Application Of Real-time PCR Assay In The Early Diagnosis Of Tuberculosis

[Objective]Tuberculosis (TB) remains one of the major causes of death from a single infections agent worldwide. The WHO has estimated that approximately one-third of the population is infected with Mycobacterium tuberculosis. Between 8 and 9 million people develop TB disease each year, and approximately 2 million die from TB each year. This situation is worsened by the HIV pandemic. Tuberculosis is a major public health threat in China where has the second highest TB prevalence worldwide, and has one of the world's highest TB burdens. A key approach to the control of tuberculosis remains the rapid identification, isolation, and treatment of patients with active pulmonary tuberculosis. Conventional TB diagnostic approaches utilize tests such as sputum microscopy, culture, chest radiography and tuberculosis skin testing. These tools have been in use for decades, but there are lack both sensitivity and specificity. There is a widely felt need for more rapid, accurate and convenient tests. In recent times, attentions has been devoted to devoted to developing nucleic acid amplification diagnostic technologies owing to their rapidity and sensitivity. In the diagnosis of TB, the first major advance was rapid identification of mycobacteria isolates. This assay significantly diminished the time to identification, but the recommended use of this product requires growth of an organism in culture. The next major advance was the detection of M. tuberculosis by nucleic acid amplification tests (NAA) from clinical specimens. There are the amplified M. tuberculosis direct test (AMTD),the Amplicor M. tuberculosis (Amplicor PCR) and ligase chain reaction (LCR). Recently, real-time PCR method have been proposed for the detection of a variety of microorganisms, including mycobacteria.The present study evaluated the real-time PCR and the amplified M. tuberculosis direct test (AMTD) for mycobacterium tuberculosis complex detection in comparison to the 'gold standard' of a combination of culture and clinical diagnosis, and discussed the clinical application of Real-time PCR assay in the early diagnosis of tuberculosis.[Methods]Five hundred and forty clinical specimens were collected, followed by culturing with BACTEC MGIT-960 system and in Lowenstein-Jensen(L-J) medium respectively. Culture positive for acid-fast bacilli were identified by Accuprobe assay,Real-time PCR assay and conventional phenotype testing method. One hundred and fifty-three respiratory specimens were tested for the presence of mycobacterium tuberculosis complex by Real-time PCR assay, AMTD, Z-N staining and MGIT-960 liquid culture. The AMTD and Real-time PCR assay were compared to the " gold standard" of a combination of culture and clinical diagnosis.[Results]1. The MGIT-960 was able to detect 153 mycobacterium isolates from 540 clinical specimens, the positive rate was 28.3%, the average detection time was 10. 5±3. 3 d. In comparison to this, culture on L-J medium revealed 104 isolates, the positive rate was 19.3%, the average detection time was 26. 1±5. 6d.2. In identificated 153 isolates, the Accuprobe assay detected 127 MTB strains, the detection time was 2h; the Real-time PCR assay revealed 127 MTB strains, the detection time was 2h; the traditional phenotype method detected 123 Mycobacterium tuberculosis complex (MTB) strains, the mean detection time was 28. 2±4. 8d; ; Compared to the Accuprobe assay, the sensitivity and specificity of Real-time PCR assay for identification strains were both 100%.3. There were 70 staining positive and 83 staining negative in 153 clinical respiratory specimens. In total, the Accuprobe identification followed MGIT-960 culture revealed 69 MTB isolates. In 153 clinical respiratory specimens, the AMTD assay was 73 positive and 80 negative while the Real-time PCR assay was 68 positive and 85 negative. When compared with that for culture, the sensitivities of the Real-time PCR assay and Gen-probe AMTD were 90.8% and 94.5%, the specificities were 92.7% and 90.5%.4. After resolution of discrepant results by review of the patients' clinical data, 75 specimens were considered positive, and the overall sensitivities, specificities, and positive and negative predictive values were 91.5%, 100%, 100% and 90.9% for Real-time PCR; 94.9%, 97.2%, 97.3% and 94.4% for AMTD; 92.6%, 100%, 100% and 92.1% for culture, respectively.[Conclusions]Real-time PCR is rapid, sensitive and specific method for the identification strains and detection of M. tuberculosis in respiratory specimens. Combination MIGT-960 culture with Real-time PCR identification and direct test of pulmonary specimens with Real-time PCR for detection of M. tuberculosis significantly diminished the time of clinic diagnosis. It is an important method of Real-time PCR assay in the early diagnosis of tuberculosis.