Progress in resistance mechanisms and diagnosis of non-first line anti-TB drugs.

Tuberculosis (TB) is one of the three major infectious diseases in China and all over the world. In 2014, for the first time, TB killed more people than HIV did. Non-first line anti-TB drugs are used as main drugs in the treatment of MDR-TB. However, MDR-TB can gradually develop as extensively drug-resistant TB (XDR-TB) because of poor diagnosis, the unreasonable treatment, poor medical conditions and so on. The death rate of XDR-TB is close to lung cancer. Research on the mechanism of drug resistance of Mycobacterium tuberculosis has turned to non first-line anti-TB drugs: second and third line drugs and some new anti-TB drugs in development. In this review, we summarized the drug resistance mechanisms of the common non-first line anti-TB drugs. Most of drug resistant TB patients can't get timely diagnosis and correct treatment. So at the end of this article, we also summarized the common methods to diagnose drug-resistant TB.

[Comparison of P-nitrobenzoic acid and hydroxylamine hydrochloride for the primary screening of mycobacterial species].

OBJECTIVES: To examine the screening methods for mycobacteria recommended by the China Anti-tuberculosis Association, in order to increase laboratory diagnostic accuracy for mycobacterial screening. METHODS: Using P-nitrobenzoic acid (PNB 0.5 g/L) as the control group, and hydroxylamine hydrochloride (HA, in 125, 150 and 175 mg/L concentrations) as the study group, laboratory preserved strains of H37Rv M.tuberculosis, and standard and clinically isolated strains of M.nontuberculosis (NTM) from Guangzhou Chest Hospital were tested for both PNB and HA sensitivity. Differences between groups were analyzed by χ(2) test. RESULTS: Among the 2529 MTB strains, the resistance rate to PNB was 3.0% (76/2529), to HA was 12.2% (308/2529), 4.8% (121/2529), and 0.9% (23/2529), respectively, corresponding to the aforementioned 3 different concentrations of HA. Among the 1766 NTM strains, the sensitive rate to PNB was 8.3% (147/1766), to HA was 0.1% (2/1766), 0.5% (9/1766), and 0.9% (16/1766), respectively, corresponding to the aforementioned 3 different concentrations of HA. There was significant difference (χ(2) = 5.44-83.50, P < 0.05). CONCLUSION: HA at 175 mg/L concentration was the optimal condition for laboratory tuberculosis preliminary screening.

[The clinical significance of changes in CD4+ T cell counts by peripheral blood from patients with pulmonary tuberculosis after antitubercular treatment].

AIM: To explore the clinical significance of changes in CD4+ T cell counts by peripheral blood from patients with pulmonary tuberculosis after antitubercular treatment. METHODS: CD4+ T cell counts in the peripheral blood of 62 pulmonary TB patients were counted by flow cytometry from groups auording to the radiologic extent of disease, and compared with those obtained from 30 controls. RESULTS: (1) The baseline levels of CD4+ T cell counts (439.21+/-210.56)/mm3 in pulmonary tuberculosis group before antituberculosis therapy were significantly lower than those (748.47+/-261.85)/mm3 in the control group (P<0.01). (2) The baseline levels of CD4+ T cell counts (399.83+/-194.17)/mm3 before antituberculosis therapy in diffuse group were significantly lower than those (521.90+/-224.40)/mm3 in limited group (P<0.05). (3) After two months of antituberculosis therapy, the levels of CD4+ T cell counts in the improved group increased from (480.75+/-228.49)/mm3 to (616.75+/-280.57)/mm3, and the values were not significantly different from those in controls (P>0.05). Whereas, the levels of CD4+ T cell counts in the stable group increased from (412.97+/-197.00)/mm3 to (447.55+/-204.60)/mm3, which were still significantly lower than those in the control group (P<0.01). CONCLUSION: These results suggested that peripheral CD4+ lymphopenia was demonstrated in patients with pulmonary tuberculosis, and such lymphopenia was reversible partly with antitubercular treatment. The numbers of peripheral CD4+ T cell counts were related to the severity of the disease, the more severe of the disease was, the less numbers of the CD4+ T cell counts were.

[Evaluation of strand displacement amplification in the diagnosis of tuberculosis].

OBJECTIVE: To study the clinical significance and reliability of strand displacement amplification (SDA) in detecting Mycobacterium tuberculosis complex. METHODS: SDA and fluorescence quantitative polymerase chain reaction (FQ-PCR) were employed to detect samples from 453 cases of tuberculosis, including 332 sputum samples, 78 samples of pleural effusion, and 43 samples of cerebrospinal fluid. RESULTS: In the 332 sputum samples, 131 were culture-positive, of which 110 samples (88 smear-positive) were Mycobacterium tuberculosis complex and 21 samples (20 smear-positive) were nontuberculous Mycobacteria. The sensitivity and specificity of SDA for the 110 samples of Mycobacterium tuberculosis complex and 21 samples of nontuberculous Mycobacteria were 99.1%, 95.2% and 94.6%, 95.2%, respectively. The positive rates in the 311 cases of Mycobacterium tuberculosis complex for SDA and FQ-PCR were 55.3% (172/311) and 47.0% (146/311), respectively. There were 20 smear positive samples in the 121 samples of pleural effusion and cerebrospinal fluid, of which 19 were Mycobacterium tuberculosis, 1 nontuberculous Mycobacterium. The positive rates for SDA and FQ-PCR were 43.4% (52/120) and 33.4% (40/120), respectively. Internal amplification control (IAC) was designed for SDA to achieve accuracy of the results. CONCLUSION: The automatic assay system of SDA is a rapid and specific test for detecting Mycobacterium tuberculosis complex.

