Diagnostic performance and problem analysis of commercial tuberculosis antibody detection kits in China

Background: The diagnosis of bacterium-negative pulmonary tuberculosis(TB) and extra-pulmonary TB is challenging clinically. The detection of the anti-TB antibody has an important, auxiliary, clinical diagnostic value. Therefore, TB antibody detection kits should be screened and evaluated, and the reagents with the highest sensitivity and specificity should be chosen and used clinically.Methods: The diagnostic performance of 7 commercially available TB antibody detection kits(kits A, B, C, D, E, F and G) based on the gold immunoassay detection of immunoglobulin(Ig) G or IgM antibodies were simultaneously evaluated and compared in 62 TB cases and 56 non-TB cases in a laboratory. A retrospective analysis including 2549 cases was carried out to assess the clinical diagnosis values of bacteriological examinations and TB antibody tests(kits B and H used in the clinic).Results: The sensitivities of TB antibody kits A, B, C, D, E, F and G in the sera from 62 TB patients were 50.0%, 83.9%, 38.7%, 9.7%, 48.4%, 69.4% and 79.0%, respectively; the sensitivities in the sera from 24 smear-negative TB patients were 29.2%, 79.2%, 29.2%, 12.5%, 29.2%, 54.2% and 79.2%, respectively; the specificities in the sera from 56 nonTB patients were 73.2%, 25.0%, 85.7%, 96.4%, 78.6%, 78.6% and 50.0%, respectively. Of the 2549 clinically diagnosed cases, there were 1752 pulmonary TB cases, 505 extra-pulmonary TB cases, 87 old pulmonary TB cases and 205 non-TB cases. The positive results for smear, culture, TB antibody kit B and kit H in pulmonary TB cases were 39.8%(543/1365), 48.6%(372/765), 45.8%(802/1752) and 25.2%(442/1752), respectively; the results in extra-pulmonary TB cases were 3.4%(6/178), 5.8%(4/69), 35.4%(179/505), and 11.3%(57/505), respectively; the results in old pulmonary TB cases were 0%(0/64), 0%(0/30), 32.2%(28/87), and 9.2%(8/87), respectively; and the results in non-TB cases were 0%(0/121), 0%(0/56), 21.5%(44/205), and 2.4%(5/205), respectively. Of 624 smear-positive and/or culture-positive pulmonary TB cases, the sensitivities of antibody test kits B and H were 53.0% and 36.4%, respectively. Of 901 smear-negative and/or culture-negative pulmonary TB cases, the sensitivities of antibody test kits B and H were 42.5% and 19.0%, respectively. The positive rate of antibody detection in the bacterium-positive pulmonary TB cases was significantly higher than that in the bacterium-negative pulmonary TB cases(P<0.05).Conclusion: The colloidal gold-labeled TB antibody IgG detection assay is a simple, rapid and economical method that provides a better clinical auxiliary diagnosis value on TB, especially in smear-negative pulmonary TB and extrapulmonary TB. The production, quality control, screening and evaluation of antibody detection kits are very important for its clinical application.

Effects of Mycobacterium vaccae vaccine in a mouse model of tuberculosis: protective action and differentially expressed genes

Background:Tuberculosis is a leading cause of death worldwide.BCG is an effective vaccine,but not widely used in many parts of the world due to a variety of issues.Mycobacterium vaccae(M.vaccae)is another vaccine used in human subjects to prevent tuberculosis.In the current study,we investigated the potential mechanisms of M.vaccae vaccination by determining differentially expressed genes in mice infected with M.tuberculosis before and after M.vaccae vaccination.Methods:Three days after exposure to M.tuberculosis H37 Rv strain(5×10~5 CFU),adult BALB/c mice randomly received either M.vaccae vaccine(22.5μg)or vehicle via intramuscular injection(n=8).Booster immunization was conducted 14 and 28 days after the primary immunization.Differentially expressed genes were identified by microarray followed by standard bioinformatics analysis.Results:M.vaccae vaccination provided protection against M.tuberculosis infection(most prominent in the lungs).We identified 2,326 upregulated and 2,221 downregulated genes in vaccinated mice.These changes could be mapped to a total of 123 signaling pathways(68 upregulated and 55 downregulated).Further analysis pinpointed to the MyD88-dependent TLR signaling pathway and PI3 K-Akt signaling pathway as most likely to be functional.Conclusions:M.vaccae vaccine provided good protection in mice against M.tuberculosis infection,via a highly complex set of molecular changes.Our findings may provide clue to guide development of more effective vaccine against tuberculosis.

