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Identifying Drug-Resistant Tuberculosis—Commentary

11/27/2017 Matthew E. Levison, MD, Former Professor, School of Public Health, Drexel University; Adjunct Professor of Medicine, Drexel University College of Medicine

Drug-resistant tuberculosis (TB) is a continuing threat that significantly complicates TB eradication efforts.The rising number of TB infections resistant to multiple drugs (MDR TB) in many countries has led to the demand for rapid and accurate same-day drug susceptibility testing (DST) for both first- and second-line anti-TB drugs, which would allow point-of-care therapeutic decision-making. MDR TB is defined as resistance to the 2 most effective first-line TB medications: isoniazid (INH) and rifampin (RIF).Extensively drug-resistant TB (XDR TB) is a type of MDR TB that is resistant to INH and RIF, <em>plus</em> any fluoroquinolone (FQ) and at least one of three injectable second-line drugs, amikacin (AMK), kanamycin (KAN), or capreomycin (CAP). In 2016, there were 600 000 new cases with resistance to rifampin (RR-TB) worldwide, of which 490 000 had MDR TB. Almost half (47%) of these cases were in India, China, and the Russian Federation.

Drug resistance in M. tuberculosis is exclusively due to mutations in specific genes and has been associated with > 20 mutations in > 11 genes and promoter regions. Resistance to multiple drugs is the consequence of the accumulation of several of these mutations. However, not all mutations conferring resistance are known.

Over 95% of RIF-resistant M. tuberculosis strains have mutations within the RIF resistance-determining region (RRDR) of the rpoB gene, and several molecular genetic tests to detect RRDR mutations have been developed.

Drug susceptibility testing is essential to

  • Guide drug selection
  • Determine whether poor clinical response is due to development of resistance
  • Survey for drug resistance

Traditionally, the gold standard for DST is culture-based phenotypic methods. But these methods have the major disadvantage of requiring an extremely long interval before results are available. Due to the slow growth of M. tuberculosis, results take six to eight weeks using solid media and four to five weeks using liquid media. Cultured-based DSTs also involve significant biohazard that necessitates high levels of biosafety practices and trained personnel.

To overcome these drawbacks, rapid genetic DSTs have been developed that shorten the turn-around time from months to several hours.The rapid diagnosis of drug resistance by molecular methods is essential to immediately initiate effective antibiotic therapy, and thus improve treatment outcomes and reduce the transmission of MDR TB. In addition to being speedy, genetic DST should also be accurate, and, to be useful in resource-poor countries where most MDR TB patients reside, should be simple to use and cost-effective.

Genetic determination of RIF resistance is now commonly available in developed countries and is becoming increasingly available in the developing world. The WHO-endorsed GeneXpert MTB/RIF (Cepheid) is an automated closed system that uses rapid PCR techniques to detect DNA sequences in sputum samples that are specific for M. tuberculosis, as well as certain mutations that confer resistance to RIF. RIF resistance is used as a surrogate marker for MDR TB, because 95% of the strains resistant to RIF are also resistant to INH. The test has much better accuracy than sputum acid-fast smear microscopy; testing is completed within 2 hours, with minimal biohazard, and can be performed by less-skilled workers who require minimal technical training.

GeneXpert MTB/RIF is highly accurate across diverse geographic regions and has contributed to the global increase in detection of RR-TB.The test can detect > 97% of patients with RIF resistance identified by culture (1) and has specificity close to 99%.

However, rapid detection of resistance to second-line drugs has lagged behind. Rapid identification of resistance to FQs and the second-line injectable drugs AMK, KAN, and CAP is urgently needed for detection of XDT-TB. Using the GeneXpert platform, an investigational assay has been developed for rapid detection of genes associated with resistance to INH, FQs (moxifloxacin and ofloxacin), and aminoglycosides (amikacin and kanamycin).

