Serological Diagnosis Of Tuberculosis Research Articles

Seroconversion against antigen MPB83 in badgers (Meles meles) vaccinated with multiple doses of BCG strain Sofia

Serological diagnosis of Mycobacterium bovis infection in badgers (Meles meles) has relied primarily on antibody recognition of MPB83, a sero-dominant antigen of M. bovis. Most vaccine studies in badgers to date have used the Bacille Calmette-Guerin (BCG) Danish strain, a low producer of MPB83. Due to a supply shortage of the BCG Danish strain, the BCG Sofia SL222 strain has been considered as an alternative vaccine. This strain is a high producer of MPB83 raising the possibility that vaccinated animals will test sero-positive in diagnostic assays that use this antigen. In this study we vaccinated a group of eleven badgers with BCG Sofia SL222 by injection via the intramuscular route and a booster vaccine dose was similarly delivered at 12 weeks and 64 weeks. Primary vaccination did not result in measured detection of antibodies against MPB83 in any badger during the first twelve weeks using serum or whole blood tested by the Dual Path Platform (DPP) VetTB, however, MPB83 antibodies were detected in a semi-quantitative ELISA assay. Following delivery of booster BCG at 12 weeks and 64 weeks, antibody responses against MPB83 were recorded in badgers using whole blood and serum on DPP VetTB and by ELISA. At all time points, vaccination was also associated with the in vitro production of gamma interferon (IFN-γ) following stimulation of lymphocytes with bovine and avian tuberculin (PPD) but not with MPB83 or M. bovis specific antigen CFP-10. The results indicate that serological diagnosis of tuberculosis using tests that target MPB83 may be compromised if badgers are repeatedly vaccinated with BCG Sofia.

Cloning expression of antigens Rv2654c, Rv1985c and Rv3868 of Mycobacterium tuberculosis and value in serological diagnosis of tuberculosis

Objective To evaluate the value of three antigens Rv2654c, Rv1985c and Rv3868 of Mycobacterium tuberculosis in the serological diagnosis of tuberculosis (TB). Methods The whole genes of Rv2654c, Rv1985c and Rv3868 were amplified from the genomic DNA of M. tuberculosis strain H37Rv by using PCR and the recombinant plasmid was constructed with expression vector pET-32a. The proteins were expressed by prokaryotic expression and purified by affinity chromatography. The best reactive conditions of enzyme linked immunosorbent assay (ELISA) of each antigen were determined by chessboard titration method. The antibodies IgG against M. tuberculosis were tested with 190 serum samples by using ELISA and the diagnostic efficiency of each antigen was analyzed by means of receiver operating characteristic curve (ROC). Results The results showed that the recombinant antigens were cloned and expressed stably and the DNA sequences of the protein antigens were completely consistent with the sequences in Genbank of NCBI respectively by BLAST alignment. The serological results of the three antigens which had been purified were analyzed with statistical software. The diagnostic efficiency of Rv2654c, Rv1985c and Rv3868 was 73.16%, 56.84% and 71.05% respectively. The best combination from the three antigens was Rv2654c+Rv3868, the sensitivity, specificity and efficiency were 78.95%, 72.63% and 75.79%. Conclusion The recombinant antigens Rv2654c, Rv1985c and Rv3868 have good diagnostic value and can be applied as candidate antigen for TB diagnosis. The combination of antigens Rv2654c and Rv3868 has good performance in TB diagnosis and possesses a certain application value in clinical practice.

Serologic diagnosis of tuberculosis by combining Ig classes against selected mycobacterial targets

Accurate, simple and cost-effective diagnostic tests are needed for diagnosis of active tuberculosis (TB). Serodiagnosis is attractive as it can be harnessed for point-of-care tests. We evaluated, in a blinded fashion, the sensitivity and specificity of serologic immunoglobulin (Ig)G, IgA and/or IgM responses to Apa, heat shock protein (HSP) 16.3, HSP20, PE35, probable thiol peroxidase Tpx and lipoarabinomannan (LAM) in 42 HIV-negative South African pulmonary TB patients and 67 control individuals. The status of latent Mycobacterium tuberculosis infection (LTBI) among controls was defined through the TST and IFN-γ release assays (IGRAs). We evaluated 47 definite LTBI (IGRA(+)/LTBI), 8 putative LTBI (IGRA(-)/TST(+)) and 12 TB-uninfected (non-LTBI) subjects. In contrast to anti-PE35 IgA, anti-PE35 IgG and particularly anti-Apa IgA, performances of anti-LAM IgG and selected anti-protein antibodies were less affected by inclusion of LTBI participants into the analysis. Anti-LAM IgG showed with a sensitivity/specificity of 71.4%/86.6% (p < 0.001) the best discrimination between TB and non-TB subjects. Selected five-antibody-combinations (including anti-LAM IgG, anti-LAM IgA and anti-Tpx IgG) further improved this performance to an accuracy exceeding 86%. Antibody responses to some Mycobacterium tuberculosis antigens often also reflect latent infection explaining the poor performance of antibody-based tests for active TB in TB-endemic settings. Our results suggest that rather a combination of serological responses against selected protein and non-protein antigens and different Ig classes should be investigated for TB serodiagnostics.

