MPEP Mycobacterium Tuberculosis Drug Susceptibility Testing – Reports

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

Isolate 2022A is susceptible to all first- and second-line drugs.

Pyrazinamide

Isolate 2022A was expected to be susceptible to PZA. DNA sequence analysis of pncA in Isolate 2022A revealed a A>T point mutation resulting in wild-type glutamate being replaced by valine at codon 37 (Glu37Val). However, isolates with the non-synonymous Glu37Val mutation have been reported to test susceptible to PZA in growth-based assays ^[12]. As noted with Isolate 2022A, issues with false-resistance to PZA have been reported ^[13] and remain a concern.

Of those testing PZA for Isolate 2022A, resistance was reported by:

• 56% (33/59) of the results when using MGIT™

Of the 5 molecular results reported for PZA, four (80%) laboratories reported detection of a mutation, with three specifically noting the Glu37Val mutation.

For internal comparison purposes, this isolate was previously sent as MPEP 2019I where 23% (15/65) 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 2022A are listed in Tables 3–9.

Expected Result: Susceptible to RMP* at 1.0 µg/ml by agar proportion

*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.

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 ^[16]. 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 ^[17]. 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 ^{[16, 17]}. 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 ^[7]. 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 ^[7]. 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 2022B revealed an A>T point mutation resulting in histidine being replaced by leucine at codon 445 (His445Leu). Isolates with His445Leu mutations are associated with low-level RMP resistance and often test as susceptible in growth-based assays at currently used critical concentrations ^{[4, 14, 15]}.

For Isolate 2022B, 70 results for RMP were reported and the majority (69%) of reported results differed from the expected result. This isolate was reported resistant to RMP by method, as follows:

• 87% (13/15) of the results when using AP
• 63% (33/52) of the results when using MGIT™
• 67% (2/3) of the results when using Sensititre^®

Of the 12 molecular results reported for RMP, all (100%) laboratories reported detection of a mutation with seven laboratories specifically noting the His445Leu mutation.

Three of the laboratories performing Sensititre® reported RMP MIC values as 1 µg/ml (n=1), 2 µg/ml (n=-1), and 16 µg/ml (n=1).

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2022B are listed in Tables 10-16.

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

Expected Result: Resistant to INH at 0.2 µg/ml, OFL at 2.0 µg/ml, CIP at 2.0 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 ^{[5, 16]}. 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 ^{[5, 16, 17]}. 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 ^{[18, 19]}. 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 ^{[20, 21]}.

DNA sequence analysis of inhA, katG, fabG1, and ahpC of Isolate 2022C 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 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 2022C, 83 INH results were reported. This isolate was reported resistant to INH by method, as follows:

• 100% (15/15) of the results when using AP
• 98% (58/59) of the results when using MGIT^™

Two (4%) results were reported resistant at the higher concentrations of INH. Only 32 (54%) 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 9 molecular results reported for INH, all (100%) laboratories reported detection of a mutation with seven specifically noting the inhA C-15T mutation.

Two of the laboratories performing Sensititre® reported INH MIC values as 0.25 µg/ml (n=2). A third laboratory reported INH MIC value as 0.5 µg/ml (n=1) and noted resistance but since an interpretation was not indicated for INH—Low and INH—High, the result was excluded from Table 19.

Ofloxacin and Ciprofloxacin

Fluoroquinolones (FQs) are one of the most commonly prescribed classes of antibiotic in the United States due to their activity against various types of bacteria. They are an important class of drugs used to treat tuberculosis (TB) ^{[9, 22, 23]}. Prolonged treatment with a FQ (>10 days) before a diagnosis of TB is associated with a higher risk of resistance and diagnostic delays ^{[22, 24]}. The primary mechanism of action for FQs is the inhibition of DNA synthesis ^[25] by inhibiting DNA gyrase. The enzyme DNA gyrase generates the activity for cleaving and resealing double-stranded DNA. This action is necessary for DNA replication, transcription, and recombination.

Resistance to FQs has been attributed to point mutations in a 21-bp region, known as the quinolone resistance determining region (QRDR), of the MTBC gyrA gene. Mutations in the QRDR, commonly occurring at codons 90, 91, and 94, prevent the drugs from effectively binding DNA gyrase ^{[5, 17, 25]}. Mutations in the gyrB gene have also been noted with varying rates of resistance, but high-level resistance is less common without a concurrent gyrA mutation ^[25-27].

