Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in
 Mycobacterium tuberculosis

Michael G. Whitfield; David M. Engelthaler; Christopher Allender; Megan Folkerts; Tim H. Heupink; Jason Limberis; Robin M. Warren; Annelies Van Rie; John Z. Metcalfe

doi:10.1128/jcm.01907-21

Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis

Michael G. Whitfield, David M. Engelthaler, Christopher Allender, Megan Folkerts, Tim H. Heupink, Jason Limberis, Robin M. Warren, Annelies Van Rie, John Z. Metcalfe

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

$\textit{Background:}$ Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampicin resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods, and explored the occurrence of pyrazinamide heteroresistance. $\textit{Methods:}$ We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the $\textit{pncA}$ gene, whole genome sequencing (WGS), and phenotypic drug-susceptibility testing. We calculated the diagnostic accuracy of the different methods, and investigated the prevalence and clinical impact of $\textit{pncA}$ heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Koser classification and expert rules. $\textit{Results:}$ We observed 100% agreement across genotypic methods for detection of $\textit{pncA}$ fixed mutations, only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% CI: 93.8-99.3%) and specificity of 100% (95% CI: 100-100%); both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI: 94.6-99.6%) and specificity of 97.8% (95% CI: 95.7-99.9%); and TDS, sensitivity of 98.8% (95% CI: 97.2-100%) and specificity of 97.8% (95% CI: 95.7-99.9%). $\textit{Conclusions:}$ We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in $\textit{Mtb}$ isolates was lower than that identified for other first-line drugs.

Original language	English (US)
Journal	Journal of Clinical Microbiology
DOIs	https://doi.org/10.1128/jcm.01907-21
State	Published - Nov 10 2021
Externally published	Yes

Bibliographical note

KAUST Repository Item: Exported on 2021-11-25
Acknowledgements: We would like to thank members of the international TORCH consortium, as well as Arnab Pain at King Abdullah University of Science and Technology for performing the whole genome sequencing. We would like to thank additional members of the TGen team, including Andrew Goedderz, Jason Agundez, Meagan Papineau, Cassidy Danbury and Darrin Lemmer for performing targeted deep sequencing and data analysis support. Lastly, we thank the participants and the healthcare workers for their dedication to this study.
This publication acknowledges KAUST support, but has no KAUST affiliated authors.

ASJC Scopus subject areas

Microbiology (medical)

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1128/jcm.01907-21

Cite this

@article{93e7d44171554770bc0dbcb646b445c5,

title = "Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis",

abstract = "$\textit{Background:}$ Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampicin resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods, and explored the occurrence of pyrazinamide heteroresistance. $\textit{Methods:}$ We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the $\textit{pncA}$ gene, whole genome sequencing (WGS), and phenotypic drug-susceptibility testing. We calculated the diagnostic accuracy of the different methods, and investigated the prevalence and clinical impact of $\textit{pncA}$ heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Koser classification and expert rules. $\textit{Results:}$ We observed 100% agreement across genotypic methods for detection of $\textit{pncA}$ fixed mutations, only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% CI: 93.8-99.3%) and specificity of 100% (95% CI: 100-100%); both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI: 94.6-99.6%) and specificity of 97.8% (95% CI: 95.7-99.9%); and TDS, sensitivity of 98.8% (95% CI: 97.2-100%) and specificity of 97.8% (95% CI: 95.7-99.9%). $\textit{Conclusions:}$ We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in $\textit{Mtb}$ isolates was lower than that identified for other first-line drugs.",

author = "Whitfield, {Michael G.} and Engelthaler, {David M.} and Christopher Allender and Megan Folkerts and Heupink, {Tim H.} and Jason Limberis and Warren, {Robin M.} and {Van Rie}, Annelies and Metcalfe, {John Z.}",

