Christophe Sola

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections.

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.

Resolving lineage assignation on Mycobacterium tuberculosis clinical isolates classified by spoligotyping with a new high-throughput 3R SNPs based method

We developed a new multiplexed-PCR assay to accurately classify Mycobacterium tuberculosis complex (MTC) isolates at the sublineage level by single nucleotide polymorphisms (SNPs). This method relies on 7 SNPs located in different genes of the MTC strains (recC, rec0, recR, ligB, ligC, alkA, and mgtC). Most of these genes are involved in replication, repair and recombination (3R) functions of M. tuberculosis strains, four of the mutations are synonymous, and thus neutral. Genes were chosen as a first empirical approach to assess the congruence between spoligotyping-based phylogeographical classification and SNP typing. This scheme efficiently classifies most of MTC phylogeographical groups: (1) confirming and identifying new sublineage-specific SNPs, (2) unraveling phylogenetical relationships between spoligotyping-defined MTC sublineages, (3) appropriately assigning sublineages to some spoligotypes and reassigning sublineages to other mis-labeled spoligotype signatures. This study opens the way to a more meaningful taxonomic, evolutionary and epidemiological classification. It also allows evaluation of spoligotype-signature significance towards a more comprehensive understanding of the evolutionary mechanisms of the clustered regularly interspaced short palindromic repeat (CRISPR) locus in MTC.

Combined Species Identification, Genotyping, and Drug Resistance Detection of Mycobacterium tuberculosis Cultures by MLPA on a Bead-Based Array

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

ABSTRACT As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.

A Molecular Epidemiological and Genetic Diversity Study of Tuberculosis in Ibadan, Nnewi and Abuja, Nigeria

Background Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. Methodology/Principal Findings DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other “modern” genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria.

“Spoligoriftyping,” a Dual-Priming-Oligonucleotide-Based Direct-Hybridization Assay for Tuberculosis Control with a Multianalyte Microbead-Based Hybridization System

ABSTRACT We developed “spoligoriftyping,” a 53-plex assay based on two preexisting methods, the spoligotyping and “rifoligotyping” assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

Strain Classification of Mycobacterium tuberculosis Isolates in Brazil Based on Genotypes Obtained by Spoligotyping, Mycobacterial Interspersed Repetitive Unit Typing and the Presence of Large Sequence and Single Nucleotide Polymorphism

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as “LAM-like”, 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

Molecular Analysis of the embCAB Locus and embR Gene Involved in Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis in France

ABSTRACT Modification of codon 306 in embB is regarded as the main mechanism leading to ethambutol (ETB) resistance in clinical isolates of Mycobacterium tuberculosis. However, numerous mutations elsewhere in the embCAB locus and in embR, a putative transcriptional activator of this locus, have been reported to be involved in ETB resistance. Here, we investigated the diversity of nucleotide variations observed in embCAB and embR in M. tuberculosis complex isolates from France. These regions were sequenced in 71 ETB-resistant (ETB-R) and 60 ETB-susceptible (ETB-S) clinical isolates of known phylogenetic lineages. The 131 isolates had 12 mutations corresponding to phylogenetic markers. Among the 60 ETB-S isolates, only 3 (5%) had nonsynonymous mutations that were not phylogenetic markers. Among the 71 ETB-R isolates, 98% had mutations in embCAB that likely contribute to ETB resistance: 70% had mutations located in embB codon 306, 406, or 497; 13% had mutations located outside these three positions between codons 296 and 426; and 15% had mutations corresponding to mutations in the embC-embA intergenic region. We found a strong association between resistance to ETB and the presence of mutations in embB and the embC-embA intergenic region (P < 0.001). In contrast, the mutations detected in embC and embA were not involved in ETB resistance, and no mutation was detected in embR. These results strongly suggest that the sensitivity of diagnostic assays for detecting ETB resistance based on testing of embB codon 306 can be increased by testing of the embB region between codons 296 and 497 and by including the embC-embA intergenic region between positions −8 and −21.

Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis

To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.A GWAS of multi- and extensively drug-resistant tuberculosis using 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries identifies novel mutations associated with resistance. The capacity to detect resistance in particular to ethionamide, pyrazinamide, capreomycin, cycloserine and paraaminosalicylic acid was enhanced by inclusion of insertions and deletions.

Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam

SETTINGnThe molecular diagnosis of tuberculosis (TB) in Viet Nam is often based on the detection of insertion sequence (IS) 6110 in Mycobacterium tuberculosis. However, 8-11% of M. tuberculosis strains in South-East Asia do not contain this target and this undermines the validity of these molecular tests.nnnOBJECTIVEnWe quantified the frequency of M. tuberculosis strains lacking IS6110 in rural Viet Nam and studied their epidemiological and clinical characteristics.nnnDESIGNnConsecutively diagnosed adult TB patients in rural Southern Viet Nam submitted two sputum samples for culture, IS6110 restriction fragment length polymorphism (RFLP) spoligotyping and 15-loci variable number tandem repeat typing. Polymerase chain reaction (PCR) was performed to confirm the absence of IS6110 elements in strains lacking IS6110 hybridisation in RFLP.nnnRESULTSnAmong 2664 TB patient isolates examined, 109 (4.1%) had no IS6110 element. Compared to other strains, these no-copy strains were less often resistant to anti-tuberculosis drugs, particularly to streptomycin (adjusted OR 0.2, 95%CI 0.1-0.5), and showed significant geographic variation. No associations with TB history or demographic factors were found.nnnCONCLUSIONSnStrains without the IS6110 target pose a problem in Viet Nam as regards false-negative molecular TB diagnosis in PCR. Compared to other strains circulating in Viet Nam, no-copy strains are more susceptible to anti-tuberculosis drugs.