Journal Article > ResearchFull Text
Int J Tuberc Lung Dis. 2023 January 1; Volume 27 (Issue 1); 41-48.; DOI:10.5588/ijtld.22.0138
Mansoor H, Hirani N, Chavan VV, Das M, Sharma J, et al.
Int J Tuberc Lung Dis. 2023 January 1; Volume 27 (Issue 1); 41-48.; DOI:10.5588/ijtld.22.0138
BACKGROUND
In high TB burden countries, access to drug susceptibility testing is a major bottleneck. Targeted next-generation sequencing (tNGS) is a promising technology for rapid resistance detection. This study assessed the role of tNGS for the diagnosis of drug-resistant TB (DR-TB).
METHODS
A total of 161 samples from bacteriologically confirmed TB cases were subjected to tNGS using the Deeplex® Myc-TB kit and sequenced using the MiSeq platform. These samples were also processed for conventional phenotypic DST (pDST) using 13 drugs on Mycobacteria Growth Indicator Tube and line-probe assays (MTBDR plus and MTBDRsl).
RESULTS
There were 146 DR-TB and 15 drug-susceptible TB (DS-TB) samples. About 70% of patients with DR-TB had no previous TB treatment history. Overall, 88.2% had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB), 58.5% pre-extensively drug-resistant TB (pre-XDR-TB) and 9.2% had XDR-TB as defined by the WHO (2020). Around 8% (n=13) of samples were non-culturable; however, identified 8 were resistant to first and second-line drugs using tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable using pDST or with limited mutational representation within databases. Sensitivities were aligned with literature reports for most drugs. We observed 10% heteroresistance, while 75% of strains were of Lineages 2 and 3.
CONCLUSIONS
Programme data supported tNGS in the diagnosis of DR-TB for early treatment using individualised regimens.
In high TB burden countries, access to drug susceptibility testing is a major bottleneck. Targeted next-generation sequencing (tNGS) is a promising technology for rapid resistance detection. This study assessed the role of tNGS for the diagnosis of drug-resistant TB (DR-TB).
METHODS
A total of 161 samples from bacteriologically confirmed TB cases were subjected to tNGS using the Deeplex® Myc-TB kit and sequenced using the MiSeq platform. These samples were also processed for conventional phenotypic DST (pDST) using 13 drugs on Mycobacteria Growth Indicator Tube and line-probe assays (MTBDR plus and MTBDRsl).
RESULTS
There were 146 DR-TB and 15 drug-susceptible TB (DS-TB) samples. About 70% of patients with DR-TB had no previous TB treatment history. Overall, 88.2% had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB), 58.5% pre-extensively drug-resistant TB (pre-XDR-TB) and 9.2% had XDR-TB as defined by the WHO (2020). Around 8% (n=13) of samples were non-culturable; however, identified 8 were resistant to first and second-line drugs using tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable using pDST or with limited mutational representation within databases. Sensitivities were aligned with literature reports for most drugs. We observed 10% heteroresistance, while 75% of strains were of Lineages 2 and 3.
CONCLUSIONS
Programme data supported tNGS in the diagnosis of DR-TB for early treatment using individualised regimens.
Conference Material > Slide Presentation
Mansoor H, Hirani N, Chavan VV, Joshi A, Oswal V, et al.
MSF Scientific Days International 2022. 2022 May 11; DOI:10.57740/x7k7-xj13
Conference Material > Video
Mansoor H, Hirani N, Chavan VV, Joshi A, Oswal V, et al.
MSF Scientific Days International 2022. 2022 June 7; DOI:10.57740/3gdn-kn91
Conference Material > Abstract
Mansoor H, Hirani N, Chavan VV, Joshi A, Oswal V, et al.
