Antibodies against native proteins of Mycobacterium tuberculosis can detect pulmonary tuberculosis patients.

Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn't distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734-0.890, p < 0.0001) and 0.821 (95% CI 0.752-0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB's POCT by antibody-based diagnosis.

Monitoring IgG against Mycobacterium tuberculosis proteins in an Asian elephant cured of tuberculosis that developed from long-term latency.

Tuberculosis (TB) is fatal in elephants, hence protecting elephants from TB is key not only in the conservation of this endangered animal, but also to prevent TB transmission from elephants to humans. Most human TB cases arise from long-term asymptomatic infections. Significant diagnostic challenges remain in the detection of both infection and disease development from latency in elephants due to their huge bodies. In this study, we assessed cryopreserved sera collected for over 16 years, from the first Japanese treatment case of elephant TB. Semi-quantification of IgG levels to 11 proteins showed high detection levels of 3 proteins, namely ESAT6/CFP10, MPB83 and Ag85B. The level of IgG specific to these 3 antigens was measured longitudinally, revealing high and stable ESAT6/CFP10 IgG levels regardless of onset or treatment. Ag85B-specifc IgG levels were largely responsive to onset or treatment, while those of MPB83 showed intermediate responses. These results suggest that ESAT6/CFP10 is immunodominant in both asymptomatic and symptomatic phases, making it useful in the detection of infection. On the other hand, Ag85B has the potential to be a marker for the prediction of disease onset and in the evaluation of treatment effectiveness in elephants.

SEVERITY OF TB CLASSIFIED BY MODIFIED BANDIM TB SCORING ASSOCIATES WITH THE SPECIFIC SEQUENCE OF ESXA GENES IN MDR-TB PATIENTS.

BACKGROUND: The severity of pulmonary TB and detection of multidrug-resistant (MDR-TB) TB strains as potential causative agents could be crucial for the determination of treatment success. This study aimed to analyze the association between the specific sequences of the full esxA gene from MDR-TB sputum isolates and the severity class of MDR-TB patients. MATERIAL AND METHODS: A total of 98 sputum samples that were suspected to be MDR-TB were collected from Dr. Soetomo, Surabaya, Indonesia, from September to December 2016. A total of 24 isolates from the 98 patients were confirmed to have positive MDR-TB based on the GeneXpert test. MDR-TB isolates were tested using PCR targeting 580 bp encompassing the full esxA gene, and the resulting amplicon was sequenced. The severity class of the pulmonary TB patients was assessed using modified Bandim TB scoring. RESULTS: The patient severity classification resulted in a moderate and severe degree of TB in 38% and a mild degree of TB in 63% of patients. Visualization of the PCR results showed that all MDR-TB samples were positive for the 580 bp band, and the sequence results showed 100% homology with that of the virulent wild-type M. tuberculosis H37Rv (NC_000962.3). CONCLUSIONS: In the current study, an association between the characteristics of the full esxA gene and the severity class of MDR-TB patients is yet to be found. However, the homologous sequence of all samples, associated with various degrees of disease severity, possess 100% identity with that of wild-type M. tuberculosis H37Rv.

Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia.

Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.

Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia

Abstract Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.

T CELL EPITOPES OF THE ESXA FULL GENE OF MYCOBACTERIUM TUBERCULOSIS FROM SPUTUM OF MDR-TB PATIENTS.

BACKGROUND: In 2015, World Health Organization (WHO) discovered 10.4 million tuberculosis (TB) cases around the world. Multidrug-resistant tuberculosis (MDR-TB) became a threat because it has high mortality number. There were 480,000 new MDR-TB cases in 2015. Based on those problems, diagnostic development to detect M. tuberculosis rapidly and accurately is needed. The importance of detecting epitope expression of esxA full gene because there was a potential of complexity over the protein structure and might affect the protein concentration. By knowing epitope prediction, there's an expectation that it can help the development of TB diagnostic. This research was aimed to determine the T cell epitope prediction of esxA full gene from MDR-TB patients. MATERIAL AND METHODS: Total of 24 MDR-TB sputum isolate from TB patients at Dr. Soetomo Hospital were collected from September to December 2016. Samples were confirmed as MDR-TB using GeneXpert and Bactec MGIT 960. Those samples tested using PCR targeted 580 bp of esxA gene and sequencing. Gene sequence was aligned against wild type using Bioedit program version 7.2.5 and NCBI BLAST. T cell epitope prediction was analyzed by GENETYX version 10. RESULTS: Epitope predictions that could be obtained were IEAAAS, ASAIQG, VTSIHS, TKLAAA, VTGMFA based IAd Pattern Position and EAAAS based Rothbard/Taylor Pattern Position. Those prediction epitopes can determine the severity of disease, therefore full gene of esxA could be used as diagnostic target. CONCLUSION: This research discovered five specific T cell epitope prediction based on IAd Pattern Position and one epitope prediction according to Rothbard/Taylor Pattern Position.

Nontuberculous mycobacterial species and Mycobacterium tuberculosis complex coinfection in patients with pulmonary tuberculosis in Dr. Soetomo Hospital, Surabaya, Indonesia.

OBJECTIVE/BACKGROUND: The aim of this study was to analyze the detection of nontuberculous mycobacterial (NTM) species derived from sputum specimens of pulmonary tuberculosis (TB) suspects. Increasing prevalence and incidence of pulmonary infection by NTM species have widely been reported in several countries with geographical variation. MATERIALS AND METHODS: Between January 2014 and September 2015, sputum specimens from chronic pulmonary TB suspect patients were analyzed. Laboratory examination of mycobacteria was conducted in the TB laboratory, Department of Clinical Microbiology, Dr. Soetomo Hospital, Surabaya. Detection and identification of mycobacteria were performed by the standard culture method using the BACTEC MGIT 960 system (BD) and Lowenstein-Jensen medium. Identification of positive Mycobacterium tuberculosis complex (MTBC) was based on positive acid-fast bacilli microscopic smear, positive niacin accumulation, and positive TB Ag MPT 64 test results (SD Bioline). If the growth of positive cultures and acid-fast bacilli microscopic smear was positive, but niacin accumulation and TB Ag MPT 64 (SD Bioline) results were negative, then the isolates were categorized as NTM species. MTBC isolates were also tested for their sensitivity toward first-line anti-TB drugs, using isoniazid, rifampin, ethambutol, and streptomycin. RESULTS: From 2440 sputum specimens of pulmonary TB suspect patients, 459 isolates (18.81%) were detected as MTBC and 141 (5.78%) as NTM species. CONCLUSION: From the analyzed sputum specimens, 18.81% were detected as MTBC and 5.78% as NTM species. Each pulmonary TB suspect patient needed clinical settings to suspect causative agents of MTBC and/or NTM species; clinicians have to understand the local epidemiological data for the evaluation of causes of lung infection to determine appropriate therapy.