Comparison of IS6110 and ‘short fragment’ devR (Rv3133c) gene targets with phenotypic methods for diagnosis of Mycobacterium tuberculosis

Background

Tuberculosis (TB), declared a global emergency in 1993 by the WHO, remains a worldwide public health problem. Rapid diagnosis is required for treatment and prevention. This study compares genotypic methods using two gene targets [IS6110 and 'short fragment' devR (Rv3133c)] with phenotypic methods [Lowenstein Jensen (LJ) and BACTEC 460] for the diagnosis of TB while using Ziehl Neelsen (ZN) staining as the gold standard.

Methods

56 clinical TB samples from a tertiary care apex center along with 50 healthy control samples, excluding samples from patients already on ATT were processed by routine USP methodology. Smears were graded by ZN stain. Solid media (LJ) and liquid media (BACTEC 460) were used along with IS6110 and 'short fragment' devR (Rv3133c) specific gene amplification and comparatively analyzed.

Results

50/56 samples were positive by phenotypic methods, 53 by IS6110 and 45 by devR (Rv3133c) amplification. 38 samples were positive by both phenotypic and genotypic methods. IS6110 detected six and devR (Rv3133c) detected five phenotypically negative samples. Both IS6110 and devR (Rv3133c) were positive in 42 samples. 11 devR (Rv3133c) negative samples were positive by IS6110 and three IS6110 negative samples were positive by 'short fragment' devR (Rv3133c). Compared to phenotypic methods, the sensitivity, specificity, positive and negative predictive values of IS6110 was 94%, 89.29%, 88.68% and 94.34% while that of devR (Rv3133c) was 80%, 91.07%, 88.89% and 83.61% respectively.

Conclusion

Simultaneous use of both phenotypic and genotypic methods increases the yield of positive results.     

Comparison of IS6110 and ‘short fragment’ devR (Rv3133c) gene targets with phenotypic methods for diagnosis of Mycobacterium tuberculosis

Background

Tuberculosis (TB), declared a global emergency in 1993 by the WHO, remains a worldwide public health problem. Rapid diagnosis is required for treatment and prevention. This study compares genotypic methods using two gene targets [IS6110 and ''short fragment'' devR (Rv3133c)] with phenotypic methods [Lowenstein Jensen (LJ) and BACTEC 460] for the diagnosis of TB while using Ziehl Neelsen (ZN) staining as the gold standard.

Methods

56 clinical TB samples from a tertiary care apex center along with 50 healthy control samples, excluding samples from patients already on ATT were processed by routine USP methodology. Smears were graded by ZN stain. Solid media (LJ) and liquid media (BACTEC 460) were used along with IS6110 and ''short fragment'' devR (Rv3133c) specific gene amplification and comparatively analyzed.

Results

50/56 samples were positive by phenotypic methods, 53 by IS6110 and 45 by devR (Rv3133c) amplification. 38 samples were positive by both phenotypic and genotypic methods. IS6110 detected six and devR (Rv3133c) detected five phenotypically negative samples. Both IS6110 and devR (Rv3133c) were positive in 42 samples. 11 devR (Rv3133c) negative samples were positive by IS6110 and three IS6110 negative samples were positive by ''short fragment'' devR (Rv3133c). Compared to phenotypic methods, the sensitivity, specificity, positive and negative predictive values of IS6110 was 94%, 89.29%, 88.68% and 94.34% while that of devR (Rv3133c) was 80%, 91.07%, 88.89% and 83.61% respectively.

Conclusion

Simultaneous use of both phenotypic and genotypic methods increases the yield of positive results.     

结核分枝杆菌Rv3881c抗原鼠单克隆抗体的制备及初步鉴定

目的:制备抗结核分枝杆菌Rv3881c抗原鼠mAb。方法:采用杂交瘤技术,获得了11株针对结核分枝杆菌Rv3881c抗原鼠mAb杂交瘤细胞株,对其中的5株进行了小鼠腹水的制备及相关鉴定。结果:5株mAb的腹水效价达到1∶32 000～1∶512 000,将这5株mAb进行了纯化,纯化后纯度大于90%,抗体亚类(型)均为IgG1/κ型,ELISA结果显示制备的mAb与结核分枝杆菌Rv3881c抗原可发生特异反应。结论:制备了抗结核分枝杆菌Rv3881c抗原鼠mAb,为结核分枝杆菌Rv3881c生物学功能的研究奠定基础。     

