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.

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.

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.

Evaluation of a new method for rapid drug susceptibility testing of Mycobacterium avium complex isolates by using the mycobacteria growth indicator tube.

The reliability of the Mycobacteria Growth Indicator Tube (MGIT [BBL]) for rapid drug susceptibility testing of Mycobacterium avium complex (MAC) isolates was evaluated. MICs of amikacin, clarithromycin, clofazimine, ethambutol, and rifabutin were determined by the MGIT system for 16 MAC strains. The results were compared with those obtained by the BACTEC broth macrodilution method. The turnaround times were 6 to 8 days (median, 7 days) for the MGIT and 5 to 7 days (median, 6 days) for the BACTEC system. Agreements with BACTEC system-determined MICs, within +/-1 log2 dilution, were 100, 100, 88, 63, and 44% for amikacin, clofazimine, rifabutin, clarithromycin, and ethambutol, respectively. Within +/-2 log2 dilutions, agreement with BACTEC system-determined MICs increased to 100% for all the tested drugs. In addition, if MGIT-determined MICs were evaluated according to the thresholds adopted for the interpretation of BACTEC system-determined ones, ethambutol was the only drug for which susceptible strains were frequently misclassified as resistant. It is concluded that the MGIT system is a promising, nonradiometric alternative to the BACTEC method for rapid susceptibility testing of MAC isolates; however, additional studies are required to confirm our results and to determine the optimal criteria for the interpretation of ethambutol MICs.

Isolation of Mycobacterium celatum from patients infected with human immunodeficiency virus.

Mycobacterium celatum is a recently described, slowly growing mycobacterium of still undefined clinical relevance. A retrospective study of seven patients was conducted to further elucidate the clinical presentation and prognosis of infection due to M. celatum in patients with AIDS. Three patients had an exclusively pulmonary infection and 3 had disseminated infection (including 2 patients with pulmonary and extrapulmonary involvement), and 1 patient had an exclusively extrapulmonary disease. Fever, weight loss, and productive cough lasting for >2 weeks were the most common symptoms. Chest radiographs showed diffuse or focal interstitial infiltrates without cavitation. The recovery of M. celatum from one patient was definitively determined to be clinically irrelevant. Our findings indicate that M. celatum may cause serious disease in patients with advanced human immunodeficiency virus-related immunosuppression. M. celatum infection appears to be responsive to antimycobacterial chemotherapy; however, further studies are needed to establish the optimal drug combination for this indication.

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

Two commercial assays detecting the presence of Mycobacterium tuberculosis complex in clinical specimens by rRNA target amplification (Gen-Probe Amplified M. tuberculosis Direct Test [AMTD]) and PCR (Amplicor) were evaluated. The tests were applied to 327 digested, decontaminated respiratory specimens collected from 236 patients. Results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered the "gold standard." A total of 60 specimens were collected from 27 patients with a diagnosis of pulmonary tuberculosis. Thirteen of these specimens were from patients receiving standard antituberculosis therapy and therefore were not included in the comparison. Of the remaining 47 specimens, 33 were smear positive, 40 were culture positive, 45 were AMTD positive, and 39 were Amplicor positive. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values were 77, 100, 100, and 95 for staining; 87, 100, 100, and 97.4 for culture; 95.9, 98.9, 94, and 99.2 for AMTD; and 85.4, 99.6, 97.9, and 97.1 for Amplicor, respectively. Agreement between AMTD and Amplicor assay results was 96.8%. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection of M. tuberculosis complex in respiratory specimens, AMTD appeared to be more sensitive than Amplicor.

Activity of seven antimicrobial agents, alone and in combination, against AIDS-associated isolates of Mycobacterium avium complex.

The activity of seven antimicrobial agents (and five two-drug combinations and five three-drug combinations) was investigated against 37 clinical isolates of Mycobacterium avium recovered from blood cultures of AIDS patients. The susceptibility tests were performed in Middlebrook 7H12 broth using a radiometric method. MICs of amikacin, ciprofloxacin, clarithromycin, clofazimine, ethambutol, rifabutin and sparfloxacin were determined. Five antimicrobial agents were tested in combination with clarithromycin and also with clarithromycin plus amikacin to look for possible synergic activity. Synergic activity in combination with clarithromycin and with clarithromycin plus amikacin, was detected for rifabutin (54% and 51% of isolates, respectively), clofazimine (38% and 35%), ethambutol (16% and 32%), ciprofloxacin (8% and 14%) and sparfloxacin (3% and 8%). No antagonism was observed. We conclude that clarithromycin is an essential component in the chemotherapy of M. avium complex disease.

Comparison of two selective media and a membrane filter technique for isolation of Campylobacter species from diarrhoeal stools.

Diarrhoeal stool specimens from 415 patients were examined for Campylobacter spp. by culture on charcoal cefoperazone deoxycholate agar (CCDA), Skirrow medium and Columbia blood agar overlaid with a 0.65 micron pore size membrane filter. Forty-eight Campylobacter strains were isolated from 45 (10.8%) specimens by all media; 44 were Campylobacter jejuni (91.7%), three were Campylobacter coli (6.3%) and one was Campylobacter hyointestinalis (2.0%). The percentages of Campylobacter-positive specimens isolated on Skirrow medium, CCDA and the membrane filter were 62, 82 and 95%, respectively. The recovery of more Campylobacter spp. from the same stool sample was achieved by the membrane filter method only. The highest isolation rate (100%) was observed when culture on CCDA and the membrane filter method were combined.

Bacteriostatic and bactericidal activities of paromomycin against Mycobacterium avium complex isolates.

The MICs and MBCs of paromomycin for 32 Mycobacterium avium complex (MAC) strains isolated from patients with the acquired immunodeficiency syndrome were determined by a radiometric broth dilution method. The MICs for the majority of strains were either 8 or 16 mg/L and the MBCs were four- to eight-fold higher. Paromomycin merits further evaluation as oral prophylaxis against disseminated MAC infection.

In vitro activity of the new quinolone lomefloxacin against Mycobacterium tuberculosis.

Minimal inhibitory concentrations (MIC) of ciprofloxacin, ofloxacin and lomefloxacin were determined for 90 Mycobacterium tuberculosis strains isolated from both AIDS and other patients. Eleven (12.2%) of these strains showed in vitro resistance to one or more first-line antituberculosis drugs. Susceptibility tests were done in 7H12 broth by the radiometric method. The MIC range for ciprofloxacin was 0.125 to 4.0 micrograms/ml; for ofloxacin, 0.25 to 4.0; and for lomefloxacin 0.5 to 4.0 micrograms/ml. On the basis of our data, we believe that the following MIC, when determined in 7H12 broth radiometrically, should be used as break points to classify the strain as susceptible: ciprofloxacin and ofloxacin, 1 microgram/ml or less; lomefloxacin, 2 micrograms/ml or less. Lomefloxacin on a once-daily basis deserves further evaluation as a potential supplementary drug for the treatment of tuberculosis.