Elsevier

Tuberculosis

Volume 111, July 2018, Pages 109-113
Tuberculosis

Characterization of pncA mutations in multi-drug and pyrazinamide resistant Mycobacterium tuberculosis isolates cultured from Queensland migrants and Papua New Guinea residents

https://doi.org/10.1016/j.tube.2018.06.006Get rights and content

Abstract

Outbreak of drug resistant tuberculosis in the Western province, Papua New Guinea is a concern to Queensland, Australia due to migration. We performed pncA mutation analysis and genotyping of multi-drug/pyrazinamide (MDR/PZA) resistant isolates from 18 Queensland (Qld) migrants and 81 Papua New Guinea (PNG) residents, to compare with phenotypic evidence of PZA resistance and to evaluate the genotypes obtained from the two countries. Seven different mutations were seen from Qld isolates of which 2 have not been described previously. A cluster of mutations were found between amino acids L35 and S65. Amongst the PNG isolates, 10 mutations were identified, of which 6 were unique and have not been described previously. Majority of the mutations formed 2 clusters, between amino acids Q10 to A20 and W68 to W119. Mutations identified at nucleotide (nt) position 202 and 307 were found to be the most common types, occurring in 25% and 51% of the PNG isolates respectively. The majority of the mutations were seen in MDR/PZA resistant isolates. These mutations could be utilized for direct screening of PZA resistance from PNG patient samples. Genotypic analysis of the isolates showed strong clustering amongst the PNG isolates as opposed to Qld isolates. A diversity of mutations and genotypes were seen amongst the Qld migrant isolates. Majority of PNG isolates had one genotype with two distinct pncA mutation patterns (T202C and T307G) which highlight on-going transmission. pncA mutation analysis provided a satisfactory alternative to PZA culture DST with high positive predictive value and an improved result turnaround time.

Introduction

Tuberculosis (TB), due to members of the Mycobacterium tuberculosis complex, remains a common human infectious disease with an estimate of 8.6 million new cases in 2012 and 1.3 million deaths worldwide [1]. Australia is fortunate to have a low incidence of TB (6.5/100, 000 population) whereas Papua New Guinea (PNG) has one of the highest incidence of TB in the Western Pacific Region (432/100,000 population) [1]. Of great concern, the regional incidence in PNG may be significantly higher with incidence of 500–600/100,000 population estimated for the Western Province [2]. Australia and PNG share a common border where people from both sides are allowed to travel across the border through the Torres Strait (TS) for socio-economics or health purposes. PNG residents from the Western Province and TS seek health care support from Australia. Transmission of TB including Multi-Drug Resistant (MDR) TB has been recognised among the PNG nationals accessing Australian health care support [3]. This poses a significant challenge to advancing Australia's public health interest whilst supporting the customary rights of people from both sides of the boarder.

The emergence of MDR-TB and extensively drug resistance (XDR) TB adds further to the complexity of TB control in Australia and PNG. Pyrazinamide (PZA) is considered as one of the corner stone drugs in the multi-drug regimen for treatment of MDR TB. It is an important first line anti-TB drug and with its unique ability to kill semi-dormant tuberculi in acidic environments, such as that found in host macrophages, it enables the treatment course to be shortened from 9-12 months–6 months [4]. There are various potential anti-TB drugs in clinical development [5] that have been shown to have improved treatment outcome of drug resistant TB when administrated in combination of PZA [[6], [7], [8]]. PZA is a pro-drug which is converted to bacterially toxic pyrazinoic acid (POA) intracellularly by bacterial pyrazinamidase (PZase) that is constitutively expressed in TB [9,10]. Though the exact mechanism of action of POA is unknown it is postulated that POA has inhibitory effects on the cellular metabolism in acidic environments [11,12]. Mutations in the pncA gene encoding PZase have been shown to confer reduction or loss in PZase activity [13]. Structural analysis of Pzase has revealed that mutations in the pncA gene can have detrimental effect of enzymatic functions. This can range from either complete loss of enzymatic functions due to mutations causing frameshift or terminations, affecting substrate or metal binding sites and or the stability of the enzymes [14]. However, there are certain neutral mutations that do not have any impact on the enzymatic activity and are believed to be either silent mutations or in the regions that do not have a major role in enzymatic activity or structural stability [14]. A great diversity of pncA gene mutations has been discovered in recent years that has an impact on PZase activity [15], however, a substantial number of PZA resistant isolates have also been discovered without pncA gene mutations suggesting the existence of alternative pathways for PZA resistance [16,17]. Mutations in 30S ribosomal protein S1 (rpsA) and aspartate 1-decarboxylase precursor (panD) have recently been reported to be concomitant with PZA resistance [18,19]. A recent multinational study identified a 2% increase in sensitivity of PZA resistance with addition of mutations identified in rpsA and panD [20].

