Interpretation of restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from a state with a large rural population.

Epidemiologic relatedness of Mycobacterium tuberculosis isolates from Arkansas residents diagnosed with tuberculosis in 1992-1993 was assessed using IS6110- and pTBN12-based restriction fragment length polymorphism (RFLP) and epidemiologic investigation. Patients with isolates having similar IS6110 patterns had medical records reviewed and were interviewed to identify epidemiologic links. Complete RFLP analyses were obtained for isolates of 235 patients; 78 (33%) matched the pattern of > or = 1 other isolate, forming 24 clusters. Epidemiologic connections were found for 33 (42%) of 78 patients in 11 clusters. Transmission of M. tuberculosis likely occurred many years in the past for 5 patients in 2 clusters. Of clusters based only on IS6110 analyses, those with > or = 6 IS6110 copies had both a significantly greater proportion of isolates that matched by pTBN12 analysis and patients with epidemiologic connections, indicating IS6110 patterns with few bands lack strain specificity. Secondary RFLP analysis increased specificity, but most clustered patients still did not appear to be epidemiologically related. RFLP clustering in rural areas may not represent recent transmission.

Retrospective detection of laboratory cross-contamination of Mycobacterium tuberculosis cultures with use of DNA fingerprint analysis.

In 1992-1993, we investigated possible cross-contamination of Mycobacterium tuberculosis cultures as part of a study of tuberculosis in Arkansas by using DNA fingerprint analysis. Of patients whose isolates were matched, those for whom smears were negative and only one culture was positive were identified from laboratory records. Clinical, laboratory, DNA fingerprint, and epidemiological data were reviewed. Of 259 patients, nine (3.5%) were judged to be due to cross-contamination. None of these patients had a clinical course consistent with tuberculosis. All nine specimens were processed with another isolate with a matching DNA fingerprint, and epidemiological connections were not identified among any of the patients. To avoid erroneous diagnoses and unnecessary therapy and public health investigations, specimens from patients with tuberculosis whose smears are negative and only one culture is positive should be investigated for cross-contamination. An inconsistent clinical course and a DNA fingerprint that matches those of other culture-positive specimens processed concurrently, coupled with the lack of an epidemiological connection, suggest cross-contamination.

Strand displacement amplification and the polymerase chain reaction for monitoring response to treatment in patients with pulmonary tuberculosis.

Specific amplification of Mycobacterium tuberculosis DNA was investigated as an alternative to conventional microbiologic follow-up in 31 cases of smear- and culture-positive pulmonary tuberculosis. Strand displacement amplification (SDA) and the polymerase chain reaction (PCR) were applied to 438 sequential sputum specimens: 67 (15%) were positive by culture, 248 (57%) by SDA, and 231 (53%) by PCR (chi2=3.94, P=.05). Of 200 specimens collected >180 days after treatment started, none yielded positive cultures, while 50 (25%), representing 16 patients, were positive by both DNA assays. A weak correlation was demonstrated between DNA persistence in sputum and duration of culture positivity (r=0.45, P=.01), although no correlation was found with the radiographic extent of disease. The inability to distinguish live and dead organisms precludes DNA amplification from use in therapeutic monitoring. For this purpose, quantitative RNA assays are needed if such techniques are to supplant conventional microbiology.

The risk for transmission of Mycobacterium tuberculosis at the bedside and during autopsy.

OBJECTIVE: To emphasize the differing infectious potentials of a patient with tuberculosis. SETTING: Hospital ward and autopsy room. DESIGN: An epidemiologic investigation of tuberculin skin test conversions in a clinical setting and during autopsy when results of tuberculin tests done before exposure were available for all participants. MEASUREMENTS: Tuberculin skin test results after the discovery of tuberculosis exposure from a patient with unsuspected tuberculosis for comparison with the test results before exposure; culture of sputum and autopsy material for Mycobacterium tuberculosis; and DNA fingerprinting of organisms. INTERVENTION: Preventive therapy for persons with skin test conversion. RESULTS: None of the 40 skin test-negative health care workers caring for the patient for 3 weeks on an open medical ward showed a skin test conversion, even though they had not used respiratory precautions. By contrast, among personnel present during the 3-hour autopsy, the test results of all five nonreactors converted from negative to positive (mean reaction, 24 mm). Two of these persons had a positive sputum culture 8 weeks later. The DNA fingerprints of all three isolates were identical. CONCLUSIONS: A patient who did not transmit tuberculosis before death released a prodigious number of tubercle bacilli during autopsy.