Abstract
Background: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis. Methods: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing. Findings: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87–94) for rpoB (rifampicin resistance), 86% (74–93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39–68) for pncA (pyrazinamide resistance), 85% (77–91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81–92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing. Interpretation: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation. Funding: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.
| Original language | English |
|---|---|
| Pages (from-to) | 675-683 |
| Number of pages | 9 |
| Journal | The Lancet Infectious Diseases |
| Volume | 18 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - Jun 2018 |
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In: The Lancet Infectious Diseases, Vol. 18, No. 6, 06.2018, p. 675-683.
Research output: Contribution to journal › Article › peer-review
TY - JOUR
T1 - Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries
T2 - a multi-country population-based surveillance study
AU - Zignol, Matteo
AU - Cabibbe, Andrea Maurizio
AU - Dean, Anna S.
AU - Glaziou, Philippe
AU - Alikhanova, Natavan
AU - Ama, Cecilia
AU - Andres, Sönke
AU - Barbova, Anna
AU - Borbe-Reyes, Angeli
AU - Chin, Daniel P.
AU - Cirillo, Daniela Maria
AU - Colvin, Charlotte
AU - Dadu, Andrei
AU - Dreyer, Andries
AU - Driesen, Michèle
AU - Gilpin, Christopher
AU - Hasan, Rumina
AU - Hasan, Zahra
AU - Hoffner, Sven
AU - Hussain, Alamdar
AU - Ismail, Nazir
AU - Kamal, S. M.Mostofa
AU - Khanzada, Faisal Masood
AU - Kimerling, Michael
AU - Kohl, Thomas Andreas
AU - Mansjö, Mikael
AU - Miotto, Paolo
AU - Mukadi, Ya Diul
AU - Mvusi, Lindiwe
AU - Niemann, Stefan
AU - Omar, Shaheed V.
AU - Rigouts, Leen
AU - Schito, Marco
AU - Sela, Ivita
AU - Seyfaddinova, Mehriban
AU - Skenders, Girts
AU - Skrahina, Alena
AU - Tahseen, Sabira
AU - Wells, William A.
AU - Zhurilo, Alexander
AU - Weyer, Karin
AU - Floyd, Katherine
AU - Raviglione, Mario C.
N1 - Funding Information: We have presented the results of a surveillance project of more than 7000 patients across several countries to assess the accuracy of genetic sequencing in determining the prevalence of resistance to the most commonly used first-line and second-line anti-tuberculosis drugs when compared with phenotypic testing. We found that genetic sequencing can be a valuable surveillance tool to accurately predict drug resistance in low-income and middle-income countries. The value of this work is that the isolates tested are representative of the entire tuberculosis patient population in seven resource-limited countries, all of which are classified as having a high burden of tuberculosis or multidrug-resistant tuberculosis. The settings also differed in their risk of and extent of drug resistance. These patients were managed under several programmatic and epidemiological situations, and included patients who had been newly diagnosed with tuberculosis and patients who had previously been treated for tuberculosis. Our results show that the accuracy of genetic sequencing is very good at predicting phenotypic resistance to rifampicin, isoniazid, the fluoroquinolones, and (among rifampicin-resistant cases) injectable drugs. These findings imply that the sensitivity values of sequencing compared with phenotypic testing ( table ) can be applied to sequencing results to estimate the true prevalence of drug resistance for surveillance purposes. These sensitivity values are consistent with previously published evidence. 6,7 One of the most difficult aspects of any study on accuracy of a diagnostic technology is defining the gold-standard test to be used as comparator. Phenotypic tests are typically used as comparator to access the accuracy of genetic tests. In assessing the drug resistance of tuberculosis, the reliability and reproducibility of phenotypic tests are suboptimal for some drugs, and clinical decisions are often made by use of a combination of phenotypic and genotypic test results. 21–23 In our Article, we considered the phenotypic test to be the gold-standard test; however, in the event of phenotypic test results finding drug susceptibly alongside the presence of mutations considered to be markers of resistance, the genetic test result was assumed to be correct. Given the nature of this large surveillance project, the discrepancies between phenotypic and sequencing results could not be investigated by repeating the phenotypic test. For all discrepancies, the sequencing data were thoroughly reviewed. For pyrazinamide, all strains with discrepant results were reassessed by use of a third method (Wayne's test) on the basis of the poor reproducibility of phenotypic tests. The breadth of sensitivity values observed across sites ( appendix ) reflects differences in the quality of phenotypic testing, random fluctuations due to test errors (particularly when the number of resistant cases was very small), and variation in the prevalence of resistance to rifampicin. Some of these differences could be partly explained by the M tuberculosis genetic background, correlation with specific drug resistance mutations, and clonal spread within geographical areas. Variations in the sensitivity of sequencing, which were most pronounced for pyrazinamide and among rifampicin-susceptible cases, resulted in