-Jensen medium for detection and recovery of acid-fast bacilli.

MB-Redox is a new manual culture system designed for the recovery of mycobacteria from clinical specimens. It consists of a liquid medium (modified Kirchner medium) containing a redox indicator, a colorless tetrazolium salt, which is reduced to colored formazan by actively growing mycobacteria. Acid fast bacilli (AFB) are easily detected in the medium as pink to purple pinhead-sized particles. We report the results of a multicenter study (involving four Italian microbiology laboratories processing 2370 clinical specimens) aiming to evaluate the recovery rates of AFB and time required for their detection by using the MB-Redox medium. Two different protocols were set up: in Protocol A (1580 specimens) the performance of MB-Redox was compared with those of the radiometric BACTEC 460 TB system (B460) and Löwenstein-Jensen medium (L-J), whereas in Protocol B (790 specimens) it was compared with those of the Mycobacteria Growth Indicator Tube (MGIT) and L-J. A total of 213 mycobacteria were recovered, including 172 Mycobacterium tuberculosis complex (MTB) isolates and 41 nontuberculous mycobacteria (NTM) isolates. In Protocol A, recovery rates were 81% for MB-Redox system, 84% for B460 system, and 77% for L-J. In Protocol B the recovery rates by individual system were 87, 83, and 76% for MB-Redox, MGIT, and L-J, respectively. Differences in both the protocols were not statistically significant. The MB-Redox system plus L-J (Combination 1) recovered 94% of the isolates in Protocol A and 93% in Protocol B, while B460 plus L-J (Combination 2) and MGIT plus L-J (Combination 3) detected 91 and 89% of all mycobacteria isolates respectively. No statistically significant differences were found among the combinations. The mean time to detection of mycobacteria was 16.3 days in Protocol A and 19.1 days in Protocol B with the MB-Redox system, 22.4 and 25.9 days with L-J, 13.2 days with B460, and 18.2 days with MGIT. The contamination rates were 2.1, 2.0, 1.9, and 3.6 for MB-Redox, B460, MGIT, and L-J respectively. The MB-Redox is a reliable, nonradiometric system for growth and detection of mycobacteria. When used in combination with a solid medium it proved to be an effective replacement for B460. The MB Redox system is a labor-intensive method requiring much handling during the visual reading procedures.

Activity of 16 antimicrobial agents against drug-resistant strains of Mycobacterium tuberculosis.

The in vitro activity of 16 antimicrobial agents against 46 drug-resistant strains of Mycobacterium tuberculosis recently isolated from Italian patients was determined. As for first-line antituberculosis drugs, while isoniazid was ineffective against all the strains tested, resistance to streptomycin, rifampicin, pyrazinamide, and ethambutol was 80.4%, 71.7%, 39.1%, and 8.7%, respectively. Among second-line antituberculous drugs, resistance to ciprofloxacin, ofloxacin, and sparfloxacin and to amikacin and kanamycin was around 20%. About 10% of the strains were resistant to capreomycin and cycloserine and 4.3% were resistant to ethionamide; no strain was found to be resistant to thiacetazone, para-aminosalicylic acid, and viomycin. Although all strains displayed a rather continuous distribution of minimal inhibitory concentrations (MICs), a bimodal distribution was observed for rifampicin, amikacin, and kanamicin, with very high MIC values for resistant strains; relatively low MICs were found for fluoroquinolone-resistant strains. Among the small number of strains resistant to second-line agents, low resistant levels were observed. Restriction fragment length polymorphism analysis showed few strain clusters with resistance to first-line antituberculous drugs and aminoglycosides, fluoroquinolones, or both. Altogether, these results showed that second-line agents were still active against the isoniazid-resistant and multiply first-line resistant strains tested, with none or low resistance levels; these observations can be of importance for the treatment of multidrug-resistant tuberculosis in Italy.

Comparative evaluation of the new gen-probe Mycobacterium tuberculosis amplified direct test and the semiautomated abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens.

Two commercial assays that detect Mycobacterium tuberculosis complex (MTB) in clinical specimens by rRNA target amplification (AMTDII) and ligase chain reaction (LCx) were evaluated. The tests were applied to 457 respiratory (n = 273) and extrapulmonary (n = 184) specimens collected from 357 patients. The results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered to be the "gold standard." Seventy specimens were from patients with pulmonary tuberculosis and 28 specimens were from patients with extrapulmonary tuberculosis. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens were 92.8, 99.4, 98.5, and 97%, respectively, for AMTDII and 75.7, 98.8, 96.4, and 90.5%, respectively, for LCx. With extrapulmonary specimens, the overall sensitivities, specificities, and positive and negative predictive values were 78.6, 99.3, 95.6, and 96.2%, respectively, for AMTDII and 53.6, 99.3, 93.7, and 92.1%, respectively, for LCx. The level of agreement between AMTDII and LCx assay results was 78.2%. We conclude that although both nucleic acid amplification methods are rapid and specific for the detection of MTB in clinical specimens, AMTDII is significantly more sensitive than LCx with both respiratory (P = 0.005) and extrapulmonary (P = 0.048) specimens.