Evaluation of IFIT3 and ORM1 as Biomarkers for Discriminating Active Tuberculosis from Latent Infection

Objective Current commercially available immunological tests cannot be used for discriminating active tuberculosis(TB)from latent TB infection.To evaluate the value of biomarker candidates in the diagnosis of active TB,this study aimed to identify differentially expressed genes in peripheral blood mononuclear cells(PBMCs)between patients with active TB and individuals with latent TB infection by transcriptome sequencing.Methods The differentially expressed genes in unstimulated PBMCs and in Mycobacterium tuberculosis(Mtb)antigen-stimulated PBMCs from patients with active TB and individuals with latent TB infection were identified by transcriptome sequencing.Selected candidate genes were evaluated in cohorts consisting of 110 patients with TB,30 individuals with latent TB infections,and 50 healthy controls by quantitative real-time RT-PCR.Receiver operating characteristic(ROC)curve analysis was performed to calculate the diagnostic value of the biomarker candidates.Results Among the differentially expressed genes in PBMCs without Mtb antigen stimulation,interferon-induced protein with tetratricopeptide repeats 3(IFIT3)had the highest area under curve(AUC)value(0.918,95%CI:0.852-0.984,P<0.0001)in discriminating patients with active TB from individuals with latent TB infection,with a sensitivity of 91.86%and a specificity of 84.00%.In Mtb antigen-stimulated PBMCs,orosomucoid 1(ORM1)had a high AUC value(0.833,95%CI:0.752-0.915,P<0.0001),with a sensitivity of 81.94%and a specificity of 70.00%.Conclusion IFIT3 and ORM1 might be potential biomarkers for discriminating active TB from latent TB infection.

Comparative study on the anti-tuberculous effect and mechanism of the traditional Chinese medicines NiuBeiXiao He extract and JieHeWan

Background:The traditional Chinese medicine NiuBeiXiaoHe(NBXH)extract and Chinese medicine preparation JieHeWan(JHW)exhibit anti-tuberculosis(TB)effects.The anti-TB effect of NBXH was compared with that of JHW to elucidate the mechanism of action of NBXH.Methods:BALB/c mice aged 6-8 weeks were randomly divided into a normal control group,TB model group,JHW treatment group,and NBXH treatment group.After 3 and 13 weeks of treatment,the therapeutic effect in each group was evaluated by comparing lung histopathology,lung and liver colony counts,the number of spots representing effector T cells secreting IFN-γin an enzyme-linked immunospot assay(ELISPOT),and the levels of Th1,Th2,and Th17 cytokines,which were measured by a cytometric bead array(CBA).Mouse RNA samples were subjected to transcriptome sequencing.Results:After 13 weeks of treatment,the mean histopathological lesion area of the NBXH group was significantly smaller than that of the TB model group(P<0.05).Compared with those in the TB model group,the lung colony counts in the JHW and NBXH groups were significantly decreased(P<0.05),and the IL-2 and IL-4 levels in the NBXH group were significantly increased(P<0.05).NBXH partly restored significant changes in gene expression caused by Mycobacterium tuberculosis(M.tuberculosis)infection.According to GO and KEGG analyses,the changes in biological process(BP),cell composition(CC)and molecular function(MF)terms and in signaling pathways caused by NBXH and JHW treatment were not completely consistent,but they were mainly related to the immune response and inflammatory response in the mouse TB model.Conclusions:NBXH had therapeutic effects similar to those of JHW in improving lung histopathology,reducing lung colony counts,and regulating the levels of cytokines.NBXH restored significant changes in gene expression and repaired cell damage caused by M.tuberculosis infection by regulating immune-related pathways,which clarified the mechanism of action of NBXH.