large uncertainty bounds around the estimates of prevalence by sequencing. To accurately monitor trends in drug resistance, more work should be done to improve the sensitivity of sequencing, particularly among rifampicin-susceptible cases. A larger difference between the prevalence of resistance as estimated by phenotypic testing and the adjusted prevalence by sequencing occurred when the sensitivity of genotypic testing was either notably higher or lower than in the other countries. For example, in Pakistan, the sensitivity of sequencing for isoniazid was lower than in other countries ( figure 2 ), whereas the sensitivity of genotypic testing for pyrazinamide in Belarus ( figure 4 ) was higher than elsewhere. Pyrazinamide is the drug for which the ability of genetic sequencing to predict phenotypic resistance was most problematic, as shown in figure 4 by the poor overlap of prevalence estimates generated by phenotypic testing and by sequencing, particularly among rifampicin-susceptible strains from Belarus and Pakistan, and by large uncertainty bounds around the sequencing-based prevalence estimates. This finding was unsurprising because our understanding of the role of mutations in conferring resistance to pyrazinamide is incomplete: 42% of all pncA mutations were unclassified in our dataset. Although most of these mutations have already been reported in the literature to be associated with resistance, their infrequent occurrence means that there is insufficient statistical power to classify them. 6,22 Additionally, the phenotypic test for pyrazinamide has inadequate reproducibility, making it a weak test with which to make comparisons. 22,23 The frequency of mutations and associated phenotypic drug susceptibility results from our study can be used for genome-based predictions of resistance ( appendix ). To further improve our understanding of genotypic markers of resistance, phenotypic and genotypic data should be considered in the context of clinical outcome data, given the suboptimal reliability and reproducibility of phenotypic tests for some anti-tuberculosis drugs and uncertainty around the most appropriate critical concentrations. 24 To accelerate the transition from reliance on phenotypic results to genotypic results for resistance prediction, it is crucial that genotypic, phenotypic, and outcome data be shared as soon as they become available. The main purpose of surveillance is to estimate the burden of drug resistance and monitor its trends, to enable a prompt and effective public health response. Our findings show that genotypic methods have an important role in surveillance, especially given the limitations of conventional phenotypic methods, and that available molecular diagnostic tools can only detect resistance to a small number of drugs. Targeted gene sequencing or whole-genome sequencing directly on sputum samples would bypass the need for culture, standardise the approach, and accelerate the availability of results. 25 When genetic sequencing is properly standardised and made economically feasible, this technology could be particularly impactful in countries with low laboratory and sample referral capacity, and would offer an opportunity to monitor the development of drug resistance more effectively during tuberculosis epidemics. Enabling use of gene sequencing would also represent a breakthrough in improving rapid surveillance of both drug-resistant tuberculosis and broader antimicrobial resistance. The cost of genetic sequencing will be a major factor in determining the feasibility of introduction and the speed of expansion. Costs of sequencing are progressively decreasing and are already lower than the cost of phenotypic testing to first-line and second-line anti-tuberculosis drugs in most settings. 26 This trend has also been confirmed in our study. In the context of surveillance, the possibility of grouping specimens to genome sequence many isolates (up to 200) in one single run offers the potential for further cost savings and a reduction in laboratory workload. Besides cost, there are several other technical challenges that need to be addressed to allow widespread use of genetic sequencing for surveillance of drug resistance in tuberculosis. DNA extraction and sample preparation methods need to be consistent, standardised nomenclature to record and report sequencing information must be developed, an external quality assurance system for genome sequencing (similar to the system available for phenotypic testing) should be established, and a standardised and common approach to analysis must be defined. 3,27–29 To support the expansion of the use of sequencing technologies in low-income and middle-income countries, molecular biology and bioinformatics skills should be developed locally and supplemented with continuous mentoring from supranational reference laboratories. Although constraints for the expansion of genetic sequencing exist, the value of whole genome sequencing data is enormous for any national tuberculosis control programme. Data can be re-analysed at a later stage to investigate newly discovered resistance-associated loci, to predict resistance to new drugs, and to inform development of more tailored molecular diagnostics as soon as knowledge on the genetic marker of resistance becomes available. Our study has some limitations. First, excepting isolates with graded mutations, which were all assumed to be phenotypically resistant as previously described, 6 we considered phenotypic drug susceptibility testing to be the gold-standard test, and we treated this test as a comparator for genetic sequencing. However, there is evidence that phenotypic drug susceptibility testing might not always be the most accurate test. Treatment outcome data should be used to guide interpretation of genotypic and phenotypic testing results 21 and, unfortunately, these data were not available in our study. Second, phenotypic testing was done at specific critical concentrations, as currently recommended by WHO, 30 but more recent evidence suggests that some of these concentrations might need to be reassessed. 