MPEP Mycobacterium Tuberculosis Drug Susceptibility Testing – Reports

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 [4, 8]. 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 [4, 8, 9]. 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 promoter region of the inhA gene, which are generally associated with low-level resistance to INH and are less frequent than katG mutations. Approximately 10–15% of isolates found to be INH-resistant have no mutations detected in either of these loci. 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 has been associated with low-level INH resistance [10, 11]. In MTBC, ahpC codes for an alkyl hydroperoxide reductase that is associated with resistance to reactive oxygen and reactive nitrogen intermediates; consequently, it is believed that mutations in the promoter region could be surrogate markers for INH resistance [8].

DNA sequence analysis of inhA, katG, fabG1, and ahpC for Isolate 2020F revealed a G>A point mutation in fabG1 at codon 203 resulting in the synonymous/silent mutation Leu203Leu; inhA, katG, and ahpC were wild-type (i.e., no mutations were detected).

Within fabG1, the silent/synonymous mutation (i.e., nucleotide change but no corresponding change in amino acid) Leu203Leu has been found to confer INH resistance through the formation of an alternative promoter, thereby increasing the transcriptional levels of inhA [11]. Although silent mutations were previously believed to not play a role in drug resistance, the Leu203Leu mutation demonstrates that silent mutations could be associated with resistance depending on the specific gene and the location of the mutation.

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 2020F, 78 INH results were reported for the critical concentration. This isolate was reported resistant to INH by method, as follows:

• 94% (15/16) of the results when using AP
• 49% (28/57) of the results when using MGIT
• 0% (0/3) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

No results were reported as resistant at the higher concentrations of INH. Only 28 (48%) laboratories performing MGIT DST reported a result for the higher concentration of INH, although some may have tested the higher concentration by a different method.

Of the 6 molecular results reported for INH, 3 (50%) laboratories reported detection of a mutation in fabG1, all specifically noting the Leu203Leu mutation.

Three of the laboratories performing Sensititre reported INH MIC values as 0.12 µg/ml (n=2) and 0.25 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 [12].

Sequencing analysis of ethA was not performed and as previously noted, sequencing of the inhA gene revealed wild-type (i.e., no mutations were detected). The synonymous/silent mutation Leu203Leu was detected in the fabG1 locus for Isolate 2020F.

For Isolate 2020F, 17 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

• 84% (11/13) of the results when using AP
• 67% (2/3) of the results when using MGIT
• 0% (0/1) of the results when using Sensititre

One laboratory performing Sensititre reported an ETA MIC value as 2.5 µg/ml (n=1) with a categorical result of susceptible. One additional laboratory reported an ETA MIC value as 10 µg/ml (n=1) but as no interpretation was indicated by this laboratory, the result was excluded from Table 9.

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 [8]. The primary mechanism of resistance is a mutation within the 81-bp central region of the rpoB gene that encodes the β-subunit of the bacterial DNA-dependent RNA polymerase [9]. 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 [8, 9]. 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 of rpoB [13]. 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 [14].  Sequencing of rpoB will allow 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 2020F revealed a C>T point mutation in codon 447 (E. coli numbering 528) of the rpoB locus.  However, this mutation does not result in an amino acid change; arginine remains arginine (Arg447Arg). Unlike the fabG1 silent mutation in this isolate that was associated with INH resistance, 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 2020F, 80 results for RMP were reported. This isolate was reported as susceptible to RMP by method, as follows:

• 100% (16/16) of the results when using AP
• 100% (59/59) of the results when using MGIT
• 100% (3/3) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Of the 8 molecular results reported for RMP, 5 (63%) laboratories reported mutation detected with 4 laboratories specifically noting the Arg447Arg silent mutation. Three laboratories reported mutation not detected, however this may be due to the detection of a silent mutation not associated with resistance.

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

Complete first-line DST, second-line DST and molecular results submitted by all participants for Isolate 2020F are listed in the tables below.

Three laboratories noted no growth for at least one antituberculosis drug tested for Isolate 2020F.

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

DNA sequence analysis of inhA, katG, fabG1, and ahpC of Isolate 2020G 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). Mutations in the promoter region of the inhA gene are generally associated with low-level resistance to INH.

