2D-DIGE based urinary proteomics and functional enrichment studies to reveal novel Mycobacterium tuberculosis and human protein biomarker candidates for pulmonary tuberculosis.

In the absence of a sensitive and specific diagnostic modality capable of detecting all forms of tuberculosis (TB), proteomics may identify specific Mycobacterium tuberculosis (M.tb) proteins in urine, with a potential as biomarkers. To identify candidate biomarkers for TB, proteome profile of urine from pulmonary TB patients was compared with non-disease controls (NDC) and disease controls (DC, Streptococcus pneumonia infected patients) using a combination of two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LCMS/MS). Eleven differentially expressed host proteins and Eighteen high abundant M.tb proteins were identified. Protein-protein interactome (PPI) and functional enrichment analyses like Gene Ontologies, Reactome pathway etc. demonstrated that the human proteins mainly belong to extracellular space and show physiological pathways for immune response and hematological disorders. Whereas, M.tb proteins belong to the cell periphery, plasma membrane and cell wall, and demonstrated catalytic, nucleotide binding and ATPase activities along with other functional processes. The study findings provide valuable inputs about the biomarkers of TB and shed light on the probable disease consequences as an outcome of the bacterial pathogenicity.

Genotyping Mycobacterium tuberculosis isolates with few copies of IS6110: Value of additional genetic markers.

PURPOSE: IS6110 restriction fragment length polymorphism (RFLP) analysis is widely used for molecular epidemiological studies of tuberculosis. Role of spoligotyping and Fluorescent Amplified Fragment Length Polymorphism (FAFLP) was studied in low-copy number IS6110 strains of Mycobacterium tuberculosis complex (Mtbc). METHODS: The study isolates included 70 strains of Mtbc collected from different regions of India. IS6110 restriction fragment, spoligotyping and FAFLP were performed for genotypic analysis. RESULTS: A single copy of IS6110 was found in 30% of isolates with 90.5% of them harboring characteristic 1.5-Kb IS6110 restriction fragment.IS6110RFLP identified 51 different types, FAFLP 41 types, and spoligotyping 31 types. Combination of all three techniques identified 67 different types.IS6110 RFLP analysis was found sensitive for genotyping isolates with more than one copy of IS6110 (Hunter Gaston Discriminatory Index (HGDI-1) while, neither spoligotyping (HGI-0.89) nor FAFLP (HGDI-0.92) or their combinations were as good. The discriminatory power of spoligotyping (HGDI- 0.89) in isolates with a single copy of IS6110 was higher than IS6110-RFLP.Clustering was reduced to 67% using spoligotyping and to 38% with FAFLP. CONCLUSION: Combination of FAFLP and Spoligotyping may prove to be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of IS6110 element.

Tuberculosis as an Etiological Factor in Liver Abscess in Adults.

Background. Tuberculosis of the liver without active pulmonary or miliary tuberculosis is considered as an uncommon diagnosis. The aim of the present study was to determine the etiological role of tuberculosis in adult patients presenting with features of liver abscess. Methods. A total of 40 patients with liver abscess were included in the study. The liver abscess aspirate was subjected to microscopy, culture, and polymerase chain reaction to determine the role of tuberculosis as an etiological factor in liver abscess. Results. Of the 40 patients enrolled, 25% (10/40) were diagnosed with having tubercular liver abscess. In a total of 40 specimens, 2.5% (1/40) were positive for acid fast bacilli by Ziehl-Neelsen method, while 10% (4/40) were positive for M. tuberculosis by culture using BACTEC 460 and the yield increased to 25% (10/40) by polymerase chain reaction for M. tuberculosis. Conclusion. 25% of the patients presenting with liver abscess had tubercular etiology without features of active pulmonary or miliary tuberculosis. Liver can act as the primary site of involvement in the absence of activity elsewhere in the body. Tuberculosis should be considered as an important differential diagnosis of liver abscess irrespective of evidence of active tuberculosis elsewhere in the body.

Incidence and risk factors for extensively drug-resistant tuberculosis in Delhi region.

BACKGROUND: India with a major burden of multidrug-resistant tuberculosis (MDR-TB) does not have national level data on this hazardous disease. Since 2006, emergence of extensively drug-resistant TB (XDR-TB) is considered a serious threat to global TB control. This study highlights the demographic and clinical risk factors associated with XDR-TB in Delhi. METHODS: The study was conducted during April 2007 to May 2010. Six hundred eleven MDR-TB suspects were enrolled from four tertiary care hospitals, treating TB patients in Delhi and the demographic details recorded. Sputum samples were cultured using rapid, automated liquid culture system (MGIT 960). Drug susceptibility testing (DST) for Rifampicin (RIF) and Isoniazid (INH) was performed for all positive M. tuberculosis (M.tb) cultures. All MDR-TB isolates were tested for sensitivity to second-line drugs [Amikacin (AMK), Capreomycin (CAP), Ofloxacin (OFX), Ethionamide (ETA)]. RESULTS/FINDINGS: Of 611, 483 patients were infected with MDR M. tuberculosis (M.tb) strains. Eighteen MDR-TB isolates (3.7%) were XDR M.tb strains. Family history of TB (p 0.045), socioeconomic status (p 0.013), concomitant illness (p 0.001) and previous intake of 2(nd) line injectable drugs (p 0.001) were significantly associated with occurrence of XDR-TB. Only two of the patients enrolled were HIV seropositive, but had a negative culture for M. tuberculosis. 56/483 isolates were pre-XDR M. tuberculosis, though the occurrence of pre-XDR-TB did not show any significant demographical associations. CONCLUSIONS: The actual incidence and prevalence rate of XDR-TB in India is not available, although some scattered data is available. This study raises a concern about existence of XDR-TB in India, though small, signaling a need to strengthen the TB control program for early diagnosis of both tuberculosis and drug resistance in order to break the chains of transmission.

