Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures.

BACKGROUND: A rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes.
OBJECTIVES: A PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013-2015.
METHODS: Eighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR.
RESULTS: The PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9-100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%).
CONCLUSIONS: PCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use.

Introduction

Zoonotic Tuberculosis (zTB) is caused principally by Mycobacterium bovis and other species of Mycobacterium tuberculosis complex (MTBC) e.g; M. caprae, M. pinnipedii, M. microti, M. orygis 1,2,3,4,5.

The World Health Organization (WHO) estimates 147,000 new human cases in 2016 due to zTB with 12,500 deaths 6. In Tunisia, lymph node TB incidence was increased from 2.3 cases/100,000 inhabitants in 1993 to 18.0 cases/100,000 inhabitants in 2017 and M. bovis could be responsible for 78.9% of lymph node TB cases 7. M. bovis is intrinsically resistant to pyrazinamide (PZA) due to the mutation C169G of pncA gene (codon 57:H57D) 8.

Phenotypic and biochemical tests traditionally used to identify this species are time-consuming and inaccurate9. The WHO recommended identifying this species to estimate the burden of zTB in each setting and prescribe an adequate treatment 1. Various methods have been developed for this purpose.

Sequencing based genotyping methods have been used as a reference standard to well differentiate between MTBC species. A set of molecular markers has been used for this aim, such as 16S rRNA, oxyR, katG, pncA, gyrA, gyrB and hsp65 10–11. However, sequencing-based genotyping methods are expensive and require specific equipment.

At the national reference laboratory (NRL) for mycobacteria in Tunisia, the line probe assay: Genotype MTBC (Hain Lifescience, Germany) is used for molecular identification of M. bovis strains, whereas, this method is costly (34 $ for one test)

Herein, two cost-effective PCR approaches are evaluated: a PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of three Region of Difference (RD9, RD4 and RD1) for the detection of M. bovis in lymph nodes cultures, in comparison with the line probe assay: GenoType MTBC assay.

Materials and methods

Ethical approval

This study is approved by the ethics committee of A. Mami pneumology hospital, Ariana, Tunisia.

Clinical specimens, strains identification and phenotypic Drug susceptibility testing (DST)

Two hundred sixty-four lymph nodes samples (n=264) were tested at the NRL for mycobacteria in Tunisia, during 2013- 2015. They were subjected to: acid-fast bacilli smear examination, a culture in liquid medium Mycobacteria Growth Indicator Tube 960 “MGIT960” (BD, USA), a culture in solid medium: Lowenstein Jensen “LJ”, and a molecular diagnosis by GeneXpert MTB/RIF (Cepheid, USA).

MTBC species identification was carried out by SD TB Ag MPT64 Rapid kit (Standard Diagnostics, South Korea), biochemical tests: niacin production, nitrate reductase activity, growth on thiophene-2-carboxylic acid hydrazide and the molecular assay GenoType MTBC.

To study the specificity of evaluated methods, different MTBC species selected from our strains bank: M. caprae, M. bovis, M. bovis BCG and M. tuberculosis H37Rv were included in addition to 7 species of non-tuberculous mycobacteria (NTM): M. chelonae, M. abscessus, M. kansasii, M. intracellulare, M. fortuitum, M. marinum, M. peregrinum. The NTM were identified by GenoType Mycobacterium CM/AS assay (Hain Lifescience, Germany). The phenotypic DST for first-line drugs was performed on MGIT 960 or LJ. For PZA, it was performed on MGIT 960 PZA kit (BD, USA).

PCR pncA-RFLP and RD-PCR

DNAs were extracted from MGIT 960 cultures. One ml of MGIT was centrifuged at 12.000 rpm for 10 min. The pellets were suspended in 200 µl of Tris EDTA Buffer (10 mMTris-Cl pH 8.0, 1 mM EDTA) and heated at 95°C for 30 min. The suspensions were then centrifuged at 13,000 rpm for 15 min and the supernatants were kept and frozen at -20°C.

The PCR mixture (25µl) for PCR pncA-RFLP method was prepared using 2.5 µl of buffer (10×), 0.1 µl of primers pncA F et R (25µM) 11, 2 µl of dNTP (10 mM), 2.5 µl of MgCl2 (25 mM), 2.5 µl of DNA, 0.15 µl of Bioamtik Taq polymerase (500U) and water. The amplification was performed, according to Huard et al.11. The PCR products (664bp) were digested by BstEII enzyme (New England, UK).

If 2 bands were obtained (170 bp and 494 bp): a mutation at position 169 of pncA is present.

If 3 bands were found (103 bp, 170 bp, and 391 bp): no mutation at position 169 of pncA gene.

For the RD-PCR method, the mix (25 µl) was composed of 2.5 µl of buffer (10×), 0.5 µl of primers F, R and int for each region RD1, RD4 and RD9 (25 µM)12,13, 4 µl of dNTP (10 mM), 1 µl of MgCl2 (50mM), 2.5 µl of DNA, 0.15 µl of Platinum Taq polymerase (Invitrogen, USA) and water.

The amplification was performed, according to Warren et al. 13. The size of the bands, after electrophoresis, allows to deduce the absence (-) or presence (+) of the target regions: RD1+: 146bp; RD1-: 196bp; RD4+: 172bp; RD4-: 268bp; RD9+: 235bp; RD9-: 108bp 13.

The species of MTBC, including M. bovis, were identified based on the presence or absence of these 3 RD.

Data analysis

Sensitivity, Specificity, Positive and Negative Predictive Values (PPV/NPV) were calculated using Open Epi version 3.01 with a confidence interval (CI) of 95%.