note = "KAUST Repository Item: Exported on 2021-11-25 Acknowledgements: We would like to thank members of the international TORCH consortium, as well as Arnab Pain at King Abdullah University of Science and Technology for performing the whole genome sequencing. We would like to thank additional members of the TGen team, including Andrew Goedderz, Jason Agundez, Meagan Papineau, Cassidy Danbury and Darrin Lemmer for performing targeted deep sequencing and data analysis support. Lastly, we thank the participants and the healthcare workers for their dedication to this study. This publication acknowledges KAUST support, but has no KAUST affiliated authors.",

year = "2021",

month = nov,

day = "10",

doi = "10.1128/jcm.01907-21",

language = "English (US)",

journal = "Journal of Clinical Microbiology",

issn = "0095-1137",

publisher = "American Society for Microbiology",

}

TY - JOUR

T1 - Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis

AU - Whitfield, Michael G.

AU - Engelthaler, David M.

AU - Allender, Christopher

AU - Folkerts, Megan

AU - Heupink, Tim H.

AU - Limberis, Jason

AU - Warren, Robin M.

AU - Van Rie, Annelies

AU - Metcalfe, John Z.

N1 - KAUST Repository Item: Exported on 2021-11-25 Acknowledgements: We would like to thank members of the international TORCH consortium, as well as Arnab Pain at King Abdullah University of Science and Technology for performing the whole genome sequencing. We would like to thank additional members of the TGen team, including Andrew Goedderz, Jason Agundez, Meagan Papineau, Cassidy Danbury and Darrin Lemmer for performing targeted deep sequencing and data analysis support. Lastly, we thank the participants and the healthcare workers for their dedication to this study. This publication acknowledges KAUST support, but has no KAUST affiliated authors.

PY - 2021/11/10

Y1 - 2021/11/10

N2 - $\textit{Background:}$ Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampicin resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods, and explored the occurrence of pyrazinamide heteroresistance. $\textit{Methods:}$ We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the $\textit{pncA}$ gene, whole genome sequencing (WGS), and phenotypic drug-susceptibility testing. We calculated the diagnostic accuracy of the different methods, and investigated the prevalence and clinical impact of $\textit{pncA}$ heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Koser classification and expert rules. $\textit{Results:}$ We observed 100% agreement across genotypic methods for detection of $\textit{pncA}$ fixed mutations, only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% CI: 93.8-99.3%) and specificity of 100% (95% CI: 100-100%); both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI: 94.6-99.6%) and specificity of 97.8% (95% CI: 95.7-99.9%); and TDS, sensitivity of 98.8% (95% CI: 97.2-100%) and specificity of 97.8% (95% CI: 95.7-99.9%). $\textit{Conclusions:}$ We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in $\textit{Mtb}$ isolates was lower than that identified for other first-line drugs.

AB - $\textit{Background:}$ Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampicin resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods, and explored the occurrence of pyrazinamide heteroresistance. $\textit{Methods:}$ We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the $\textit{pncA}$ gene, whole genome sequencing (WGS), and phenotypic drug-susceptibility testing. We calculated the diagnostic accuracy of the different methods, and investigated the prevalence and clinical impact of $\textit{pncA}$ heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Koser classification and expert rules. $\textit{Results:}$ We observed 100% agreement across genotypic methods for detection of $\textit{pncA}$ fixed mutations, only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% CI: 93.8-99.3%) and specificity of 100% (95% CI: 100-100%); both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI: 94.6-99.6%) and specificity of 97.8% (95% CI: 95.7-99.9%); and TDS, sensitivity of 98.8% (95% CI: 97.2-100%) and specificity of 97.8% (95% CI: 95.7-99.9%). $\textit{Conclusions:}$ We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in $\textit{Mtb}$ isolates was lower than that identified for other first-line drugs.

UR - http://hdl.handle.net/10754/673762

UR - https://journals.asm.org/doi/10.1128/JCM.01907-21

U2 - 10.1128/jcm.01907-21

DO - 10.1128/jcm.01907-21

M3 - Article

SN - 0095-1137

JO - Journal of Clinical Microbiology

JF - Journal of Clinical Microbiology

ER -