Diagnostic potential of 16 kDa (HspX, α-crystalline) antigen for serodiagnosis of tuberculosis.

BACKGROUND & OBJECTIVES: Tuberculosis (TB) is a public health problem worldwide. Rapid and accurate diagnosis of tuberculosis is crucial to facilitate early treatment of infectious cases and to reduce its spread. The present study was aimed to evaluation of 16 kDa antigen as a serodiagnostic tool in pulmonary and extra-pulmonary tuberculosis patients in an effort to improve diagnostic algorithm for tuberculosis. METHODS: In this study, 200 serum samples were collected from smear positive and culture confirmed pulmonary tuberculosis patients, 30 tubercular pleural effusions and 21 tubercular meningitis (TBM) patients. Serum samples from 36 healthy, age matched controls (hospital staff), along with 60 patients with non-tubercular respiratory diseases were also collected and evaluated. Humoral response (both IgG and IgA) was looked for 16 kDa antigen using indirect ELISA. RESULTS: Sensitivity of detection in various categories of pulmonary TB patients ranged between 73.8 and 81.2 per cent. While in the extra-pulmonary TB samples the sensitivity was 42.8 per cent (TBM) and 63.3 per cent (tubercular pleural effusion). The test specificity in both the groups was high (94.7%). All of the non-disease controls were negative. Among non-tubercular disease controls, five patients gave a positive humoral response against 16 kDa. INTERPRETATION & CONCLUSIONS: Serodiagnostic tests for TB have always had drawbacks of suboptimal sensitivity and specificity. The antigen used in this study gave encouraging results in pulmonary TB only, while in extra-pulmonary TB (tubercular meningitis and tubercular pleural effusion), this has shown a limited role in terms of sensitivity. Further work is required to validate its role in serodiagnosis of TB especially extra-pulmonary TB.

Utility of reverse transcriptase PCR and DNA-PCR in the diagnosis of female genital tuberculosis.

This study was designed to test the utility of mRNA-based RT-PCR to detect viable bacilli, indicating active tubercular involvement, and DNA-PCR to detect present or past infection in the diagnosis of active female genital tuberculosis (TB) infection. A total of 200 subjects with complaints of infertility were enrolled in the study. Multiple sampling was done. One hundred and forty-three endometrial aspirate (EA), 94 peritoneal fluid/peritoneal washing (PF/PW) and six cornual biopsy (CB) specimens were collected for diagnosis using microscopy, culture, RT-PCR and DNA-PCR and results were compared with laparoscopic findings. RT-PCR and culture were concordant [positive in four (2.8%) EA specimens] signalling sampling from the site of active infection. Smear microscopy showed a poor detection rate while DNA-PCR showed high positivity. Sixty-one (44.85%) EA specimens, nine (9.57%) PF/PW specimens and two (33.33%) CB specimens were positive by DNA-PCR. Genital TB causing infertility (localized or secondary to TB elsewhere) can be picked up early by DNA-PCR, when it can be completely cured prior to the appearance of florid disease.

Characterization of predominant Mycobacterium tuberculosis strains from different subpopulations of India.

The predominant strains from India belong to Central-Asian (CAS) and the East-African-Indian (EAI) clade of Mycobacterium tuberculosis. The two clades have also been shown to be geographically partitioned. The study of such strains may help to understand the characteristics that make M. tuberculosis an effective pathogen and its overrepresentation in certain populations. M. tuberculosis isolates characterized by spoligotyping under a population based tuberculosis study covering different regions from the North and South India were further analyzed by restriction fragment length polymorphism (RFLP) and by deletion analysis of M. tuberculosis specific deletion region 1 (TbD1). The genetic relationship of the two clades inferred using different genetic markers showed good correlation. In the North where the CAS clade predominates the isolates are characterized by presence of high IS6110 copy number and absence of TbD1 region whereas in the South where the EAI clade predominates the isolates are characterized by low copy number of IS6110 and presence of TbD1 region. The ancestral EAI strains were found to be less often associated with drug resistance or young age as compared to the CAS clade. The study highlights that the EAI lineage is well established in India and that CAS may be emerging or more recently introduced to India. The results depict a distinction in the lineage of strains from the North versus South India indicating a need to study if the pathogen has adapted to specific human populations.