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. 2025 Mar 5;12(4):ofaf132.
doi: 10.1093/ofid/ofaf132. eCollection 2025 Apr.

Limitations of the MTB/RIF Assay: An Xpert Review of 4 Clinical Cases

Allison L Haas  1   2 , Angela Ma  1   2 , Jonathan Pham  1   2 , Punam Verma  3 , Uma Malhotra  4   5 , E Chandler Church  5   6   7 , Masahiro Narita  7   8 , Vincent Escuyer  9 , Salika M Shakir  1   2
Affiliations

Limitations of the MTB/RIF Assay: An Xpert Review of 4 Clinical Cases

Allison L Haas et al. Open Forum Infect Dis. .

Abstract

Current U.S. Centers for Disease Control and Prevention tuberculosis (TB) guidelines recommend molecular testing for initial diagnosis of TB and detection of rifampin resistance to expedite initiation of proper treatment. The Cepheid Xpert MTB/RIF assay can detect members of the Mycobacterium tuberculosis complex and rifampin resistance by evaluating for mutations in the rpoB gene. However, false-positive and false-negative detection of M tuberculosis and rifampin resistance results can lead to incorrect treatment of patients, including overuse of second-line anti-TB drugs, and may result in patient harm and increased healthcare cost. We present a series of 4 cases to demonstrate the limitations of the Xpert MTB/RIF assay in the diagnosis of TB, emphasizing the importance of follow-up confirmatory testing and laboratory oversight in reporting accurate results.

Keywords: Mycobacterium tuberculosis; Xpert MTB/RIF; rapid diagnostics; rifampin resistance; rpoB mutation.

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Conflict of interest statement

Potential conflicts of interest. All authors: No reported conflicts of interest

Figures

Figure 1.
Figure 1.
Xpert MTB/RIF PCR curves from (A) case 1, demonstrating false-positive detection of a silent mutation with no amplification in probe B. (b-c) Case 2, demonstrating false prediction of rifampin resistance due to high MTB bacterial load from sputum (B) and from pelvis cyst fluid (C). (D) Case 3, demonstrating false-positive detection of MTB and rifampin resistance due to cross-reaction with an atypical NTM. (E) Case 4, demonstrating a rifampin resistance-associated mutation present outside of the RRDR. Ct values of probes are as follows: a. case 1: A: 31.8, B: 33.6, C: 32.6. D: 33.9, E: 32.9, SPC: 29.7; b. case 2 (sputum): A: 0.0, B: 9.6, C: 9.8, D: 8.7, E: 0.0, SPC: 26.1; c. case 2 (pelvis cyst fluid): A: 0.0, B: 9.0, C: 9.1, D: 8.8, E: 10.0, SPC: 26.2; d. case 3: A: 24.4, B: 0.0, C: 25.3, D: 0.0, E: 0.0, SPC: 21.8; e. case 4: A: 14.2, B: 16.0, C: 14.5, D: 15.6, E: 16.5, SPC: 23.1. MTB/RIF PCR, Mycobacterium tuberculosis complex detection and rifampin resistance by polymerase chain reaction; Ct, cycle threshold; NTM, non-TB mycobacteria; RRDR, rifampin resistance determining region; SPC, specimen processing control.
Figure 2.
Figure 2.
Xpert detection of rifampin resistance in Mycobacterium tuberculosis rpoB. The 5 probes utilized by the Xpert MTB/RIF PCR and their approximate binding location within the rifampin resistance determining region (RRDR) are illustrated (A-E) along with the known rifampin resistance-associated mutations present outside of the RRDR at the codons listed [15]. MTB/RIF PCR, Mycobacterium tuberculosis complex detection and rifampin resistance by polymerase chain reaction.
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References

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