A colorimetric method for quantitative measurement of the susceptibility of Mycobacterium tuberculosis to antimicrobial agents is described. The method utilizes an oxidation-reduction dye, Alamar blue, as an indicator of growth. By this method, MICs of isoniazid, rifampin, streptomycin, and ethambutol were determined for 50 strains of M. tuberculosis. Colorimetric MIC results were available on the 7th, 10th, or 14th day of incubation for 29 (58%), 14 (28%), and 7 (14%) of the 50 strains, respectively. When MIC susceptibility results were compared with results obtained by the agar proportion method, increased levels of resistance detected by agar proportion were associated with higher MICs obtained by the colorimetric method. Tentative interpretive criteria for colorimetric MIC results which showed good agreement with results obtained by the agar proportion method were established. Interpretive agreement between the two methods was 98% for isoniazid, rifampin, and ethambutol and 94% for streptomycin. Overall, there was agreement between the two methods for 194 of 200 test results (97%). The colorimetric method is a rapid, quantitative, nonradiometric method for determining the antimicrobial susceptibility of M. tuberculosis.

Several studies have reported using methods based on polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in respiratory tract specimens. However, little is known about the actual clinical utility of PCR-based tests, and it is uncertain if PCR technology can be transferred to the clinical laboratory. To determine its utility, we evaluated a commercially developed PCR test system in a clinical laboratory using consecutive respiratory tract specimens. Microscopic examination of smears stained with acid-fast bacilli (AFB), culture, and a PCR-based test (Amplicor Mycobacterium tuberculosis assay; Roche Molecular Systems) were used to evaluate 535 consecutive sputum and bronchoalveolar lavage specimens from 227 patients. A clinical case definition of tuberculosis was used as the reference-standard to determine the utility of all diagnostic tests. For all specimens from patients with a new or a treatment-failure case of pulmonary tuberculosis, the positivity rate of PCR (58%) was similar to that of culture (56%) (p > 0.90) and substantially greater than microscopic examination of AFB-stained smears (22%) (p < 0.001). PCR and culture detected M. tuberculosis in 46 and 43%, respectively, of the specimens from patients who did not have AFB on microscopic examination of their respiratory tract specimens (p > 0.90). PCR had a false positive rate of 0.8%. In several instances, PCR detected M. tuberculosis when culture did not; and vice versa. The clinical utility of this PCR-based test is similar to that of culture for detecting M. tuberculosis in respiratory tract specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

The value of the smear for acid-fast bacilli in predicting pulmonary tuberculosis is unclear in a setting where there is a high prevalence of Mycobacterium avium complex in respiratory specimens. To evaluate the impact of a high prevalence of M. avium complex on the predictive value of the acid-fast bacilli smear for tuberculosis, we reviewed findings on smears and results of cultures over a 3-year period at a hospital where M. avium complex is the predominant mycobacterial isolate. In this setting, the predictive value of the acid-fast bacilli smear for Mycobacterium tuberculosis was 92% for expectorated sputum specimens, 71% for induced sputum specimens, and 71% for bronchoalveolar lavage specimens. When multiple specimens collected from the same patient were excluded from the data base, the predictive values were 87%, 70%, and 71%, respectively. Smears of sputum samples were positive at the same rate for patients with tuberculosis who had AIDS and for patients with tuberculosis who did not have AIDS.

A commercially available enzyme immunoassay for the diagnosis of giardiasis was evaluated in a clinical trial. The ProSpecT/Giardia diagnostic test (Alexon, Inc., Mountain View, Calif.) was compared with the standard ova and parasite (O&P) microscopic examination. Additionally, several widely used stool fixatives and a commonly used transport medium were assessed for compatibility with the immunoassay. A total of 325 stool specimens were collected and used to evaluate assay performance. Of those, 93 specimens were collected from symptomatic Giardia O&P-positive patients and 232 specimens were randomly collected from patients as part of a routine health screening procedure. All 93 Giardia O&P-positive stool specimens were strongly positive by visual and spectrophotometric examination using the immunoassay. Of the 232 randomly collected specimens, 16 were positive by O&P examination and immunoassay, 6 were negative by O&P examination but positive by immunoassay, and 1 was positive by O&P examination and negative by immunoassay. There was substantial supportive evidence that indicated that the six immunoassay-positive, O&P-negative specimens were true-positives. When these six specimens were accepted as true-positives, the immunoassay detected almost 30% more cases of Giardia infection than did O&P examination. Its sensitivity and specificity were 96 and 100%, respectively, while the sensitivity and specificity of O&P examination were 74 and 100%, respectively. The immunoassay also performed well on specimens treated with 10% neutral Formalin, sodium acetate-Formalin fixative, and Cary-Blair transport medium. However, the test was not compatible with polyvinyl alcohol-treated specimens. Overall, the ProSpecT/Giardia test was a sensitive, specific immunoassay which was easy to run and interpret. It offers a simple solution to traditional difficulties encountered in diagnosing Giardia infection.