Expression and serological diagnosis of Mycobacterium tuberculosis CFP-10 and Rv2626c proteins.

We constructed a Mycobacterium tuberculosis vector expressing CFP-10 and Rv2626c to examine the expression of these proteins in Escherichia coli as well as their immunoreactivity. The CFP-10 and Rv2626c genes were amplified from tuberculosis H37Rv genomic DNA using polymerase chain reaction. They were ligated into the expression vector PET30a and expressed in E. coli. Histidine tag nickel column chromatography was used to purify the recombinant protein. An enzyme-linked immunosorbent assay (ELISA) was used for detection. In our E. coli-engineered bacteria containing a CFP10 and Rv2626c plasmid, the target protein was found mainly to be in the soluble form. We formed mixed antigens of the recombinant CFP10 and Rv2626c proteins. ELISA results showed that in 214 blood samples, the positive rate was 77.1%. The target gene was successfully expressed in the host strain. Mixed antigens of the recombinant CFP-10 and Rv2626c proteins can be used as a combination antigen in the serological diagnosis of tuberculosis.

The progress of serological diagnosis of tuberculosis

结核病是由结核分枝杆菌感染引起的传染性疾病,在历史上曾经夺去过数以万计的生命.伴随着减毒牛分枝杆菌疫苗的诞生,以及一系列抗生素和抗结核药物的发现与应用,科学家曾乐观地认为结核病会被人类消灭.然而,2008年全球仍有940万例新增结核患者,全世界每年有170万人死于结核病,其中90%以上的患者分布在发展中国家.同时,结核病也是引起HIV感染患者死亡的重要原因之一,估计2008年全世界200万HIV阳性死亡患者中,有50万患者同时患有结核病。

The research of MPT64 antibodies aptamer of Mycobacterium tuberculosis in the serological diagnosis

Objective To establish mixed-sandwich ELISA detection system by screening aptam-ers of MPT64 antibodies with SELEX to detect clinical serum samples, and explore the potential laboratory diagnosis value of this method. Methods To detect the affinity of the final round ssDNA library to MPT64 antibodies inhibited by MPT64 antigen with the competitive ELISA method, optimize the mixed-sandwich ELISA detection method that was aptamer-serum-horseradish peroxidase labeled goat anti-human IgG anti-body detection system to detect 230 cases of clinical serum samples as well as the lowest concentration of MPT64 antibodies and the linear range. Results In competitive ELISA test results, the percentage of inhi-bition effect of MPT64 antigen to final round ssDNA library is from 0.25% to 80% when the MPT64 antigen concentration rised from 2 μg/ml to 256 μg/ml. The Optimized detection system of mixed-sandwich ELISA was constitute of the concentration of ssDNA coated with 0.1μg/hole, serum dilution of 1/200, horseradish peroxidase labeled goat anti-human IgG antibody concentration of 1/40 000. The lowest concentration of MPT64 antibody is 3 mg/L and the linear range is between 10 mg/L and 1000 mg/L. The serum samples of 100 cases of tuberculosis patients, 100 healthy individuals and 30 cases of non-tuberculesis were tested in this system and the test result was analyzed with Graphpad Prism, the difference of tuberculosis group and healthy group was statistically significant (P<0.001 ), the difference of TB group and non-TB control group was also statistically significant (P<0.001). The specificity and the sensitivity was 96.1% and 31.0% re-spectively. Conclusion The aptamer mixed-sandwich ELISA method will play an important role in the sero-logical diagnosis of tuberculosis. Key words: MPT64 antibodies; SELEX; Aptamer; ssDNA; Serological detection

Serological diagnosis of tuberculosis

To evaluate the efficacy of ELISA for the detection of IgG antibodies against antigen 85 complex (Ag 85 complex) of Mycobacterium tuberculosis. Children of either sex, 0-18 years of age, attending the outpatient department and admitted in the casualty and wards of the Department of Pediatrics, S.N. Medical College, Agra, were included in present study. The study was carried out on children with pulmonary and CNS tuberculosis along with matching controls (83 cases and 32 controls). Informed consents of their parents or guardians were taken. They were subjected to clinical examination, relevant laboratory investigations, tuberculin test and chest radiograph. Relevant body fluids were subjected to bacteriological tests; ELISA was applied to serum samples for detection of IgG antibodies against antigen 85 complex (Ag85). The result of ELISA was compared with bacteriological tests [Ziehl Neelson (ZN) staining for acid-fast bacilli, culture on Lowenstein Jensen (LJ) medium and culture on BacT/Alert 3D system]. ELISA tests showed a significantly higher sensitivity (59.1%) as compared with LJ medium culture method (19.3%), BacT/Alert 3D system (24.1%) and ZN staining (16.9%) in all patients (p<0.001). Specificity of ELISA test was 71.9%. In view of the convenience, low cost and good sensitivity, ELISA tests have a promising future in the diagnosis of childhood tuberculosis.