MSF Scientific Days International 2022. 2022 May 11; DOI:10.57740/atfq-6s03
INTRODUCTION
In countries with a high tuberculosis (TB) burden, poor access to drug susceptibility testing is a major bottleneck in diagnosing drug-resistant (DR) TB. India is estimated to account for a quarter of multidrug-resistant (MDR)-TB patients globally, with around 124,000 cases in 2020. Mumbai, a densely populated city in Maharashtra State, is a DR-TB hotspot with 24% of treatment- naïve cases, and 41% of previously-treated cases, having MDR-TB, and a high frequency of fluoroquinolone resistance occurring among these MDR-TB cases. Targeted next- generation sequencing (tNGS) is a promising technology for rapid detection of resistance. We assessed the role of tNGS for diagnosis of DR-TB.
METHODS
We performed a laboratory-based study involving Mycobacterium tuberculosis (MTB)-positive samples from patients with presumptive TB or DR-TB identified by GeneXpert in Shatabdi Hospital, Mumbai. A total of 161 sputum samples from bacteriologically-confirmed TB cases were included in the study. The study was conducted at Sir JJ Hospital’s TB lab, with sample collection occurring from patients living in M-East Ward (MEW), Mumbai. Two sputum samples were collected from each presumptive TB patient at MEW. Spot samples with a positive result on Xpert MTB/Rif were sent for tNGS and conventional testing (phenotypic drug sensitivity testing (pDST), line probe assays (LPA), and mycobacteria growth indicator tubes (MGIT)) at Sir JJ Hospital’s TB lab. tNGS samples were processed using Deeplex MycTB-kit (GenoScreen, France) and sequenced on a MiSeq platform (Illumina, USA). These samples were also processed for pDST using 16 drugs on MGIT (Becton Dickinson, USA) and LPA (MTBDRplus and MTBDRsl, Hain Lifesciences, Germany). To ensure sequence quality, Xpert results with cycle threshold values <20 or direct smear results >2+ were prepared for tNGS using direct sputum sediments. Primary cultures were prepared for samples with lower bacterial loads.
ETHICS
This study was approved by the ethics committee of the Grant Medical College & Sir J J Group of Hospitals, Mumbai, India. Permission was granted by the Medical Director of MSF, Operational Centre Brussels.
RESULTS
The median age of patients with samples included was 24 years (interquartile range, 20-40), and 57% were female. Approximately 70% of cases had no previous history of TB. Of 161 samples evaluated, 15 (9.3%) were rifampicin-sensitive and 146 (90.7%) were rifampicin-resistant (RR). 161 samples with completed pDST, tNGS and LPA were analysed. Of these, 88.2% had RR/MDR-TB resistance per WHO definitions, 58.5% had additional fluoroquinolone-resistance (pre-XDR) and 9.2% had fluoroquinolone resistance plus resistance to either linezolid or bedaquiline (extensively drug-resistant (XDR). Thirteen of 161 samples (8%) were culture-negative, yet resistance to one or more drugs was demonstrated in 8/13 samples with tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable in pDST or limited mutational representation within databases. Sensitivities aligned with the WHO catalogue for most drugs. 10% of the sample showed hetero-resistance and 75% of strains were of lineages 2 and 3.
CONCLUSION
In countries with a high burden of DR-TB, and high transmission rates, tNGS can provide information to rapidly design individualised regimens for early initiation and effective case management. It also gives information regarding lineages, uncharacterized mutations, hetero-resistance and mixed infection status of TB cases. Potentially tNGS could provide a diagnostic tool for rapid initiation of treatment in high DR-TB settings.
CONFLICTS OF INTEREST
None declared.
In countries with a high tuberculosis (TB) burden, poor access to drug susceptibility testing is a major bottleneck in diagnosing drug-resistant (DR) TB. India is estimated to account for a quarter of multidrug-resistant (MDR)-TB patients globally, with around 124,000 cases in 2020. Mumbai, a densely populated city in Maharashtra State, is a DR-TB hotspot with 24% of treatment- naïve cases, and 41% of previously-treated cases, having MDR-TB, and a high frequency of fluoroquinolone resistance occurring among these MDR-TB cases. Targeted next- generation sequencing (tNGS) is a promising technology for rapid detection of resistance. We assessed the role of tNGS for diagnosis of DR-TB.
