Mycobacterial Drug Susceptibility Unit Procedure Change Notification. Additional Modifications to PZA QC, Testing, Reflexing, and Reporting

Procedure: PZA Testing Algorithm, reporting.

Date effective: 04/12/2024

Pyrazinamide (PZA) is a critical drug in antituberculosis treatment. However, phenotypic testing is plagued by issues with repeatability [1, 2]. PZA must be tested at an acidic pH, and an inoculum that is too high may result in false resistance. Reagent variability is also a major issue, and issues with drug and media lot combinations failing QC have intermittently delayed testing since at least October, 2023.

Due to these issues and to ensure continuity of testing given the critical nature of the drug, we previously made changes to our QC and testing procedures as well as to our reporting language and reflexing protocol. However, the issues remain, and since we now have a reliable WGS assay available, the workflow is being modified to better utilize resources while ensuring accurate results.

Refer to the attached FAQ for a discussion of the issues necessitating the change and justification of the new workflow.

Please note the following procedural changes:

Samples that have been submitted prior to submitter notification will under parallel WGS-DST and pDST for PZA and reported as indicated in the FAQ and in the figures below.
Once submitters are officially notified and an effective date is determined, we will perform phenotypic DST (pDST) for PZA only as a reflex test after WGS-DST is completed. It will be performed for all samples with “Uncertain” mutations since susceptible results are considered trustworthy. For “Resistant” mutations, pDST for PZA will be performed on a case-by-case basis, serving as a check for sample mix-ups. However, the results will not be reported.
In addition, since false resistance is very common with the PZA assay, samples that have 50≤GU<100 will not undergo repeat testing. This is considered a susceptible result.

The reportable comments for indeterminate results for CA submitters are below:

WGS=S, pDST=R (applicable only to interim protocol for the isolates received prior to new workflow effective date):

“Indeterminate” Result Interpretation:

This isolate has tested PZA-resistant using the BACTEC MGIT platform; however, NO pncA mutations known to be associated with resistance were detected by the WGS assay as reported on XX/XX/XXXX. These discordant results may be caused by phenotypic DST assay variability combined with strain characteristics such as strain lineage or unknown factors which could lead to phenotypic resistance. For California cases, please consult with the California MDR-TB Service (510-620-3000) for additional treatment guidance. For cases outside California, consult your state TB program or the TB Centers of Excellence (TB COE) phone number 877-390-6682.

WGS=U, pDST=R (under new reflex workflow for isolates received after the announcement):

“Indeterminate” Result Interpretation:

This isolate has tested PZA-resistant using the BACTEC MGIT platform; however, the pncA mutation(s) detected by the WGS assay and reported on XX/XX/XXXX have uncertain significance. The effect of this pncA mutation is unknown and PZA resistance cannot be confirmed by phenotypic DST due to assay variability combined with strain characteristics such as strain lineage or unknown factors which could lead to phenotypic resistance. For California cases, please consult with the California MDR-TB Service (510-620-3000) for additional treatment guidance. For cases outside California, consult your state TB program or TB Centers of Excellence (TB COE) phone number 877-390-6682.

The reportable comment for Indeterminate results for out-of-state submitters is below:

This isolate has tested PZA-resistant using the BACTEC MGIT platform; however, NO pncA mutations known to be associated with resistance were detected by the WGS assay as reported on XX/XX/XXXX. These discordant results may be caused by inherent assay variability combined with strain characteristics such as strain lineage or unknown factors which could lead to phenotypic resistance. For California cases, please consult with the California MDR-TB Service (510-620-3000) for additional treatment guidance. For cases outside California, consult your state TB program or TB Centers of Excellence (TB COE) phone number 877-390-6682.

Other procedure changes are delineated below:

Quality Control of PZA

	Current	New
Initial Lot QC	MIC only tested at 100µg/ml as instructed by the manufacturer (BD)[3].	MIC testing is performed at 12.5, 25, 50, and 100µg/ml on H37Rv (ATCC 27294). Passing is considered 100µg/ml as instructed by the manufacturer (BD)[3]. However, we have noted a higher rate of false resistance with drug and media lot combinations with MIC > 25-50µg/ml.

