MPEP Mycobacterium Tuberculosis Drug Susceptibility Testing – Reports

Expected Result: Resistant to RMP at 1.0 µg/ml 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 [4]. 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 DNAdependent RNA polymerase [5]. Mutations in codons 531, 526 and 516 (E. coli numbering system corresponding to 450, 445 and 435 in MTBC) are among the most frequent mutations in RMP-resistant isolates and serve as predictors of RMP resistance [4, 5].

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 [6]. 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 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 2020 revealed a C>T point mutation in codon 531 resulting in wild-type serine being replaced by leucine (Ser531Leu). Isolates with Ser531Leu (Ser450Leu in MTBC numbering system) mutations consistently test resistant to RMP in growth-based assays.

Among four methods, 84 results for RMP were reported for Isolate 2020A. This isolate was reported as resistant to RMP by method, as follows:

• 100% (17/17) of the results when using AP
• 97% (59/61) of the results when using MGIT
• 100% (4/4) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Of the 10 molecular results reported for RMP, all (100%) laboratories reported detection of a mutation with 4 laboratories specifically noting the Ser531Leu mutation.

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

Rifabutin

Participant results are consistent with rifabutin (RBT) results based on the presence of the rpoB Ser531Leu mutation[8].

Among three methods, 12 results for RBT were reported for Isolate 2020A. This isolate was reported as resistant to RBT by method, as follows:

• 100% (7/7) of the results when using AP
• 100% (2/2) of the results when using MGIT
• 100% (3/3) of the results when using Sensititre

Three of the laboratories performing Sensititre reported RBT MIC values as 2 µg/ml (n=2) and 8 µg/ml (n=1).

Streptomycin

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[4, 5]. In MTBC, the genetic basis of the majority of resistance to STR is usually due to mutations in rrs or rpsL[5, 9]. 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, 10].

Among three methods, 51 results for STR were reported for Isolate 2020A. This isolate was reported as resistant to STR by method, as follows:

• 24% (4/17) of the results when using AP
• 77% (24/31) of the results when using MGIT
• 0% (0/3) of the results when using Sensititre

Three of the laboratories performing Sensititre reported STR MIC values as 0.5 µg/ml, 1 µg/ml and 2 µg/ml.

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

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

Expected Result: Resistant to INH at 0.2 µg/ml and 1.0 µg/ml and EMB 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 [2, 4]. 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 [2, 4, 5]. 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 [8, 9]. In MTBC, ahpC codes for an alkyl hydroperoxide reductase that is associated with resistance to reactive oxygen and reactive nitrogen intermediates; consequently, it was initially believed that mutations in the promoter region could be surrogate markers for INH resistance [4].

DNA sequence analysis of inhA, katG, fabG1 and ahpC of Isolate 2020B detected a C>G point mutation at codon 315 in the katG locus resulting in wild-type serine being replaced by threonine (Ser315Thr) and a G>A point mutation at nucleotide position -88 of the intergenic region of oxyR’-ahpC (G-88A); inhA and fabG1 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 2020B, 88 INH results were reported. This isolate was reported resistant to INH by method, as follows:

• 100% (19/19) of the results when using AP
• 98% (62/63) of the results when using MGIT
• 100% (4/4) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Sixty (98%) results were reported as resistant at the higher concentrations of INH. Only 35 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 7 molecular results reported for INH, all (100%) laboratories reported detection of a mutation with 4 laboratories specifically noting the Ser315Thr mutation.

Three of the laboratories performing Sensititre reported INH MIC values as 4 µg/ml (n=2) and >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 [4, 5]. EMB targets the arabinosyl transferases (embCAB operon), thereby inhibiting the biosynthesis of the cell wall components arabinogalactan and lipoarabinomannan [10].

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 [2, 10]. False susceptibility with some growth–based methods for EMB have been reported [11, 12].

DNA sequence analysis of embB of Isolate 2020B revealed a G>A point mutation at codon 306 in the embB gene resulting in wild-type methionine being replaced by isoleucine (Met306Ile). While certain embB mutations at the 306 codon, such as Met306Val and Met306Leu, are associated with EMB resistance, isolates with Met306Ile have been reported to show variable resistance [2]. This may be due to an increased MIC close to the critical concentration tested.

For Isolate 2020B, 86 EMB results were reported. This isolate was reported resistant to EMB by method, as follows:

• 40% (8/20) of the results when using AP
• 2% (1/61) of the results when using MGIT
• 33% (1/3) of the results when using Sensititre
• 0% (0/2) of the results when using VersaTREK

Of the 3 molecular results reported for EMB, all (100%) laboratories reported detection of a mutation and specifically noted the Met306Ile mutation.

Three of the laboratories performing Sensititre reported EMB MIC values as 2 µg/ml (n=1) and 4 µg/ml (n=2).

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

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

Rifampin

DNA sequence analysis of rpoB in Isolate 2020C revealed a C>G point mutation in codon 511 resulting in wild-type leucine being replaced by valine (Leu511Val). The effects of a Leu511Val (Leu430Val in MTBC numbering system) mutation on rifampin susceptibility are currently unknown.

Among four methods, 86 results for RMP were reported for Isolate 2020C. This isolate was reported as susceptible to RMP by method, as follows:

• 100% (17/17) of the results when using AP
• 100% (63/63) of the results when using MGIT
• 100% (4/4) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Of the 8 molecular results reported for RMP, 6 (75%) laboratories reported detection of a mutation with 3 laboratories specifically noting the Leu511Val 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 participants for Isolate 2020C are listed in tables below.

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

Rifampin

DNA sequence analysis of rpoB in Isolate 2020D revealed a G>T point mutation in codon 146 of rpoB (E. coli numbering system and also sometimes indicated as codon 176—M. tuberculosis codon 170) resulting in wild-type valine being replaced by phenylalanine (Val146Phe or Val176Phe). Isolates with Val146Phe mutation have been shown to confer resistance [16].

Among four methods, 65 results for RMP were reported for Isolate 2020D; 17 laboratories reported no growth due to growth issues during testing (15 using MGIT and 2 using AP). This isolate was reported as resistant to RMP by method, as follows:

• 94% (16/17) of the results when using AP
• 90% (38/42) of the results when using MGIT
• 100% (4/4) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Of the 10 molecular results reported for RMP, 2 (20%) laboratories reported detection of a mutation with 1 laboratory specifically noting the Val146Phe mutation.

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

Pyrazinamide

Isolate 2020D was expected to be susceptible to PZA. DNA sequence analysis of pncA in Isolate 2020D revealed a T>C point mutation in codon 135 resulting in wild-type threonine being replaced by alanine (Thr135Ala). Mutations in pncA gene are typically associated with PZA resistance, however it has been reported that not all pncA mutations confer resistance [9, 17]. The effects of a Thr135Ala mutation on PZA susceptibility are currently unknown.

Of the 58 laboratories reporting results for PZA for Isolate 2020D, susceptible was reported by:

• 98% (55/56) of the results when using MGIT
• 100% (1/1) of the results when using VersaTREK

Of the 2 molecular results reported for PZA, both (100%) laboratories reported detection of a mutation, specifically noting the Thr135Ala mutation.

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

Seventeen laboratories noted no growth and one laboratory noted contamination for at least one antituberculosis drug tested for Isolate 2020D.