MPEP Mycobacterium Tuberculosis Drug Susceptibility Testing – Reports

Expected Results

Note—S=susceptible, R=resistant
^*Growth-based expected results performed by agar proportion, except for PZA which was performed by MGIT. Molecular expected results performed by whole genome sequencing.
^†Resistant at 0.2 µg/ml and 1.0 µg/ml by agar proportion. See Equivalent Critical Concentration table on page 8 for more information.
^§Mutation not associated with resistance. [9]

Isoniazid

DNA sequence analysis of inhA, katG, fabG1, and ahpC of Isolate 2023A revealed a G>C point mutation in the katG locus resulting in wild-type serine being replaced by threonine at codon 315 (Ser315Thr); inhA, fabG1, and ahpC were wild-type (i.e., no mutations were detected). The Ser315Thr mutation confers resistance to INH at both the low and high concentrations [6, 9, 13].

For internal comparison purposes, this isolate was previously sent as MPEP 2020H where comparable results, by method, were reported as resistant for INH.

Figure 6. Isolate 2023A: Percent of laboratories reporting INH-Low and INH-High resistance, by growth-based method.

Note—Two laboratories performing Sensititre® reported INH MIC value as 4.0 µg/ml (n=2).

Ethambutol

DNA sequence analysis of embB of Isolate 2023A revealed a A>G point mutation in the embB gene resulting in wild-type methionine being replaced by valine at codon 306 (Met306Val). Certain embB mutations at the 306 codon, such as Met306Val and Met306Leu, are associated with EMB resistance [6, 9].

For internal comparison purposes, this isolate was previously sent as MPEP 2020H where comparable results, by method, were reported.

Figure 7. Isolate 2023A: Percent of laboratories reporting EMB resistance, by growth-based method.

Note—Two of the laboratories performing Sensititre® reported EMB MIC values as 2.5 µg/ml (n=1) and 8 µg/ml (n=1).

Ethionamide

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 internal comparison purposes, this isolate was previously sent as MPEP 2020H where 64% (9/14) of AP results, 100% (3/3) of MGIT™ results, and 0% (0/1) of Sensititre^® results were reported as resistant.

Figure 8. Isolate 2023A: Percent of laboratories reporting ETA resistance, by growth-based method.

Note—One of the laboratories performing Sensititre® reported an ETA MIC value as 10 µg/ml (n=1).

Streptomycin

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

For internal comparison purposes, this isolate was previously sent as MPEP 2020H where 76% (11/14) of AP results, 48% (16/33) of MGIT™ results, and 100% (1/1) of Sensititre^® results were reported as resistant.

Figure 9. Isolate 2023A: Percent of laboratories reporting STR resistance, by growth-based method.

Note—Two of the laboratories performing Sensititre® reported STR MIC values as 2 µg/ml (n=2).

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2023A are listed below.

Expected Results:

Note—S=susceptible, R=resistant
^*Growth-based expected results performed by agar proportion, except for PZA which was performed by MGIT. Molecular expected results performed by whole genome sequencing.

Rifampin

DNA sequence analysis of rpoB in Isolate 2023B revealed a C>T point mutation in codon 445 resulting in wild-type histidine being replaced by tyrosine (His445Tyr). Isolates with His445Tyr mutations consistently test resistant to RIF in growth-based assays [9, 13-15].

For internal comparison purposes, this isolate was previously sent as MPEP 2019H where comparable results, by method, were reported for RIF.

Figure 10. Isolate 2023B: Percent of laboratories reporting RIF resistance, by growth-based method.

Note—Two of the laboratories performing Sensititre® reported RIF MIC values as 0.25 µg/ml (n=1) and 16 µg/ml (n=1).

Pyrazinamide

For Isolate 2023B, DNA sequencing of the pncA gene did not reveal a mutation. There may be additional mechanisms of resistance to PZA besides nucleotide changes in the pncA gene that are still unknown [16]. Issues with false-resistance to PZA have been reported as well [17] and remain a potential concern.

Isolate 2023B was expected to be susceptible to PZA; however, of those testing PZA, resistance was reported.

For internal comparison purposes, this isolate was previously sent as MPEP 2019H where 60% (39/65) of MGIT™ results and 0% (0/1) of VersaTREK™ results were reported as resistant.

Figure 11. Isolate 2023B: Percent of laboratories reporting PZA resistance, by growth-based method.

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2023B are listed below.

Two laboratories noted contaminated/no growth for Isolate 2023B and did not report results for at least one antituberculosis drug tested.

Expected Results:

Note—S=susceptible, R=resistant
^*Growth-based expected results performed by agar proportion, except for PZA which was performed by MGIT. Molecular expected results performed by whole genome sequencing.

Rifampin

DNA sequence analysis of rpoB in Isolate 2023C revealed a C>T point mutation in codon 450 in wild-type serine being replaced by leucine (Ser450Leu). Isolates with Ser450Leu mutations consistently test resistant to RIF in growth-based assays [9, 13-15].

For internal comparison purposes, this isolate was previously sent as MPEP 2020J where 88% (15/17) of AP results, 98% (58/59) of MGIT™ results, 100% (3/3) of Sensititre^® results, and 100% (2/2) of VersaTREK™ results were reported as resistant.

Figure 12. Isolate 2023C: Percent of laboratories reporting RIF resistance, by growth-based method.

Note—Two of the laboratories performing Sensititre® reported RIF MIC values as 0.5 µg/ml (n=1) and 16 µg/ml (n=1).

Complete first-line DST, second-line DST, and molecular results submitted by all participant for Isolate 2023C are listed below.

Expected Results:

Note—S=susceptible, R=resistant
^*Growth-based expected results performed by agar proportion, except for PZA which was performed by MGIT. Molecular expected results performed by whole genome sequencing.
^⟡Effect of mutation is unknown.

Rifampin

DNA sequence analysis of rpoB in Isolate 2023D revealed a G>T point mutation in codon 170 of rpoB resulting in wild-type valine being replaced by phenylalanine (Val170Phe). Isolates with Val170Phe mutation have been shown to confer resistance [9, 18]. The Val170Phe mutation is outside the rifampin resistance determining region tested by Cepheid^® Xpert^® MTB/RIF assay.

For internal comparison purposes, this isolate was previously sent as MPEP 2020D where 94% (16/17) of AP results, 90% (38/42) of MGIT™ results, 100% (4/4) of Sensititre® results, and 100% (2/2) of VersaTREK™ results were reported as resistant.

Figure 13. Isolate 2023D: Percent of laboratories reporting RIF resistance, by growth-based method.

Note—Two of the laboratories performing Sensititre® reported RIF MIC values as 0.5 µg/ml (n=1) and 16 µg/ml (n=1).

Pyrazinamide

DNA sequence analysis of pncA in Isolate 2023D revealed a A>G point mutation in codon 135 resulting in wild-type threonine being replaced by alanine (Thr135Ala). The effect of the pncA Thr135Ala mutation for this isolate is unknown and 49/50 (98%) of laboratories performing MGIT reported PZA susceptible.

For internal comparison purposes, this isolate was previously sent as MPEP 2020D where comparable results were reported.

Complete first-line DST, second-line DST, and molecular results submitted by all participants for Isolate 2023D are listed below.

Nine laboratories noted contaminated/no growth for Isolate 2023D and did not report results for at least one antituberculosis drug tested.