A simplified pyrazinamidase test for Mycobacterium tuberculosis pyrazinamide antimicrobial susceptibility testing

ABSTRACT

Pyrazinamide (PZA) is an important first-line drug for tuberculosis (TB) treatment by eradicating the persisting Mycobacterium tuberculosis complex (MTBC). Due to cost and technical challenges, end TB strategies are hampered by the lack of a simple and reliable culture-based PZA antimicrobial susceptibility testing (AST) for routine use. We initially developed a simplified chromogenic pyrazinamidase (PZase) test in the TB reference laboratory using a training set MTBC isolates with various drug-resistant profiles, and validated its performance using consecutive BACTEC MGIT 960 (MGIT)-culture-positive culture in 10 clinical laboratories. The pncA gene Sanger sequencing results were used as the reference, and compared to the MGIT-PZA AST. Differential diagnosis of Mycobacterium bovis was conducted using patented in-house real-time PCR. Of the 106 training isolates, the PZase test and MGIT-PZA AST showed 100.0% and 99.1% concordance as compared to Sanger sequencing, respectively. We found 32.1% (34/106) isolates harbored pncA mutations, including one isolate with silent mutation S65S. For validation, 1,793 clinical isolates were tested including 150 duplicate isolates from specimens of the same cases and 16 isolates with uncharacterized drug resistance (UDR)-associated mutations. Excluding duplicated and UDR isolates, we identified 2.6% (43/1,627) PZA-resistant isolates, including 1.3% (21/1,627) M. bovis isolates. The kappa values were 0.851–1.000. In addition, the accuracy of the PZase test conducted by 10 laboratories was 98.5%–100.0%. Our simplified PZase test demonstrated high concordance with Sanger sequencing and MGIT-PZA AST. Integrating the PZase test into routine first-line AST is effortless and represents an improvement in laboratory services for ending TB.

IMPORTANCE

We developed and validated a simple pyrazinamidase (PZase) test for pyrazinamide (PZA) antimicrobial susceptibility testing (AST). Our results demonstrated that the PZase test had high agreement with the pncA gene sequencing and MGIT-PZA AST. Integrating PZase test into routine AST is effortless and represents an improvement in laboratory services for ending TB.

INTRODUCTION

Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), remains the world’s second leading cause of death from a single infectious agent. Pyrazinamide (PZA) has been included in TB treatment regimens since the 1960s, reducing the treatment duration to 6 months, owing to its unique mechanism of action, which is effective against semi-dormant bacilli (1). PZA is a prodrug hydrolyzed to weakly acidic pyrazinoic acid (POA) active form by pyrazinamidase/nicotinamidase (PZase), the enzyme encoded by the pncA gene. The intracellular bactericidal activity of PZA increases as bacterial metabolic activity decreases (2). Therefore, PZA is an important component of the standard first-line regimen for TB (3), as well as for the treatments for multidrug-resistant (MDR) and extensively drug-resistant TB, according to the WHO consolidated guidelines (4). Understanding PZA susceptibility can guide the selection and design of effective regimens.

A wide variation in resistance-associated pncA mutation rates among general TB cases was revealed across different countries and settings, ranging from 42.1% in Belarus, 12.6% in Azerbaijan, 5.1% in Bangladesh, 3.1%–3.9% in South Africa, to 3.0% in Pakistan (5). The genetic surveillance of PZA resistance among general TB cases in Taiwan was 4.4% (6). It is suggested that routine PZA antimicrobial susceptibility testing (AST) is important in the end TB program.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is one of the concerning zoonosis worldwide. PZA resistance is commonly regarded as a marker of M. bovis, while resistance to isoniazid (INH) and/or rifampin (RIF) has also been documented (7). This could deplete resources for treatment and hamper comprehensive TB control efforts (6). Deploying a simplified PZA AST for clinical use can not only enhance the screening and identification of M. bovis but also enhance the One Health approach for zoonotic bTB surveillance.