Mannose-binding lectin 2 gene polymorphisms and their association with tuberculosis in a Chinese population

Background:Immune-and inflammation-related genes(IIRGs)play an important role in the pathogenesis of tuberculosis(TB).However,the relationship between IIRG polymorphisms and TB risk remains unknown.In this study,the gene polymorphisms and their association with tuberculosis were determined in a Chinese population.Methods:We performed a case-control study involving 1016 patients with TB and 507 healthy controls of Han Chinese origin.Sixty-four single-nucleotide polymorphisms(SNPs)belonging to 18 IIRGs were genotyped by the PCR-MassArray assay,and the obtained data was analyzed withχ2-test,Bonferroni correction,and unconditional logistic regression analysis.Results:We observed significant differences in the allele frequency of LTA rs2229094*C(P=0.015),MBL2 rs2099902*C(P=0.001),MBL2 rs930507*G(P=0.004),MBL2 rs10824793*G(P=0.004),and IL12RB1 rs2305740*G(P=0.040)between the TB and healthy groups.Increased TB risk was identified in the rs930507 G/G genotype(Padjusted=0.027)under a codominant genetic model as well as in the rs2099902(C/T+C/C)vs T/T genotype(Padjusted=0.020),rs930507(C/G+G/G)vs C/C genotype(Padjusted=0.027),and rs10824793(G/A+G/G)vs A/A genotype(Padjusted=0.017)under a dominant genetic model after Bonferroni correction in the analysis of the overall TB group rather than the TB subgroups.Furthermore,the rs10824793_rs7916582*GT and rs10824793_rs7916582*GC haplotypes were significantly associated with increased TB risk(P=0.001,odds ratio[OR]=1.421,95%confidence interval[CI]:1.152-1.753;and P=0.018,OR=1.364,95%CI:1.055-1.765,respectively).Moreover,the rs10824793_rs7916582*AT/AT or rs10824793_rs7916582*GT/GT diplotype showed a protective(P=0.003,OR=0.530,95%CI:0.349-0.805)or harmful(P=0.009,OR=1.396,95%CI:1.087-1.793)effect against the development of TB.Conclusions:This study indicated that MBL2 polymorphisms,haplotypes,and diplotypes were associated with TB susceptibility in the Han Chinese population.Additionally,larger sample size studies are needed to further confirm these findings in the future.

Mesenchymal-epithelial Transition Factor Regulates Monocyte Function during Mycobacterial Infection via Indoleamine 2,3-dioxygenase

Objective Mycobacterium tuberculosis(Mtb),the causative agent of tuberculosis(TB),causes an estimated 1.6 million human deaths annually,but the pathogenesis of TB remains unclear.Immunity plays a critical role in the onset and outcome of TB.This study aimed to uncover the roles of innate and adaptive immunity in TB.Methods The gene expression profiles generated by RNA sequencing from human peripheral blood mononuclear cells(PBMCs)stimulated with or without Mtb strain H37Rv antigens were analyzed.A total of 973 differentially expressed mRNAs were identified.Results The differentially expressed genes were enriched in innate immunity signaling functions.The mesenchymal-epithelial transition factor(MET)gene was significantly upregulated in CD14^(+)monocytes.A MET inhibitor improved the uptake of the BCG strain by monocytes and macrophages as well as inhibited the expression of indoleamine 2,3-dioxygenase(IDO).The expression of IDO was increased in PBMCs stimulated with Mtb antigens,and the IDO inhibitor promoted the expression of CD40,CD83,and CD86.Conclusion Our results might provide clues regarding the immunomodulatory mechanisms used by Mtb to evade the host defense system.