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 6-month 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 [10]. 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 [11]. 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 2021A revealed a C>T point mutation in codon 450 (E. coli numbering 531) resulting in wild-type serine being replaced by leucine (Ser450Leu). Isolates with Ser450Leu (Ser531Leu in E. coli numbering system) mutations consistently test resistant to RMP in growth-based assays.

For Isolate 2021A, 85 results for RMP were reported. This isolate was reported as resistant 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% (3/3) 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 six laboratories specifically noting the Ser450Leu 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 Ser450Leu mutation [12].

Among three methods, 12 results for RBT were reported for Isolate 2021A. This isolate was reported as resistant to RBT by method, as follows:

• 100% (7/7) of the results when using AP
• 100% (3/3) of the results when using MGIT
• 50% (1/2) of the results when using Sensititre

Of the four molecular results reported specifically for RBT, two (50%) laboratories reported detection of a mutation.

Two of the laboratories performing Sensititre reported RBT MIC values as 0.25 µg/ml (n=1) and 4 µg/ml (n=1). Another laboratory reported a RBT MIC value as 8 µg/ml (n=1) but as no categorical interpretation was provided, the data were excluded from Table 9.

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2021A are listed in Tables 3–10.

Expected Result: Resistant to EMB at 5.0 µg/ml and STR at 2.0 µg/ml by agar proportion

Ethambutol

Ethambutol (EMB) is an important first-line drug for the treatment of TB and is used in combination with RMP, INH, 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 [13].

Issues of 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 [14-16].

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, 13].

DNA sequence analysis of embB of Isolate 2021B revealed a A>G point mutation at codon 497 in the embB gene resulting in wild-type glutamine being replaced by arginine (Gln497Arg). Mutations in the 497 codon of embB have been associated with EMB resistance [4, 17].

For Isolate 2021B, 86 EMB results were reported. This isolate was reported resistant to EMB by method, as follows:

• 94% (16/17) of the results when using AP
• 55% (35/64) of the results when using MGIT
• 67% (2/3) of the results when using Sensititre
• 50% (1/2) of the results when using VersaTREK

Of the three molecular results reported for EMB, all (100%) laboratories reported detection of a mutation with two specifically noting the Gln497Arg mutation.

Three of the laboratories performing Sensititre reported EMB MIC values as 2 µg/ml (n=1), 8 µg/ml (n=1), and 16 µg/ml (n=1).

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, 18]. 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, 19].

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 2021B. This isolate was reported as resistant to STR by method, as follows:

• 81% (13/16) of the results when using AP
• 90% (27/30) of the results when using MGIT
• 100% (2/2) of the results when using Sensititre

Two of the laboratories performing Sensititre reported STR MIC values as 4.0 µg/ml (n=1) and 8.0 µg/ml (n=1). A third laboratory reported a STR MIC value as 4 µg/ml (n=1) and indicated borderline resistance.

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2021B are listed in Tables 11–18.

Expected Result: Mycobacterium bovis; Resistant to PZA at 100 µg/ml by MGIT

Pyrazinamide

Unlike M. tuberculosis, M. bovis has an inherent resistance to PZA caused by a characteristic single point mutation of C>G at nucleotide position 169 of the pncA gene resulting in aspartic acid replacing histidine at codon 57 (His57Asp). This substitution causes defective pyrazinamidase activity and confers natural PZA resistance in M. bovis strains, including BCG substrains [20, 21]. DNA sequence analysis of pncA in Isolate 2021C confirmed the His57Asp mutation.

Among two methods, 64 results for PZA were reported for Isolate 2021C. This isolate was reported as resistant to PZA by method, as follows:

• 95% (60/63) of the results when using MGIT
• 100% (1/1) of the results when using VersaTREK

Of the five molecular results reported for PZA, all (100%) laboratories reported detection of a mutation with four laboratories specifically noting the His57Asp mutation or M.bovis.

Complete first-line DST, second-line DST, and molecular results submitted by all participant for Isolate 2021C are listed in Tables 19–26.

