Urvashi B. Singh

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology

BackgroundThe Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.ResultsThe fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network.ConclusionOur results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Clinical, Endoscopic, and Histological Differentiations Between Crohn's Disease and Intestinal Tuberculosis

OBJECTIVES:The clinical, endoscopic, and histological features of Crohns disease (CD) and intestinal tuberculosis mimic each other so much that it becomes difficult to differentiate between them. The aim was to find out clinical, endoscopic, and histological predictor features for differentiation between CD and intestinal tuberculosis.METHODS:We recruited 106 patients, 53 each with CD and intestinal tuberculosis, in this study. The clinical, histological, and endoscopic features were subjected to univariate, bivariate, and multivariate analyses. On the basis of regression coefficients of the final multivariate logistic model, a score to discriminate between CD and intestinal tuberculosis was devised. For the validation of the score, the same model was tested on 20 new patients, each with CD and intestinal tuberculosis.RESULTS:On univariate analysis, although longer duration of disease, chronic diarrhea, blood in stool, perianal disease, extra-intestinal manifestations, involvement of left colon, skip lesions, aphthous ulcers, cobblestoning, longitudinal ulcers, focally enhanced colitis, and microgranulomas were significantly more common in CD, partial intestinal obstruction, constipation, presence of nodular lesions, higher number, and larger granulomas were significantly more common in intestinal tuberculosis. On multivariate analysis, blood in stool (odds ratio (OR) 0.1 (confidence interval (CI) 0.04–0.5)), weight loss (OR 9.8 (CI 2.2–43.9)), histologically focally enhanced colitis (OR 0.1 (CI 0.03–0.5)), and involvement of sigmoid colon (OR 0.07(0.01–0.3)) were independent predictors of intestinal tuberculosis. On the basis of regression coefficients of the final multivariate logistic model, a score that varied from 0.3 to 9.3 was devised. Higher score predicted more likelihood of intestinal tuberculosis. Once the cutoff was set at 5.1, then the sensitivity, specificity, and ability to correctly classify the two diseases were 83.0, 79.2, and 81.1%, respectively. Area under the curve for receiver-operating characteristic (ROC) to assess the ability of these features to discriminate between CD and intestinal tuberculosis was 0.9089. The area under ROC in the validation data set was 89.2% (95% CI 0.79–0.99). With a similar cutoff score of 5.1, sensitivity and specificity in the validation model were 90% (95% CI 66.9–98.2) and 60% (95% CI 36.4–80.0), respectively.CONCLUSIONS:Blood in stool, weight loss, focally enhanced colitis, and involvement of the sigmoid colon were the most important features in differentiating CD from intestinal tuberculosis.

Predominant Tuberculosis Spoligotypes, Delhi, India

One hundred five Mycobacterium tuberculosis clinical isolates from the Delhi area were typed by spoligotyping; 45 patterns were identified. Comparison with an international spoligotype database showed type 26, Delhi type (22%), type 54 (12%), and type 1, Beijing type (8%), as the most common. Eighteen spoligotypes did not match any existing database pattern.

Partial Nucleotide Sequencing and Molecular Evolution of Epidemic Causing Dengue 2 Strains

To study the genetic variability and to detect evolutionary changes and movement of dengue 2 (DEN-2) strains, nucleotide sequencing of the envelope protein gene and the nonstructural protein 1 gene junction was performed for 9 isolates from the 1996 Delhi epidemic and 1 isolate from the 1967 Delhi epidemic. The epidemic strains had a divergence of 10%-11% from the 1967 strains, but were quite similar to DEN-2 isolates from Seychelles, Somalia, and Torres Strait. In addition, the sequence data were compared to the prototype DEN-2 strain, New Guinea C, and other published DEN-2 sequences from different parts of the world. The phylogenetic analysis by the Molecular Evolutionary Genetics Analysis program suggests that the 1996 Delhi isolates of DEN-2 were genotype IV. The 1967 isolate was similar to a 1957 isolate of DEN-2, P9-122, from India, and was classified as genotype V. This study indicates that earlier DEN-2 strains of genotype V have been replaced by genotype IV.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus.

ABSTRACT An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. abscessus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients.

Genital tuberculosis among infertile women and fertility outcome after antitubercular therapy.

To compare modalities for diagnosing genital tuberculosis (GTB) and to assess fertility outcome after antitubercular therapy (ATT).

Tubercular infection after arthroscopic anterior cruciate ligament reconstruction.

PURPOSE Tubercular infection has not been described, to our knowledge, in the literature after anterior cruciate ligament (ACL) reconstruction, and, hence, the purpose of our case series was to describe our experience, evaluate the clinical and laboratory findings, and assess the treatment outcome. METHODS We performed a retrospective analysis of 1,152 cases of arthroscopic ACL reconstruction with autografts performed at our institution between January 1998 and May 2007. Tubercular infection was considered to be present in the setting of recurrent negative bacterial cultures but a positive result on microscopy, culture, histopathology, or polymerase chain reaction (PCR). All patients underwent arthroscopic lavage and synovectomy, followed by antitubercular therapy for 12 months. RESULTS We identified 8 patients (0.69%) with infection. Bone-patellar tendon-bone graft was used in 1 and hamstring graft in 7. All patients were immunocompetent. The mean time from surgery to presentation was 64.4 days (range, 23 to 152 days). Aspirate fluid staining and culture for acid-fast bacilli was negative in all cases, synovial tissue culture was positive in 3, characteristic histopathology was positive in 7, and PCR was positive in 6. A mean of 1.25 surgeries (range, 1 to 2) were performed. The mean length of follow-up in our series was 43.6 months (range, 25 to 73 months), with no reinfections. The mean postoperative Lysholm knee score was 80. CONCLUSIONS Tubercular infection as a complication after arthroscopic ACL reconstruction, though rare, should be kept in mind as a possible cause of infection in immunocompetent patients in zones endemic for tuberculosis. It should also be kept in mind in nonendemic areas, among immigrants from endemic areas, and in cases with persistent swelling and discharge, effusion with minimal inflammatory signs, and negative cultures. We recommend deoxyribonucleic acid-PCR testing for early diagnosis of tuberculosis. Arthroscopic debridement and antitubercular chemotherapy together are the mainstay of treatment.

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 2nd 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.

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.