Testing

	Current	New
Repeats	Repeat 1x	No repeat testing (provided there is no contamination and QC passes)
Reporting	Report consistent with both pDST repeats. If mono-resistant, add reportable comment (see below). If the two tests differ, report as “Indeterminate” with reportable comment (see below).	Report based on pDST 1x taking into account the WGS-DST results as indicated in the FAQ and flowcharts for final interpretation.

FAQs:

What is causing the current issues with the phenotypic PZA assay?

MDL and other laboratories around the country have continued to experience significant intermittent issues with lots of PZA DST reagents used in the BD BACTEC MGIT testing system since late 2023. These issues have compounded longstanding issues with repeatability and false resistance due to the requirement for maintaining an acidic pH and the strong dependence on inoculum size [1]. Reagent quality issues have exacerbated these intrinsic problems plaguing the assay, resulting in delayed PZA reporting due to intermittent QC failures, the need for extensive repeat testing, and an increased number of non-M. bovis PZA-monoresistant samples, suggestive of false resistance.

What evidence supports use of pncA gene sequencing results in lieu of phenotypic susceptibility testing for PZA?

Utilization of WGS for molecular DST for PZA will reduce false-resistant results that may be encountered with the phenotypic assay due to inherent assay variability or commercial reagent issues.
The positive predictive value for pncA sequencing is high [1,2]; i.e. detected resistance-conferring mutations highly correlate with resistant phenotype.
The negative predictive value for pncA sequencing is high for non-MDR TB samples [1]; i.e. absence of resistance-conferring mutations in pncA correlates well with phenotypic susceptibility. Even though we cannot exclude the possibility of a mutation conferring PZA resistance outside of pncA, it is not common. Considering the low pretest probability for PZA resistance in non-MDR samples, the risk of PZA false-susceptibility in the WGS assay is much smaller than the risk associated with an increase in PZA false-resistance that is currently observed with phenotypic DST. For multidrug resistant (MDR) TB isolates, PZA resistance occurs more often; however, PZA is not part of BPaL / BPaLM regimens currently used for most patients with MDR TB disease.

What is the sensitivity and specificity for prediction of resistance based on the pncA sequence?

The estimates for sensitivity and specificity for PZA resistance prediction based on the presence of resistant mutations in the pncA gene vary among studies largely based on the tested population, abundance of MDR, and lineage 1 strains in the dataset, which have an elevated MIC close to the critical concentration [6]. Below are some numbers from different sources:

	All isolates	All isolates	MDR isolates only	MDR isolates only
Source	Sensitivity	Specificity	Sensitivity	Specificity
APHL[1] (= WHO 2021)	72.3%	98.8%	N/A	N/A
CDC [3]	51.9%	99.7%	95.5%	94.2%
WHO 20234	78.0%	97.9%	N/A	N/A
MDL WGS validation study (n=189)	61.9%	100%	N/A	N/A
Chang et al, 2011 [5]	85%	88%	N/A	N/A

It is important to note that the low sensitivity in the comparisons between sequencing-based resistance prediction and phenotypic DST for PZA are an artifact of the low reproducibility and propensity for false-resistance of the phenotypic DST assay, thereby underestimating the true sensitivity.

Notably, in a meta-analysis performed by Chang et al [5], the negative predictive value (NPV) of pncA sequencing for non-MDR M. tuberculosis was >99% which strongly supports use of pncA sequencing for ruling out PZA resistance.

The NPV for pncA sequencing for MDR-TB can be estimated based on the sensitivity and specificity values for MDR isolates provided by the CDC [3] and prevalence values of MDR-TB for PZA resistance from two meta-analyses, as follows:

Meta-analysis	Prevalence	NPV
Chang et al, 20115	51%	95%
Whitfield et al, 20157	61%	93%

Considering that the NPV of pncA in MDR isolates has been estimated at >90%, the absence of a resistance-conferring mutation in pncA is a good predictor of PZA susceptibility in MDR strains. Additionally, PZA is normally not used for treatment in MDR cases, further reducing the utility of phenotypic PZA testing in these situations.

What does the modified reporting workflow look like for WGS and pDST?