Results

During 2013–2015, lymphadenitis TB was confirmed in 164 cases (62.12%) by microscopy and /or culture and/or GeneXpert MTB/RIF. The culture was positive in 82 cases (50.0%). GenoType MTBC assay showed that TB lymphadenitis was due to M. bovis (n=54), M. tuberculosis (n=27) and M. caprae (n=1).

All M. bovis strains were resistant to PZA by MGIT960.

Molecular identification by PCR pncA-RFLP

PCR pncA-RFLP showed that 54 M. bovis strains presented 2 bands of 170 bp and 494 bp after digestion by BstEII (Figure 1). It showed that 28 strains presented 3 bands of 103bp, 170bp and 391bp (Figure 1). Twenty-seven were M. tuberculosis and one strain was M. caprae, according to GenoType MTBC.

Figure 1

Results of PCR pncA-RFLP

M: 100bp DNA ladder, 1: M. kansasii (NTM: control strain), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11: M. bovis, 12:M. bovis BCG (control strain), 13: M. tuberculosis

PCR-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0), a specificity of 100.0 % (95 CI: 87.9–100.0), a PPV of 100.0% (95%CI:93.3 -100.0) and a NPV of 100.0% (95% CI:87.9–100.0) for detecting M. bovis.

As regards the control strains: M. bovis and M. bovis BCG presented 2 bands after the digestion, whereas, M. tuberculosis H37Rv and M. caprae showed 3 bands.

No amplification of pncA was detected for NTM species.

Molecular identification by Regions of Difference

All M. bovis strains (n=54) had RD9 and RD4 deleted. Our results showed that 27 strains presented the 3 RD targeted (RD9+/RD4+/RD1+) (Figure 2).

Figure 2

Results of the identification by RD

M: 100bp DNA ladder, 1 (NTM: control strain), 2 and 3 : 268bp (RD4-), 146bp (RD1+), 108bp (RD9-), 4 : 235bp (RD9+) 172bp, (RD4+), 146 bp (RD1+), 5M. caprae:172 bp (RD4+), 146bp (RD1+), 108bp (RD9-), 6 (control strain): 268bp (RD4 -), 196bp (RD1-), 108 bp (RD9-)

These strains belonged to (M. tuberculosis/ M. canettii) group. The biochemical tests and the GenoType MTBC identify these strains as M. tuberculosis. The M. caprae strain showed the absence of RD9 and the presence of RD4 and RD1 and was classified in (M. caprae/ M. africanum /M. pinnipedii and M. microti) group.

The sensitivity of RD-PCR for identifying M. bovis was 100.0% (95%CI: 93.3 -100.0) with a specificity of 100.0% (95% CI: 87.9–100.0).

As concerns the control strains: M. tuberculosis H37Rv had the 3 RD studied, M. caprae presented only RD9, M. bovis had RD9 and RD4 deleted and M. bovis BCG had the 3 RD deleted. No amplification was found for the NTM species.

Discussion

Mycobacterium bovis is an important cause of TB in humans. Accurate, rapid identification of this species is required to allow appropriate treatment and set a strategy to monitor the cattle's disease. For this purpose, two cost-effective PCR approaches were evaluated in comparison with the molecular assay: GenoType MTBC.

The molecular identification based on the polymorphism at position 169 of pncA presented very high sensitivity and specificity in detecting M. bovis strains (100.0%). This method could also represent a rapid tool to detect the natural resistance to PZA. In fact, it is known that three MTBC species are intrinsically resistant to this drug: M. bovis, M. bovis BCG, due to the pncA C169G substitution and M. canettii 14,15.

The allelic variation at oxyR position 285 has also been proposed to differentiate M. bovis from M. tuberculosis but did not distinguish between BCG and non-BCG M. bovis strains 11,16,17.

In addition, a multiplex PCR was tested to detect the presence or absence of 3RD: RD9, RD4 and RD112,13. The RDs represent the loss of genetic materials in M. bovis BCG compared to M. tuberculosis H37Rv genome11. All M. bovis strains in this study (n=54) had RD9 and RD4 deleted but presented RD1. Consequently, the RD-PCR showed excellent sensitivity and specificity (100.0%) for identifying M. bovis isolates.

Compared with the conventional methods, pncA-RFLP and RD-PCR represent accurate and fast tools (few hours versus many weeks for biochemical tests) to identify and differentiate M. bovis from other MTBC members and NTM species. Furthermore, they have a low cost compared to GenoType MTBC (1.8$ versus 34$ for one test) and do not require expensive equipment and reagents as squencing.

This study had some limitations: first, the two methods were tested using MTBC isolates and were not evaluated directly in lymph nodes samples. Second, mutation pncA C169G was also found in M. bovis BCG strains 8,14. In addition, some PZA resistant M. tuberculosis isolates could display a mutation at this position. Consequently, these strains could be misidentified by pncA-RFLP as M. bovis.

However, M. bovis BCG is rarely isolated from lymph node samples. In addition, a recent study in Tunisia has not reported any mutation at this position in PZA resistant M. tuberculosis isolates 18.

Finally, it was shown that some M. caprae strains and some M. tuberculosis isolates belonging to lineage 3 displayed the RD4 deleted 2,19,20. Despite this finding, RD4 cannot be ruled out until further genomic deletion will be found to well distinguish between these species 19.

Conclusions

pncA-RFLP and RD-PCR represent a rapid, accurate tools to detect M. bovis in tuberculosis lymph nodes cultures compared with phenotypic and biochemical tests. They could be implemented easily in each laboratory owing to their easy use and low cost, in comparison with the DNA strip assay: GenoType MTBC and sequencing.