For Isolate 2020G, 82 INH results were reported. This isolate was reported resistant to INH by method, as follows:

• 100% (17/17) of the results when using AP
• 100% (61/61) of the results when using MGIT
• 50% (1/2) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Five (9%) results were reported as resistant at the higher concentrations of INH. Only 34 (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 6 molecular results reported for INH, all (100%) laboratories reported detection of a mutation with 5 laboratories specifically noting the C-15T mutation.

Two of the laboratories performing Sensititre reported INH MIC values as 0.25 µg/ml (n=1) and 0.5 µg/ml (n=1). A third laboratory reported an INH MIC value as 0.25 µg/ml (n=1) and indicated a result of borderline.

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2020G are listed in the tables below.

Expected Result: Resistant to INH at 0.2 µg/ml and 1.0 µg/ml, EMB at 5.0 µg/ml, ETA at 5.0 µg/ml and STR at 2.0 µg/ml by agar proportion

Isoniazid

As previously noted, resistance to INH most commonly occurs due to mutations in the katG gene or the promoter region of the inhA gene, however, mutations in fabG1 and ahpC can also cause resistance. DNA sequence analysis of Isolate 2020H revealed a G>C point mutation at codon 315 in the katG locus resulting in wild-type serine being replaced by threonine (Ser315Thr); inhA, fabG1, and ahpC were wild-type (i.e., no mutations were detected).

For Isolate 2020H, 82 INH results were reported. This isolate was reported resistant to INH by method, as follows:

• 100% (17/17) of the results when using AP
• 100% (61/61) of the results when using MGIT
• 100% (2/2) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Fifty-five or 100% of results at the higher concentrations of INH were reported as resistant. Only 34 (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 6 molecular results reported for INH, all (100%) laboratories reported detection of a mutation with 5 laboratories specifically noting the Ser315Thr mutation.

Two of the laboratories performing Sensititre reported INH MIC values as 4 µg/ml (n=2). One additional laboratory reported an INH MIC value as 4 µg/ml (n=1).

Ethambutol

Ethambutol (EMB) is an important first-line drug for the treatment of TB and is used in combination with INH, RMP and PZA to prevent emergence of drug resistance. EMB is a bacteriostatic agent that is active against growing bacilli and has no effect on non-replicating bacilli [8, 9]. EMB targets the arabinosyl transferases (embCAB operon), thereby inhibiting the biosynthesis of the cell wall components arabinogalactan and lipoarabinomannan [15].

Issues with false-susceptibility with some growth-based methods for EMB, particularly in broth-based media, have been reported and remain a potential concern. Probable causes include the bacteriostatic nature of the drug, reduced drug activity in culture, and an organism’s MIC for EMB falling too close to the critical concentration tested [16-18].

Sequence analysis of EMB-resistant clinical isolates has shown that EMB resistance is associated primarily with missense (non-synonymous) mutations within the EMB resistance determining region of the gene embB at codons 306, 406 and 497 [4, 15].

DNA sequence analysis of embB of Isolate 2020H revealed a A>G point mutation at codon 306 in the embB gene resulting in wild-type methionine being replaced by valine (Met306Val). Certain embB mutations at the 306 codon, such as Met306Val and Met306Leu, are associated with EMB resistance [4].

For Isolate 2020H, 83 EMB results were reported. This isolate was reported resistant to EMB by method, as follows:

• 88% (15/17) of the results when using AP
• 16% (10/61) of the results when using MGIT
• 100% (3/3) of the results when using Sensititre
• 50% (1/2) of the results when using VersaTREK

Of the 4 molecular results reported for EMB, all laboratories reported detection of a mutation and specifically noted the Met306Val mutation.

Three of the laboratories performing Sensititre reported EMB MIC values as 8 µg/ml (n=3).

Ethionamide

As previously noted, resistance to ETA is commonly due to mutations in the ethA gene or mutations in fabG1 or inhA resulting in cross-resistance with INH.

DNA sequencing analysis revealed a partial deletion of ethA; inhA and fabG1 were wild-type (i.e., no mutations were detected).

For Isolate 2020H, 18 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

• 64% (9/14) of the results when using AP
• 100% (3/3) of the results when using MGIT
• 0% (0/1) of the results when using Sensititre

Of the 2 molecular results reported for ETA, 1 (50%) laboratory reported detection of a mutation and specifically noted an ethA deletion.