Phenotypic PZA susceptibility testing is carried out under acidic conditions in order to reproduce the in vivo environment in which it is expected to exert its mycobacteriocidal effect. However, acidic conditions hinder in vitro susceptibility testing by limiting the growth of the bacilli [21]. Mutational analysis offers an alternative method to overcome these technical difficulties. DNA sequencing studies of the pncA gene from PZA resistant and PZA susceptible strains, as well as PZA resistant mutants constructed in vitro, demonstrate excellent correlation between pncA mutations, the loss of PZase activity, and PZA resistance [22,23]. Some studies have rebutted mutations in rpsA and panD to be responsible for PZA resistance [23,24].

We sought to investigate the utility of pncA mutational analysis for the detection of pyrazinamide resistance. We compared sequencing of the pncA gene from selected isolates of pyrazinamide resistant and susceptible M. tuberculosis strains from two populations; Queensland migrants and Papua New Guinea patients of the South Fly District, Western Province. Relatedness between strains was examined using the 12 loci MIRU (Mycobacterial Interspersed Repetitive Unit) method and comparing this with heterogeneity of pncA mutations identified.

Section snippets

Materials and methods

All Qld and PNG clinical isolates of M. tuberculosis cultured at, or submitted to, our laboratory and identified as MDR and or PZA resistant by phenotypic method between 2009-2013 were included in this study. Some pan-susceptible isolates were also included as a negative control. Isolates that were identified as M. bovis were excluded from this study. Queensland Mycobacteria Reference laboratory carries out susceptibility testing on all Qld M. tuberculosis isolates and receives sputum specimens

Results

Ninety-nine PZA resistant or susceptible TB isolates from Qld (18 isolates) and PNG (81 isolates) underwent pncA mutational analysis. Of the 18 Qld isolates, 5 were MDR/PZA resistant, 3 were MDR and 10 were pan-susceptible. Seven different pncA mutations were identified in Qld isolates (Table 1, Fig. 1). Two of the 7 mutations at nt position (amino acid position or mutations type) C28G (Q10E) and T192G (Y64Stop) have not been described previously. The remainder of the mutations at nt positions

Discussion

The rise in MDR and XDR TB cases globally necessitates a rapid and accurate diagnosis of drug resistance for effective TB management. PZA is an important first line drug for current treatment regimens for both drug susceptible and drug resistant tuberculosis and is showing promising results when administered with new drugs such as PA824 and bedaquiline that are undergoing clinical trials [6,7,33]. It has been shown that PZA is a pro-drug that needs to be activated by PZase encoded by pncA [25].

Ethics

This work received ethical approval from the Children's Health Queensland Hospital and Health Service Human Research Ethics Committee (approval number: HREC/14/QRCH/133) and Papua New Guinea Medical Research Advisory Committee (approval number: 16.42).

Acknowledgments

A special thanks to the staff members of Queensland Mycobacterium Reference Laboratory, Australia and Central Public Health Laboratory, Papua New Guinea.

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