Molecular Detection Of M. Tuberculosis Complex In Clinical Lung Tissue Samples

Objective: We attempted to apply the nested PCR technique for the molecular diagnosis of Mycobacterium tuberculosis directly from clinical lung tissue samples. To evaluate the sensitivity and specificity of this method. To assess the relationship between MTBC and pulmonary sarcoidosis by examination of mycobacterial DNA in lung tissue samples of sarcoidosis,and to assess the differential diagnostic value of this method in pulmonary sarcoidosis and pulmonary tuberculosis.Method: 110 lung tissue specimens from 110 patients at Peking Union Medical Hospital from 2008 to 2009 were collected. Among them, 47 cases were diagnosed as pulmonary TB (AFB stain and/or mycobacetrial culture were positive in 20 cases, 20 cases were diagnosed by typical histopathological findings, and 6 were diagnosed clinically) , 23 were pulmonary sarcoidosis, 10 were lung cancer, 19 were pulmonary fungal infection and 11 were pulmonary bacterial infection except MTBC. DNA was extracted and MTBC DNA was tested using nested PCR, the target for the amplification being a segment of IS6110 gene. Their medical records were examined to gather the microbiogic and histopathologic data.Results:The minimum amount of DNA necessary for a positive results was 100fg, ,corresponding to 20 mycobacterial cells. nPCR was positive in 36(76.6%) of 47 patients with pulmonary TB, and nPCR was negative in 52(82.5%) of 63 patients with non-pulmonary TB. 82.6% of nPCR-positive patients in pulmonary TB group demonstrated granulomatous inflammation, higher than nPCR-negative patients in the same group. However the differences between the rate of positive AFB stain and mycobacterial culture, TBLB or percutaneous lung biopsy samples, and fresh tissue samples were not significant. nPCR was positive in 11(17.5%) of 63 patients with non-pulmonary TB. Among them 5 were from pulmonary fungal infection group and 6 were from pulmonary sarcoidosis group. The genome of MTBC can be detected in 26.1% of patients with pulmonary sarcoidosis. But the Frequency was significantly lower than that of pulmonary TB group.Conclusion:The sensitivity and specificity of this method was 76.6% and 82.5% respectively. A higher proportion of paitienls with pulmonary TB was diagnosed when nPCR was combined with histopathologic results. The frequency of positive nPCR results was higher in the pulmoary TB patients whose hi slopathol ogi c findings showed granulomatous inflammation. However the nPCR examine results has no relation to the microbiologic findings, sampling method and whether fresh tissue or FFPE tissue. False positive rate i s 17.5%, False positive was seen in the patients with pulmonary sarcoidosis or fungal infection. There was association between MTBC and some cases of pulmonary sarcoidosis. nPCR amplifing the TS6110 gene of V1TBC is a valuable molecular diagnostic method for differential ion between sarcoidosis and tuberculosis. Objective: To investigate species distribution, clinical features, drug-susceptibility, treatment outcome, and prgnostic factors of pulmonary mycobacterium infection. To identify risk factors for pulmonary NTM and drug-resistant M. tuberculosis infection. To compare the difference between non-HIV immunosuppressed patients and non-immunosuppressed patients.Method: A total of 38 mycobacterium clinical strains isolated from respiratory specimens were obtained from patients hospitalized at Peking Union Medical College Hospital from Jan 2009 to July 2010.Their medical records were examined to gather clinical, radiological,and follow-up data. 1dentification of mycobacterial species was performed through using differential culture medium and sequencing analysis of rpoB gene. Drug susceptibility testing was performed by using proportion method. DNA was extracted by CTAB method. Sequencing analysis of mutation conferring resistance to RFP, INH, SM, and EMB in rpoB gene, katG gene, inhA gene, inhA promoter, rrs gene, rpsL gene, and embB gene was performed in drug-resistant M. tuberculosis strains.Results: 16 strains were isolated from sputum, 8 from bronchoalveolar washings or tracheal aspirates, 10 from pleural fluid, and 4 from lung tissue specimens. 18 strains (47.4%) were from nor-HIV immunosuppressed patients. 6 strains (15. 8%) were identified as NTM. Compared wi th pulmonary TB patients, patients with pulmonary NTM infection had a higher average age, more structural lung disease, more masslike shadow on lung CT, less pleural effusion, and less extra-pulmonary organ involving. Masslike/nodular shadow was more frequently seen in male patients. On the contrary, bronchiectasis was more frequently seen in female patients. 40% of NTM infection cases had treatment failure. Compared with non-immunosuppressed patients, the immnunosuppressed pat ients had more complication of organ failure and higer mortalityrate. The infection rate of NTM and drug-resistant M. tuberculosis had no significant difference between the two groups. 6 strains (18.8%) of M. tuberculosis were resistant to at least one of four first-line antituberculosis drugs. Initial drug-resistant rate in descending order is SM 18.8%, INH 9.4%, RFP and EMB 3.1%, respectively. 33.3% of drug resistant patients had treatment failure, significant higher than drug sensitive patients. Average follow-up period was 14.3 months. 23(65.7%) of patients improved, 4(11.4%) failed, and 8(22.9%) died. Dead patients had a higer average age, more bilateral multiple/diffuse lesion on lung CT, more complication of organ failure espcially respi ratory failure or septic shock, more immunosuppressed cases, and higher treatment default or no treatment rate. The mutation S531L in rpoB gene, S315T in katG gene, K43R in rpsl gene were detected only in RFP, INH, and SM-resistant strains. The mutation R436L in katG gene was found in both INH-resi slant and INH-sensitive strains.Conclusion: 15.8% of patients were infected by NTM. The most common species were MCAG and M. intracellulare. Fever, pleural effusion, and extra-pulmonary involving were less common in NTM lung disease. Masslike/nodular shadow was male predominance and bronchi ectasis was female predominance. Risk factors for pulmonary NTM infection was old age and structural lung diseases. Non-HIV immnunosuppressed patients has no specific symptoms and plumonary manifestat ion, and were not risk factor for pulmonary NTM and drug-resistant M. tuberculosis infection. However, the risk for complications of organ failure and mortality elevated. Other risk factors for mortality included old age, bilateral multiple/diffuse lesions, treatment default or no treatment. There were good agreement between the mutation in rpoB gene, katG gene, rpsL gene and phenotypic resistance.