One of the laboratories performing Sensititre reported an ETA MIC value as 2.5 µg/ml (n=1). One laboratory reported an ETA MIC value as 5 µg/ml (n=1) but as no interpretation was indicated by this laboratory, the result was excluded from Table 25.

Streptoymycin

Streptomycin (STR) belongs to the aminoglycoside class of drugs and its primary mechanism of action is to inhibit protein synthesis by preventing the initiation of translation by binding to the 16s rRNA [8, 9]. In MTBC, the genetic basis of the majority of resistance to STR is usually due to mutations in rrs or rpsL [9, 19]. CLSI recommended testing STR as a second-line drug based on American Thoracic Society’s categorization of STR as a second-line drug for treatment due to increased resistance in many parts of the world [1, 20].

DNA sequencing analysis did not reveal a mutation in rrs or rpsL; other mechanisms of resistance may exist.

Among three methods, 48 results for STR were reported for Isolate 2020H. This isolate was reported as resistant to STR by method, as follows:

• 76% (11/14) of the results when using AP
• 48% (16/33) of the results when using MGIT
• 100% (1/1) of the results when using Sensititre

One of the laboratories performing Sensititre reported an STR MIC value as 4.0 µg/ml (n=1). A second laboratory reported a STR MIC value as 2 µg/ml (n=1) and indicated borderline resistance. A third laboratory reported STR MIC value as 4 µg/ml (n=1) but as no interpretation was indicated, the result was excluded from Table 25.

Complete first-line DST, second-line DST and molecular results submitted by all participant for Isolate 2020H are listed in the tables below.

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

Isolate 2020I is a duplicate of Isolate 2020F. Therefore, laboratories should have the same results for both isolates. Overall, laboratories reported similar results for INH, ETA, and RMP. Laboratories should consider performing an internal comparison of results between these two isolates.

Isoniazid

DNA sequence analysis for Isolate 2020I revealed a fabG1 G>A point mutation at codon 203 resulting in the synonymous/silent mutation Leu203Leu; inhA, katG, 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 2020I, 80 INH results were reported. This isolate was reported resistant to INH by method, as follows:

• 82% (14/17) of the results when using AP
• 55% (32/58) of the results when using MGIT
• 0% (0/3) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

No results were reported as resistant at the higher concentrations of INH. Only 29 (49%) laboratories performing MGIT DST reported a result for the higher concentration of INH, although some may have tested the higher concentration by a different method.

Of the 6 molecular results reported for INH, 3 (50%) laboratories reported detection of a mutation, specifically noting the Leu203Leu mutation.

Three of the laboratories performing Sensititre reported INH MIC values as 0.25 µg/ml (n=1), 0.50 µg/ml (n=1) and 1.2 µg/ml (n=1). 

Ethionamide

DNA sequencing analysis of ethA was not performed and as previously noted, sequencing of the inhA gene revealed wild-type (i.e., no mutations were detected). The synonymous/silent mutation Leu203Leu was detected in the fabG1 locus for Isolate 2020I.

For Isolate 2020I, 18 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

• 71% (10/14) of the results when using AP
• 67% (2/3) of the results when using MGIT
• 0% (0/1) of the results when using Sensititre

One of the laboratories performing Sensititre reported an ETA MIC value as 5 µg/ml (n=1). Another laboratory reported an ETA MIC value as 10 µg/ml (n=1) but as no categorical interpretation was provided, the data were not included in Table 33.

Rifampin

DNA sequence analysis of rpoB in Isolate 2020I revealed a C>T point mutation in codon 447 (E. coli numbering 528) of the rpoB locus.  However, this mutation does not result in an amino acid change; arginine remains arginine (Arg447Arg). Unlike the fabG1 silent mutation in this isolate that was associated with INH resistance, 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 2020I, 82 results for RMP were reported. This isolate was reported as susceptible to RMP by method, as follows:

• 100% (17/17) of the results when using AP
• 100% (60/60) of the results when using MGIT
• 100% (3/3) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Of the 8 molecular results reported for RMP, 5 (63%) laboratories reported mutation detected with 4 laboratories specifically noting the Arg447Arg silent mutation. Three laboratories reported mutation not detected, however this may be due to the detection of a silent mutation not associated with resistance.

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

Complete first-line DST, second-line DST and molecular results submitted by all participants for Isolate 2020I are listed in the tables below.

Two laboratories noted no growth for at least one antituberculosis drug tested for Isolate 2020I.