Use of potassium tellurite for rapid-drug susceptibility testing of Mycobacterium avium complex.

BACKGROUND: Mycobacterium avium complex (MAC) is a major cause of infection in immunocompromised patients. MAC possesses an enzyme that reduces potassium tellurite in less than 3 days and results in formation of a black precipitate. The objective of this study was to determine whether reduction of potassium tellurite by mycobacteria can be used as a means of testing the susceptibility of MAC to clarithromycin. METHODS: Minimum inhibitory concentrations (MICs) for 104 clinical isolates of MAC were determined by the tellurite method and compared with those tested by a recommended microdilution method. Microdilution breakpoints were used for interpretation of susceptibility. MIC of less than 8 microg/mL was considered as susceptible, and MIC of greater than or equal to 8 microg/mL was resistant. RESULTS: There was agreement within a 2-fold dilution between MICs for 89% of isolates. Of the 53 isolates that had discrepant MICs by the two methods, 70% had higher MICs by the tellurite method. When the MICs were classified into interpretive categories, there was 100% agreement by the two methods. The MIC tested by the tellurite method was available within 5 days. CONCLUSIONS: These data suggest that use of potassium tellurite is a more rapid, reliable, and inexpensive method of testing the susceptibility of MAC to clarithromycin.

Rapid differentiation of Mycobacterium tuberculosis and Mycobacterium bovis using glycerol susceptibility and quantitative polymerase chain reaction.

BACKGROUND: The differentiation between Mycobacterium tuberculosis (MTB) and Mycobacterium bovis has significant epidemiological and therapeutic implications, such as their susceptibility to different drugs and the need for different approaches to patient isolation. However, currently this differentiation is based on laboratory methods that take 8 to 16 weeks. Simple qualitative polymerase chain reaction (PCR) can not differentiate between MTB and M bovis because of their genetic similarities. METHODS: We evaluated the use of quantitative PCR (QPCR) and glycerol susceptibility to differentiate MTB from M bovis. Three patient isolates of M bovis and 5 patient isolates of MTB were suspended in 7H9 broth containing 0.0%, 0.2%, or 0.6% glycerol. These suspensions were inoculated on 7H11 plates and incubated at 37 degrees C and 5% CO2. QPCR was performed after 0, 3, and 6 days of incubation. RESULTS: After 6 days of incubation for M bovis isolates, the mean DNA concentration decreased by one log in the presence of 0.6% glycerol while for MTB isolates, the mean DNA concentration increased by one log regardless of the glycerol concentration. CONCLUSION: These data suggest that QPCR and glycerol susceptibility may be used to differentiate between MTB and M bovis within 1 week.

Comparison of quantitative polymerase chain reaction, acid fast bacilli smear, and culture results in patients receiving therapy for pulmonary tuberculosis.

Quantitative-competitive polymerase chain reaction (QPCR) was performed on serial sputum samples from 22 consecutive cases of acid fast bacilli (AFB) smear-positive pulmonary tuberculosis. Of 94 specimens, 55, 72, and 83% were positive by culture, AFB smear, and QPCR, respectively. Of 52 culture-positive specimens, 6% were negative by PCR, and 13% were negative by AFB smear. Of 42 culture-negative specimens, AFB smear and QPCR were positive in 55 and 61%, respectively. AFB smear and QPCR results were strongly correlated (r = 0.75, p < 0.001), but each correlated less strongly with culture (r = 0.54, p < 0.005 for smear and r = 0.52, p < 0.005 for QPCR). When patients were classified by microbiologic response, responders tended to have less DNA in their sputum and shorter time to a negative PCR result compared to nonresponders. These data do not suggest a great advantage of QPCR over AFB smear for predicting culture results in patients with pulmonary tuberculosis.

Quantitative-competitive polymerase chain reaction for rapid susceptibility testing of Mycobacterium tuberculosis to isoniazid.

The objective of this study was to determine whether quantitative-competitive polymerase chain reaction (QC-PCR) can be used for rapid susceptibility testing of Mycobacterium tuberculosis (MTB). QC-PCR was used to determine relative amounts of mycobacterial DNA inoculated at different isoniazid (INH) concentrations. A total of six different INH-sensitive (INH-S) and five INH-resistant (INH-R) strains were inoculated in the presence of 0.0, 0.2, 1.0, and 10.0 micrograms/ml of INH. DNA was quantified using QC-PCR at Week 0 and weekly thereafter for 3 weeks. For the QC-PCR, 10-fold dilutions of control (240 bp) DNA having the same primer set as the target DNA (123 bp) were used. The amount of target DNA was estimated by using known amounts of the internal standard. For INH-S isolates there was > or = 1 log difference in DNA concentration in the presence of each INH concentration compared to that of the control within 1 to 3 weeks. In contrast, for INH-R isolates there were no apparent differences in DNA concentration between the control suspensions and those containing 0.2 and 1.0 microgram/ml INH during the 3-week incubation period. The highest INH concentration (10 micrograms/ml), however, did abolish the DNA increase seen in the other MTB suspensions. This preliminary study suggests that by using concentrations of 0.2 or 1.0 microgram/ml of INH, QC-PCR may differentiate INH-R and INH-S MTB isolates within 1 week. This method may be of particular value when applied directly to clinical specimens with varying numbers of bacilli.

Polymerase chain reaction for diagnosis of M. tuberculosis: comparison of simple boiling and a conventional method for DNA extraction.

Although the polymerase chain reaction (PCR) has been used increasingly for rapid diagnosis of tuberculosis (TB) in clinical specimens, no consensus exists regarding DNA extraction protocols. We compared a simple boiling method to a conventional, labor-intensive chemical method using lysozyme and silica particles. The boiling method was performed in less than 30 min; the chemical method required at least 6 h. A total of 82 clinical specimens (mostly respiratory) from 77 patients were obtained after routine processing from the microbiology laboratory. After DNA extraction by each method, PCR was performed to detect the 123-bp segment of IS6110, and results were compared to culture. Of 20 culture-positive specimens, 17 (85%) and 12 (60%) were positive by boiling and chemical methods, respectively. Of 62 culture-negative specimens, 61 (98%) and 57 (92%) were negative by boiling and chemical methods, respectively. The sensitivity was 100 and 92% for the boiling and chemical methods, respectively, for smears containing more than rare AFB. Our results suggest that boiling method of DNA extraction is more sensitive and no less specific than a conventional chemical method. Larger studies including a variety of clinical specimens are necessary to standardize the optimal conditions of PCR for diagnosis of M. tuberculosis.

Paradoxical enlargement or development of intracranial tuberculomas during therapy: case report and review.

Intracranial tuberculomas can sometimes develop or increase in size despite administration of appropriate therapy. We report the case of a child whose intracranial tuberculomas paradoxically enlarged while therapy was being administered, and we review 23 other cases in which tuberculomas increased in size or number and 17 cases in which tuberculomas appeared during therapy. These phenomena generally occurred within 3 months of the start of therapy. All but four patients had neurological deterioration that prompted obtaining a repeated computed tomographic scan. One patient died, about one-fourth of patients had residual neurological symptoms, and less than one-third of the patients required surgical intervention. Most patients received a 12-18 month course of antituberculous therapy. Adjunctive therapy with steroids appears to diminish neurological symptoms and may improve outcome. Paradoxical enlargement or development of tuberculomas usually does not represent failure of antituberculous therapy; the most likely explanation for these phenomena is an interaction between the host's immune response and the direct effects of mycobacterial products.