Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda

Ssengooba, Willy; Meehan, Conor J.; Lukoye, Deus; Kasule, George William; Musisi, Kenneth; Joloba, Moses; Cobelens, Frank; Jong, Bouke C. de

doi:10.1016/j.meegid.2016.02.019

Cited by 36 publications

(35 citation statements)

References 46 publications

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“…18 Within the MDR sub-cluster, ethambutol and pyrazinamide was acquired and transmitted. This finding was similar to that of a study in Uganda, 47 where pyrazinamide resistance typically arose in MDR strains with several different causative mutations. This highlights the importance of testing for pyrazinamide resistance in order to determine benefit of its use in MDR-TB treatment regimens.…”

Section: Discussionsupporting

confidence: 90%

Genotypic clustering does not imply recent tuberculosis transmission in a high prevalence setting: A genomic epidemiology study in Lima, Peru

Dixit

Freschi

Vargas

et al. 2018

Preprint

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BackgroundWhole genome sequencing (WGS) can elucidate Mycobacterium tuberculosis (Mtb) transmission patterns but more data is needed to guide its use in high-burden settings. In a household-based transmissibility study of 4,000 TB patients in Lima, Peru, we identified a large MIRU-VNTR Mtb cluster with a range of resistance phenotypes and studied host and bacterial factors contributing to its spread.MethodsWGS was performed on 61 of 148 isolates in the cluster. We compared transmission link inference using epidemiological or genomic data with and without the inclusion of controversial variants, and estimated the dates of emergence of the cluster and antimicrobial drug resistance acquisition events by generating a time-calibrated phylogeny. We validated our findings in genomic data from an outbreak of 325 TB cases in London. Using a larger set of 12,032 public Mtb genomes, we determined bacterial factors characterizing this cluster and under positive selection in other Mtb lineages.FindingsFour isolates were distantly related and the remaining 57 isolates diverged ca. 1968 (95% HPD: 1945-1985). Isoniazid resistance arose once, whereas rifampicin resistance emerged subsequently at least three times. Amplification of other drug resistance occurred as recently as within the last year of sampling. High quality PE/PPE variants and indels added information for transmission inference. We identified five cluster-defining SNPs, including esxV S23L to be potentially contributing to transmissibility.InterpretationClusters defined by MIRU-VNTR typing, could be circulating for decades in a high-burden setting. WGS allows for an improved understanding of transmission, as well as bacterial resistance and fitness factors.FundingThe study was funded by the National Institutes of Health (Peru Epi study U19-AI076217 and K01-ES026835 to MRF). The funding sources had no role in any aspect of the study, manuscript or decision to submit it for publication.Research in contextEvidence before this studyUse of whole genome sequencing (WGS) to study tuberculosis (TB) transmission has proven to have higher resolution that traditional typing methods in low-burden settings. The implications of its use in high-burden settings are not well understood.Added value of this studyUsing WGS, we found that TB clusters defined by traditional typing methods may be circulating for several decades. Genomic regions typically excluded from WGS analysis contain large amount of genetic variation that may affect interpretation of transmission events. We also identified five bacterial mutations that may contribute to transmission fitness.Implications of all the available evidenceAdded value of WGS for understanding TB transmission may be even higher in high-burden vs. low-burden settings. Methods integrating variants found in polymorphic sites and insertions and deletions are likely to have higher resolution. Several host and bacterial factors may be responsible for higher transmissibility that can be targets of intervention to interrupt TB transmission in communities.