The BACTEC MGIT 960 PZA (MGIT-PZA) assay is the only WHO-recommended phenotypic antimicrobial susceptibility testing (pAST) (8). Although an acidic pH is required for the activity of PZA and is utilized in the MGIT-PZA assay, such an acidic medium may inhibit bacterial growth. Additionally, false resistance in PZA pAST may occur due to large bacterial inoculum sizes (9, 10). In terms of genotypic AST (gAST), while it is possible to assess the entire pncA gene using targeted genome sequencing or whole genome sequencing (WGS), identifying resistance-conferring mutations remains challenging due to the absence of a hotspot region for pncA gene mutations (11). Therefore, pAST remains necessary as an initial step to confirm whether rare mutation sites lead to PZA resistance.

The classic Wayne test is a biochemical colorimetric test, with PZA incorporated into agar culture medium at pH 6.4–6.8, where the presence of a red color ring on the surface of the medium indicates sensitivity, while no discoloration indicates resistance (12). The subjective interpretation of color change makes it impractical for routine clinical use. Although the quantitative Wayne test using a spectrophotometer provides high sensitivity and specificity, it requires a large inoculum size (4 McFarland) (13). However, increasing the bacterial density does not alter the drug susceptibility profile of MTBC, and preparing such a high-density inoculum poses a risk of infection (14). An indirect competitive enzyme-linked immunosorbent assay (ELISA) quantifies POA by fixing a construct made of bovine serum albumin linked to pure POA at the bottom of wells. This test was incompatible with the microscopic-observation drug susceptibility (MODS) culture medium because 7H9-oleic acid-albumin-dextrose-catalase (OADC)-polymixin-B-amphotericin-B-nalidixic-acid-trimethoprim-azlocillin in the medium poses a risk of interference in the competitive system, leading to improper ELISA curves (15). A colorimetric adaptation of the MODS test (MODS-Wayne) characterized drug resistance directly from sputum in a liquid culture medium. Growth was observed on different days using an inverted microscope. Although it is a cost-effective alternative, the sensitivity and specificity vary depending on the contamination rate (16, 17).

Since conventional PZA AST faces challenges in clinical TB laboratories due to operational techniques, inoculum size, instrumentation, and difficulties in assessing its activity in vitro (16), we aimed to develop a simplified PZase test for clinical PZA AST and for bTB screening.

MATERIALS AND METHODS

Study design

This was a retrospective and prospective study conducted in the TB reference laboratory (TRL) for the development and training of the PZase test, and evaluated in 10 Taiwan CDC-contract clinical laboratories for validation of the test. The development and training of the PZase test was conducted in January to August 2022, and the validation was carried out from August 2022 to January 2024.

Study materials

The training set was 111 clinical MTBC isolates, including 42 isolates from MDR/rifampin-resistant (RR) TB cases, 34 isolates from non-MDR/RR-TB, 8 M. bovis, 8 M. bovis BCG, and 19 isolates from the 2022 WHO proficiency testing panel. We also tested four reference isolates, including one PZA-susceptible M. tuberculosis subsp. tuberculosis (ATCC 27294) and three PZA-resistant isolates: M. bovis Karlson and Lessel (ATCC 35720), M. bovis Karlson and Lessel (ATCC 35738), and MDR Vertullo (National Jewish Health, Denver, Colorado, USA) strain. The validation set was 1,793 consecutive MGIT culture-positive MTBC isolates from 10 clinical TB laboratories (Fig. 1).

Fig 1.

Distribution and numbers of Mycobacterium tuberculosis complex isolates in the validation set. Numbers and percentages of M. bovis-infected cases were outlined with dashed circle. The base map is from https://whgis-nlsc.moi.gov.tw/Opendata/Files.aspx.

Phenotypic antimicrobial susceptibility testing

First-line AST

MTBC isolates were subjected to AST using the agar proportion method with 7H10 medium (Becton, Dickinson and Company, Sparks, MD, USA). Drug resistance was defined as the growth of 1% of colonies in a drug-containing medium. According to WHO recommendations, the critical concentrations were as follows: RIF, 1 µg/mL; INH, 0.2 µg/mL; ethambutol (EMB), 5 µg/mL; streptomycin (SM), 2 µg/mL in the 7H10 medium (8). The tests were validated by determining the susceptibility of M. tuberculosis H37Rv. MDR-TB is defined as an MTBC isolate that is resistant to at least INH and RIF.

