Detection of Mycobacterium avium in blood samples of patients with AIDS by using PCR.
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ABSTRACT:
SUBMITTER: Rodrigues Vde F
PROVIDER: S-EPMC130774 | biostudies-literature | 2002 Jun
REPOSITORIES: biostudies-literature
Publications
Detection of Mycobacterium avium in blood samples of patients with AIDS by using PCR.
Rodrigues Vívian de F Sumnienski Vde F Queiroz Mello Fernanda C FC Ribeiro Marta Osório MO Fonseca Leila L Kritski Afrânio Lineu AL Rossetti Maria Lucia ML Zaha Arnaldo A
Journal of clinical microbiology 20020601 6
Sixty-nine blood samples from 47 patients infected with human immunodeficiency virus were analyzed by using PCR to detect Mycobacterium avium. The sensitivity can be up to 95.7%, depending on the detection method used and the number of blood samples analyzed from each patient. The procedure can be helpful in the diagnosis of mycobacterial disease. ...[more]
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Project description: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 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.
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