Detection of mutations in Mycobacterium tuberculosis genome determining resistance to fluoroquinolones by hybridization on biological microchips

We developed a method of identification of Mycobacterium tuberculosis with simultaneous evaluation of the sensitivity to fluoroquinolones on a biological microchip array. The method of multiplex two-staged PCR followed by hybridization of a biochip makes it possible to detect 8 mutant variants of gyrA gene occurring in fluoroquinolone-resistant strains (∼85% all resistant forms) within 1 day. Using this method we analyzed 107 cultures isolated from patients with tuberculosis and 78 sputum samples. Mutations in gyrA gene were detected in 48 (92%) resistant strains. Natural S95T polymorphism in gyrA gene was detected in all resistant and in 76% sensitive strains. The sensitivity and specificity of the proposed method calculated on the basis of the analysis of sputum samples (n=78) were 94 and 100%, respectively. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 1, pp. 115–120, January, 2008     

HIV coinfections with tuberculosis among HIV-1 infected individuals in old cross river state,Nigeria

ABSTRACT

Human immunodeficiency virus (HIV) and Tuberculosis (TB) are two main global public health threats that dent development in low and middle-income countries. This study evaluated the HIV/TB co-infection rate among HIV-1 infected individuals in old Cross River State, Nigeria. A total of 417 HIV-infected individuals participated in this study, 241 (57.8%) from Calabar, Cross River State, Nigeria and 176 (42.2%) from Uyo, Akwa-Ibom State, Nigeria. The age range of the 417 HIV-1 positive individuals who participated in the study was 4–72 years with an average age of 39.1 years. Plasma samples were analyzed for HIV and TB using fourth-generation Enzyme-Linked immunosorbent Assay. The CD4 count was enumerated using the Partec CyFlow® Counter. Plasma viral loads (PVL) were determined using the Abbott Real-Time HIV-1 assay. Results showed that 230 (55.2%) of the participants were in the 31–45 years age range. The majority (67.4%) of the HIV-1 infected individuals were females and 32.6% were males. An overall prevalence of HIV/TB coinfection in Old Cross River State, Nigeria was 1.4%, with Akwa Ibom State (0.6%) and Cross River State (1.2%). A higher prevalence of HIV/TB coinfection was observed among females (1.8%) than in males (0.7%). Higher prevalences of HIV/TB coinfections was observed in patients above 45 years of age (2.2%), married (2.3%), tertiary education (1.8%) followed by those with secondary education (1.4%), traders and civil servants (3.1%), patients with CD4 counts 200–349 and ≥500 cells/μl (1.9%), and those with viral load <40 copies/mL (2.7%). This study confirmed the presence of HIV/TB co-infection in old Cross River State, Nigeria. Although the prevalence rate of HIV/TB coinfection was low, its presence alone among HIV-1 infected individuals makes it a major source of concern. This finding highlights the need for a well-structured approach to the management of co-infection, and this includes both the social and medical aspects of the problem.     

结核分枝杆菌Ag85B基因疫苗免疫保护作用的初步研究

目的:研究编码结核分枝杆菌分泌蛋白Ag85B的基因疫苗pTB30m和pTB30s对免疫动物的保护作用。方法:以基因疫苗pTB30m和pTB30s肌注免疫BALB/c小鼠。免疫完成6 wk后,用5×105 CFU的MTB H37Rv毒株经小鼠尾静脉攻击感染。同时用尼龙毛柱分离基因免疫BALB/c小鼠的T细胞,并以5×106 T细胞/只小鼠过继免疫正常BALB/c小鼠,立即用105 CFU的MTB毒株经小鼠尾静脉攻击感染。4 wk后分别计数脾脏中的细菌负荷。结果:与生理盐水对照组相比较,pTB30m及pTB30s质粒免疫组BALB/c小鼠脾脏中的细菌负荷均减少,分别为0.645(log10 CFU,P<0.01)和0.839(log10CFU,P<0.001);而空质粒对照组小鼠脾脏中的细菌负荷减少较少。经质粒pTB30m和pTB30s免疫的BALB/c小鼠的T细胞,过继免疫的正常BALB/c小鼠,对攻击感染的MTB H37Rv毒株在脾脏中的增殖具有部分抑制作用。结论:pTB30s免疫的BALB/c小鼠,对MTB H37 Rv毒株攻击的保护作用优于pTB30m质粒免疫,有望进一步用于结核病的防治研究。     

High antibody levels to the mycobacterial fibronectin-binding antigen of 30-31 kD in tuberculosis and lepromatous leprosy.

