Use of a multiplex PCR to detect and identify Mycobacterium avium and M. intracellulare in blood culture fluids of AIDS patients

Abstract

The presence of mycobacteria in blood culture fluids (BACTEC) of AIDS patients with positive growth indices (GIs, >20 U) was investigated by using a multiplex PCR to detect and identify members of the genus Mycobacterium, M. avium, M. intracellulare, and M. tuberculosis. Three different methods of extracting mycobacterial DNA from blood culture fluid were compared for use with the multiplex PCR. Mycobacterial cells were pelleted from a small aliquot of blood culture fluid by centrifugation, and the DNA was extracted from cells by heat lysis or a sodium iodide-isopropanol or a phenol-chloroform method. DNAs of different sizes were amplified from a region of the MPB70 gene of M. tuberculosis (372 bp) and from a region of the 16S rRNA gene of members of the genus Mycobacterium (1,030 bp), M. intracellulare (850 bp), or M. avium (180 bp) as a multiplex PCR in a single tube. The amplified DNA products were detected by agarose gel electrophoresis and ethidium bromide staining in all 41 (100%) positive cultures after sodium iodide-isopropanol extraction, in 18 (44%) after heat lysis, and in 5 (12%) after phenol-chloroform extraction. Of the 41 positive cultures, 38 were identified as M. avium and 2 were identified as M. intracellulare by both routine methods and multiplex PCR. The remaining mycobacterium was identified as M. intracellulare by routine methods and as M. avium by the multiplex PCR. Another six blood cultures that were negative for the presence of acid-fast bacilli after Ziehl-Neelson staining were also negative by PCR. The study shows that the multiplex PCR is a useful method for the detection and identification of either M. avium or M. intracellulare in small samples of cultured BACTEC 13A fluid with positive GIs ranging from 21 to 999 U. The average time to a positive GI was 18 days (median, 13 days) and ranged between 8 and 42 days. The multiplex PCR may permit cultured mycobacteria to be identified at an earlier stage than the routine methods which have been adapted for use with the BACTEC system. The results also show that the method selected for extracting mycobacterial DNA from blood culture fluids is crucial for providing sensitive and accurate PCR results.

Identification and differentiation of Mycobacterium avium and M. intracellulare by PCR

“…Our data support the findings of Kulski et al (9) that M. avium and M. intracellulare may be differentiated by specific 16S rRNA primers and PCR technology. Additionally, our data indicate that the currently identified MAC includes strains genetically diverse from M. avium and M. intracellulare.…”

“…In their system, 2 of 32 isolates of M. intracellulare were identified as M. avium by PCR. In their blood culture testing, one of their strains identified as M. intracellulare by a routine method was found to be M. avium (9).…”

Genetic relatedness of Mycobacterium avium-intracellulare complex isolates from patients with pulmonary MAC disease and their residential soils

“…PCR-positive cultures were subcultured on Middlebrook 7H11 agar plates to obtain single colonies. Two colonies were isolated for extraction of DNA to identify Mycobacterial species using PCR [15] and to perform VNTR analysis.…”

Committee Report: Emerging pathogens–bacteria

“…Characteristic mycolic acids and other cell wall lipids of M. avium can be identified using capillary gas chromatography or high‐performance liquid chromatography methods. Commercial DNA probes 34 and multiplex polymerase chain reaction (PCR) techniques 35 can also be used.…”