Biomedical subjects
Publications and source records attributed to Erica Engelke.
Concern has been raised about the efficacy of the heat killing of mycobacteria during the isolation of DNA. We demonstrate a method that allows for the efficient killing of Mycobacterium tuberculosis without compromising DNA integrity for subsequent molecular investigation. This method reduces the risk of infection to the research scientist.
In an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled. Mycobacterium tuberculosis strains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple M. tuberculosis strains in the context of the molecular epidemiology of the study setting.
Our laboratory, engaged in a prospective study of adult pulmonary tuberculosis, processed on average 1186 sputum samples per year for the detection of Mycobacterium tuberculosis (M. tuberculosis). Approximately 55% of all sputum samples were culture-positive. The study protocol required that all patients had their M. tuberculosis isolates DNA fingerprinted at diagnosis, and at subsequent time points if the patients either failed treatment or presented again with tuberculosis. Over a 22-month period, there were 14 apparent treatment failures from 109 patients who had completed 6 months of therapy. Only two of these were true treatment failures, while the other 12 had DNA fingerprints that were different from those obtained at diagnosis. It was concluded that these 12 cultures represented episodes of laboratory cross-contamination. Retrospective DNA fingerprinting of patient isolates was done so that each patient had at least two independent isolates fingerprinted. This survey revealed that 7.3% of DNA fingerprints were discordant. False-positive cultures with discordant DNA fingerprints generally arose late in chemotherapy and the isolates were usually co-processed with other strongly smear-positive sputum samples. Simple modifications of laboratory procedures were made, and over a following 10.5-month period the false-positive rate was reduced to 2.1%. These modifications did not increase the workload or the cost of processing samples and can thus be used successfully by any laboratory, and particularly by those in resource-poor settings.