Philip Noel Suffys

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.

Snapshot of Moving and Expanding Clones of Mycobacterium tuberculosis and Their Global Distribution Assessed by Spoligotyping in an International Study

ABSTRACT The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.

Global Distribution of Mycobacterium tuberculosis Spoligotypes

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

Role of Tumor Necrosis Factor–α and Interleukin-10 Promoter Gene Polymorphisms in Leprosy

Single-nucleotide polymorphismswithin thegenescodingfortumornecrosisfactor(TNF)‐a and interleukin (IL)‐10 have been associated with several infectious diseases. To determine whether such polymorphisms are associated with leprosy, genotyping was performed at the 308 and 238 positions of the promoter of the TNF-a gene in 210 and 191 patients with multibacillary (MB) leprosy, respectively; 90 and 79 patients with paucibacillary (PB) leprosy; and 92 control subjects. For the 592 and 819 positions within the promoter of the IL-10 gene, 143 patients with MB leprosy, 79 patients with PB leprosy, and 62 control subjects were included in the analysis. TNF2 allele frequency was significantly higheramong controlsubjects than among all patients with leprosy or in the MB group ( and ). For the ILP ! .05 P ! .01 10 gene, the frequency of the homozygous 819TT genotype was significantly higher among patients than among control subjects. These data indicate that a relationship exists between TNF-a and IL-10 promoter polymorphisms and the development of PB leprosy. The more benign paucibacillary (PB) forms of leprosy— borderline tuberculoid (BT) and tuberculoid tuberculoid (TT) leprosy—are characterized by the predominance of a Th1-type immune response, the presence of well-formed granulomas at the site of the lesion, and control of mycobacterial replication. In contrast, in the multibacillary (MB) forms—borderline borderline (BB) and borderline lepromatous (BL) leprosy and lepromatous leprosy (LL)—no granuloma is seen, and high bacterial load and antibody levels are detected. Cytokines evidently play a critical role in triggering host-pathogen interactions. On one hand, greater tumor necrosis factor (TNF)–a production,

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.

Correlations of mutations in katG, oxyR-ahpC and inhA genes and in vitro susceptibility in Mycobacterium tuberculosis clinical strains segregated by spoligotype families from tuberculosis prevalent countries in South America.

BackgroundMutations associated with resistance to rifampin or streptomycin have been reported for W/Beijing and Latin American Mediterranean (LAM) strain families of Mycobacterium tuberculosis. A few studies with limited sample sizes have separately evaluated mutations in katG, ahpC and inhA genes that are associated with isoniazid (INH) resistance. Increasing prevalence of INH resistance, especially in high tuberculosis (TB) prevalent countries is worsening the burden of TB control programs, since similar transmission rates are noted for INH susceptible and resistant M. tuberculosis strains.ResultsWe, therefore, conducted a comprehensive evaluation of INH resistant M. tuberculosis strains (n = 224) from three South American countries with high burden of drug resistant TB to characterize mutations in katG, ahpC and inhA gene loci and correlate with minimal inhibitory concentrations (MIC) levels and spoligotype strain family. Mutations in katG were observed in 181 (80.8%) of the isolates of which 178 (98.3%) was contributed by the katG S315T mutation. Additional mutations seen included oxyR-ahpC; inhA regulatory region and inhA structural gene. The S315T katG mutation was significantly more likely to be associated with MIC for INH ≥2 μg/mL. The S315T katG mutation was also more frequent in Haarlem family strains than LAM (n = 81) and T strain families.ConclusionOur data suggests that genetic screening for the S315T katG mutation may provide rapid information for anti-TB regimen selection, epidemiological monitoring of INH resistance and, possibly, to track transmission of INH resistant strains.

Discovery of a Novel Mycobacterium tuberculosis Lineage That Is a Major Cause of Tuberculosis in Rio de Janeiro, Brazil

ABSTRACT The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for genomic deletions. One locus in our panel of PCR targets failed to amplify in ∼30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RDRio. Homologous recombination between two similar protein-coding genes is proposed as the mechanism for deleting or modifying 10 genes, including two potentially immunogenic PPE proteins. The flanking regions of the RDRio locus were identical in all strains bearing the deletion. Genetic testing by principal genetic group, spoligotyping, variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR), and IS6110-based restriction fragment length polymorphism analysis cumulatively support the idea that RDRio strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RDRio lineage is therefore the predominant clade causing tuberculosis (TB) in Rio de Janeiro and, as indicated by genotypic clustering in MIRU-VNTR analysis, the most significant source of recent transmission. Limited retrospective reviews of bacteriological and patient records showed a lack of association with multidrug resistance or specific risk factors for TB. However, trends in the data did suggest that RDRio strains may cause a form of TB with a distinct clinical presentation. Overall, the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a Euro-Latin American host population. The identification of RDRio strains outside of Brazil points to the ongoing intercontinental dissemination of this important genotype. Further studies are needed to determine the differential strain-specific features, pathobiology, and worldwide prevalence of RDRioM. tuberculosis.

Rapid Identification of Mycobacteria to the Species Level Using INNO-LiPA Mycobacteria, a Reverse Hybridization Assay

ABSTRACT INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification ofMycobacterium species in culture and identifies theMycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacteriumspecies: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with theMycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species- or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA asM. avium (n = 18) or M. intracellulare (n = 7) or as belonging to theM. avium-M. intracellulare-M. scrofulaceum complex (n = 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.

Application of Sensitive and Specific Molecular Methods To Uncover Global Dissemination of the Major RDRio Sublineage of the Latin American-Mediterranean Mycobacterium tuberculosis Spoligotype Family

ABSTRACT The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of ∼15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RDRio. Using the Brazilian strains, we pinpoint an Ag85C103 single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RDRio strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between “wild-type” and RDRio strains, were then used to analyze an international collection of M. tuberculosis strains. RDRioM. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RDRio and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C103 SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RDRio strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RDRio sublineage.

Genetic polymorphisms of NAT2, CYP2E1 and GST enzymes and the occurrence of antituberculosis drug-induced hepatitis in Brazilian TB patients

Isoniazid (INH), one of the most important drugs used in antituberculosis (anti-TB) treatment, is also the major drug involved in hepatotoxicity. Differences in INH-induced toxicity have been attributed to genetic variability at several loci, such as NAT2, CYP2E1, GSTM1 and GSTT1, that code for drug-metabolising enzymes. Our goal was to examine the polymorphisms in these enzymes as susceptibility factors to anti-TB drug-induced hepatitis in Brazilian individuals. In a case-control design, 167 unrelated active tuberculosis patients from the University Hospital of the Federal University of Rio de Janeiro, Brazil, were enrolled in this study. Patients with a history of anti-TB drug-induced acute hepatitis (cases with an increase to 3 times the upper limit of normal serum transaminases and symptoms of hepatitis) and patients with no evidence of anti-TB hepatic side effects (controls) were genotyped for NAT2, CYP2E1, GSTM1 and GSTT1 polymorphisms. Slow acetylators had a higher incidence of hepatitis than intermediate/rapid acetylators [22% (18/82) vs. 9.8% (6/61), odds ratio (OR), 2.86, 95% confidence interval (CI), 1.06-7.68, p = 0.04). Logistic regression showed that slow acetylation status was the only independent risk factor (OR 3.59, 95% CI, 2.53-4.64, p = 0.02) for the occurrence of anti-TB drug-induced hepatitis during anti-TB treatment with INH-containing schemes in Brazilian individuals.