结核分枝杆菌Rv2660c蛋白结构分析及抗原表位预测

目的 分析结核分枝杆菌Rv2660c蛋白的结构并预测其抗原表位,为研发针对结核潜伏性感染的治疗性疫苗和药物提供新的靶点。方法 用BLAST软件分析Rv2660c蛋白与人类蛋白的同源性,然后运用生物信息学方法预测其二级结构、跨膜结构、信号肽序列及T细胞和B细胞抗原表位。结果 BLAST结果显示,Rv2660c蛋白与人类蛋白同源性不高,同源性最高的人类蛋白是MAD 6蛋白,同源性仅为16%。Rv2660c蛋白无跨膜区,为胞外蛋白,不含信号肽序列;该蛋白含丰富的B细胞和T细胞抗原表位,19-35、48-54、28-44和58-73位氨基酸残基可能存在优势线性B细胞表位;56-64位和65-74位氨基酸可能存在优势辅助性T细胞表位,66-74、41-49、63-71位氨基酸可能存在优势细胞毒性T细胞表位。结论 Rv2660c蛋白含丰富抗原表位,具有较强的体液免疫和细胞免疫原性,可望作为潜伏性感染结核的治疗性疫苗和药物的作用靶点。     

结核分枝杆菌NrdF1、PE_PGRS35、Rv1985c和Rv1986的原核表达及检测牛结核病抗体之应用

目的 在大肠杆菌中表达结核分枝杆菌RD2区域的NrdF1、PE_PGRS35、Rv1985c和Rv1986蛋白,并评价诊断牛结核病中的应用潜能。方法 以结核分枝杆菌H37Rv基因组DNA为模板,PCR 扩增nrdF1,pe_pgrs35,rv1985c 和 rv1986基因,并将其克隆到表达载体中,重组表达质粒转化E.coli BL21(DE3),IPTG诱导目的蛋白表达,镍柱亲和纯化重组蛋白,SDS-PAGE和Western blotting试验鉴定目的蛋白的表达及其反应原性。以纯化的重组蛋白作为包被抗原,通过ELISA方法测定牛血清中特异性抗体,以之评价这些蛋白用于牛结核病抗体检测的价值。结果 成功表达了NrdF1、PE_PGRS35、Rv1985c和Rv1986蛋白,相对分子质量为42 ku、63 ku、46 ku和41 ku,对表达产物进行镍柱亲和纯化,获得了纯度较高的融合蛋白。重组蛋白均能与抗HIS单抗发生特异反应,表现出良好的反应原性。通过间接ELISA方法检测牛血清中特异性抗体,阳性检出率分别为7.35%、22.06%、16.18% 和16.18%。结论 结核分枝杆菌原核表达的NrdF1、PE_PGRS35、Rv1985c和Rv1986蛋白具有用作牛结核病血清学诊断试剂的潜能。     

Development and evaluation of a novel multiple-antigen ELISA for serodiagnosis of tuberculosis

In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB patients and 57 healthy subjects against antigens Rv3425, 38 kDa and lipoarabinomannan (LAM) from Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i) Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425 antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38 kDa antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15 extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%, respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii) Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425, 38 kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB. Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.     

Xenotropic murine leukemia virus-related virus (XMRV) in prostate cancer cells likely represents a laboratory artifact

The prevalence of xenotropic murine leukemia virus-related virus (XMRV) in human population and its involvement in prostate cancer are subjects of ongoing research and debate. 22Rv1, which is a human cell line that serves as a common model of androgen-independent prostate cancer, was recently reported to carry infectious copies of XMRV. 22Rv1 was derived from a prostate cancer xenograft CWR22 that was serially passaged in immunodeficient mice. Based on the analysis of the DNA from CWR22 and 22Rv1, we present evidence against the presence of XMRV in CWR22 and, by inference, the tumor, from which CWR22 and 22Rv1 were established. While the presence of XMRV in 22Rv1 is likely to be an artifact, it may be a significant factor in determining the biological properties of this cell line. This consideration warrants additional caution for the interpretation of the relevance of the studies, which utilize this popular cell line as a model. It also invites a closer look at the sources of viral contamination in xenografts and cultured cells, as well as in the experiments that allege the presence of this virus in human cells and populations.