Pyrazinamide susceptibility by BACTEC MGIT 960:

Are the discrepancies always due to the test method?

Introduction

• Pyrazinamide (PZA) is an important first-line drug used in

combination with other tuberculosis (TB) drugs and has a

critical role in shortening TB therapy. Susceptibility

results must be accurate.

• Culture-based drug susceptibility testing (DST) using the

BACTEC MGIT 960 system and a PZA breakpoint of

100μg/ml has been the gold standard for years to test for

PZA resistance.

• This assay has been thought to give poor reproducibility

and false resistance when DST results don’t correlate with

pncA sequencing results. These problems are often

ascribed to inoculum density.

• Our objective is to show that some Mycobacterium tuberculosis (MTB) strain lineage(s) may contribute to the

disparities observed in PZA susceptibility testing.

Methods

Data / Observations Results

• 60% of strains displaying discrepant PZA 100 µg/ml

susceptibility results belonged to the EAI lineage.

• Within these discrepant EAI strains, a majority belonged to the

EAI1 (26%) and EAI2 (36%) clades.

• When tested at a concentration of 75 µg/ml, 42.7% of the initial

124 PZA discrepant strains showed resistance. Among them

92.5% belonged to the EAI lineage.

• Incidence of resistance to PZA 75 µg/ml was high within the EAI

lineage (58%) and among the different EAI clades (50-87.5%)

with the exception of the EAI2 clade (27%).

• Incidence of resistance to PZA 75 µg/ml was low within the other

lineages (0-13%).

Conclusions

• While the BACTEC MGIT 960 PZA susceptibility assay is not

perfect, some of the discrepant results observed during testing

might be attributed to the genetic background of certain strains

exhibiting intrinsic low level resistance to PZA.

• When a TB isolate gives discrepant PZA results upon repeat

testing, genotype information should be obtained before

concluding that this discrepancy is due to the limitations of the

BACTEC MGIT 960 PZA 100 µg/ml susceptibility assay.

• We suggest that the critical concentration of 100 μg/ml currently

in use should be revised to a higher concentration to

compensate for the low level PZA resistance found in certain

strains.

Relevant Literature1. M. Aminian, D. Couvin, A. Shabbeer, et al., Predicting Mycobacterium tuberculosis Complex Clades Using Knowledge-Based Bayesian Networks, Biomed

Research International, vol. 2014, Article ID 398484, 11 pages, 2014. https://doi.org/10.1155/2014/398484.

2. Budzik JM, Jarlsberg LG, Higashi J, et al. Pyrazinamide resistance, Mycobacterium tuberculosis lineage and treatment outcomes in San Francisco, California.

PLoS One. 2014;9(4):e95645. Published 2014 Apr 23. doi:10.1371/journal.pone.0095645.

3. Ekaterina V. Kurbatova, Joseph S. Cavanaugh, Tracy Dalton, Eleanor S. Click, J. Peter Cegielski, Epidemiology of Pyrazinamide-Resistant Tuberculosis in the United States, 1999–2009, Clinical Infectious Diseases, Volume 57, Issue 8, 15 October 2013, Pages 1081–1093, https://doi.org/10.1093/cid/cit452.

4. G. Morlock, F. Tyrrell, D. Baynham, et al., Using Reduced Inoculum Densities of Mycbacterium tuberculosis in MGIT Pyrazinamide Susceptibility Testing to Prevent False-Resistant Results and Improve Accuracy: A Multicenter Evaluation, Tuberculosis Research and Treatment, vol 2017, ArticleID 3748163, 9 pages,

2017. https://doi.org/10.1155/2017/3748163.

5. J. Wemgren, E Sturegard, P. Jureen et al., Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide using Wild-Type MIC Distributions and pncA Gene Sequencing, Antimicrob Agents Chemother. 2012 Mar, 56 (3): 1253-1257.

D. Kohlerschmidt, S. Wolfe, M. Isabelle, A. Fiero, J. Shea, T.A. Halse, K.A. Musser, V.E. Escuyer

• 124 prospective and retrospective strains known to give

discrepant culture-based PZA susceptibility results at

100μg/ml when repeated (RES/SUSC) were tested.

• Culture-based PZA DST was performed in triplicate using

a drug concentration of 75 µg/ml.

• DST inoculum was diluted 1:5 for reduced inoculum

density.

• Spoligotyping was performed by Luminex or whole

genome sequencing (WGS).

• Spoligotyping results were analyzed with the TB-lineage

program http://tbinsight.cs.rpi.edu/run_tb_lineage.html to

determine SITVIT clade by knowledge-based Bayesian

network (KBBN).

Figure 1

Lineage distribution of TB isolates in

New York State by WGS

Table 1

A disproportionate number of strains showing

discrepant MGIT PZA 100µg/ml DST results belong

to the Indo-Oceanic Lineage

Table 2

Percent of isolates from each lineage showing

resistance to PZA at 75µg/ml

Figure 2

Phylogenetic analysis of a subset of

EAI clades by WGS

• Strains in green are susceptible to PZA 75 µg/ml• Strains in red are resistant to PZA 75 µg/ml

Acknowledgements• We thank Linda Gebhardt for performing spoligotyping on some of the strains in this study and the Wadsworth

Center Applied Genomics Technologies Core for performing WGS.

• This work is part funded by award P15-1501, Tuberculosis Elimination and Laboratory Cooperative Agreement, U.S.

Centers for Disease Control and Prevention (CDC) and Cooperative agreement #1U60OE000103 (CFDA NO.

93.322) with CDC.

Major Lineage

Clade within major

lineage

# Tested

# Resistant to PZA

(75µg/mL)

% Resistant to PZA

(75µg/mL)

Indo-Oceanic (Lineage 1)N=84

EAI 7 4 57.1%

EAI1 22 19 86.4%

EAI2 30 8 26.7%

EAI3 8 7 87.5%

EAI4 4 3 75%

EAI6 6 4 66.7%

EAI7 5 4 80%

MANU 2 1 50%

Total 49 58.3%East Asian (Lineage 2) Beijing 8 0 0%

Central Asian (Lineage 3) CAS1 2 0 0%

Euro-American (Lineage 4)

LAM, T, X, S,Haarlem 30 4 13.3%

Lineage for all isolates that had BACTEC MGIT 960 PZA 100 µg/ml testing from 3/1/16 to 9/18/18

Lineage Number of isolates tested Percent

Indo-Oceanic (Lineage 1) 104 17.2%

Beijing (Lineage 2) 104 17.2%

Central Asian (Lineage 3) 51 8.4%

Euro-American (Lineage 4) 345 57.1%

Total # isolates 604

Lineage for all “discrepant” isolates from 3/1/16 to 9/18/18, where resistant PZA 100µg/ml was susceptible upon repeat

Lineage Number of isolates tested Percent

Indo-Oceanic (Lineage 1) 18 60%

Beijing (Lineage 2) 3 10%

Central Asian (Lineage 3) 0 0%

Euro-American (Lineage 4) 9 30%

Total # isolates 30