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Section: Discussionsupporting

confidence: 90%

Genotypic clustering does not imply recent tuberculosis transmission in a high prevalence setting: A genomic epidemiology study in Lima, Peru

Dixit

Freschi

Vargas

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…Within the MDR subcluster, ethambutol and pyrazinamide resistance was acquired and transmitted. This finding is similar to that of a study in Uganda 64 , where pyrazinamide resistance typically arose in MDR strains with several different causative mutations. This highlights the importance of testing for pyrazinamide resistance in order to determine benefit of its use in MDR-TB treatment regimens 65 .…”

Section: Discussionsupporting

confidence: 89%

Genotypic Clustering Does Not Imply Recent Tuberculosis Transmission in a High Prevalence Setting: A Genomic Epidemiology Study in Lima, Peru

et al. 2018

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Whole genome sequencing (WGS) can elucidate Mycobacterium tuberculosis (Mtb) transmission patterns but more data is needed to guide its use in high-burden settings. In a household-based TB transmissibility study in Peru, we identified a large MIRU-VNTR Mtb cluster (148 isolates) with a range of resistance phenotypes, and studied host and bacterial factors contributing to its spread. WGS was performed on 61 of the 148 isolates. We compared transmission link inference using epidemiological or genomic data and estimated the dates of emergence of the cluster and antimicrobial drug resistance (DR) acquisition events by generating a time-calibrated phylogeny. Using a set of 12,032 public Mtb genomes, we determined bacterial factors characterizing this cluster and under positive selection in other Mtb lineages. Four of the 61 isolates were distantly related and the remaining 57 isolates diverged ca. 1968 (95%HPD: 1945-1985). Isoniazid resistance arose once and rifampin resistance emerged subsequently at least three times. Emergence of other DR types occurred as recently as within the last year of sampling. We identified five cluster-defining SNPs potentially contributing to transmissibility. In conclusion, clusters (as defined by MIRU-VNTR typing) may be circulating for decades in a high-burden setting. WGS allows for an enhanced understanding of transmission, drug resistance, and bacterial fitness factors.

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“…Estos resultados difieren de trabajos previos publicados en los cuales solo se reporta la presencia de perfiles de mutación en la posición S315T en aislados resistentes 7,19,20,22,31 , el 25,5% de aislados fenotipicamente resistentes que no presentaron la mutación estudiada, pudo deberse a la presencia de polimorfismos en regiones diferente a la posición S315T [15][16][17][18][19][20] en el gen katG u otros genes que aporten resistencia a Isoniacida 9-14 indiciendo a la sobreexpresión que superen el poder inhibitorio de INH 26,31 ; otra causa puede ser la presencia de mecanismos de resistencia diferente a las mutaciones tales como mecanismos de barrera (reducción de la permeabilidad 32-33 y bomba de eflujo [34][35][36][37] ). En cuanto a la presencia de un aislado sensible con la mutación S315T, posiblemente se deba a que el límite de la detección por PCR-RFLP empleada se encuentre cercano al 1% de resistentes que basado en el método de las proporciones pasa imperceptible y se considera como sensible, tambien podría deberse a una inadecuada identificación del perfil de resistencia mediante la prueba de las proporciones en el aislado sensible, y/o contaminación del aislado después de su conservación.…”

Section: Nuestros Resultados Mostraron Que Al Emplear Las Enzimasunclassified

“…La INH actúa inhibiendo la biosíntesis de los ácidos micólicos de la pared celular, e ingresa en el Mycobacterium tuberculosis como un profárma-co por difusión pasiva y es activada por la enzima catalasaperoxidasa codificada por katG, para generar radicales libres, que atacan a continuación múltiples objetivos en las células 8 . Se ha reportado en varios estudios que la resistencia a isoniacida está relacionada con mutaciones específicas en varios genes importantes de Mycobacterium tuberculosis, existiendo variantes genéticas tales como: katG (S315T, S315N, S315D, S315I, S315R, V61G, Q247H, A62T, G383A, C701G, G1388T), inhA (S94A, I21V, I21T, I47T, I194T, 3, 258, 190) y su región promotora (-15 C-T, -8 T-C, T-A ó T-G,-17 G-T), ahpC, nhd, kasA, oxyR, furA, fabG1 [9][10][11][12][13][14][15][16][17][18][19][20][21] , de igual manera, se ha podido mostrar que la aplicación de PCR-RFLP del gen katG permite la identificación eficiente de aislados resistentes a isoniacida pudiendo emplearse como una prueba rápida de detección [22][23][24][25] .…”