24 Third, although only laboratory methods recommended by WHO were used and all laboratories passed proficiency testing before beginning the project, some variability in phenotypic results between laboratories and between media types could have affected outcomes. Fourth, different platforms were used for DNA sequencing, including Sanger platforms, which could have slightly different coverage of some genomic regions. 31 Finally, given that testing for second-line injectable drugs was limited to strains with rifampicin resistance, the number of isolates tested for kanamycin, amikacin, and capreomycin is not large enough to generate conclusive evidence. Our work shows that genetic sequencing can be a valuable tool in surveillance of drug resistance, enabling new ways to monitor drug resistance in tuberculosis in low-income and middle-income countries. The findings of this study can also be used to guide the development and introduction of new diagnostic technologies, including genetic sequencing, in different geographical areas and patient groups and contribute to our knowledge on the role of genotypic markers in conferring resistance to anti-tuberculosis drugs. This online publication has been corrected. The corrected version first appeared at thelancet.com/infection on March 27, 2018 Contributors MZ, AMC, ASD, DMC, CG, RH, SH, NI, SN, LR, and KW contributed to the study design. NA, CA, SA, AB, AB-R, ADr, MD, ZH, AH, AZ, SMMK, FMK, TAK, MM, PM, SVO, IS, MSe, GS, AS, and ST collected the data and did the laboratory testing. MZ, AMC, ASD, and PG analysed the data, which was interpreted by all authors. DPC, CC, ADa, MK, YDM, LM, MSc, and WAW provided technical and policy input. MZ, AMC, ASD, KF, and MCR drafted the report with input from all other authors. MZ, AMC, and ASD contributed equally to the manuscript. All authors have seen and approved the final report. Declaration of interests DPC is an employee of the Bill & Melinda Gates Foundation, which co-funded the study. CC, YDM, and WAW are employees of the United States Agency for International Development, which also co-funded the study. These authors were acting as subject matter experts rather than agency representatives, and did not have veto power over any study decision. All other authors declare no competing interests. Acknowledgments We thank Mussarat Ashraf, Samreen Shafiq, and Joveria Farooqi (Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan) for their support in data collection and interpretation and Andrew Siroka (World Health Organization, Geneva, Switzerland), Maria Rosaria De Filippo (San Raffaele Scientific Institute, Milan, Italy), and Christian Utpatel (Molecular and Experimental Mycobacteriology, Borstel Research Centre, Borstel, Germany) for support with data analysis. This study was supported by the Bill & Melinda Gates Foundation, the United States Agency for International Development, and the TB Alliance. The views and opinions expressed in this paper are those of the authors and not necessarily the views and opinions of the World Health Organization or of the United States Agency for International Development. Publisher Copyright: © 2018 World Health Organization
PY - 2018/6
Y1 - 2018/6
N2 - Background: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis. Methods: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing. Findings: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87–94) for rpoB (rifampicin resistance), 86% (74–93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39–68) for pncA (pyrazinamide resistance), 85% (77–91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81–92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing. Interpretation: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation. Funding: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.
AB - Background: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis. Methods: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing. Findings: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87–94) for rpoB (rifampicin resistance), 86% (74–93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39–68) for pncA (pyrazinamide resistance), 85% (77–91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81–92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing. Interpretation: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation. Funding: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.
UR - http://www.scopus.com/inward/record.url?scp=85044251701&partnerID=8YFLogxK
U2 - 10.1016/S1473-3099(18)30073-2
DO - 10.1016/S1473-3099(18)30073-2
M3 - Article
C2 - 29574065
AN - SCOPUS:85044251701
SN - 1473-3099
VL - 18
SP - 675
EP - 683
JO - The Lancet Infectious Diseases
JF - The Lancet Infectious Diseases
IS - 6
ER -