A recent study in the New England Journal of Medicine assessed the diagnostic accuracy of this investigational assay in patients in China and South Korea (2). When using DNA sequencing as the reference standard, the investigational assay met the WHO's 95% sensitivity target for INH, the FQs, and AMK and missed the sensitivity target for KAN by approximately 2 percentage points. However, using a culture-based standard, the sensitivity of the investigational genetic assay to detect resistance was lower—83.3% for INH; 88.4% and 87.6% for ofloxacin and moxifloxacin; and 71.4% and 70.7% for kanamycin and amikacin. However, the specificity ranged from 94.3 to 99.6% compared to the WHO target of 98%.

Limitations and Concerns

This study did not evaluate resistance to CAP, although the rrs mutation detected by the assay was said to account for the "majority" of CAP resistance. This study also did not evaluate resistance to other first-line drugs, streptomycin, ethambutol, and pyrazinamide (PZA), that may be useful in MDR TB. Another limitation of this study was the limited geographic representation of participants and M. tuberculosis strains. It is hoped that future studies will document its usefulness worldwide.

The concordance between the results of genetic DST and culture-based DST is known to not always be close to 100%. Genetic tests detect only mutations that are screened for, and some resistant strains have known mutations not included in the assay or have unknown mutations. Thus, it would seem prudent to use culture-based DST to confirm susceptibility identified by genetic DST. In the future, incorporating additional mutations associated with resistance might improve the sensitivity of this investigational assay. It is hoped that with greater access to improved rapid molecular testing, especially in resource-poor communities, a greater proportion of patients with MDR TB will be identified and promptly treated with effective multidrug regimens.

References

 1. Boehme CC, Nabeta P, Hillemann D, et al: Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 363:1005–1015, 2010.

2. Xie YL, Chakravorty S, Armstrong DT, et al: Evaluation of a rapid molecular drug-susceptibility test for tuberculosis. N Engl J Med 377:1043–1054, 2017. doi: 10.1056/NEJMoa1614915.

Matthew Levison, MD

Identifying Drug-Resistant Tuberculosis—Commentary

11/27/2017 Matthew E. Levison, MD, Former Professor, School of Public Health, Drexel University; Adjunct Professor of Medicine, Drexel University College of Medicine

Drug-resistant tuberculosis (TB) is a continuing threat that significantly complicates TB eradication efforts.The rising number of TB infections resistant to multiple drugs (MDR TB) in many countries has led to the demand for rapid and accurate same-day drug susceptibility testing (DST) for both first- and second-line anti-TB drugs, which would allow point-of-care therapeutic decision-making. MDR TB is defined as resistance to the 2 most effective first-line TB medications: isoniazid (INH) and rifampin (RIF).Extensively drug-resistant TB (XDR TB) is a type of MDR TB that is resistant to INH and RIF, <em>plus</em> any fluoroquinolone (FQ) and at least one of three injectable second-line drugs, amikacin (AMK), kanamycin (KAN), or capreomycin (CAP). In 2016, there were 600 000 new cases with resistance to rifampin (RR-TB) worldwide, of which 490 000 had MDR TB. Almost half (47%) of these cases were in India, China, and the Russian Federation.

Drug resistance in M. tuberculosis is exclusively due to mutations in specific genes and has been associated with > 20 mutations in > 11 genes and promoter regions. Resistance to multiple drugs is the consequence of the accumulation of several of these mutations. However, not all mutations conferring resistance are known.

Over 95% of RIF-resistant M. tuberculosis strains have mutations within the RIF resistance-determining region (RRDR) of the rpoB gene, and several molecular genetic tests to detect RRDR mutations have been developed.

Drug susceptibility testing is essential to

  • Guide drug selection
  • Determine whether poor clinical response is due to development of resistance
  • Survey for drug resistance

Traditionally, the gold standard for DST is culture-based phenotypic methods. But these methods have the major disadvantage of requiring an extremely long interval before results are available. Due to the slow growth of M. tuberculosis, results take six to eight weeks using solid media and four to five weeks using liquid media. Cultured-based DSTs also involve significant biohazard that necessitates high levels of biosafety practices and trained personnel.