Evaluation of the ICT Tuberculosis test for the routine diagnosis of tuberculosis

BackgroundRapid and accurate diagnosis of tuberculosis (TB) is crucial to facilitate early treatment of infectious cases and thus to reduce its spread. To improve the diagnosis of TB, more rapid diagnostic techniques such as antibody detection methods including enzyme-linked immunosorbent assay (ELISA)-based serological tests and immunochromatographic methods were developed. This study was designed to evaluate the validity of an immunochromatographic assay, ICT Tuberculosis test for the serologic diagnosis of TB in Antalya, Turkey.MethodsSera from 72 patients with active pulmonary (53 smear-positive and 19 smear-negative cases) and eight extrapulmonary (6 smear-positive and 2 smear-negative cases) TB, and 54 controls from different outpatient clinics with similar demographic characteristics as patients were tested by ICT Tuberculosis test.ResultsThe sensitivity, specificity, and negative predictive value of the ICT Tuberculosis test for pulmonary TB were 33.3%, 100%, and 52.9%, respectively. Smear-positive pulmonary TB patients showed a higher positivity rate for antibodies than smear-negative patients, but the difference was not statistically significant. Of the eight patients with extrapulmonary TB, antibody was detected in four patients.ConclusionOur results suggest that ICT Tuberculosis test can be used to aid TB diagnosis in smear-positive patients until the culture results are available.

Potential serological use of a recombinant protein that is a replica of a Mycobacterium tuberculosis protein found in the urine of infected mice.

The recent availability of numerous well-characterized Mycobacterium tuberculosis recombinant proteins has revived interest in the serological diagnosis of tuberculosis. Several promising results have been reported, particularly when more than one antigen is used in the test. However, thus far these antigens have not been used in routine diagnostic tests because they lack sufficient sensitivity. In addition, with the exception of one antigen, most recombinant M. tuberculosis proteins do not identify the majority of tuberculosis patients coinfected with human immunodeficiency virus (HIV). Here, we report a newer M. tuberculosis protein that is a promising candidate for increasing the sensitivity of the serological tests, in particular for patients coinfected with HIV. The protein was found in the urine of mice during the early stages of infection with M. tuberculosis (10 to 14 days), thus suggesting that the antigen is abundantly released during the in vivo growth of the mycobacterium. Reverse genetics was used to produce the recombinant protein, which we named U1 (for urine protein 1). Using a conventional enzyme-linked immunosorbent assay (ELISA), antibody to U1 could be detected in 60% of patients with pulmonary tuberculosis with no signs of coinfection with HIV (n = 83). Conversely, anti-U1 antibody was detected in 87% of the sera from tuberculosis patients coinfected with HIV (n = 47). Out of 12 HIV-infected nontuberculosis patients' sera, 9 did not react with U1 and three sera gave borderline ELISA signals (signal/cutoff of < or =1.75). These results suggest that the high efficiency of U1 in identifying tuberculosis patients coinfected with HIV may be related to abundant release of this protein during the initial phase of the HIV coinfection. The immediate availability of the antigen at a time point in which the patient's immune system is still competent would lead to a secondary immune response to U1 that persists for months in the patient's serum.

Evaluation of a novel tuberculosis complex-specific 34 kDa protein in the serological diagnosis of tuberculosis

Tuberculosis (TB) serological testing with antigen complexes, although very sensitive, is not always as specific due to reactive serum antibodies in patients with inactive TB or nontuberculous infections. Since the use of recombinant M. tuberculosis proteins may enhance specificity, this study was designed to evaluate a novel 34 kDa tuberculosis complex-specific protein as a component of an antigen panel of recombinant proteins. Seventy patients with active TB (41 positive and 29 negative for acid-fast bacilli (AFB) in sputum) were evaluated, in comparison with 30 tuberculin purified protein derivative skin test positive (PPD+) and 30 PPD- normals, 20 subjects with inactive TB and 20 PPD+ subjects with nontuberculous pneumonia as controls. Serum antibody levels were quantified using enzyme linked immunosorbent assay (ELISA) tests with MS2-34, a fusion protein comprising the NH2-terminal 16 kDa of the 34 kDa protein, a recombinant 38 kDa protein (p38), and PPD. Using MS2-34 and p38 as an antigen panel in active TB patients yielded higher sensitivity and negative predictive value (sensitivity 86%; negative predictive value 91%) than using PPD (sensitivity 66%; negative predictive value 81%). Importantly, the MS2-34+p38 panel yielded a higher sensitivity (83%) than PPD (66%) in the subset of AFB- active TB patients. Thus, this novel protein increases sensitivity and specificity of serological testing for TB when used in panels of recombinant proteins.