Heteroresistance is the result of varying levels of resistance within a population of MTBC due to the presence of sub-populations with differing nucleotides at a locus associated with drug resistance, resulting in both drug-resistant and drug-susceptible organisms ^{[28, 29]}. This phenomenon is not limited to FQs but is commonly noted with this drug class.

Studies suggest that the level of resistance, as measured by MIC testing, to ofloxacin (OFL), ciprofloxacin (CIP), levofloxacin (LVX), and moxifloxacin (MOX) is dependent on the mutation and can vary among the FQ ^{[26, 30, 31]}. CLSI and WHO currently recommend testing LVX and/or MOX; however, the preferred FQ to be tested is the one used in the treatment regimen ^{[1, 10]}.

DNA sequencing of gyrA in Isolate 2022C detected a C>T point mutation in gyrA resulting in wild-type alanine being replaced with valine at codon 90 (Ala90Val). The Ala90Val mutation has been associated with low-level FQ resistance, and the MIC for isolates with this mutation could be close to the critical concentration tested thereby impacting reproducibility ^{[3, 5, 32]}. Sequencing of the gyrB locus for this isolate revealed no mutations (i.e., wild-type sequence).

Among three growth-based methods, 11 results for OFL were reported for Isolate 2022C. This isolate was reported resistant to OFL by method, as follows:

• 100% (7/7) of the results when using AP
• 100% (3/3) of the results when using MGIT™
• 100% (1/1) of the results when using Sensititre^®

Participating laboratories also reported results for other FQs (e.g., CIP, LVX, and MOX) for Isolate 2022C; 80% (16/20) of results noted resistance to these additional FQs. The isolate was reported resistant to three other FQs by method, as follows:

CIP

• 75% (3/4) of the results when using AP

LVX

• 100% (3/3) of the results when using AP
• 100% (3/3) of the results when using MGIT™

MOX

• 75% (3/4) of the results when using AP
• 80% (4/5) of the results when using MGIT™
• 0% (0/1) of the results when using Sensititre^®

A mutation in the gyrA gene was detected by six (75%) laboratories that reported molecular testing for OFL and CIP [five (71%) laboratories reported molecular testing for MOX and LVX], with four laboratories noting the Ala90Val mutation.

Two of the laboratories performing Sensititre® reported MIC values for FQs; one of these did not report interpretations. Reported MIC values were as follows: OFL at 8 µg/ml (n=1); MOX at 2 µg/ml (n=1) and 4 µg/ml (n=1); and LVX at 4 µg/ml (n=1).

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 ^[33]. 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 ^[34].

Issues with reproducibility of DST results for ETA have been reported [35] and remain a potential concern.

For Isolate 2022C, 15 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

• 77% (10/13) of the results when using AP
• 100% (1/1) 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 >40 µg/ml (n=1).

Complete first-line DST, second-line DST, and molecular results submitted by all participant for Isolate 2022C are listed in Tables 17-23.

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

Expected Result: Susceptible to RMP* at 1.0 µg/ml by agar proportion

^*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.

Rifampin

DNA sequence analysis of rpoB in Isolate 2022D revealed a G>T point mutation resulting in wild-type serine being replaced by leucine in MTB codon 435 (Asp435Tyr). Isolates with Asp435Tyr (Asp516Tyr in E. coli numbering system) mutations are associated with low-level RMP resistance and can test as susceptible in growth-based assays ^{[14, 15]}.

For Isolate 2022D, 74 results for RMP were reported. This isolate was reported susceptible to RMP by method, as follows:

• 100% (12/12) of the results when using AP
• 100% (59/59) of the results when using MGIT™
• 67% (2/3) of the results when using Sensititre^®

Of the 11 molecular results reported for RMP, all (100%) laboratories reported detection of a mutation in rpoB. Seven laboratories specifically noted the Asp435Tyr mutation.

Three of the laboratories performing Sensititre® reported RMP MIC values as 0.25 µg/ml (n=1), 1 µg/ml (n=1), and 16 µg/ml (n=1).

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2022D are listed in Tables 24-30.