One laboratory noted no growth for at least one antituberculosis drug tested for Isolate 2021C.

Expected Result: Resistant to INH at 0.2 µg/ml and 1.0 µg/ml, OFL at 2.0 µg/ml, and CIP at 2.0 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 [22, 23]. 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 Isolate 2020D 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).

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 2021D, 85 INH results were reported for the critical concentration. This isolate was reported resistant to INH by method, as follows:

• 100% (17/17) of the results when using AP
• 98% (63/64) 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 33 (52%) 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 eight molecular results reported for INH, all (100%) laboratories reported detection of a mutation with six laboratories specifically noting the Ser315Thr mutation.

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

Ofloxacin and Ciprofloxacin

Fluoroquinolones (FQ) are one of the most commonly prescribed classes of antibiotic in the United States due to their activity against various types of bacteria. FQ are an important class of drugs used to treat tuberculosis (TB) cases that are resistant to first-line drugs, yet this class of antibiotics also have become an important part of newer TB regimens [24, 25]. In the U.S., resistance to FQ is relatively uncommon in strains of MTBC susceptible to first-line drugs, however prolonged treatment with a FQ (>10 days) before a diagnosis of TB is associated with a higher risk for FQ resistance and diagnostic delays [24, 26]. The primary mechanism of FQ action is the inhibition of DNA synthesis [18] 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 FQ has mainly been attributed to point mutations in a 21-bp region of the MTBC gyrA gene, often called the quinolone resistance determining region (QRDR). These mutations, commonly occurring at codons 90, 91, and 94, prevent the drugs from effectively binding DNA gyrase [4, 9, 18]. Mutations in the gyrB gene have been noted with varying rates of resistance, but high-level resistance is less common without a concurrent gyrA mutation [18].

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 [27, 28]. This phenomenon is not limited to FQ, but is commonly noted with this class of drugs.

Studies suggest that there may not be full cross-resistance between ofloxacin (OFL), ciprofloxacin (CIP), levofloxacin (LEV), and moxifloxacin (MOX) at the defined critical concentrations [29, 30]. CLSI currently recommends testing LEV and/or MOX [1].

DNA sequencing of gyrA in Isolate 2021D detected a A>G point mutation in codon 94 of gyrA for 90% of alleles, resulting in wild-type aspartic acid being replaced with asparagine (Asp94Asn). A second gyrA mutation was detected in codon 90 for 10% of alleles, a C>T point mutation resulting in wild-type alanine being replaced with valine (Ala90Val). Both Asp94Asn and Ala90Val mutations have been associated with FQ resistance [4, 31]. Sequencing of gyrB for this isolate was wild-type (i.e., no mutations were detected).

Among three methods, 15 results for OFL were reported for Isolate 2021D. This isolate was reported as resistant to OFL by method, as follows:

• 90% (9/10) of the results when using AP
• 100% (4/4) of the results when using MGIT
• 100% (1/1) of the results when using Sensititre

Participating laboratories also reported results for other FQ drugs (e.g., CIP, LVF, and MOX) for Isolate 2021D; 100% (21/21) of results noted resistance to these additional FQ. The isolate was reported resistant to three other FQ by method, as follows:

A mutation in the gyrA gene was detected by all (100%) laboratories that reported molecular testing for FQ drugs; five laboratories specifically noted the Asp94Asn mutation.

Three laboratories performing Sensititre reported MIC values for FQ drugs; two of these did not report interpretations. Reported MIC values were as follows: OFL at 16 µg/ml (n=2); LEV at 8 µg/ml (n=1); and MOX at 4 µg/ml (n=1) and 8 µg/ml (n=1).

Streptoymycin

Among three methods, 48 results for STR were reported for Isolate 2021D. This isolate was reported as resistant to STR by method, as follows:

• 81% (13/16) of the results when using AP
• 100% (29/29) of the results when using MGIT
• 100% (3/3) of the results when using Sensititre

Three of the laboratories performing Sensititre reported STR MIC values as 4.0 µg/ml (n=2) and 8.0 µg/ml (n=1).

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2021D are listed in Tables 27–34.