MPEP Mycobacterium Tuberculosis Drug Susceptibility Testing – Reports

Expected Result: Resistant to OFL at 2.0 µg/ml and CIP at 2.0 µg/ml by agar proportion

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. They are an important class of drugs used to treat tuberculosis (TB) resistant to first-line drugs but also have the potential to become an important part of new TB regimens including as part of treatment regimens for drug-susceptible TB [11, 12]. Prolonged treatment with a FQ (>10 days) before a diagnosis of TB is associated with a higher risk for FQ resistance and diagnostic delays [11, 13]. The primary mechanism of action of FQ is the inhibition of DNA synthesis [14] 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, 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, 14, 15]. 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 [14].

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 [16, 17]. This phenomenon is not limited to FQ but is commonly noted with this drug class.

Studies suggest that the level of resistance, as measured by MIC, to ofloxacin (OFL), ciprofloxacin (CIP), levofloxacin (LVX), and moxifloxacin (MOX) is dependent on the mutation and can vary among the FQs [18, 19]. CLSI and WHO currently recommends testing LVX and/or MOX, however, the preferred FQ to be tested is the one used in the treatment regimen [1, 9].

DNA sequencing of gyrA in Isolate 2021F detected a C>T point mutation in codon 90 of gyrA resulting in wild-type alanine being replaced with valine (Ala90Val). The Ala90Val mutation has been associated with FQ resistance [5, 20]. Sequencing of gyrB locus for this isolate revealed no mutations (i.e., wild-type sequence).

Among three growth-based methods, 12 results for OFL were reported for Isolate 2021F. This isolate was reported as 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% (2/2) of the results when using Sensititre

Participating laboratories also reported results for other FQ drugs (e.g., CIP, LVX, and MOX) for Isolate 2021F; 88% (14/16) of results noted resistance to these additional FQ. The isolate was reported resistant to three other FQ by method, as follows:

CIP

• 100% (2/2) of the results when using AP

LVX

• 100% (1/1) 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
• 75% (3/4) of the results when using MGIT
• 100% (2/2) of the results when using Sensititre

A mutation in the gyrA gene was detected by all (100%) laboratories that reported molecular testing for FQ, with five laboratories noting the Ala90Val mutation.

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

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

Seven laboratories noted no growth for Isolate 2021F 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 STR 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 [5, 21]. 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, 15, 21]. 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 [22, 23]. 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 [24, 25].

DNA sequence analysis of inhA, katG, fabG1, and ahpC of Isolate 2021G detected a C>T point mutation at codon 94 in the katG locus resulting in wild-type aspartic acid being replaced with asparagine (Asp94Asn); inhA, fabG1, and ahpC were wild-type (i.e., no mutations were detected). The katG Asp94Asn mutation’s role in INH resistance is not fully characterized at this time, but it appears to confer low-level resistance. For internal comparison purposes, this isolate was previously sent as MPEP Isolate 2019A and most participating laboratories reported resistance at the low concentration of INH.

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 2021G, 83 INH results were reported. This isolate was reported resistant to INH by method, as follows:

• 88% (14/16) of the results when using AP
• 97% (60/62) of the results when using MGIT
• 33% (1/3) of the results when using Sensititre
• 100% (2/2) of the results when using VersaTREK

Four (8%) results were reported as resistant at the higher concentrations of INH. Only 32 (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 8 molecular results reported for INH, one (13%) laboratory reported detection of a mutation and specifically noted the katG Asp94Asn mutation.

Two of the laboratories performing Sensititre reported INH MIC values as ≤0.12 µg/ml and 0.12 µg/ml.

Ofloxacin and Ciprofloxacin

DNA sequencing of gyrA in Isolate 2021G detected an A>G point mutation in codon 94 of gyrA resulting in wild-type aspartic acid being replaced with glycine (Asp94Gly). The Asp94Gly mutation has been associated with FQ resistance [5, 20]. Sequencing of gyrB locus for this isolate revealed no mutations (i.e., wild-type sequence).

Among three methods, 13 results for OFL were reported for Isolate 2021G. This isolate was reported as resistant to OFL by method, as follows:

• 100% (8/8) of the results when using AP
• 100% (3/3) of the results when using MGIT
• 100% (2/2) of the results when using Sensititre

Participating laboratories also reported results for other FQ (e.g., CIP, LVX, and MOX) for Isolate 2021G; 100% (19/19) of results noted resistance. The isolate was reported resistant to three other FQ by method, as follows:

CIP

• 100% (3/3) of the results when using AP
• 100% (1/1) of the results when using MGIT

LVX

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

MOX

• 100% (4/4) of the results when using AP
• 100% (3/3) of the results when using MGIT
• 100% (2/2) of the results when using Sensititre

A mutation in the gyrA gene was detected by all (100%) laboratories that reported molecular testing for FQ drugs with five laboratories specifically noting the Asp94Gly mutation.

Three of the laboratories performing Sensititre reported MIC values for FQ drugs; one of these did not report interpretations. Reported MIC values were as follows: OFL at 16 µg/ml (n=2); MOX at 4 µg/ml (n=2) and 8 µg/ml (n=1); and LVX at 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 [15, 21]. In MTBC, the genetic basis of the majority of resistance to STR is usually due to mutations in rrs or rpsL [14, 15].

DNA sequencing of rpsL analysis revealed a A>G point mutation resulting in wild-type lysine being replaced by arginine (Lys43Arg). The Lys43Arg mutation has been associated with resistance to STR [26].

