scholarly journals Does Choice Matter? Reference-Based Alignment for Molecular Epidemiology of Tuberculosis

2016 ◽  
Vol 54 (7) ◽  
pp. 1891-1895 ◽  
Author(s):  
Robyn S. Lee ◽  
Marcel A. Behr
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Epidemiology ◽  
Phylogenetic Trees ◽  
Reference Strain ◽  
Reference Genome ◽  
Nucleotide Polymorphisms ◽  
Short Sequence ◽  
Single Nucleotide ◽  
Content Type ◽  
Genome Selection

When using genome sequencing for molecular epidemiology, short sequence reads are aligned to an arbitrary reference strain to detect single nucleotide polymorphisms. We investigated whether reference genome selection influences epidemiological inferences ofMycobacterium tuberculosistransmission by aligning sequence reads from 162 closely related lineage 4 (Euro-American) isolates to 7 different genomes. Phylogenetic trees were consistent with use of all but the most divergent genomes, suggesting that reference choice can be based on considerations other thanM. tuberculosislineage.

scholarly journals Using Mycobacterium tuberculosis Single-Nucleotide Polymorphisms To Predict Fluoroquinolone Treatment Response

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Marva Seifert ◽  
Edmund Capparelli ◽  
Donald G. Catanzaro ◽  
Timothy C. Rodwell
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Single Nucleotide Polymorphisms ◽  
Specific Resistance ◽  
Therapeutic Targets ◽  
World Health ◽  
Population Pharmacokinetic ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type ◽  
Level Increase

ABSTRACT Clinical phenotypic fluoroquinolone susceptibility testing of Mycobacterium tuberculosis is currently based on M. tuberculosis growth at a single critical concentration, which provides limited information for a nuanced clinical response. We propose using specific resistance-conferring M. tuberculosis mutations in gyrA together with population pharmacokinetic and pharmacodynamic modeling as a novel tool to better inform fluoroquinolone treatment decisions. We sequenced the gyrA resistance-determining region of 138 clinical M. tuberculosis isolates collected from India, Moldova, Philippines, and South Africa and then determined each strain’s MIC against ofloxacin, moxifloxacin, levofloxacin, and gatifloxacin. Strains with specific gyrA single-nucleotide polymorphisms (SNPs) were grouped into high or low drug-specific resistance categories based on their empirically measured MICs. Published population pharmacokinetic models were then used to explore the pharmacokinetics and pharmacodynamics of each fluoroquinolone relative to the empirical MIC distribution for each resistance category to make predictions about the likelihood of patients achieving defined therapeutic targets. In patients infected with M. tuberculosis isolates containing SNPs associated with a fluoroquinolone-specific low-level increase in MIC, models suggest increased fluoroquinolone dosing improved the probability of achieving therapeutic targets for gatifloxacin and moxifloxacin but not for levofloxacin and ofloxacin. In contrast, among patients with isolates harboring SNPs associated with a high-level increase in MIC, increased dosing of levofloxacin, moxifloxacin, gatifloxacin, or ofloxacin did not meaningfully improve the probability of therapeutic target attainment. We demonstrated that quantifiable fluoroquinolone drug resistance phenotypes could be predicted from rapidly detectable gyrA SNPs and used to support dosing decisions based on the likelihood of patients reaching therapeutic targets. Our findings provide further supporting evidence for the moxifloxacin clinical breakpoint recently established by the World Health Organization.

scholarly journals Comparative Genomics ofesxGenes from Clinical Isolates of Mycobacterium tuberculosis Provides Evidence for Gene Conversion and Epitope Variation

2011 ◽  
Vol 79 (10) ◽  
pp. 4042-4049 ◽  
Author(s):  
Swapna Uplekar ◽  
Beate Heym ◽  
Véronique Friocourt ◽  
Jacques Rougemont ◽  
Stewart T. Cole
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Sequences ◽  
Antigenic Variation ◽  
Clinical Isolates ◽  
Nucleotide Polymorphisms ◽  
Secretion Systems ◽  
Human Immune System ◽  
Single Nucleotide ◽  
Content Type ◽  
Type Vii Secretion

ABSTRACTThe 23-membered Esx protein family is involved in the host-pathogen interactions ofMycobacterium tuberculosis. These secreted proteins are among the most immunodominant antigens recognized by the human immune system and have thus been used to develop vaccines and immunodiagnostic tests for tuberculosis (TB). Gene pairs for 10 Esx proteins are contained in the ESX-1 to ESX-5 loci, encoding type VII secretion systems. A subset of Esx proteins can be further classified into the Mtb9.9, QILSS, and TB10.4 subfamilies. To survey genetic diversity in the Esx family and its potential for antigenic variation, we sequenced allesxgenes from 108 clinical isolates ofM. tuberculosisfrom different clades by using a targeted approach. A total of 109 unique single nucleotide polymorphisms (SNPs) were observed, and 59 of these were nonsynonymous. Some of the resultant amino acid substitutions affect known Esx epitopes, including two in the EsxB (CFP-10) and EsxH (TB10.4) antigens. Assessment of the SNP distribution across the Esx proteins revealed high genetic variability, especially in the Mtb9.9 and QILSS subfamilies, and more conservation in the ESX-1 to ESX-4 loci. Comparison of the DNA sequences of variableesxgenes provided clear evidence for recombination events between different genes in the same strain, some of which are predicted to truncate the corresponding protein. Many of these polymorphisms escape detection by ultrahigh-throughput sequencing using short sequence reads, as such approaches cannot distinguish between closely related genes. Theesxgene family is dynamic, and sequence changes likely lead to immune variation.