Immunoblot assays showed that mycobacterial fibronectin-binding antigens are important targets of the humoral immune response in tuberculosis and leprosy. Using culture filtrate antigens of Mycobacterium tuberculosis, strong reactivity with the fibronectin-binding of 30-31 kD (Fn 30-31) was demonstrated in 55.9% of tuberculosis sera and in 56.5% of lepromatous leprosy sera. Sera from patients with tuberculoid leprosy and control sera gave very weak binding. Reactivity of tuberculosis and lepromatous leprosy sera with the fibronectin-binding antigen of 58-60 kD (Fn 58-60) was less conspicuous. The ability to react with fibronectin of the antigens of 58-60 and 30-31 kD was demonstrated by parallel labelling with a fibronectin-biotin conjugate. Fn 30-31 was purified to homogeneity by a two-step procedure and used for ELISA. Positive titres were found in 63% out of 65 tuberculosis sera and in 60.5% out of 43 lepromatous leprosy sera. Antibody titres in lepromatous leprosy sera were higher than in tuberculosis sera. Our observations indicate indirectly that M. leprae possess a highly immunogenic molecule homologous to M. tuberculosis Fn 30-31, which elicits a high antibody response in lepromatous leprosy but not in tuberculoid leprosy. In this investigation, direct evidence for the presence of this antigen in M. leprae was obtained by immunochemistry of lepromatous leprosy lesions with a monospecific antibody raised against M. tuberculosis Fn 30-31.     

Generalized AA-amyloidosis in a 58-year-old Caucasian woman with an 18-month history of gastrointestinal tuberculosis

 We report on a 58-year-old Caucasian woman who went to a general practitioner about recurrent abdominal pain, night sweats and weight loss of a few weeks’ duration. Once gynaecological disease had been ruled out, the patient was admitted to hospital with severe abdominal pain and intestinal obstruction and a right-sided hemicolectomy was performed. Following the investigation of osteolytic lumbar vertebrae, 18 months after visiting the general practitioner the patient was finally found to be suffering from generalized AA-amyloidosis secondary to gastrointestinal tuberculosis. This had been misinterpreted as Crohn’s disease. Re-examination of the specimens from the right-sided hemicolectomy demonstrated that scanty deposits of AA-amyloid were present 9 months after the first presentation. AA-amyloid can thus be present in serious inflammatory disease even during the first 9 months after the initial clinical presentation. Received: 23 June 1998 / Accepted: 19 August 1998     

Differential regulation of Th1 and Th2 cells by p91–110 and p21–40 peptides of the 16-kD α-crystallin antigen of Mycobacterium tuberculosis

Permissively recognized peptides which can activate lymphocytes from subjects with a variety of class II HLA types are interesting diagnostic and vaccine candidates. In this study we generated T helper clones reactive to the permissively recognized p21–40 and p91–110 peptides of the 16-kD heat shock protein of Mycobacterium tuberculosis. All the clones specific for p91–110 secreted interferon-gamma (IFN-γ) and were of the Th1 phenotype. By contrast, the p21–40 peptide favoured the generation of IL-4-producing clones. Antibody blockade established that the peptide-specific Th clones could either be DR-, DP- or DQ-restricted. Thus, two permissively recognized sequences p21–40 and p91–110 from the same mycobacterial antigen can drive the differentiation of functionally distinct T helper subsets. Attempts to immunize against tuberculosis should bear in mind epitope specificity if a favourable Th subtype response is to be generated.     

Influence of Mycobacterium tuberculosis on differential activation of helper T-cells

Host defence against tuberculosis infection involves T-lymphocyte mediated cellular immune responses. In this study we assessed T-cell activation by studying the early signal transduction events and production of cytokines by human CD4+ T-cells. The study constituted of five groups of subjects: (a) untreated acid fast bacilli (AFB)+ve TB patients who have not started anti-tuberculosis therapy (ATT) [New]; (b) patients who have taken ATT for two months [2T]; (c) patients who have taken ATT for six months [6T]; (d) mantoux positive healthy controls [T+ve]; (e) mantoux negative healthy controls [T-ve]. We found that mantoux positive healthy controls produced significantly higher levels of IP3, intracellular Ca2+ and presented increased PKC activity when CD4+ T-cells were stimulated with M. tuberculosis H37Rv cell lysate as compared to mantoux negative controls. Furthermore, decreased expression of CD54 (ICAM-1) and reduced [Ca2+]i were seen in TB patients as compared to T+ve healthy controls. TB patients showed significantly lower levels of IL-2 and IFNgamma and higher levels of IL-4 as compared to normal healthy controls, suggesting a diminished Th1 response. Thus, the reciprocal changes in cytokines, reduced [Ca2+]i levels, and CD54 expression in patients imply phenotype shifting of Th precursors to Th2 type in TB patients.     

Pulmonary tuberculosis due to bacille Calmette-Guérin

Summary Immunotherapy using bacille Calmette-Guérin (BCG) has gained increasing acceptance in the management of superficial bladder cancer. Systemic reactions after intravesical instillation of BCG are rare. However, when the therapy is complicated, the lung often becomes involved. Since the pathogenesis of lung infiltrates after immunotherapy is unknown, we report on a patient who developed a lung infiltrate after receiving BCG immunotherapy for bladder cancer. The infectious etiology was established by culture confirmation of a BCG strain in the broncheoalveolar lavage fluid.Abbreviation BCG bacille Calmette-Guérin