A modified PZase test

The inoculum can be either prepared from fresh subculture or the MGIT culture. The isolate was quantitatively adjusted 0.5 to 1 McFarland using 7H9 culture medium and continued culturing in a 37°C incubator for at least 1 day. Two milliliters of bacterial solution was pipetted into a 15 mL centrifuge tube, and 0.1 mL of PZA drug (8 mg/mL) (Sigma-Aldrich, USA) was added to the final concentration of 0.4 mg/mL. The solution was cultured in a 37°C incubator for another 4 days, and was centrifuged at 3,000 × g for 2 min or settled overnight. The supernatant was transferred to a glass tube (13 × 100 mm) and 0.2 mL of 1% ferrous ammonium sulfate (Sigma-Aldrich, USA) was added. The color change was evaluated by the naked eye and using a spectrophotometer [optical density at 490 nm (OD₄₉₀)].

MGIT-PZA antimicrobial susceptibility testing

PZA pAST was performed using BACTEC MGIT 960 PZA Kit with PZA 100 µg/mL according to the manufacturer’s instructions (8). Briefly, add 0.8 mL PZA supplement to each PZA-MGIT tube. Add 100 µL of 0.8 mg/mL PZA to the drug-containing tube. Adjust the bacterial solution of the test strain to 0.5 McFarland. Dilute the bacterial solution 1:5 with 0.85% sterile saline and add 0.5 mL to the drug-containing tube. Then dilute the bacterial solution 1:10 with 0.85% sterile saline and add 0.5 mL to the growth control tube. Culture for 4–20 days, and fluorescent analysis of the growth control values is used by machine result interpretation to obtain a complete drug susceptibility report.

Genotypic antimicrobial susceptibility testing

PZA susceptibility testing

PZA gAST was performed by sequencing the drug resistance-associated genes, pncA, using Sanger sequencing as described previously (6). Briefly, the pncA gene and its promoter were amplified using a pair of specific primers (pncA-F, 5′-GCTGGTCATGTTCGCGATCG-3′, pncA-R, 5′-CGCTTGCGGCGAGCGCTCCA-3′) to detect mutations associated with PZA resistance as described previously. PCR conditions were as follows: 95°C denaturation for 10 min, 35 cycles of 95°C denaturation for 1 min, 65°C annealing for 1 min, extension at 72°C for 1 min, and a final extension cycle at 72°C for 6 min. PCR products were verified using a high-performance DNA analyzer (QIAxcel Advanced System, Qiagen, Hilden, North Rhine-Westphalia, Germany) and sent for sequencing (Genomics, New Taipei City, Taiwan). Sequences were analyzed using Sequencher 4.7 (Gene Codes Corporation, Ann Arbor, MI, USA) by comparison with the M. tuberculosis H37Rv sequence (NC_000962.3). For drug resistance genetic assessment, the WHO catalog was used as a reference standard for the interpretation of mutations conferring resistance to PZA (18). The uncharacterized mutations were classified according to the WHO catalog, SUSPECT-PZA webtool (https://biosig.lab.uq.edu.au/suspect_pza/) (19), and CRyPTIC tuberculosis data set (https://github.com/oxfordmmm/tuberculosis_amr_catalogues) (20).

M. bovis identification

Differential diagnosis of M. bovis was conducted using patented single-tube triplex real-time PCR with primers targeting IS6110, RD4, and a BCG-specific sequence for the identification of MTBC, the M. bovis and M. bovis BCG, respectively. Cycling conditions were one cycle at 50°C for 2 min and one cycle at 95°C for 10 min followed by a two-step PCR (45 cycles of 95°C for 15  s and 60°C for 1  min). Signals were separated by labeling with different colors of fluorescence (7).

Statistical analysis

Data entry and analysis were done using Microsoft Excel. The sensitivity and specificity of the training set of isolates and 10 clinical laboratories for validation were estimated using a 2-by-2 contingency table, and 95% confidence intervals (CIs) were calculated to estimate the correlation between the variables (http://vassarstats.net/).

RESULTS

Development and training of the PZase test

In the analysis, we excluded five isolates with uncharacterized drug resistance-associated mutations identified using the Sanger sequencing. Of the 106 isolates, 31.1% (33/106) harbored mutations, 67.9% (72/106) were wild-type isolates, and one isolate had a pncA S65S mutation. The PZase test and MGIT-PZA showed concordance with Sanger sequencing were 100.0% and 99.1%, respectively. Besides, the color change to orange-red was easily identified by the naked eye (Fig. 2). We observed MGIT-PZA false-susceptible result in the pncA Q141* mutation. In addition, to determine the optimal cut-off of OD₄₉₀ for PZA resistance, the Sanger sequencing was used as the reference. The concordance rates were 98.1% and 97.2% for 0.1 and 0.03, respectively (Table S1). However, we found two susceptible isolates showing color changes despite OD₄₉₀ values <0.1, specifically 0.033 and 0.047 (Fig. 3). The sensitivity and specificity of OD₄₉₀ values <0.1 were 100.0% and 97.3%, respectively, compared to the Sanger sequencing.

Fig 2.

Resistant and susceptible strain results of modified pyrazinamidase tests. (1) Resistance, 0.009 (OD₄₉₀). (2) Susceptible, 0.124 (OD₄₉₀). (3) Susceptible (strong positive), 0.311 (OD₄₉₀).

Fig 3.

Optical density values (OD₄₉₀) of the tested M. tuberculosis isolates, categorized based on the results of the pyrazinamidase test.

Validation of the PZase test

Of the 1,793 consecutive MGIT culture-positive isolates tested, 1,643 were clinical isolates and 150 were repeated isolates from the same cases. The PZase results were 100.0% reproducible among repeated isolates. Of the 1,643 isolates, 1.8% (29/1643) and 0.7% (11/1643) were MDR and RR isolates, respectively. Excluding 16 isolates with uncharacterized drug-resistant-associated mutations, 2.6% (43/1627) isolates were PZA-resistant. Of the 43 PZA-resistant isolates, 48.8% (21/43) were M. bovis with typical pncA H57D mutation, followed by 37.2% (16/43) M. tuberculosis and 14.0% (6/43) M. bovis-BCG. In the study, we timely identified 1.3% (21/1627) bTB cases, of which 47.6% (10/21) isolates were concurrently resistant to INH.

Table 1 showed the sensitivity, specificity, concordance, and agreement kappa of the 10 clinical laboratories (Lab-A to Lab-J). Using the Sanger sequencing as a reference, the concordance of three laboratories were 100.0%, and that of seven laboratories were 94.0% to 99.3% (95% CI, 0.323 to 0.885) (Table S2). Lab-A identified nine false-resistant isolates; Lab-B identified three false-resistant isolates; Lab-C identified three false-resistant isolates and one false-susceptible isolate; Lab-F identified five false-resistant isolates; and Lab-D, H, and J each identified one false-resistant isolate. The isolates with discordant results were retested in seven laboratories: the concordance of Lab-A, B, C, D, and H were 97.9%, 99.0%, 97.7%, 99.3%, and 98.8%, respectively, all of which increased to 100.0%; the concordance of Lab-F was 94.0% increased to 98.8%; the concordance of Lab-J remained unchanged. Two discordant PZA-resistant clinical isolates, after being retested in clinical laboratories, were identified as PZA-susceptible by the TRL. Compared to Sanger sequencing, the sensitivity and specificity of 10 clinical TB laboratories were 100.0% and 98.5%–100.0%, respectively (Table 1). Even though all the medical technologists in the Taiwan Centers for Disease Control (TCDC)-authorized TB laboratories were authorized to perform routine first-line AST, they might be not familiar with the PZase test procedures and were reluctant to interpret borderline AST results in the early stage of the validation study.

TABLE 1.

Validation of the pyrazinamidase test in 10 TB clinical laboratories, after repeat testing^a

Clinical laboratories	pncA gAST (no. of isolates)					Total (N = 1,643)	Performance (excluding uncharacterized mutations)
	TP (43)	FP (2)	FN (0)	TN (1,582)	U (16)		Sensitivity %	Specificity %	Concordance %	Agreement kappa (95% CI)
A	21	0	0	404	2	427	100.0	100.0	100.0	1.000 (1.000–1.000)
B	6	0	0	289	4	299	100.0	100.0	100.0	1.000 (1.000–1.000)
C	2	0	0	171	1	174	100.0	100.0	100.0	1.000 (1.000–1.000)
D	4	0	0	132	1	137	100.0	100.0	100.0	1.000 (1.000–1.000)
E	0	0	0	137	1	138	NA	100.0	100.0	NA
F	3	1	0	80	0	84	100.0	98.8	98.8	0.851 (0.564–1.000)
G	1	0	0	98	1	100	100.0	100.0	100.0	1.000 (1.000–1.000)
H	1	0	0	82	2	85	100.0	100.0	100.0	1.000 (1.000–1.000)
I	5	0	0	125	1	131	100.0	100.0	100.0	1.000 (1.000–1.000)
J	0	1	0	64	3	68	NA	98.5	98.5	NA

^a gAST, genotypic antimicrobial susceptibility testing; TP, true positives; FP, false positives; FN, false negatives; TN, true negatives; U, uncharacterized mutations, detection of at least one novel nonsynonymous mutation; NA, not available; CI, confidence interval.

Mutations of PZA-resistant isolates

In this study, the most prevalent pncA mutation was H57D (N = 43), with all isolates further identified as M. bovis, followed by T76P (N = 5), –11a > g (N = 3), D40fs (N = 3), T142K (N = 2), V155G (N = 2), L172P (N = 2), and one isolate for each of the other 16 pncA mutations (Table 2). A discordant AST result was detected in one phenotypically PZA-MGIT-susceptible isolate harboring pncA Q141* (Table 2). One isolate with pncA G78V mutation is classified as “associated with resistance–interim” in the 2023 updated WHO mutation catalog.

TABLE 2.

Characterization of the pncA gene mutations and drug-resistant profiles of the study isolates^a

	Mutation	Frequency	pncA gAST	PZA pAST		Drug-resistant profile (no. of isolates)
			Based on the WHO catalog	MGIT	PZase test
Training set	H57D	16	Assoc w R	R	R	PZA (13), PZA + INH (2), PZA + INH + RIF (1)
	T76P	2	Assoc w R	R	R	PZA + INH + RIF + EMB + SM (2)b
	−11a > g	2	Assoc w R	R	R	PZA + INH + RIF + EMB + SM (2)b
	V155G	2	Assoc w R	R	R	PZA + INH + RIF + EMB + SM
	L27P	1	Assoc w R	R	R	PZA + INH + RIF + EMB + SM
	G97D	1	Assoc w R	R	R	PZA + INH + RIF + EMB + SMb
	S104R	1	Assoc w R	R	R	PZA + RIF
	T142K	1	Assoc w R - Interim	R	R	PZA + INH + RIF + EMB + SM
	V155M	1	Assoc w R	R	R	PZA + INH + RIF + EMB
	Q10c	1	Assoc w R	R	R	PZA + INH + RIF
	K96c	1	Assoc w R	R	R	PZA + INH + RIF + EMB + SM
	Q141c	1	Assoc w R	S	R	PZA + INH + RIF
	D40fs	1	Assoc w R - Interim	R	R	PZA + RIF + EMB
	V131fs	1	Assoc w R	R	R	PZA + INH + RIF + EMB + SM
	T87fs	1	Assoc w R - Interim	R	R	PZA + INH + RIF + EMB + SM
Validation set	H57D	27	Assoc w R	–	R	PZA (17), PZA + INH (10)
	T76P	3	Assoc w R	–	R	PZA (2), PZA + INH + RIF (1)
	L172P	2	Assoc w R	–	R	PZA + INH + RIF + EMB + SM (2)
	D40fs	2	Assoc w R - Interim	–	R	PZA + INH + RIF + EMB (2)
	−11a > g	1	Assoc w R	–	R	PZA + INH + SM
	I5S	1	Assoc w R	–	R	PZA + INH + RIF + EMB
	G78Vd	1	Assoc w R - Interim	R	R	PZA + INH + RIF + SM
	I90S	1	Assoc w R	–	R	PZA
	T135P	1	Assoc w R	–	R	PZA + INH + RIF + EMB + SM
	T142K	1	Assoc w R - Interim	–	R	PZA + INH + EMB + SM
	A171E	1	Assoc w R	–	R	PZA + INH + RIF + EMB + SM
	V180G	1	Assoc w R	–	R	PZA
	K96fs	1	Assoc w R - Interim	–	R	PZA + INH + RIF + EMB + SM

^a gAST, genotypic antimicrobial susceptibility testing; pAST, phenotypic antimicrobial susceptibility testing; RIF, rifampin; INH, isoniazid; EMB, ethambutol; PZA, pyrazinamide; SM, streptomycin; NA, not available; fs, frameshift mutation; Assoc w R, associated with resistance; Assoc w R - Interim, associated with resistance–interim; –, the MGIT-PZA AST were not conducted in the analysis.

^b WHO proficiency test isolates.

^c Stop codon (nonsense mutation).

^d 2023 updated WHO mutation catalog.

We identified 21 isolates carrying uncharacterized mutations. For pAST-susceptible isolates, we found one each of pncA S67L, N149S, −5g > a, G16S, G23D, E144D, T167A, and pncA PCR-negative. For pAST-resistant isolates, we identified pncA A134D, D136N, V163G, P69del, and a.a. 10–13 del. Notably, two isolates with discordant results between the PZase test and MGIT-PZA AST, pncA L35R and T100I, were detected, respectively (Table 3). Of the 14 isolates with novel or rare mutations identified, 50.0% (7/14) were concordance, 28.6% (4/14) dis-concordance, and 21.4% (3/14) uncharacterized results between phenotypic PZase AST and that predicted by SUSPECT-PZA or CRyPTIC tuberculosis data set.

TABLE 3.

Characterization of novel or rare pncA mutations of the study isolates^a

	Mutation	Frequency	pncA gAST			PZA pAST		Drug-resistant profile excluding PZA AST (no. of isolates)
			WHO catalog	SUSPECT-PZA	CRyPTIC	MGIT	PZase test
Training set	S67L	1	–	S	–	S	S	RIF + EMB
	A134D	1	–	S	S	R	R	INH + RIF + SM
	D136N	1	Uncertain significance	R	R	R	R	INH + RIF + EMB
	N149S	1	–	S	–	S	S	INH + SM
	V163G	1	Uncertain significance	S	S	R	R	RIF + EMB + SM
Validation set	P69del	6	–	–	–	R	R	Pansusceptible (4), INH (2)
	−5g > a	1	–	–	–	S	S	Pansusceptible
	a.a. 10–13 del	1	–	–	–	R	R	INH + RIF + EMB + SM
	G16S	1	Uncertain significance	S	–	S	S	Pansusceptible
	G23D	1	–	S	–	S	S	Pansusceptible
	L35R	1	Not assoc w R - Interim	R	R/S	R	S	Pansusceptible
	T100I	1	Uncertain significance	S	–	R	S	INH + EMB
	E144D	1	–	S	–	S	S	INH + SM
	V163G	1	Uncertain significance	S	S	R	R	RIF + EMB + SM
	T167A	1	–	S	–	S	S	Pansusceptible
	pncA PCR negative	1	–	–	–	S	S	Pansusceptible

^a gAST, genotypic antimicrobial susceptibility testing; pAST, phenotypic antimicrobial susceptibility testing; R, resistant; S, susceptible; RIF, rifampin; INH, isoniazid; EMB, ethambutol; PZA, pyrazinamide; SM, streptomycin; Not assoc w R - Interim, not associated with resistance–interim; a.a., amino acid; del, deletion; –, no data are available for the corresponding mutation.

DISCUSSION

We developed and validated a simplified PZase test for PZA AST and surveillance of bTB. In our study, 2.6% (43/1,627) of the isolates were PZA-resistant, with 1.3% (21/1,627) identified as M. bovis, including 47.6% (10/21) that were resistant to INH. Among the PZA mono-resistant isolates (1.3%, 21/1,627), of which 19.1% (4/21) were M. tuberculosis, 52.4% (11/21) were M. bovis and 28.6% (6/21) were M. bovis-BCG. Our previous report identified 1.0% (202/20,972) bTB cases with 47.6% INH resistance, higher than that of estimated 30.2% from 2008 to 2019 (7). In our previous study, 4.4% (17/385) of MTBC isolates were resistant to PZA, with MTBC isolates collected from four TCDC-contracted TB laboratories (6), including two from central and southern Taiwan, regions with a higher burden of human bTB cases (7). Whereas, in this study, we enrolled wider range of TB cases from 10 TCDC-contracted TB laboratories across Taiwan and revealed a lower rate of PZA resistance at 2.6% (43/1,627). We successfully implemented the simplified PZase test in 10 clinical laboratories to strengthen routine PZA AST services and establish an effective laboratory-based bTB surveillance system.

Since the PZase test evaluates the presence of POA in the supernatant, it does not require an acidic pH (16), thereby addressing the issues of false-positive results and poor reproducibility associated with the acidic medium required in MGIT-PZA AST (9). A modified Wayne’s PZase test, characterized by directly adding PZA solution to the positive-flagged MGIT tube, showed a 95% concordance with the MGIT-PZA AST (21). We attempted to use positive-flagged MGIT tubes for the PZase test in clinical laboratories. However, low bacterial load in positive-flagged MGIT tubes from sputum cultures made it difficult to predict the initial inoculum size, leading to challenges in the interpretation of color change and low consistency (H.-H. Chan, W.-H. Lin, H.-C. Hsiao, S.-H. Wu, and R. Jou, unpublished data). We therefore developed and simplified the PZase test, utilizing the neutral pH Middlebrook 7H9 media enriched with OADC. The bacterial inoculum for the PZase test ranges from 0.5 to 1 McFarland according to the Clinical and Laboratory Standards Institute guidelines (22), which are also the inoculum levels used for agar-based AST in clinical laboratories. Furthermore, the continued culturing for at least 1 day allows the MTBC to reach the log phase (23), making it easier to identify with the naked eye. We even observed consistent results of the same case across different clinical laboratories. It suggests that the PZA AST has been successfully implemented in routine first-line AST.

Timely and accurate PZA AST was achieved using our simplified PZase test. It has a shorter turnaround time (5 days) than that of MGIT-PZA AST (4.2–19.3 days), the classic Wayne test (7–10 days), and the MODS-Wayne test (14–29 days). In addition, the PZase test showed 100.0% sensitivity and specificity for visual interpretation in the training set, and in the validation set, sensitivity remained at 100.0% while specificity ranged from 98.5% to 100.0%, all compared to Sanger sequencing. MGIT-PZA AST had a sensitivity and specificity of 100.0% and 85.4%, respectively (24, 25). Meanwhile, the classic Wayne test had sensitivity and specificity of 75.6%–95.7% and 88.7%–97.0%, respectively (13, 26). The MODS-Wayne test had sensitivity and specificity of 92.7% and 99.3%, respectively (16).

This was the first study using the largest validation set to confirm the feasibility of the inexpensive, simple, robust, and accurate PZase test to be implemented in clinical TB laboratories, concurrently with first-line AST, and requires minimal training of laboratory staff. We found that the PZase test showed 100.0% and 98.5%–100.0% concordance and specificity with Sanger sequencing in the TRL and clinical TB laboratories, respectively. Appropriate treatment regimens could be prescribed to improve patient outcomes.

The PZase test helps to clarify the discordance with genotypic results that arises from the high confidence-level resistance (>100 µg/mL) (8) criteria used by the MGIT-PZA AST. In the WHO catalog for predicting PZA resistance caused by pncA mutations, the combined sensitivity and specificity of Group 1 (resistance-associated) and Group 2 (resistance-interim-associated) mutations were 78.0% and 97.9%, respectively. These values were calculated by comparing the listed mutations with the total number of phenotypically resistant isolates (18). The Genoscholar PZA-TB II test, a WHO-recommended rapid diagnostic test (27), covers the entire pncA gene and promoter region to account for the widespread resistance mutations and demonstrates a sensitivity of 76.8% and a specificity of 96.8% (28). However, available data were scarce on its diagnostic accuracy and cost-effectiveness. SUSPECT-PZA, although having a sensitivity of 93.7% and a specificity of 44.3% (29), is restricted to predicting missense mutations in the coding sequence of the pncA gene. It must be considered that insertions/deletions, promoter mutations, and nonsense mutations might be associated with resistance (29). In addition, as we have identified novel or rare mutations as potential resistant genetic mutations in the pncA gene (Table 3), the PZase test can be a composite reference standard in combination with the MGIT-PZA AST.

We found six isolates that harbored PZA resistance-associated P69del mutation, identified mainly from southern Taiwan, was not yet classified in the WHO catalog, SUSPECT-PZA webtool, and CRyPTIC tuberculosis data set. Previous studies revealed that Pro-69, located in the conserved region of the PncA protein, was associated with the catalytic triad (amino acids 8, 96, and 138). PZA resistance by a mutation in Pro-69 residue caused a conformational change of the PncA protein and decreased enzyme activity (11, 30). We found that isolates harboring P69del were PZA-resistant using the PZase test.

We found that isolates harboring D136N, A134D mutations, and amino acids 10–13 deletion were resistant to PZA using pAST. Previous studies have shown that three major regions of the pncA gene, amino acids 3–17, 61–85, and 132–142, are most commonly affected by mutations, which can influence the catalytic activity of PZase (31). A134D mutation was not associated with drug resistance, as predicted by the SUSPECT-PZA web tool and the CRyPTIC tuberculosis dataset. However, A134D mutation occurs in a region where the wild-type residue is involved in hydrogen bonding within the binding pocket and may induce electrostatic and steric clashes, resulting in protein destabilization (32, 33). Another uncharacterized mutation, V163G, showed resistance to PZA, in agreement with other studies using the MGIT-PZA AST (34).

The mutation S67L was characterized as functionally neutral (35), and residue T167 within helix 164–178 exhibited the least flexibility (33) by various bioinformatics prediction tools, as mentioned above, suggesting that the PZase test for uncharacterized mutations were not associated with drug resistance. In addition, the L35R and T100I mutations, displaying either resistance or susceptibility, were observed in this study. The L35R mutation, classified as “not associated with resistance-interim” in the WHO catalog, and different pAST methodologies also produce drug-resistance/susceptibility results (18, 36). The T100 mutation is physically adjacent to known resistance-conferring mutations, and this mutation 90% occurs in the first-line or other drug-resistant isolates (37), which is consistent with our observation. However, the PZA pAST result of the T100I mutation has not been reported. Other uncharacterized mutations, −5g > a, N149S, G16S, G23D, E144D, and T167A, found in PZA-susceptible isolates were novel and distant from the functional site. Therefore, we hypothesized that these amino acid substitutions would not lead to the loss of PZase activity. In addition, one of the uncertain significances of PZA resistance by gAST results from PCR failure. Previous studies revealed that PCR failure in the pncA gene could be due to a large deletion in the primer and further verification by WGS is required (38).

Of note, the growth of some isolates was slow, resulting in low quantities of converted POA, which may lead to weak color changes and might cause false results. This issue could be mitigated by extending cultivation time before adding PZA when conducting the PZase test. Besides, when inconsistencies are observed between the PZase test and gAST results, it is crucial to first investigate whether these inconsistencies are due to false susceptibility from PZA heteroresistant isolates or contamination by other bacterial species. In addition, we found resistance results obtained with inoculum prepared from Lowenstein-Jensen (LJ) medium may cause false PZA resistance. It is recommended to avoid the use of LJ medium or to use a short incubation in Middlebrook medium that can accommodate LJ medium.

In conclusion, the PZase test demonstrated high accuracy, low cost, reproducibility, and a turnaround time of 5 days. It had strong agreement with the pncA gene sequencing and MGIT-PZA AST. One limitation of the PZase test, as a culture-based AST, needs to be performed in a high-containment biological laboratory with biosafety cautions. Integrating the PZase test into routine first-line AST is effortless and represents an improvement in laboratory services for ending TB.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jcm.01227-24.

Table S1. jcm.01227-24-s0001.docx.

Performance of different cut-off values of the training set isolates.

Table S2. jcm.01227-24-s0002.docx.

Validation of a simplified pyrazinamidase test in 10 TB clinical laboratories, before discrepancy analysis.

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