M Kumar1, S Sharma, A B Ram, I A Khan. 1. Clinical Microbiology Division, Indian Institute of Integrative Medicine (Council of Scientific & Industrial Research), Jammu, India. manojmanu80@yahoo.com
Abstract
BACKGROUND: Polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis in clinical samples requires the use of an extraction method that can efficiently lyse mycobacterial cells and recover small amounts of DNA. OBJECTIVE: To evaluate the use of a benzyl-alcohol guanidine hydrochloride (DNA extraction) method (GuHClM) on blood samples. DESIGN: GuHClM was evaluated in quantitatively spiked blood samples with M. tuberculosis. We assessed the insertion sequence (IS) 6110 region of M. tuberculosis to evaluate the efficacy of the method. The method was also applied on 102 clinical samples of suspected tuberculosis (TB) individuals and compared with smear microscopy of sputum specimens and the results of cultures. RESULTS: This method reproducibly detected as low as 4-6 bacilli. Of 102 clinical samples, 84 were human immunodeficiency virus (HIV) negative, while 18 were HIV-positive. Among the HIV-negative individuals, 58.3% were TB-positive using PCR, while respectively 47.6% and 45.2% were sputum- and culture-positive. Among the HIV-positive individuals, 55.6% were PCR-positive, whereas only 38.9% were sputum-positive and 50% were culture-positive. CONCLUSION: These results demonstrate that the identification of mycobacteria by PCR using GuHClM is very sensitive and therefore may have wide utility in the diagnosis of TB.
BACKGROUND: Polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis in clinical samples requires the use of an extraction method that can efficiently lyse mycobacterial cells and recover small amounts of DNA. OBJECTIVE: To evaluate the use of a benzyl-alcohol guanidine hydrochloride (DNA extraction) method (GuHClM) on blood samples. DESIGN: GuHClM was evaluated in quantitatively spiked blood samples with M. tuberculosis. We assessed the insertion sequence (IS) 6110 region of M. tuberculosis to evaluate the efficacy of the method. The method was also applied on 102 clinical samples of suspected tuberculosis (TB) individuals and compared with smear microscopy of sputum specimens and the results of cultures. RESULTS: This method reproducibly detected as low as 4-6 bacilli. Of 102 clinical samples, 84 were human immunodeficiency virus (HIV) negative, while 18 were HIV-positive. Among the HIV-negative individuals, 58.3% were TB-positive using PCR, while respectively 47.6% and 45.2% were sputum- and culture-positive. Among the HIV-positive individuals, 55.6% were PCR-positive, whereas only 38.9% were sputum-positive and 50% were culture-positive. CONCLUSION: These results demonstrate that the identification of mycobacteria by PCR using GuHClM is very sensitive and therefore may have wide utility in the diagnosis of TB.