Pairs of 11 antimicrobial agents were tested in vitro for their ability to act synergistically against three strains of Mycobacterium avium complex isolated from patients with acquired immune deficiency syndrome. From the combinations tested, four drugs (ethambutol, rifampin, ciprofloxacin, and erythromycin) were selected for more extensive study against 20 strains of M. avium complex. The inhibitory and killing synergism obtained with combinations of two, three, or four drugs was assessed by determining the fractional inhibitory concentration index and fractional bactericidal concentration index. Inhibitory synergism occurred against 90 to 100% of the strains for all drug combinations in which ethambutol was included. Killing synergism occurred against 85 to 95% of the strains when ethambutol was used in combinations which included either rifampin or ciprofloxacin. However, killing synergism occurred against only 45% of the strains when drugs were tested at concentrations that can be obtained in patient serum. In other experiments, rifabutin (Ansamycin) gave results that were comparable to those obtained with rifampin. Clofazimine did not show synergistic killing activity at a concentration that is achievable in serum for any of the drugs tested. Our results indicate that there is considerable variability in the antimicrobial susceptibility of M. avium isolates obtained from patients with acquired immune deficiency syndrome. This variability could have significant impact on the clinical response to various therapies.

Levofloxacin, the active L-isomer of the quinolone ofloxacin, is now widely accepted for treatment of multidrug-resistant tuberculosis. Because the drug is now widely used, we sought to establish susceptibility test conditions for Mycobacterium tuberculosis against levofloxacin by the traditional reference method, agar proportion (AP), the commonly used BACTEC 460 radiometric system, and the newer BACTEC MGIT 960 method. To determine the stability of levofloxacin in the two newer test systems (BACTEC 460 and BACTEC MGIT 960), media containing subinhibitory levels of levofloxacin were prepared and stored at 4 and 37°C for 14 days. The stored media were inoculated with H37Rv, and the drug activity was compared to freshly prepared media. Results show that levofloxacin is stable over the course of testing. Next, optimum levofloxacin test concentrations were determined for AP, BACTEC 460, and BACTEC MGIT 960 methods. MICs were determined for 32 pan-susceptible isolates of M. tuberculosis obtained from presumably untreated patients and 14 quinolone-resistant isolates. The levofloxacin-resistant strains either were isolated from patients who remained culture-positive despite treatment with a quinolone agent (six strains) or contained known mutations in gyrA (eight strains). Levofloxacin MICs resulted in a bimodal pattern with values for resistant strains consistently higher than those for pan-susceptible strains. Results show that levofloxacin concentrations of 2 g/ml (BACTEC 460 and BACTEC MGIT 960) and 1 g/ml (AP) inhibited the growth of all pan-susceptible strains while permitting the growth of all levofloxacin-resistant strains. Confirmatory tests with a subset of pan-susceptible and levofloxacin-resistant isolates validated the selected test concentrations.Increasing reports of multidrug-resistant tuberculosis (MDR-TB) and the emergence of TB in human immunodeficiency virus-infected persons have resulted in the need for new anti-TB agents (3). The fluoroquinolones, especially ofloxacin, have been shown to be highly useful as second-line anti-TB agents against drug-resistant strains. Studies have demonstrated that levofloxacin (LVX), the L-isomer of ofloxacin, is nearly twice as active against Mycobacterium tuberculosis in vitro as its parent compound ofloxacin (8,9,15). Because of its effectiveness, LVX has become widely used as a second-line anti-TB agent to treat patients with MDR-TB.In response to the use of LVX for treatment of MDR-TB, the need to establish laboratory protocols for susceptibility testing of LVX has developed. To address this need, we investigated the conditions necessary to obtain valid LVX susceptibility results with the BACTEC 460 radiometric system (10) and the newer BACTEC MGIT 960 nonradiometric system (13), based on the previously published drug stability studies done by the agar proportion (AP) method (4).Once susceptibility test conditions were determined, critical testing concentration levels for LVX for the three methods were established with fluoroquinolone-resistant and -susceptibl...

Organ donors are not routinely screened for tuberculosis (TB) in the United States. We investigated a case of pulmonary TB in a double-lung transplant recipient. We reviewed the donor's and recipient's records, and used molecular methods to compare the lung recipient's isolate with others from three sources: her hospital, the California state health department's genotyping database, and the donor's resident-nation of Guatemala. A respiratory specimen obtained from the lung recipient 1 day after transplantation grew Mycobacterium tuberculosis. Donor chest radiograph had a previously unnoticed pulmonary opacity that was present on post-transplant recipient chest radiographs and computed tomographs. The recipient's isolate was molecularly distinct from others at her hospital and in the state database, but was identical to two isolates from Guatemala. Tuberculosis was transmitted from lung donor to recipient. As organ transplantation becomes more common worldwide, similar cases could occur. Screening for TB in potential organ donors should be considered.

No abstract