LETTER
Rifampicin plays a pivotal role in the first-line treatment of tuberculosis. Mutations in the rpoB gene confer varying levels of rifampicin resistance (1). Historically, phenotypic drug susceptibility testing based on bacilli growth in media containing the critical concentration (CC) of rifampicin was considered the gold standard for detecting rifampicin resistance. CC is the lowest concentration of an anti-tuberculosis drug that inhibits 99% phenotypically wild-type MTBc in vitro (2). However, recent studies have underscored its limitations, particularly in Mycobacteria Growth Indicator Tube (MGIT), which frequently misses borderline rpoB mutations associated with impaired bacterial growth (3–5). Consequently, WHO now recommends sequencing the entire rpoB gene as the new reference standard and revised the CC in MGIT from 1.0 to 0.5 µg/mL to boost its ability to detect the borderline rpoB mutations (2, 6). This study assesses whether the revised CC increases MGIT’s ability to detect borderline rpoB mutations.
In all, 37 pre-treatment isolates from participants of the Unitaid-funded endTB and endTB-Q clinical trials in Pakistan (n = 33), and Peru (n = 4) were included in this analysis. These isolates were rifampicin resistant by the MTBDRplus assay but rifampicin susceptible at 1.0 µg/mL in MGIT (2). We performed indirect rpoB sequencing on the isolates and direct rpoB sequencing on the sediments from which the isolates originated. Meanwhile, the isolates were retested in MGIT at the revised CC (0.5 µg/mL) in single replicates.
In all, 36 of the 37 isolates carried rpoB mutations: 32 Group-1, four Group-2, and one Group-5 (7) (Table 1). In total, 28 of the 32 isolates with Group-1 rpoB mutations had a borderline rpoB mutation, alone or together with a Group-1 or Group-2 rpoB mutation. In all, 30 of the 37 sediments had valid direct rpoB sequences: all were concordant with the indirect rpoB sequences. At the revised CC, 2/32 (6.2%) of Group-1 rpoB mutants were classified as resistant, with only 1/28 (3.6%) Group-1-borderline rpoB mutations; this is much less than the WHO’s predicted 21% reduction in misclassifying borderline rpoB mutations by reducing the rifampicin CC in MGIT (6).
TABLE 1.
| Isolate ID | Direct rpoB sequencing using the sputum sediment | Indirect rpoB sequencing using the culture isolate |
WHO grouping of the rpoB mutation (7) | pDST results at the revised CC for rifampicin (0.5 µg/mL) in MGIT |
|---|---|---|---|---|
| PE-1 | Asp435Val | Asp435Val | Group-1 | S |
| PK-1 | His445Arg | His445Arg | Group-1 | S |
| PE-2 | His445Cys | His445Cys | Group-1 | R |
| PK-2 | NA | His445Tyr | Group-1 | S |
| PK-3 | Asp435Tyr | Asp435Tyr | Group-1-borderline | S |
| PK-4 | Asp435Tyrc | Asp435Tyr | Group-1-borderline | S |
| PK-5 | Asp435Tyr | Asp435Tyr | Group-1-borderline | S |
| PK-6 | Asp435Tyr | Asp435Tyr | Group-1-borderline | S |
| PK-7 | Asp435Tyr | Asp435Tyr | Group-1-borderline | S |
| PK-8 | Asp435Tyr | Asp435Tyr | Group-1-borderline | R |
| PK-9 | Asp435Tyr | Asp435Tyrd | Group-1-borderline | S |
| PK-10 | His445Asn | His445Asn | Group-1-borderline | S |
| PK-11 | His445Asn | His445Asn | Group-1-borderline | S |
| PK-12 | His445Asnc | His445Asn | Group-1-borderline | S |
| PK-13 | His445Asn | His445Asn | Group-1-borderline | S |
| PK-14 | His445Asnc | His445Asn | Group-1-borderline | S |
| PK-15 | His445Asn | His445Asn | Group-1-borderline | S |
| PE-3 | His445Asn | His445Asn | Group-1-borderline | S |
| PK-16 | Leu430Pro | Leu430Pro | Group-1-borderline | S |
| PK-17 | Leu430Pro | Leu430Pro | Group-1-borderline | S |
| PK-18 | Leu430Proc | Leu430Pro | Group-1-borderline | S |
| PK-19 | Leu430Pro | Leu430Pro | Group-1-borderline | S |
| PK-20 | Leu430Pro | Leu430Pro | Group-1-borderline | S |
| PK-21 | Leu430Pro | Leu430Pro | Group-1-borderline | S |
| PK-22 | Leu452Pro | Leu452Pro | Group-1-borderline | S |
| PK-23 | NA | Leu452Pro | Group-1-borderline | S |
| PK-24 | NA | His445Leu | Group-1-borderline | S |
| PK-25 | NA | His445Leud | Group-1-borderline | S |
| PK-26 | His445Leu | His445Leud | Group-1-borderline | S |
| PK-27 | Asp435Tyr; Met434Ile | Asp435Tyr; Met434Ile | Group-1-borderline + Group-2 | S |
| PK-28 | NA | Asp435Tyr; Met434Ile | Group-1-borderline + Group-2 | S |
| PK-29 | Leu430Pro;Met434Ile | Leu430Pro;Met434Ile | Group-1-borderline + Group-2 | S |
| PK-30 | NA | Asp435Gly | Group-2 | S |
| PK-31 | NA | Asp435Gly | Group-2 | S |
| PE-4 | His445Gln | His445Gln | Group-2 | S |
| PK-32 | Lys446Arg | Lys446Arg | Group-2 | S |
| PK-33 | Thr444Thr | Thr444Thr | Group-5 | S |
R= resistant, CC= critical concentration, pDST = phenotypic drug susceptibility testing, NA = interpretable direct rpoB sequencing results not available.
Group 1: associated with resistance; Group 2: associated with resistance—interim; Group 3: uncertain significance in resistance; Group 4: not associated with resistance—interim; Group 5: not associated with resistance; This analysis did not include His445Ser, the sixth Group-1-borderline rpoB mutation (7).
rpoB sequences by Sanger sequencing (Direct rpoB sequencing using the sediments was done using the GenoScreen Deeplex Myc-TB assay or targeted Sanger sequencing).
rpoB sequences by WGS (Indirect rpoB sequencing using the isolates was done using targeted Sanger sequencing or WGS).
In the endTB/endTBQ clinical trials, patients with rifampicin resistance detected by genotypic testing received second-line anti-tuberculosis treatment; therefore, we were unable to assess the clinical response of different borderline rpoB mutations to rifampicin-based treatment. However, it has been reported that borderline rpoB mutations may have the same clinical significance and transmission potential as high-confidence rpoB mutations; thus, misdiagnosing them can lead to treatment failure and further transmission of rifampicin-resistant tuberculosis (8, 9). Our data suggest that despite revising the CC in MGIT, genotypic tests are better at detecting these mutations. However, we tested only a limited number of isolates from just two distinct geographical locations and did not include MIC or replicate testing. A larger study including MIC and replicate testing would provide a more comprehensive assessment of the MIC distribution of the borderline rpoB mutations and whether further reduction of the CC is required.
The endTB and endTBQ trials were approved by institutional review/ethics boards of Harvard Medical School, Interactive Research and Development, Institute of Tropical Medicine, Médecins Sans Frontières, and each participating site.
Contributor Information
Praharshinie Rupasinghe, Email: prupasinghe@itg.be.
Sean Wasserman, St. George's, University of London, London, United Kingdom.
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