METHODS
We performed a laboratory-based study involving Mycobacterium tuberculosis (MTB)-positive samples from patients with presumptive TB or DR-TB identified by GeneXpert in Shatabdi Hospital, Mumbai. A total of 161 sputum samples from bacteriologically-confirmed TB cases were included in the study. The study was conducted at Sir JJ Hospital’s TB lab, with sample collection occurring from patients living in M-East Ward (MEW), Mumbai. Two sputum samples were collected from each presumptive TB patient at MEW. Spot samples with a positive result on Xpert MTB/Rif were sent for tNGS and conventional testing (phenotypic drug sensitivity testing (pDST), line probe assays (LPA), and mycobacteria growth indicator tubes (MGIT)) at Sir JJ Hospital’s TB lab. tNGS samples were processed using Deeplex MycTB-kit (GenoScreen, France) and sequenced on a MiSeq platform (Illumina, USA). These samples were also processed for pDST using 16 drugs on MGIT (Becton Dickinson, USA) and LPA (MTBDRplus and MTBDRsl, Hain Lifesciences, Germany). To ensure sequence quality, Xpert results with cycle threshold values <20 or direct smear results >2+ were prepared for tNGS using direct sputum sediments. Primary cultures were prepared for samples with lower bacterial loads.
ETHICS
This study was approved by the ethics committee of the Grant Medical College & Sir J J Group of Hospitals, Mumbai, India. Permission was granted by the Medical Director of MSF, Operational Centre Brussels.
RESULTS
The median age of patients with samples included was 24 years (interquartile range, 20-40), and 57% were female. Approximately 70% of cases had no previous history of TB. Of 161 samples evaluated, 15 (9.3%) were rifampicin-sensitive and 146 (90.7%) were rifampicin-resistant (RR). 161 samples with completed pDST, tNGS and LPA were analysed. Of these, 88.2% had RR/MDR-TB resistance per WHO definitions, 58.5% had additional fluoroquinolone-resistance (pre-XDR) and 9.2% had fluoroquinolone resistance plus resistance to either linezolid or bedaquiline (extensively drug-resistant (XDR). Thirteen of 161 samples (8%) were culture-negative, yet resistance to one or more drugs was demonstrated in 8/13 samples with tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable in pDST or limited mutational representation within databases. Sensitivities aligned with the WHO catalogue for most drugs. 10% of the sample showed hetero-resistance and 75% of strains were of lineages 2 and 3.
CONCLUSION
In countries with a high burden of DR-TB, and high transmission rates, tNGS can provide information to rapidly design individualised regimens for early initiation and effective case management. It also gives information regarding lineages, uncharacterized mutations, hetero-resistance and mixed infection status of TB cases. Potentially tNGS could provide a diagnostic tool for rapid initiation of treatment in high DR-TB settings.
CONFLICTS OF INTEREST
None declared.
Journal Article > ResearchFull Text
Public Health Action. 2023 June 21; Volume 13 (Issue 2); 43-49.; DOI:10.5588/pha.22.0041
Iyer AS, Ndlovu Z, Sharma J, Mansoor H, Bharati M, et al.
Public Health Action. 2023 June 21; Volume 13 (Issue 2); 43-49.; DOI:10.5588/pha.22.0041
English
Français
BACKGROUND
Phenotypic drug susceptibility testing (pDST) for Mycobacterium tuberculosis can take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India.
METHODS
Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below.
RESULTS
Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n = 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119.
CONCLUSIONS
Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.
Phenotypic drug susceptibility testing (pDST) for Mycobacterium tuberculosis can take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India.
METHODS
Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below.
RESULTS
Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n = 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119.
CONCLUSIONS
Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.