scholarly journals Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country

2016 ◽  
Vol 54 (7) ◽  
pp. 1862-1870 ◽  
Author(s):  
David Stucki ◽  
Marie Ballif ◽  
Matthias Egger ◽  
Hansjakob Furrer ◽  
Ekkehardt Altpeter ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Nucleotide Polymorphisms ◽  
Foreign Born ◽  
Single Nucleotide ◽  
Content Type ◽  
Weak Evidence ◽  
High Incidence ◽  
Low Incidence

Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520Mycobacterium tuberculosisisolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.

scholarly journals Ultrafast Assessment of the Presence of a High-Risk Mycobacterium tuberculosis Strain in a Population

2015 ◽  
Vol 54 (3) ◽  
pp. 779-781 ◽  
Author(s):  
Laura Pérez-Lago ◽  
Marta Herranz ◽  
Iñaki Comas ◽  
María Jesús Ruiz-Serrano ◽  
Paula López Roa ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Risk ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type ◽  
Tuberculosis Strain ◽  
Target Strain ◽  
Pcr Method ◽  
Mycobacterium Tuberculosis Strain

A persistent 8-year infection by a BeijingMycobacterium tuberculosisstrain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission—despite opportunities—and challenged some of the advantages associated with Beijing strains.

scholarly journals Draft Genome Sequence of the First Confirmed Isolate of Multidrug-Resistant Mycobacterium tuberculosis in Tasmania

2017 ◽  
Vol 5 (44) ◽  
Author(s):  
Sanjay S. Gautam ◽  
Micheál Mac Aogáin ◽  
Ronan F. O’Toole
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Genome Sequence ◽  
Draft Genome ◽  
Multidrug Resistant ◽  
Whole Genome Sequence ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type ◽  
Global Challenge ◽  
Mdr Tb

ABSTRACT The spread of multidrug-resistant (MDR) tuberculosis (TB) has become a major global challenge. In 2016, Tasmania recorded its first known incidence of MDR-TB. Here, we report the draft whole-genome sequence of the Mycobacterium tuberculosis isolate from this case, TASMDR1, and describe single-nucleotide polymorphisms associated with its drug resistance.

scholarly journals Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis

2016 ◽  
Vol 54 (12) ◽  
pp. 2969-2974 ◽  
Author(s):  
Laura Pérez-Lago ◽  
Miguel Martínez-Lirola ◽  
Sergio García ◽  
Marta Herranz ◽  
Igor Mokrousov ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Risk ◽  
Primary Cultures ◽  
Hospital Setting ◽  
Drug Resistant ◽  
Nucleotide Polymorphisms ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Incident Cases ◽  
Rule Out

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk importedMycobacterium tuberculosisstrains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR BeijingM. tuberculosisstrains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis.

scholarly journals Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing

2014 ◽  
Vol 80 (14) ◽  
pp. 4398-4413 ◽  
Author(s):  
Sam Crauwels ◽  
Bo Zhu ◽  
Jan Steensels ◽  
Pieter Busschaert ◽  
Gorik De Samblanx ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Dna Fingerprinting ◽  
Wine Industry ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type ◽  
Carbon And Nitrogen ◽  
Spoilage Yeasts ◽  
Off Flavors ◽  
Carbon And Nitrogen Metabolism

ABSTRACTBrettanomycesyeasts, with the speciesBrettanomyces(Dekkera)bruxellensisbeing the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However,B. bruxellensisis also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance,Brettanomycesyeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50Brettanomycesstrains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between theB. bruxellensisfingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate ofB. bruxellensis(VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminateBrettanomycesstrains and provides a first glimpse at the genetic diversity and genome plasticity ofB. bruxellensis.

scholarly journals Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5658-5664 ◽  
Author(s):  
Soo-Jin Yang ◽  
Nagendra N. Mishra ◽  
Aileen Rubio ◽  
Arnold S. Bayer
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Reading Frame ◽  
A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

scholarly journals Molecular Surveillance of Drug Resistance of Plasmodium falciparum Isolates Imported from Angola in Henan Province, China

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Ruimin Zhou ◽  
Chengyun Yang ◽  
Suhua Li ◽  
Yuling Zhao ◽  
Ying Liu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Henan Province ◽  
Nucleotide Polymorphisms ◽  
Combination Therapies ◽  
Antimalarial Drug Resistance ◽  
Single Nucleotide ◽  
Molecular Surveillance ◽  
Content Type

ABSTRACT Angola was the main origin country for the imported malaria in Henan Province, China. Antimalarial drug resistance has posed a threat to the control and elimination of malaria. Several molecular markers were confirmed to be associated with the antimalarial drug resistance, such as pfcrt, pfmdr1, pfdhfr, pfdhps, and K13. This study evaluated the drug resistance of the 180 imported Plasmodium falciparum isolates from Angola via nested PCR using Sanger sequencing. The prevalences of pfcrt C72V73M74N75K76, pfmdr1 N86Y184S1034N1042D1246, pfdhfr A16N51C59S108D139I164, and pfdhps S436A437A476K540A581 were 69.4%, 59.9%, 1.3% and 6.3%, respectively. Three nonsynonymous (A578S, M579I, and Q613E) and one synonymous (R471R) mutation of K13 were found, the prevalences of which were 2.5% and 1.3%, respectively. The single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, and pfdhps were generally shown as multiple mutations. The mutant prevalence of pfcrt reduced gradually, but pfdhfr and pfdhps still showed high mutant prevalence, while pfmdr1 was relatively low. The mutation of the K13 gene was rare. Molecular surveillance of artemisinin (ART) resistance will be used as a tool to evaluate the real-time efficacy of the artemisinin-based combination therapies (ACTs) and the ART resistance situation.

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