For isolates submitted for first-line pDST effective 04/12/2024, PZA pDST will not be set up by default and WGS will be performed as the primary test. WGS results will be reported as soon as available. Other first-line drugs will be set up for pDST as usual, if ordered.
If the WGS result for PZA is “No mutations associated with resistance to pyrazinamide detected", pDST confirmation will NOT be performed due to the higher likelihood of false-resistant results.
If the WGS result for PZA is “Mutation(s) associated with resistance to pyrazinamide detected", pDST confirmation will be performed on a case by case basis or as directed to collect additional data phenotypic data. In the case of discrepant results, WGS and pDST results will be investigated.
If the WGS result for PZA is “The detected mutation(s) have uncertain significance. Resistance to pyrazinamide cannot be ruled out", pDST will be performed by reflex. If an isolate with an “Uncertain” mutation tests resistant by pDST, the final interpretation for the PZA pDST result will be reported as “Indeterminate”. Since we are confident in pDST susceptible results, susceptible results be reported.

With the new workflow, PZA DST for samples that test resistant by the phenotypic PZA assay but do not have corresponding resistance-conferring mutations in pncA will be reported as “Indeterminate". What does this mean and what are my next steps if I receive this result?

For an uncertain WGS pncA result followed by a resistant phenotypic DST result after reflex testing:

“This isolate has tested PZA-resistant using the BACTEC MGIT platform; however, the pncA mutation(s) detected by the WGS assay and reported on XX/XX/XXXX have uncertain significance. The effect of this pncA mutation is unknown and PZA resistance cannot be confirmed by pDST due to inherent assay variability combined with strain characteristics such as strain lineage or unknown factors which could lead to phenotypic resistance”.

For a phenotypic PZA resistant result and no pncA mutation detected by WGS (applicable only for samples that were received prior to the new workflow; See FAQ #6 below):

“This isolate has tested PZA-resistant using the BACTEC MGIT platform; however, NO pncA mutations known to be associated with resistance were detected by the WGS assay as reported on XX/XX/XXXX. These discordant results may be caused by inherent assay variability combined with strain characteristics such as strain lineage or unknown factors which could lead to phenotypic resistance”.

As the next step, for California cases, please consult with the California MDR-TB Service (510-620-3000) for additional treatment guidance. For cases outside California, consult your state TB program or TB Centers of Excellence (TB COE) phone number 877-390-6682.

Will phenotypic DST for PZA still be performed for isolates submitted prior to this announcement?

Yes, however, susceptible/resistant phenotypic PZA results will be reported only in the following two scenarios:

When phenotypic PZA results are “susceptible”
When phenotypic PZA results are “resistant” and high-confidence resistance mutations are detected in pncA by WGS.

If the WGS-based PZA result either states “No mutations associated with resistance to pyrazinamide detected” or “The detected mutation(s) have uncertain significance", and phenotypic PZA result is “Resistant", the final interpretation will be reported as “Indeterminate” due to higher likelihood of phenotypic false-resistance.

References:

Issues in Mycobacterium tuberculosis Complex Drug Susceptibility Testing: Pyrazinamide (PDF)
Rodwell, T., P. Miotto, C. Köser, T. Walker, P. W. Fowler, J. Knaggs, Z. Iqbal et al. “Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance.” (2021).
CDC personal communications.
Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance, Second edition, WHO, 2023.
Chang KC, Yew WW, Zhang Y. Pyrazinamide susceptibility testing in Mycobacterium tuberculosis: a systematic review with meta-analyses. Antimicrob Agents Chemother. 2011 Oct;55(10):4499-505. doi: 10.1128/AAC.00630-11. Epub 2011 Jul 18. PMID: 21768515; PMCID: PMC3186960.
Mok S, Roycroft E, Flanagan PR, Montgomery L, Borroni E, Rogers TR, Fitzgibbon MM, 2021. Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis Isolates. Antimicrob Agents Chemother 65:10.1128/aac.02617-20.
Whitfield, Michael G., Heidi M. Soeters, Robin M. Warren, Talita York, Samantha L. Sampson, Elizabeth M. Streicher, Paul D. Van Helden, and Annelies Van Rie. “A global perspective on pyrazinamide resistance: systematic review and meta-analysis.” PloS one 10, no. 7 (2015): e0133869.