What Is Resistance? Impact of Phenotypic versus Molecular Drug Resistance Testing on Therapy for Multi- and Extensively Drug-Resistant Tuberculosis

ABSTRACT Rapid and accurate drug susceptibility testing (DST) is essential for the treatment of multi- and extensively drug-resistant tuberculosis (M/XDR-TB). We compared the utility of genotypic DST assays with phenotypic DST (pDST) using Bactec 960 MGIT or Löwenstein-Jensen to construct M/XDR-TB treatment regimens for a cohort of 25 consecutive M/XDR-TB patients and 15 possible anti-TB drugs. Genotypic DST results from Cepheid GeneXpert MTB/RIF (Xpert) and line probe assays (LPAs; Hain GenoType MTBDRplus 2.0 and MTBDRsl 2.0) and whole-genome sequencing (WGS) were translated into individual algorithm-derived treatment regimens for each patient. We further analyzed if discrepancies between the various methods were due to flaws in the genotypic or phenotypic test using MIC results. Compared with pDST, the average agreement in the number of drugs prescribed in genotypic regimens ranged from just 49% (95% confidence interval [CI], 39 to 59%) for Xpert and 63% (95% CI, 56 to 70%) for LPAs to 93% (95% CI, 88 to 98%) for WGS. Only the WGS regimens did not contain any drugs to which pDST showed resistance. Importantly, MIC testing revealed that pDST likely underestimated the true rate of resistance for key drugs (rifampin, levofloxacin, moxifloxacin, and kanamycin) because critical concentrations (CCs) were too high. WGS can be used to rule in resistance even in M/XDR strains with complex resistance patterns, but pDST for some drugs is still needed to confirm susceptibility and construct the final regimens. Some CCs for pDST need to be reexamined to avoid systematic false-susceptible results in low-level resistant isolates.

 Give with pyridoxine.
 Due to lack of data, it cannot be recommended at present to count high dose isoniazid among the ≥4 active drugs in the MDR-TB regimen (≥5 drugs in XDR-TB) or shorten the duration of therapy.
 With inhA promotor mutation ethionamide or prothionamide should not be included in the regimen, as inhA promotor mutations usually lead to thioamide drug resistance.
Step 3 Unless drug resistance to a later generation fluoroquinolone is suggested by genotypic of phenotypic drug susceptibility testing use moxifloxacin or levofloxacin in the drug regimen.
Comments  Fluoroquinolones are probably the most effective 2nd line antituberculosis drugs available at present.
 QTc interval prolongation may be potentiated with other drugs. Close monitoring recommended when used with other drugs that prolong the QTc interval. Concurrent use of moxifloxacin with bedaquiline or delamanid not recommended.
Step 4 In case of later generation fluoroquinolone drug resistance, use bedaquiline in the drug regimen.

Comments
Bedaquiline is approved for the treatment of MDR-TB by the FDA and EMA for use as part of an appropriate combination regimen when an effective treatment regimen is unavailable because of resistance or intolerability to other medications. QTc interval prolongation may be potentiated with other drugs. Close monitoring recommended when used with other drugs that prolong the QTc interval. Concurrent use with delamanid or moxifloxacin not recommended. -3-Step 5 Unless drug resistance to a second line injectable drug (SLID, amikacin, capreomycin or kanamycin) is suggested by genotypic or phenotypic drug susceptibility testing use a SLID as part of the drug regimen.

Comments
If possible, a SLID should be included during the first 8 months of the treatment.
Capreomycin is preferred for reasons of drug-toxicity in long-term treatment. Patients should have a CVC catheter with subcutaneous reservoir implanted for daily intravenous therapy. Monitor renal function, electrolytes and audiology exam.
Step 6 Unless drug resistance to ethionamide/prothionamide, cycloserine/terizidone, PAS or linezolid is suggested by genotypic or phenotypic drug susceptibility testing, a combination of up to 3 of these drugs is often used.

Comments
 Prothionamide or ethionamide are often not tolerated in combination with PAS. Monitor liver and thyroid function. Give with pyridoxine.
 Terizidone is less toxic than cycloserine and is the fusion product of two molecules of cycloserine and one molecule of terephtalaldehyde. Monitor mental status. Give with pyridoxine.
 PAS is often not tolerated in combination with prothionamide or ethionamide.
 Severe adverse events are frequent in long-term therapy with linezolid, close monitoring of blood count and awareness of peripheral neuropathy is mandatory Step 7 If a sufficient number of likely active drugs cannot be achieved in the regimen, add meropenem/amoxicillin-clavulanate and/or clofazimine and/or delamanid -4-Comments  For the treatment of XDR-TB with meropenem/amoxicillin-clavulanate long term intravenous access recommended.
 When treatment with clofazimine causes severe skin discoloration dose reduction to five times a week may be justified. Monitor QTc interval.
 Delamanid is approved for the treatment of MDR-TB by the EMA for use as part of an appropriate combination regimen when an effective treatment regimen is unavailable because of resistance or intolerability to other medications. QTc interval prolongation may be potentiated with other drugs.
Close monitoring recommended when used with other drugs that prolong the QTc interval.
Concurrent use with bedaquiline or moxifloxacin not recommended.
Step 8 Pyrazinamide and/or ethambutol should be added to the initial treatment regimen, but the drugs should not be counted among the ≥4 active drugs in the MDR-TB regimen (≥5 drugs in XDR-TB regimen).
Treatment with pyrazinamide and/or ethambutol should be discontinued, if results from phenotypic or genotypic drug susceptibility testing suggest resistance to either drug.

Comments
Phenotypic drug resistance to pyrazinamide and ethambutol is very common in M/XDR-TB. There are no data available to support the use of either drug when drug resistance has been documented. Due to the high pill burden of a M/XDR-TB regimen, the authors do not recommend the use of either drug in a M/XDR-TB regimen if drug resistance has been documented by a quality controlled laboratory.

Loss-of-function mutations in alanine dehydrogenase (Ald) have been shown to result in elevated
MICs, which should apply to the XDR isolate 13739-13 as a result of a premature stop at codon 271 out of the total 371 codons. 3 Another XDR isolate (811-15) had an ald mutation at codon 198, which directly interacts with its coenzyme nicotinamide adenine dinucleotide (NAD). 4 Moreover, codon 238, which was mutated in a third isolate, is within 4 Å of the NAD binding region. 4

PAS
For the isolates, for which PAS was tested, all 13 isolates that lacked mutations in any of the known PAS resistance genes tested susceptible at 4 μg/ml. Only five of the seven isolates with known resistance mutation in either the dihydrofolate synthase FolC, which is required for the activation of the PAS, or with promoter-up mutations in Rv2671, an alternative dihydrofolate reductase and one of the targets of PAS, tested resistant. [5][6][7] The susceptible result for 13255-14 (C-12T Rv2671) and 13739-13 (folC S150G) likely represented a false-susceptible result. [6][7][8][9] The remaining four isolates harboured two different amino acid changes at the same codon (P253A or P253L) of the thymidylate synthase ThyA, which is typically a signal for positive selection, yet all tested susceptible (repeatedly, in case of the former mutation). 10 This was surprising given that the latter mutation coincided with a promoter mutation in the second thymidylate synthase ThyX, which is believed to compensate for loss-offunction mutations in thyA, which, in turn, confer resistance by reducing the catalytic demand of the dihydrofolate reductase DfrA, the main target of PAS. 5,11 Table S1 Details of 25 patients in this study.