Prevalence of pyrazinamide resistance and Wayne assay performance analysis in a tuberculosis cohort in Lima, Peru

Abstract

Rationale Multidrug-resistant (MDR) tuberculosis (TB) regimens often contain pyrazinamide, despite the absence of confirmed sensitivity to the drug. This gap is due to limited availability and reliability of pyrazinamide susceptibility testing. Objectives To estimate the prevalence of pyrazinamide resistance by the Wayne assay among TB patients in Lima, Peru; to describe characteristics associated with pyrazinamide resistance; and to compare Wayne performance to BACTEC MGIT 960. Methods In patients diagnosed with culture-positive pulmonary TB from September 2009 to August 2012, we tested pyrazinamide susceptibility using the Wayne assay. We evaluated factors associated with pyrazinamide resistance. In a convenience sample, we compared Wayne assay performance to MGIT 960. Results Pyrazinamide resistance prevalence was 6.6% (95% CI: 5.8%–7.5%) among 3,277 patients and 47.7% (95% CI: 42.7%–52.6%) among the subset of 405 MDR-TB patients. In multivariable analysis, MDR-TB (OR=86.0; 95% CI: 54.0–136.9) and LAM lineage (OR=3.40; 95% CI: 2.33–4.96) were associated with pyrazinamide resistance. The Wayne assay agreed with MGIT 960 in 83.9% of samples (Kappa: 0.66; 95% CI: 0.56–0.76). Conclusion Resistance to pyrazinamide was detected by the Wayne assay in nearly half of MDR-TB patients in Lima. This test can inform selection and composition of regimens, especially those dependent on additional resistance.

MODS-Wayne, a Colorimetric Adaptation of the Microscopic-Observation Drug Susceptibility (MODS) Assay for Detection of Mycobacterium tuberculosis Pyrazinamide Resistance from Sputum Samples

“…Additionally, these isolates reported PZA resistance by MGIT-PZA. This discrepancy may be attributable to misinterpretation during the reading of the classic Wayne assay, as difficulty in the correct interpretation of weak-positive samples has been reported as a limitation of this test (8, 29, 30). On the contrary, the single wild-type strain that was falsely reported as PZA resistant by the MODS-Wayne may have been be due to the lack of PZA addition during the incubation period.…”

“…All sputum samples selected for this study were positive for TB according to the MODS assay. Given that MDR-TB isolates have a 50% chance of also being resistant to PZA (3, 14, 29), in order to select a high number of PZA-resistant sputum samples for evaluation, we selected approximately 50% of specimens that were MDR and monoresistant to isoniazid or rifampin and 50% of non-MDR specimens. Data concerning patient age, sex, and TB treatment were collected.…”

“…The classic Wayne test is a biochemical colorimetric test. It is inexpensive and simple to perform and uses an agar culture medium containing 100 µg/ml PZA at pH 6.4 to 6.8 (28, 29). PZA-susceptible strains release POA near neutral pH, and in the presence of ferrous ammonium sulfate (SAF), this produces a pink complex that can be visualized (reported as positive).…”

“…The absence of a color change indicates the absence of POA, and therefore, PZA resistance (reported as negative) (28). The classic Wayne test has a sensitivity of 75.6 to 95.7% and specificity of 88.7 to 97% (8, 14, 29–31). However, a long turnaround time due to very low mycobacterial growth rates, the requirement of a large inoculum size, and subjective interpretation make this assay impractical for routine clinical use (8, 29, 30).…”

Genotypic Clustering Does Not Imply Recent Tuberculosis Transmission in a High Prevalence Setting: A Genomic Epidemiology Study in Lima, Peru

“…All sputum cultures positive for MTB complex subspecies tuberculosis were subjected to first-line drug susceptibility testing (DST) for isoniazid (critical concentration of 1 µg/mL and 0.2 µg/mL), rifampicin (40 µg/mL), streptomycin (4 µg/mL) and ethambutol (2 µg/mL) using the proportion method. Pyrazinamide susceptibility (100 µg/mL) was measured using the Wayne method 37 . Any resistant strains underwent further DST for second-line agents performed using the indirect proportion method on 7H11 agar as previously described [38][39][40] .…”