Preliminary evaluation of a Mycobacterium tuberculosis lipooligosaccharide (LOS) antigen in the serological diagnosis of tuberculosis in HIV seropositive and seronegative patients

To assess the value of detection of specific IgG or IgM antibodies against three polar glycolipids of Mycobacterium tuberculosis in HIV seropositive and seronegative patients with documented active tuberculosis. Using an ELISA test, concentrations of IgM and IgG antibodies against phenolic glycolipid Tb1 (PGL Tb1), 2.3 diacyl trehalose (DAT) and polar lipooligosaccharide (LOS) were measured in the serum of 36 patients with active tuberculosis: 16 tuberculosis patients were coinfected with HIV (TB HIV+), and 20 were HIV seronegative (TB HIV-). 104 control sera were tested: they were collected from 50 healthy blood donors and from 54 asymptomatic HIV seropositive patients. Specificity, sensitivity and predictive positive and negative values were calculated. For the LOS antigen tested, sensitivity, specificity and predictive positive value of specific IgG antibodies were 75%, 95% and 84% respectively among the 36 tuberculosis patients. No significant results were obtained either with the IgM against the LOS antigen or with IgG against the DAT and PGL Tb1 antigens. Significant sensitivity and positive predictive values were demonstrated only with the ELISA test measuring specific IgG against the LOS antigen. The sensitivity was greater in the TB HIV+ subgroup (94%) as compared with the TB HIV- (60%) subgroup. These results were not related to different potential factors such as the site of disease (pulmonary or extrapulmonary) or by the acid-fast bacteria load as detected by the smear microscopic (Ziehl-Neelsen) examination. The results suggest that of the 3 antigens tested, only the LOS antigen is a potential marker for detecting the development of tuberculosis in HIV patients.

Preliminary evaluation of a Mycobacterium tuberculosis phenolglycolipid antigen in the serologic diagnosis of tuberculosis

Resume La presence d'IgM specifiques dirigees contre un antigene phenolglycolipidique recemment decrit chez Mycobacterium tuberculosis a ete recherchee par test ELISA dans le serum de 162 personnes (40 malades tuberculeux et 122 individus sains). Les resultats du test ELISA se sont reveles positifs chez 39 malades tuberculeux (97,5%) et 17 individus sains (13,9%). La sensibilite et la specificite du test etaient respectivement de 97,5 et de 86,1%. Ces resultats indiquent que la recherche d'IgM reagissant avec le phenolglycolipide de M. tuberculosis peut etre utile pour le diagnostic presomptif de la tuberculose active.

Serological diagnosis of tuberculosis: a report of 12 months' clinical experience.

This study reports the use of an agglutination test as a routine diagnostic aid for tuberculosis. We have found this to be both useful and reliable in clinical practice. The overall reliability was greater than 90%, 13 false results being obtained in the 222 patients tested. False negatives (4) were less common than false positives (9). The major advantage of the test is its speed, serological diagnosis being available overnight as compared with an average of one month for diagnosis by culture.

Serological Diagnosis of Tuberculosis

The past decade has seen dramatic developments in serological tests for tuberculosis. The long history of serological tests for tuberculosis is a testimony to the need for a sensitive diagnostic test, especially when the sputum smear is unhelpful. New reagents, both purified antigens and monoclonal antibodies, provide the means to obtain a sensitivity and specificity to rival the tuberculin skin test and equal other commonly used diagnostic blood tests. Evaluation with sera from patients with smear-positive pulmonary tuberculosis has identified one antigen (antigen 5, the 38 kDa antigen) as a potential screening reagent for infectious tuberculosis and another (16 kDa antigen) for monitoring compliance. A monoclonal antibody competition assay anti-38 kDa antibody is the most sensitive serological test for smear-negative tuberculosis so far. Tests for tuberculous meningitis need clinical evaluation. Serological tests for human immunodeficiency virus (HIV)-related tuberculosis are disappointing. In general, antibody levels in primary tuberculosis are low and appear directed towards cytoplasmic antigens, whilst in post-primary disease antibody levels are higher and appear to bind to secreted antigens. No single reagent gives a 100% sensitivity; future research should identify the best combination of antigens for the serodiagnosis of tuberculosis.