Among three methods, 45 results for STR were reported for Isolate 2021G. This isolate was reported as resistant to STR by method, as follows:

• 100% (14/14) of the results when using AP
• 100% (28/28) 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 ≥32 µg/ml (n=2) and 32 µg/ml (n=1).

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

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
DNA sequence analysis of inhA, katG, fabG1, and ahpC of Isolate 2021H detected a G>A point mutation at codon 203 in the fabG1 locus resulting in wild-type leucine being replaced by leucine (Leu203Leu); katG, inhA, 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 [25]. 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.

For Isolate 2021H, 81 INH results were reported. This isolate was reported resistant to INH by method, as follows:

• 100% (15/15) of the results when using AP
• 59% (36/61) of the results when using MGIT
• 0% (0/3) of the results when using Sensititre
• 50% (1/2) of the results when using VersaTREK

No results were reported as resistant at the higher concentrations of INH. Only 27 (44%) 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, 3 (38%) laboratories reported detection of a mutation and specifically noted the Leu203Leu mutation.

Two of the laboratories performing Sensititre reported INH MIC values as 0.12 µg/ml (n=2).

Ofloxacin and Ciprofloxacin

DNA sequencing of gyrA in Isolate 2021H detected a G>A point mutation in codon 94 resulting in wild-type aspartic acid being replaced with asparagine (Asp94Asn). The Asp94Asn mutation has been associated with FQ resistance [5, 20]. Sequencing of gyrB locus for this isolate revealed no mutations (i.e., wild-type sequence).

Among three methods, 13 results for OFL were reported for Isolate 2021H. This isolate was reported as resistant to OFL by method, as follows:

• 100% (8/8) of the results when using AP
• 100% (3/3) of the results when using MGIT
• 100% (2/2) of the results when using Sensititre

Participating laboratories also reported results for other FQ (e.g., CIP, LVX, and MOX) for Isolate 2021H; 95% (19/20) of results noted resistance to these additional FQ. The isolate was reported resistant to three other FQ by method, as follows:

CIP

• 100% (3/3) of the results when using AP
• 100% (1/1) of the results when using MGIT

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
• 100% (4/4) of the results when using MGIT
• 100% (2/2) of the results when using Sensititre

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

Three of the laboratories performing Sensititre reported MIC values for FQ drugs; one of these did not report interpretations. Reported MIC values were as follows: OFL at 16 µg/ml (n=1) and 32 µg/ml (n=1); MOX at 8 µg/ml (n=3); and LVX at 8 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 [27]. Sequencing analysis of ethA did not detect a mutation 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 2021H. This mutation has been associated with ETA resistance [26].

Issues with reproducibility of DST results for ETA have been reported [28] and remain a potential concern.

For Isolate 2021H, 19 ETA results were reported. This isolate was reported resistant to ETA by method, as follows:

• 79% (11/14) of the results when using AP
• 100% (3/3) of the results when using MGIT
• 50% (1/2) of the results when using Sensititre

Two of the laboratories performing Sensititre reported ETA MIC values as 5 µg/ml (n=1) and 10 µg/ml (n=1).

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 [21]. 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 [15]. 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 [15, 21]. 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 [29]. 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 [30]. 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 2021H 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). 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 2021H, 82 results for RMP were reported. This isolate was reported as susceptible to RMP by method, as follows:

• 100% (15/15) of the results when using AP
• 100% (62/62) 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 ten molecular results reported for RMP, five (50%) laboratories reported mutation detected specifically noting the Arg447Arg silent mutation. Five laboratories reported mutation not detected, however this may be due to laboratory reporting practices when a silent mutation not associated with resistance is detected.

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

Expected Result: Resistant to OFL at 2.0 µg/ml and CIP at 2.0 µg/ml by agar proportion

Ofloxacin and Ciprofloxacin

DNA sequencing of gyrA in Isolate 2021I revealed a T>C point mutation in codon 91 of gyrA resulting in wild-type serine being replaced with proline (Ser91Pro). The Ser91Pro mutation has been associated with FQ resistance [5, 20]. Sequencing of gyrB locus for this isolate revealed no mutations (i.e., wild-type sequence).

Among three methods, 12 results for OFL were reported for Isolate 2021I. This isolate was reported as 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% (2/2) of the results when using Sensititre

Participating laboratories also reported results for other FQ drugs (e.g., CIP, LVX, and MOX) for Isolate 2021I; 84% (16/19) of results noted resistance to these additional FQ. The isolate was reported resistant to three other FQ by method, as follows:

CIP

• 100% (2/2) of the results when using AP
• 0% (0/1) of the results when using MGIT

LVX

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

MOX

• 100% (4/4) of the results when using AP
• 75% (3/4) of the results when using MGIT
• 50% (1/2) of the results when using Sensititre

A mutation in the gyrA gene was detected by all (100%) laboratories that reported molecular testing for FQ drugs with five laboratories specifically noting the Ser91Pro mutation.

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

Pyrazinamide

For Isolate 2021I, DNA sequencing of the pncA gene revealed the silent/synonymous mutation (i.e., nucleotide change but no corresponding change in amino acid) Ser65Ser that is not associated with resistance. There may be additional mechanisms of resistance to PZA besides nucleotide changes in the pncA gene that are still unknown [31]. Issues with false-resistance to PZA have been reported [32] and remain a potential concern.

Isolate 2021I was expected to be susceptible to PZA; however, of those testing PZA, resistance was reported by:

• 42% (25/60) of the results when using MGIT
• 0% (0/1) of the results when using VersaTREK

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