[The change and the clinical significance of peripheral blood Th1/Th2 cells in patients with pulmonary tuberculosis].

OBJECTIVE: To study the change of peripheral blood CD4+ helper T cells and T helper subtype I and II (Th1/Th2) in patients with pulmonary tuberculosis and to explore their changes during anti-tuberculosis chemotherapy. METHODS: CD4+ T cells from 105 patients with pulmonary tuberculosis and 25 normal controls were counted by Flow Cytometry. Peripheral blood cells were stimulated in vitro by phorbol myristate acetate (PMA) (25 ng/ml) and ionomycin (250 ng/ml). Cytokines were confined to the cells by using the protein transporting inhibitor containing monensin (2 micro mol/ml). These cells were incubated in 5% CO(2) for 4 h-4.5 h. The membrane and plasma of CD4+ helper T cells were marked by CD3 -PC5 + CD8 -FITC + INF-gamma -PE/CD3 -PC5 + CD8 -FITC + IgG1 -PE, CD3 -PC5 + CD8 -FITC + IL-4 -PE/CD3 -PC5 + CD8 -FITC + IgG1 -PE monoclonal antibodies respectively. Th1 and Th2 cells were counted and the ratio of Th1/Th2 cells was calculated. The levels of Th1 and Th2 cells in the 67 patients with pulmonary tuberculosis were detected at the end of intensive chemotherapy and at the sixth month of chemotherapy. RESULTS: (1) The levels of CD4+ T and Th1 cells in patients with tuberculosis were significantly lower than those of controls. Their values were (663 +/- 160)/ microl vs (735 +/- 156)/ microl and (9.56 +/- 3.60)% vs (18.7 +/- 5.03)% respectively (P < 0.05). (2) The levels of CD4+ T and Th1 cells in patients with severe pulmonary tuberculosis were lower than those in patients with moderate or mild pulmonary tuberculosis. Their values were (579 +/- 120)/ microl vs (726 +/- 166)/ microl, (684 +/- 192)/ microl and (5.43 +/- 2.33)% vs (12.2 +/- 1.81)% and (10.9 +/- 2.30)% respectively (P < 0.05). The level of Th2 cells was in contrast to Th1 cells and their values were (5.63 +/- 1.26)% vs (2.93 +/- 0.87)% and (3.22 +/- 1.01)% (P < 0.01). (3) The level of Th1 cells increased while that of Th2 decreased in the 61 patients who gradually recovered. (4) The level of Th2 cells in patients with smear positive tuberculosis was strikingly higher than that in patients with smear negative tuberculosis and their values were (5.20 +/- 0.97)% vs (2.77 +/- 1.96)% (P < 0.05). CONCLUSION: The detection of CD4+ helper T cells and Th1/Th2 cells in the peripheral blood cells from patients with pulmonary tuberculosis is useful in evaluating the state of disease and the effect of chemotherapy.

[A study on the correlation between HLA-DR genes and susceptibility to pulmonary tuberculosis in a population of Han nationality from southern China].

OBJECTIVE: To analyze the association between HLA-DR genes and pulmonary tuberculosis and to explore susceptible genes associated with pulmonary tuberculosis in a population of Han nationality from southern China. METHODS: Using case-control study, we detected by polymerase chain reaction-sequence specific primers (PCR-SSP) technique the 23 alleles of HLA-DR gene sites in 110 tuberculosis cases and 101 controls from Guangdong, Guangxi, Hunan, Hubei, Jiangxi and Fujian provinces. Gene frequency (GF) and odds ratio (OR) were calculated and compared. RESULTS: The frequency of DR16 allele in pulmonary tuberculosis cases was strikingly higher than that in the healthy controls (chi2=5.915, Pc< 0.05). Their GFs were 12.62% and 5.60% respectively, and the OR was 2.53. Significantly decreased frequency of DR1 and DR13.3 alleles in cases were found (chi2 values were 17.847 and 14.258 respectively, Pc < 0.01). Their ratios of GFs were 8.08% vs. 23.57% and 29.29% vs. 50.24%, and their ORs were 0.26 and 0.33 respectively. CONCLUSIONS: The results suggest that DR16 allele is closely correlated to incidence of pulmonary tuberculosis in this population of Han nationality from southern China, or linked to susceptible genes which are functional. It is also suggested that expression of DR1 and DR13.3 alleles may be associated with an antagonist effect in the pathogenesis of pulmonary tuberculosis in this population.