MPEP Mycobacterium Tuberculosis Drug Susceptibility Testing – Reports

Expected Result: Susceptible to all first- and second-line drugs by agar proportion

Rifampin

Rifampin (RMP) is a bactericidal drug used as part of a standard first-line regimen for the treatment of TB. RMP’s mechanism of action is to inhibit mycobacterial transcription by targeting DNA-dependent RNA polymerase [13]. The primary mechanism of resistance is mutations within the 81-bp central region of the rpoB gene (i.e., rifampin resistance determining region or RRDR) that encodes the β-subunit of the bacterial DNA-dependent RNA polymerase [14]. Mutations in codons 450, 445, and 435 (E. coli numbering system corresponding to 531, 526, and 516) are among the most frequent mutations in RMP-resistant isolates and serve as predictors of RMP resistance [13, 14]. The activity of RMP on isolates with rpoB mutations depends on both the mutation position and the type of amino acid change.

CDC has recommended that RMP resistance detected by the Xpert^® MTB/RIF assay be confirmed by DNA sequencing [15]. The Xpert® MTB/RIF assay could generate results that falsely indicate resistance when compared to growth-based methods because of the presence of silent/synonymous mutations[16]. Sequencing of rpoB allows for clarification of the result and understanding of possible discordance between rapid molecular and growth-based testing results.

DNA sequence analysis of rpoB in Isolate 2022F revealed a C>T point mutation in codon 433 (Phe433Phe). This mutation does not result in an amino acid change; phenylalanine remains phenylalanine (Phe433Phe). The Arg447Arg synonymous (i.e., silent) mutation in rpoB is not considered clinically significant and isolates with this mutation reliably test as RMP-susceptible in growth-based systems. However, as noted above, the Xpert MTB/RIF assay could indicate RMP resistance for this isolate and sequencing of rpoB should be performed.

For Isolate 2022F, 71 results for RMP were reported. This isolate was reported susceptible to RMP by method, as follows:

• 100% (11/11) of the results when using AP
• 100% (57/57) of the results when using MGIT™
• 100% (3/3) of the results when using Sensititre^®

Of the 10 molecular results reported for RMP, 8 (80%) laboratories reported detection of a mutation with six laboratories specifically noting the Phe433Phe mutation.

Three of the laboratories performing Sensititre^® reported RMP MIC values as ≤0.12 µg/ml (n=1) and 0.25 µg/ml (n=2).

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2022F are listed below.

One laboratory noted contaminated/no growth for Isolate 2022F and did not report results for at least one antituberculosis drug tested.

Expected Result: Resistant to PZA* at 100 µg/ml by MGIT

*80% consensus for a single categorical result across all methods reported for this drug of either susceptible or resistant was not achieved for these isolates among participating laboratories.

Pyrazinamide

Pyrazinamide (PZA) is an important first-line drug for treatment of TB and is used with INH and RIF. The addition of this drug shortens TB treatment from the previous 9–12 months to 6 months because it kills a population of persistent bacilli in acidic pH environments within the lesions that are not killed by other drugs [17]. PZA is a prodrug that requires conversion to its active form, pyrazinoic acid, by the pyrazinamidase encoded by the pncA gene of M. tuberculosis. PZA-resistant M. tuberculosis strains lose pyrazinamidase activity, and resistance to PZA is usually caused by nucleotide changes scattered throughout the pncA gene. However, there may be additional mechanisms of resistance to PZA that are still unknown [18].

DNA sequence analysis of pncA in Isolate 2022G revealed a C>G point mutation in codon 82 resulting in wild-type histidine being replaced by aspartate (His82Asp). The His82Asp mutation is thought to confer PZA resistance.

Among those performing MGIT, 54 results for PZA were reported for Isolate 2022G. This isolate was reported as resistant to PZA by method, as follows:

• 74% (42/54) of the results when using MGIT™

Of the 5 molecular results reported for PZA, all (100%) laboratories reported detection of a mutation, with 4 specifically noting the His82Asp mutation.

For internal comparison purposes, this isolate was previously sent as MPEP 2019G where 85% (56/66) of MGIT™ results and 0% (0/1) of VersaTREK™ results were reported as resistant.

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2022G are listed below.

Two laboratories noted contaminated/no growth for Isolate 2022G and did not report results for at least one antituberculosis drug tested.

Expected Result: Susceptible to all first-line drugs by agar proportion

Rifampin

DNA sequence analysis of rpoB in Isolate 2022H revealed a T>C point mutation resulting in wild-type leucine being replaced by proline in MTB codon 440 (Leu430Pro). Isolates with Leu430Pro (Leu511Pro in E. coli numbering system) mutations are associated with low-level RMP resistance and can test as susceptible in growth-based assays [19-21].