To overcome these drawbacks, rapid genetic DSTs have been developed that shorten the turn-around time from months to several hours.The rapid diagnosis of drug resistance by molecular methods is essential to immediately initiate effective antibiotic therapy, and thus improve treatment outcomes and reduce the transmission of MDR TB. In addition to being speedy, genetic DST should also be accurate, and, to be useful in resource-poor countries where most MDR TB patients reside, should be simple to use and cost-effective.

Genetic determination of RIF resistance is now commonly available in developed countries and is becoming increasingly available in the developing world. The WHO-endorsed GeneXpert MTB/RIF (Cepheid) is an automated closed system that uses rapid PCR techniques to detect DNA sequences in sputum samples that are specific for M. tuberculosis, as well as certain mutations that confer resistance to RIF. RIF resistance is used as a surrogate marker for MDR TB, because 95% of the strains resistant to RIF are also resistant to INH. The test has much better accuracy than sputum acid-fast smear microscopy; testing is completed within 2 hours, with minimal biohazard, and can be performed by less-skilled workers who require minimal technical training.

GeneXpert MTB/RIF is highly accurate across diverse geographic regions and has contributed to the global increase in detection of RR-TB.The test can detect > 97% of patients with RIF resistance identified by culture (1) and has specificity close to 99%.

However, rapid detection of resistance to second-line drugs has lagged behind. Rapid identification of resistance to FQs and the second-line injectable drugs AMK, KAN, and CAP is urgently needed for detection of XDT-TB. Using the GeneXpert platform, an investigational assay has been developed for rapid detection of genes associated with resistance to INH, FQs (moxifloxacin and ofloxacin), and aminoglycosides (amikacin and kanamycin).

A recent study in the New England Journal of Medicine assessed the diagnostic accuracy of this investigational assay in patients in China and South Korea (2). When using DNA sequencing as the reference standard, the investigational assay met the WHO's 95% sensitivity target for INH, the FQs, and AMK and missed the sensitivity target for KAN by approximately 2 percentage points. However, using a culture-based standard, the sensitivity of the investigational genetic assay to detect resistance was lower—83.3% for INH; 88.4% and 87.6% for ofloxacin and moxifloxacin; and 71.4% and 70.7% for kanamycin and amikacin. However, the specificity ranged from 94.3 to 99.6% compared to the WHO target of 98%.

Limitations and Concerns

This study did not evaluate resistance to CAP, although the rrs mutation detected by the assay was said to account for the "majority" of CAP resistance. This study also did not evaluate resistance to other first-line drugs, streptomycin, ethambutol, and pyrazinamide (PZA), that may be useful in MDR TB. Another limitation of this study was the limited geographic representation of participants and M. tuberculosis strains. It is hoped that future studies will document its usefulness worldwide.

The concordance between the results of genetic DST and culture-based DST is known to not always be close to 100%. Genetic tests detect only mutations that are screened for, and some resistant strains have known mutations not included in the assay or have unknown mutations. Thus, it would seem prudent to use culture-based DST to confirm susceptibility identified by genetic DST. In the future, incorporating additional mutations associated with resistance might improve the sensitivity of this investigational assay. It is hoped that with greater access to improved rapid molecular testing, especially in resource-poor communities, a greater proportion of patients with MDR TB will be identified and promptly treated with effective multidrug regimens.

References

 1. Boehme CC, Nabeta P, Hillemann D, et al: Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 363:1005–1015, 2010.

2. Xie YL, Chakravorty S, Armstrong DT, et al: Evaluation of a rapid molecular drug-susceptibility test for tuberculosis. N Engl J Med 377:1043–1054, 2017. doi: 10.1056/NEJMoa1614915.