Accelerated Diagnosis Of Smear-Negative Pulmonary Tuberculosis With Middlebrook 7H12 Semi-liquid Culture PCR(SLCP)

There's a great need of more sensitive and specific method for the rapid diagnosis and monitoring treatment response of tuberculosis nowadays. It has proved that nucleic acid amplification techniques are rapid, sensitive and specific, but the inability of the PCR methods to detect a difference between viable and nonviable organisms limited its widespread use in clinical specimens. At present, it is considered that molecular methods have not been able to replace cultivation for the detection of mycobacteria from clinical specimens. So we developed a new method, named Middlebrook 7H12 semi-liquid culture PCR(SLCP), which combine cuture with Middlebrook 7H12 semi-liquid media and PCR to detect smear-negative sputum from patients with pulmonary tuberculosis, in order to achieve rapid diagnosis and determine viable organisms. At the same time, via this study, we try to show the clinical significance of PCR in initial diagnosis and monitoring treatment response of tuberculosis.We detected 46 specimens for rapid diagnosis and 24 specimens for monitoring treatment response of tuberculosis. We found that for smear-negative specimens, the mean time to detection of M. tuberculosis were 29.56 days in L-J media, 16 days in 7H12 semi-liquid media and 3.45 days with SLCP. As for sensitivity, at the 0 day and the 7th day, the sensitivity for SLCP is 62.07% and 86.21%, respectively. The latter is higher(P<0.05). Compared to SLCP, the sensitivity for L-J media and 7H12 semi-liquid media is 62.07% at the 8th week. We also found that the mean time to detection of M. tuberculosis in 7H12 semi-liquid media is fewer than in L-J media(P<0.01). Therefore we can use 7H12 semi-liquid media to determine viable organisms. Moreover, the support rate of L-J media or/and 7H12 semi-liquid media, and combine with clinical data is 76.09%, 80.43% and 91.11%, respectively. Besides, the results of specimens for monitoring treatment response of tuberculosis showed that the persistence of M.tuberculosis DNA in sputum as long as 7 months after the start of treatment, at the same time the specimens were smear and culture negative. From all results above, we concluded that SLCP might be useful in the initial diagnosis of tuberculosis to a certain extent, especially for smear-negative pulmonary tuberculosis. With this method, we might achieve rapid diagnosis and determine viable organisms; Under the conditions of standardization and strict quality control, PCR methods are rapid, sensitive and specific, and supported by cuture and clinical data at high rate. They could be useful in the initial diagnosis of tuberculosis, but they might be inappropriate for monitoring the response of patients to antituberculosis therapy.