Differentiation of mycobacteria in sputa by duplex polymerase chain reaction for mycobacterial hsp65 gene.
Early differentiation of mycobacteria in sputa is crucial. This study was set to evaluate the usefulness of a newly developed duplex polymerase chain reaction (PCR) for hsp65 gene-based method in differentiating mycobacteria in sputum with a positive acid-fast bacilli (AFB) smear before culturing. One hundred forty-seven sputa with positive AFB smear were included for the analysis. Mycobacterial species were identified using a newly developed duplex PCR for hsp65 gene followed by a nested PCR-direct sequencing and the conventional colony-based method. Final decision of mycobacterial species were made based on 1) results of species identification based on mycobacterial colonies or 2) results of species identification of other sputa from the same patients and clinical findings. The duplex PCR-based method correctly identified 83.2% sputa from tuberculosis patients and 82.2% sputa from nontuberculous mycobacteria patients, whereas the colony-based method correctly identified 86.1% and 77.8%, respectively. Sensitivity and specificity of the colony-based method for Mycobacterium tuberculosis were 86.1% and 100%, respectively, whereas those of the duplex PCR-based method were 83.2% and 95.6%, respectively. The duplex PCR-based method, to differentiate mycobacterial species in sputa, produced comparable results as those of the colony-based identification method.
Kim
Bum-Joon
BJ
Department of Microbiology and Immunology, Cancer Research Institute and Liver Research Institute, Seoul National University, Seoul 110-799, Republic of Korea. kbumjoon@snu.ac.kr
Park
Joo-Hee
JH
Lee
Seoung-Ae
SA
Kim
Hong
H
Cha
Chang-Yong
CY
Kook
Yoon-Hoh
YH
Kim
Eui-Chong
EC
Joo
Sei Ick
SI
Lee
Jae Seok
JS
Yim
Jae-Joon
JJ
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D003937:Diagnosis, Differential; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013183:Sputum / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins