Published online Jun 02, 2023.
https://doi.org/10.3947/ic.2023.0037
pncA Large Deletion is the Characteristic of Pyrazinamide-Resistant Mycobacterium tuberculosis belonging to the East Asian Lineage
Abstract
Background
Pyrazinamide (PZA) is often used as an add-on agent in the treatment of multidrug-resistant tuberculosis, regardless of phenotypic drug susceptibility testing (pDST) results. However, evaluating the effectiveness of PZA is challenging because of its low pH activity, which can result in unreliable pDST results. This study aimed to investigate the genomic characteristics associated with PZA resistance that can be used to develop genotypic DST.
Materials and Methods
A publicly available whole genome sequencing (WGS) dataset of 10,725 Mycobacterium tuberculosis complex genomes (3,326 phenotypically PZA-resistant and 7,399 phenotypically PZA-susceptible isolates) were analyzed.
Results
In total, 2,934 pncA non-silent mutations were identified in 2,880 isolates (26.9%). Detected mutations were found throughout the entire coding region of pncA in a scattered pattern, of which the most frequent mutation was p.Q10P (n = 278), followed by p.H57D (n = 167) and c.-11A>G (n = 122). The sensitivity and specificity of the group 1 or 2 mutations reported by the World Health Organization (WHO) mutational catalogue were 73.0% and 98.9%, respectively. We further identified 18 novel pncA mutations that were significantly associated with phenotypically PZA-resistant. In addition to these mutations, we identified 102 large deletions in the pncA gene, and all but two isolates were phenotypically resistant to PZA isolates. Notably, pncA deletions were mutually exclusive to pncA mutations, and more than half of the isolates with pncA large deletions belonged to the East Asian lineage (67.6%). The sensitivity, specificity, positive predictive value, and negative predictive value of the pooled variants (group 1 or 2 mutations, novel resistance-associated mutations, and large deletions of the pncA gene) were 79.0%, 98.9%, 97.0%, and 91.3%, respectively. The area under the curve (AUC) value for the pooled variants was significantly higher than the AUC value for the group 1 or 2 mutations (P <0.001), indicating that the pooled variants have a better discriminative ability for predicting PZA resistance.
Conclusion
Using WGS, we found that the pncA mutations are scattered without specific mutational hotspots, and large deletions associated with PZA resistance are more common in the East Asian lineage of M. tuberculosis isolates. Our data also demonstrated the reliability of group 1 or 2 mutations presented in the WHO mutation catalogue and the need for further investigation on group 3 mutations, contributing to the evaluation of the current knowledge base on mutations associated with the PZA-resistant M. tuberculosis complex.
Graphical Abstract
Introduction
Management and treatment of multidrug-resistant tuberculosis (MDR-TB) are one of the major public health concerns worldwide [1, 2]. From 2019 to 2021, the number of reported patients with MDR-TB in Korea annually was between 371 and 580, while that with extensively drug-resistant TB in Korea annually was between 17 and 33 [3]. Although phenotypic drug susceptibility testing (pDST) and genotypic DST (gDST) can accurately detect resistance to isoniazid (INH) and rifampicin (RIF), evaluating the activity of pyrazinamide (PZA), another first-line drug used in the treatment of TB, poses considerable challenges [4]. PZA is often used as an add-on agent in the treatment of MDR-TB, regardless of pDST results [5]. It helps shorten the duration of therapy and is particularly effective against latent or semi-dormant Mycobacterium tuberculosis residing in acidic environments. However, the use of PZA can result in multiple side effects, including hepatotoxicity and arthralgia. Developing a reliable and reproducible DST for PZA could help avoid unnecessary drug exposure and prevent harmful side effects.
The Löwenstein-Jensen (L-J) absolute concentration or Mycobacterium Growth Indicator Tube (MGIT) 960 system is commonly used for pDST in clinical settings [6]. However, these methods do not provide information on the level of resistance, making it challenging for clinicians to adjust drug dosage in patients with low-level resistant strains. Broth microdilution methods can assess the minimal inhibitory concentration of many drugs at various concentrations; however, they are labor-intensive, and there is a need for a global consensus on the critical concentration. Additionally, conducting pDST for PZA under acidic conditions can yield unreliable results because of its lower pH activity [7].
Whole-genome sequencing (WGS) has become an essential tool for rapidly identifying drug resistance and has grown significantly over the past decade [8]. Previous studies demonstrated that mutations and indels can accurately predict phenotypic resistance [9, 10]. In M. tuberculosis, the pncA gene encodes the enzyme pyrazinamidase (PZase), which converts the prodrug PZA into its active form, pyrazinoic acid [7]. Although pncA mutations predicted PZA resistance with high specificity, their sensitivity was found to be low at 75.8% in one study [9] and 57.3% in another [10]. In June 2021, the World Health Organization (WHO) released the first comprehensive catalogue of mutations in the M. tuberculosis complex, which assigned a confidence grade to one of five groups for each variant [11]: (1) associated with resistance; (2) associated with resistance-interim, (3) uncertain significance; (4) not associated with resistance-interim; and (5) not associated with resistance. Group 1 (associated with resistance) or 2 (associated with resistance-interim) in the catalogue accurately predicted resistance and were strongly correlated with pDST for most drugs (sensitivities up to 93.8% and specificities up to 99.0%) [11]. Despite these promising results, the sensitivity of group 1 or 2 pncA mutations for predicting PZA phenotypic resistance remains low, at 72.3%, even in the WHO catalogue.
Large-scale deletion is a type of genomic structural variation that results in the loss of genetic material and can impair proteins. Although the role of large deletions as a resistance mechanism is not well understood, it has been sporadically reported in drug resistance-associated genes such as katG and pncA [12, 13]. Such deletions can lead to the misdiagnosis of drug resistance when using conventional gDST, such as polymerase chain reaction (PCR). Because WGS can identify both large deletions and mutations/indels, it is necessary to investigate the frequency and clinical significance of large deletions using genome-based analysis.
In this study, we analyzed 10,725 M. tuberculosis complex genomes harboring pDST data for PZA to investigate pncA gene deletions and identify novel mutations associated with PZA resistance, in addition to group 1 or 2 mutations suggested by the WHO.
Materials and Methods
1. Sequencing data
WGS data of phenotypically PZA-resistant M. tuberculosis complex isolates generated using Illumina instruments (Illumina Inc., San Diego, CA, USA) were included in this study. Approximately 2.5 times more PZA-susceptible isolates than PZA-resistant isolates were randomly selected and analyzed as controls. After removing isolates that were duplicated between datasets [4, 7, 9, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29], raw sequence data (FASTQ file format) for 11,130 M. tuberculosis complex isolates (3,393 PZA-resistant isolates and 7,737 PZA-susceptible isolates) were downloaded from the European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena/browser/home), Sequence Read Archive (SRA; https://www.ncbi.nlm.nih.gov/sra), or DDBJ Sequence Read Archive (DRA; https://www.ddbj.nig.ac.jp/index-
2. Ethics statement
This study was approved by the Institutional Review Board of the Catholic University of Korea College of Medicine (approval number: MC22TNSI0059). The ethics committee waived the requirement for informed consent since we used de-identified data that were collected retrospectively.
3. Identification of genomic alterations
Sequencing reads were mapped to the H37Rv reference genome (GenBank accession number: NC_000962), and bioinformatic processing of the WGS data was performed as previously described [30]. Single nucleotide variants (SNVs) and small indels were identified using SAMtools and BCFtools [31]. SnpEff has been used to define mutations in genomic sequences and predict their functional consequences [32]. To obtain reliable and robust mutation calling, the following variants were eliminated: (1) read depth <10, (2) Phred quality score <15, (3) variant allele frequency (VAF) <10%, and (4) variants with >90% support on one strand. Synonymous or previously reported lineage-associated SNVs were excluded from further analysis [33]. The remaining variants were classified into one of five groups according to the WHO catalogue [11]: (1) associated with resistance; (2) associated with resistance-interim; (3) uncertain significance; (4) not associated with resistance-interim; and (5) not associated with resistance (neutral mutations). Group 1 mutations were defined when the following five criteria were met: (1) sum of resistant and susceptible isolates with a solo mutation ≥5, (2) lower bound of the 95% confidence interval of the positive predictive value (PPV) conditional on being solo ≥25%, (3) odds ratio (OR) >1, (4) OR of solo mutations >1, and (5) false discovery rate-corrected P ≤0.05. Group 2 mutations were present as solo in pncA in at least two resistant isolates and with a PPV of at least 50%, whereas group 4 mutations were present as solo in pncA with a PPV less than 40% and the upper bound of the 95% confidence interval below 75%. Group 3 were mutations that did not meet the criteria for inclusion in groups 1, 2, 4, or 5. The gDST of PZA was predicted based on group 1 or 2 mutations reported in the WHO catalogue. To this end, we defined genotypic resistance as SNVs or indels detected in our dataset that overlapped with the catalogue at the amino acid level. Group 3, 4, or 5 mutations in the WHO catalogue or mutations that did not meet the criteria of the WHO catalogue were considered genotypically susceptible. To identify novel targets conferring resistance to PZA, we compared genetic alterations at the variant level with pDST using Fisher’s exact test. These analyses were limited to the pncA gene, which has been reported to be associated with PZA resistance. DELLY software was used to predict large deletions (>50 bp) [34]. The depth ratio of all pncA deletions was manually inspected using the Integrative Genomics Viewer browser.
4. Statistical analysis
The sensitivity, specificity, positive PPV, negative predictive value (NPV), and OR were calculated. Mutations satisfying a P-value <0.01, PPV >85%, and NPV >85% were considered putative resistant-conferring targets. The receiver operating characteristic (ROC) and area under the curve (AUC) were used to compare the predictive value between the WHO catalogue and our dataset. Pearson’s chi-square test was used to compare the frequency of pncA large deletions between lineages. All statistical analyses were performed using SPSS (version 29.0, IBM Corp., Armonk, NY, USA).
Results
Raw sequence reads of 10,725 M. tuberculosis complex isolates were aligned to the H37Rv reference genome, and their mean coverage of the sequencing depth was 105.4x (Supplementary Table 1). The most common lineage was L2.2.1 (East Asian; n = 1,935, 18.0%), followed by L3 (Delhi-Central Asian; n = 1,483, 13.8%), L4.1.2.1 (Euro-American; n = 1,010, 9.4%), and L4.8 (Euro-American; n = 796, 7.4%) (Supplementary Table 1). Based on the pDST, 3,326 of 10,725 isolates were classified as PZA-resistant (31.0%), and the other 7,399 isolates were PZA-susceptible (69.0%) (Supplementary Table 1).
In total, 2,934 pncA non-silent mutations (2,555 SNVs and 379 indels) were identified in 2,880 isolates (26.9%) (Supplementary Table 2). Most isolates harbored only one mutation in pncA, but 52 isolates (0.5%) had two or more mutations. When gDST determined by group 1 or 2 mutations reported in the WHO catalogue was compared with the pDST, only 73.0% of the phenotypically resistant isolates were identified as genotypically resistant (Table 1). Detected mutations were found throughout the entire coding region of pncA in a scattered pattern, of which the most frequent mutation was p.Q10P (n = 278, 2.6%), followed by p.H57D (n = 167, 1.6%) and c.-11A>G (n = 122, 1.1%) (Fig. 1A). Despite the low sensitivity of gDST for PZA, its specificity was relatively high (98.9%) (Table 1). The sensitivity and specificity of the gDST in our dataset were largely consistent with those of a previous report (AUC difference = 0.004, P = 0.589) [11], indicating that the poor predictive value of PZA in our dataset was not due to biased sampling (Supplementary Fig. 1). The PPV and NPV of the group 1 or 2 mutations reported using the WHO catalogue were 96.8% and 89.1%, respectively (Table 1).
Table 1
Performances between pDST and gDST for PZA in 10,725 Mycobacterium tuberculosis isolates
Figure 1
Lollipop plots of pncA mutations.
(A) Lollipop plots show pncA mutations identified in 3,326 phenotypically resistant Mycobacterium tuberculosis genomes (upper) and 7,399 phenotypically susceptible M. tuberculosis genomes (lower). (B) Lollipop plots show pncA mutations identified in 899 M. tuberculosis genomes that were phenotypically resistant to pyrazinamide but did not harbor group 1 or 2 mutation (upper) and 7,399 phenotypically susceptible M. tuberculosis genomes (lower). The X-axis represents the amino acid position. The dot size and color represent mutation frequency and mutation types, respectively. The white asterisks represent the novel mutations significantly associated with pyrazinamide resistance.
