Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction

Abstract

The emergence of epidemic multiple-drug-resistant (MDR) strains of Mycobacterium tuberculosis in conjunction with an increase in the number of reported cases of tuberculosis (TB) represents a major public health problem. In light of a recent outbreak of MDR M. tuberculosis at our center, we began the development of a polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary TB using two sets of primers, one based on the IS6110 repeated sequence of M. tuberculosis and the other based on the protein antigen b (PAB). Reaction conditions were first optimized as to the appropriate extraction protocol and the concentrations of primer pairs, nucleotides, and MgCl2. Following a preliminary evaluation of the assay with clinical specimens, extraction and amplification procedures were further modified. PAB and IS6110 primers detected between 2 and 23 and 0.023 and 0.23 CFU of M. tuberculosis, respectively, in pooled, M. tuberculosis-negative sputa by our optimized PCR assay. After routine processing for mycobacteria, 734 specimens were subsequently amplified. DNA for amplification was obtained by boiling and beating the sediments with Tween 20. For each reaction, DNA (10 ,ul) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 JLM nucleotides, and 2.5 U of Taq polymerase and the mixture was then amplified for 40 cycles. The sensitivity and specificity of our PCR assay were 87.2 and 97.7%, respectively. We were unable to interpret the results for seven specimens (1%). In our experience, PCR proved to be a useful rapid diagnostic test for TB in a clinical setting and a valuable epidemiological tool for determining exposure groups in the hospital setting. Our findings also underscore the need for the systematic optimization of PCR assay conditions.

Multiprimer PCR system for differential identification of mycobacteria in clinical samples

“…A recent blinded study comparing the results obtained for specially prepared standardized samples by different laboratories and different PCR methods demonstrated impressive differences in the results obtained and numerous deficiencies in both sensitivity an specificity (21). The most widely used target sequence for the diagnosis of tuberculosis has been the IS6110 insertion element present in different numbers of copies in the genomes of species belonging to the M. tuberculosis complex (11,17,29). In fact, PCR tech- niques based on this sequence have been shown to be of great value.…”

Diagnosis of Infectious Diseases: a Cytopathologist’s Perspective

“…Material can be preserved in a similar fashion for molecular diagnostic methods including fluorescent in situ hybridization and PCR. Recent advances in PCR have allowed the detection of infectious agents in cytologic material (4,44,45,48,83,86,162). Although the quantity of material may be a consideration in certain circumstances, most immunodiagnostic and molecular techniques today have protocols adapted to cytologic specimens.…”

“…In these aspirates, the presence of numerous organisms both within distended histiocytes and scattered in the background tends to be from mycobacterial infections other than tuberculosis. Although material is usually obtained for culture or fluorescence microscopy, PCR can now be used for the rapid detection and identification of M. tuberculosis (44,48,86).…”

Polymerase chain reaction test in induced sputum of patients with pulmonary tuberculosis

“…[33][34][35][36] Also, when culture is used as the gold standard test, specimens containing non-cultivable organisms may lead to a positive PCR and be considered as false-positive result. 37,38 To support the hypothesis that PCR is more sensitive than culture, it would be useful to have 2 or more positive PCR results in patients with negative cultures, 36 16 demonstrated that the yield of PCR test increased with the greater number of IS samples that were evaluated. However, when we considered the clinical diagnosis together with PCR/culture results, we found that almost 90% of patients with PCR-positive/culture-negative samples had a clinical diagnosis of TB and were treated with anti-TB drugs, supporting the hypothesis that PCR is more sensitive than culture.…”