Section: Introductionunclassified

Determinación de la mutación S315T del gen katG en aislados resistentes a Isoniacida de Mycobacterium tuberculosis mediante PCR-RFLP

Sánchez-Domínguez

Nicola-Salas

Morey-León

2018

Infect

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Cómo citar este artículo: J. Sánchez-Domínguez, et al. Determinación de la mutación S315T del gen katG en aislados resistentes a Isoniacida de Mycobacterium tuberculosis mediante PCR-RFLP. Infectio 2018; 22(4): 178-184 ResumenObjetivo: determinar la mutación S315T del gen katG en aislados de Mycobacterium tuberculosis resistentes a isoniacida mediante la técnica PCR-RFLP. Materiales y métodos: A partir de 68 aislados de Mycobacterium tuberculosis se realizó el análisis de polimorfismo en productos de amplificación de 1054 y 630 pb que contenían la mutación S315T del gen katG mediante PCR-RFLP empleando las enzimas de restricción MspI y SatI. Mediante SPSS se determinó sensibilidad, especificidad, valores predictivo positivo y negativo, coeficientes de probabilidad positivo y negativo. Resultados: El 74,46% de aislados fenotípicamente resistentes y 4,76% fenotípicamente sensibles presentaron la mutación del gen katG S315T. La PCR-RFLP para S315T del gen katG presentó 85,4% de sensibilidad y 95,2% de especificidad con MspI y 85,4% de sensibilidad y 94,4% de especificidad con SatI. Discusión: La PCR-RFLP tiene una alta capacidad resolutiva que depende de la enzima que se emplee como se observó en estudios previos. La presencia de la mutación S315T en pacientes vírgenes al tratamiento sugiere la circulación de aislados resistentes a Isoniacida. Conclusión: La PCR-RFLP resultó una alternativa válida y rápida para el diagnóstico de la resistencia a isoniacida, mediante la detección de la mutación S315T del gen katG en comparación con el método convencional de las proporciones.Palabras Clave: Mutaciones, Polimorfismo, Isoniacida, PCR-RFLP, Mycobacterium tuberculosis, katG. Determination of S315T mutation within the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolate by PCR-RFLP AbstractObjetive: To determine the S315T mutation of the katG gene in Mycobacterium tuberculosis isoniazid-resistant isolates by PCR-RFLP. Materials and Methods: Polymorphism analysis of 1054 and 630 bp products containing the S315T mutation of the katG gene was performed by PCR-RFLP using the MspI and SatI restriction enzymes from 68 Mycobacterium tuberculosis isolates. Sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio were determined using SPSS. Results: 74.46% of isoniazid-resistant and 4.76% of isoniazid-sensitive isolates showed the S315T mutation in katG gene. The PCR-RFLP for S315T of the katG gene had 85.4% sensitivity and 95.2% specificity with MspI and 85.4% sensitivity and 94.4% specificity with SatI. Discussion: The PCR-RFLP has a high resolutive capacity that depends on the enzyme that is used as it was observed in previous studies. The presence of the S315T mutation in treatment-naive patients suggests the circulation of isolates resistant to isoniazid. Conclusion: PCR-RFLP is a valid and rapid alternative for the diagnosis of isoniazid resistance, by detection of S315T mutation in the katG gene compared to the conventional method of proportions.

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Whole genome sequencing to complement tuberculosis drug resistance surveys in Uganda

Cited by 36 publications

References 46 publications

Genotypic clustering does not imply recent tuberculosis transmission in a high prevalence setting: A genomic epidemiology study in Lima, Peru

Genotypic clustering does not imply recent tuberculosis transmission in a high prevalence setting: A genomic epidemiology study in Lima, Peru

Genotypic Clustering Does Not Imply Recent Tuberculosis Transmission in a High Prevalence Setting: A Genomic Epidemiology Study in Lima, Peru

Determinación de la mutación S315T del gen katG en aislados resistentes a Isoniacida de Mycobacterium tuberculosis mediante PCR-RFLP

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