For Isolate 2022H, 67 results for RMP were reported. This isolate was reported susceptible to RMP by method, as follows:

• 100% (9/9) of the results when using AP
• 98% (54/55) of the results when using MGIT™
• 100% (3/3) of the results when using Sensititre^®

Of the 10 molecular results reported for RMP, 9 (90%) laboratories reported detection of a mutation in rpoB. Seven laboratories specifically noted the Leu430Pro mutation.

Three of the laboratories performing Sensititre^® reported RMP MIC values as ≤0.12 µg/ml (n=2) and 0.25 µg/ml (n=1).

Complete first-line DST, second-line DST, and molecular results submitted by all participant for Isolate 2022H are listed below.

Three laboratories noted contaminated/no growth for Isolate 2022H and did not report results for at least one antituberculosis drug tested.

Expected Result: Resistant to INH at 0.2 µg/ml and ETA at 5.0 µg/ml by agar proportion

Isoniazid

Isoniazid (INH) is the most widely used first-line antituberculosis drug and is a cornerstone of regimens used to treat TB disease and latent TB infection. INH is a prodrug and is activated by the catalase-peroxidase enzyme encoded by the katG gene [6, 13]. The target of activated INH is enoyl-acyl-carrier protein reductase (encoded by the inhA gene); this binding inhibits cell wall mycolic acid biosynthesis. There are two mechanisms that account for the majority of INH resistance [6, 13, 22]. The most common mechanism, mutations in katG, is generally associated with high-level resistance to INH. Resistance to INH can also occur by mutations in the fabG1/inhA promoter region, which are generally associated with low-level resistance to INH and are less frequent than katG mutations. It has been reported that approximately 10–15% of isolates found to be INH-resistant have no mutations detected in either of these loci; however, this percent is decreasing due to the more comprehensive nature of whole genome sequencing [23, 24]. Numerous loci have been investigated to identify additional genes correlated with INH resistance. The fabG1 (also known as mabA) gene, like inhA, is involved in mycolic acid biosynthesis and at least one mutation in this region (Leu203Leu) has been associated with low-level INH resistance [21, 25, 26].

DNA sequence analysis of inhA, katG, fabG1, and ahpC of Isolate 2022I revealed a C>T point mutation at nucleotide position -15 of the promoter region of the inhA gene (C-15T); katG, fabG1, and ahpC were wild-type (i.e., no mutations were detected). The C-15T mutation has been associated with low-level INH resistance [21, 27].

The recommended critical concentration and additional higher concentrations for testing INH using the AP method are 0.2 µg/ml and 1.0 µg/ml, respectively. The equivalent concentrations for MGIT™ and VersaTREK™ are 0.1 µg/ml and 0.4 µg/ml [1].

For Isolate 2022I, 69 INH results were reported. This isolate was reported resistant to INH by method, as follows:

• 91% (10/11) of the results when using AP
• 100% (57/57) of the results when using MGIT™
• 0% (0/1) of the results when using Sensititre^®

Two (4%) results were reported resistant at the higher concentrations of INH. Only 32 (56%) laboratories performing MGIT™ DST reported a result for the higher concentration of INH, although some may have tested the higher concentration by a second DST method.

Of the 8 molecular results reported for INH, 7 (88%) laboratories reported detection of a mutation with all specifically noting the inhA C-15T mutation.

One of the laboratories performing Sensititre^® reported INH MIC value as 0.25 µg/ml (n=1). Another laboratory reported INH MIC value as 0.25 µg/ml (n=1) and noted ‘Intermediate’. A third laboratory reported INH MIC value as 0.25 µg/ml (n=1) and noted ‘Resistant’ but since an interpretation was not indicated for INH—Low and INH—High, the result was excluded from the table.

For internal comparison purposes, this isolate was previously sent as MPEP 2018E where 94% (17/18) of AP results, 100% (72/72) of MGIT™ results, 100% (4/4) of Sensititre^® results, and 100% (1/1) of VersaTREK™ results were reported as resistant.

Ethionamide

Resistance to INH and ethionamide (ETA) can occur by mutations in the fabG1–inhA regulatory region, which are generally associated with low-level resistance to INH. Mutations in ethA also confer resistance to ETA, without concomitant resistance to INH [27]. Sequencing analysis of ethA did not detect a mutation but sequencing of the promoter region of the inhA gene revealed a C>T point mutation at nucleotide position -15 (C-15T). This mutation has been associated with ETA resistance [21, 27].

For Isolate 2022I, 11 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

• 63% (5/8) of the results when using AP
• 100% (2/2) of the results when using MGIT™
• 100% (1/1) of the results when using Sensititre^®

One of the laboratories performing Sensititre^® reported ETA MIC value as 20 µg/ml (n=1).

For internal comparison purposes, this isolate was previously sent as MPEP 2018E where 65% (11/17) of AP results, 100% (3/3) of MGIT™ results, and 50% (1/2) of Sensititre^® results were reported as resistant.

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2022I are listed below.

One laboratory noted contaminated/no growth for Isolate 2022I and did not report results for at least one antituberculosis drug tested.