Next, we focused on 899 M. tuberculosis isolates that were phenotypically resistant to PZA but did not harbor group 1 or 2 mutations in pncA. Among them, 263 isolates harbored pncA mutations (29.3%), whereas 636 isolates did not have a pncA mutation (70.7%). The most frequent mutations were p.I6L (n = 10, 1.1%), followed by p.V139M (n = 9, 1.0%), p.T160A (n = 8, 0.9%), and p.T100I (n = 8, 0.9%) (Fig. 1B). When we compared the mutations and pDST results for PZA, 18 mutations were found to be significantly enriched in PZA-resistant M. tuberculosis isolates (Table 2 and Supplementary Table 3). All but one (p.I6L, 10 PZA-resistant and one PZA-susceptible) mutation was exclusively detected in the PZA-resistant M. tuberculosis isolates (Fig. 1B). Truncating mutations, including start loss, nonsense, and frameshift mutations, were found in 4.9% (44 of 899) of the isolates and were significantly enriched in PZA-resistant M. tuberculosis isolates (P <0.001). Overall, novel mutations significantly associated with PZA resistance, together with truncating mutations, showed a sensitivity of 3.2% and specificity of 99.9% in 10,725 M. tuberculosis isolates (Table 1).
Table 2
Putative mutations associated with pyrazinamide-resistant Mycobacterium tuberculosis complex isolates
In addition to mutations, 102 of the 10,725 M. tuberculosis complex isolates (1.0%) harbored large deletions (58 partial deletions and 44 complete deletions) ranging from 60 to 17,144 bp in the pncA genes (Fig. 2). M. tuberculosis complex isolates belonging to lineage 2 (East Asian) had the highest number of pncA large deletions (69 of 2,269, 3.0%, P <0.001), whereas those belonging to lineages 4 (Euro-American; 29 of 5,532, 0.5%) and 3 (Delhi-Central Asian; 4 of 1,934, 0.2%) had less than 1% of pncA large deletions (Supplementary Table 1). M. tuberculosis complex isolates belonging to other lineages did not harbor pncA deletions. pncA large deletions were predominantly detected in PZA-resistant isolates, and only two PZA-susceptible isolates harbored pncA large deletions (Table 1). Notably, pncA deletions were mutually exclusive to pncA mutations (17 co-occurring isolates out of 10,725 isolates, P = 0.021), which led us to incorporate group 1 or 2 mutations, novel resistance-associated mutations, and large deletions of pncA gene to predict PZA resistance. The sensitivity, specificity, PPV, and NPV of the pooled variants were 79.0%, 98.9%, 97.0%, and 91.3%, respectively (Table 1). The AUC value for the pooled variants was significantly higher than the AUC value for the group 1 or 2 mutations reported by the WHO catalogue (AUC 0.889 vs. 0.856, P <0.001), indicating that the pooled variants have a better discriminative ability for predicting PZA resistance.
Figure 2
Large deletions in the pncA gene identified across 10,725 Mycobacterium tuberculosis genomes.
Coverage depth plots of 18 representative M. tuberculosis isolates with large pncA deletions. The X-axis represents genomic position, and the Y-axis represents sequencing depth. Blue arrows indicate large deletions.
Discussion
In the present study, we identified novel pncA mutations associated with PZA resistance and evaluated pncA large deletions in 10,725 M. tuberculosis complex genomes. Our data support four major conclusions: first, pncA gene mutations are detected in a scattered pattern across the entire coding region without specific mutational hotspots; second, despite the recent publication of a comprehensive mutational catalogue, mutations associated with PZA resistance remain to be studied; third, large deletions in the pncA gene occur frequently and are closely linked to PZA resistance; and fourth, pncA large deletions are more prevalent in the East Asian lineage (lineage 2) than in other lineages. Altogether, these WGS-based findings provide a valuable resource for exploring the molecular targets and underlying resistance mechanisms of PZA.
Mutations in the pncA gene or its promoter region are the most common causes of PZA resistance in M. tuberculosis and result in decreased PZase activity [7]. Based on this, the majority of pncA mutations identified in this study (n = 2,514, 85.7%) were group 1 or 2 mutations known to be associated with PZA resistance [11]. However, owing to the dispersed nature of mutations in the entire pncA gene, developing a rapid molecular drug susceptibility assay will be challenging; therefore, the identification of PZA resistance may be facilitated through WGS. Recent advances in sequencing technology, such as targeted deep sequencing, will provide clinical applicability at a relatively low cost for the rapid diagnosis of TB and the prediction of resistance to a wide range of anti-TB drugs.
In some cases, group 1 or 2 mutations reported in the WHO catalogue and those detected in this study overlapped at the amino acid level but were discordant at the nucleotide level. For example, the nucleotide change of the pncA p.Y103* nonsense mutation is ‘c.309C>A’ in the WHO catalogue, whereas ‘c.309C>G’, causing the identical p.Y103*, was recurrently detected in our dataset (four c.309C>A mutations and 17 c.309C>G mutations). Likewise, the recurrent missense mutation p.D12E, caused by ‘c.36C>G’ (n = 6), was more prevalent than that caused by ‘c.36C>A’ (n = 4), as previously reported by the WHO. This finding suggests that clinicians who evaluate or diagnose gDST based on the current version of the WHO mutational catalogue should be careful to avoid misdiagnosis. One of the limitations acknowledged by the WHO catalogue is that the catalogue has not been validated in an independent dataset [11]. By combining larger datasets, we further identified 18 putative mutations associated with PZA resistance, of which nine (50.0%) were group 3 mutations, while the other eight mutations (44.4%) did not meet the WHO catalogue criteria (Table 2). These results indicate that the current release of the WHO catalogue may be over- or under-fitting for anti-TB drugs; therefore further evaluation and upgrading of group 3 mutations are needed.
A recent study investigated the prevalence of resistance caused by large deletions in 32,000 publicly available M. tuberculosis isolates and reported that large deletions in pncA, katG, gid, and ethA were significantly associated with resistance to PZA, INH, streptomycin, and ethionamide [22]. Consistent with this, we identified 102 large deletions in the pncA gene (102 of 10,725 isolates, 1.0%), and all but two isolates were resistant to PZA. Interestingly, more than half of the isolates with pncA large deletions belonged to the East Asian lineage (lineage 2, 69 of 102 isolates with a large pncA deletion, 67.6%). This suggests that the pncA large deletion may be a genomic characteristic specific to the East Asian lineage that has not yet emerged globally. To the best of our knowledge, this is the first data showing that pncA deletion is frequent in lineage 2 M. tuberculosis isolates. Partial or complete deletion of the pncA gene causes a decrease in PZase activity [13], which may lead to PZA resistance. Therefore, the introduction of next-generation sequencing in clinical laboratories can provide advantages for diagnosing PZA-resistant M. tuberculosis harboring a large deletion, which is challenging to detect using conventional methods such as PCR. Moreover, because the current WHO catalogue does not include large deletions, a major update to include them will be required.
An inherent limitation of this study was that the newly identified mutations linked to PZA resistance were not validated in an independent cohort. In addition, the phenotypic data were derived from various assays with varying critical concentrations. Given the absence of mutational hotspots in the pncA gene, further studies in a larger cohort where the phenotypic data were evaluated with a unified DST are required to assess the functional impact of mutations that could potentially cause PZA resistance and to elucidate the underlying resistance mechanisms.
In conclusion, we provided comprehensive results of the genomic characteristics of PZA-resistant M. tuberculosis isolates, including the high prevalence of pncA large deletions in the East Asian lineage. Our data also contribute to the evaluation of the current knowledge base on mutations associated with the PZA-resistant M. tuberculosis complex. Further investigations to determine the functions and regulatory factors of novel resistance-associated variants will help establish new therapeutic strategies for the PZA-resistant M. tuberculosis complex.
SUPPLEMENTARY MATERIALS
Mycobacterium tuberculosis isolates analyzed in this study and their phenotypic/genotypic profiles of PZA resistanceSupplementary Table 1
Single nucleotide variants (SNVs) and indels identified across 10,725 Mycobacterium tuberculosis genomesSupplementary Table 2
Mutations associated with Mycobacterium tuberculosis isolates that are phenotypically resistant to PZA but did not harbor group 1 or 2 mutationsSupplementary Table 3
Comparison of receiver operating characteristic (ROC) curves between the WHO and our datasets.Supplementary Figure 1
Funding:This study was supported by a grant from the Korea Health Technology R&D Project grant through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Korea (HI22C0226).
Conflict of Interest:No conflict of interest.
Author Contributions:
Conceptualization: SHJ.
Data curation: NYK, DYK, JC.
Funding acquisition: SHJ.
Methodology: NYK, DYK, JC, SHJ.
Software: NYK, DYK, SHJ.
Visualization: NYK, SHJ.
Writing - original draft: NYK, DYK, JC, SHJ.
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