Journal List > Ann Clin Microbiol > v.23(1) > 1144294

Cho, Kim, and Uh: Frequency of Mycobacterium tuberculosis AmongM. tuberculosis Complex Strains Isolated from Clinical Specimen

Abstract

Background

Rapid and accurate detection of Mycobacterium tuberculosis (MTB) is of primary importance for infection control and selection of anti-tuberculosis drugs. However, most clinical laboratories report MTB complex (MTC) without reporting MTB because MTC comprising MTB, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium caprae and Mycobacterium pinnipedii have 99.9% similarity at the nucleotide level and identical 16S rRNA sequences. This study was conducted to analyze the species.

Methods

Of 310 MTC isolates obtained from clinical samples in a tertiary care hospital from February 2017 to August 2018, MolecuTech Real TB-Taq (YD Diagnostics, Korea) real-time PCR was performed, specifically to detect MTB. For DNA showing MTB negative results by MTB-specific real-time PCR or pyrazinamide-resistant strains, PCR-based MTC typing, spoligotyping, and exact tandem repeat D gene sequencing were performed.

Results

All the 310 MTC isolates were identified to be MTB. Two MTB strains of East-African-Indian 4-Vietnam genotype, which have not been reported in Korea, were also found.

Conclusion

There was no zoonotic tuberculosis in this study. Since we investigated only 310 MTC isolates detected in only one medical institution, multi-center study is needed to accurately know the prevalence of zoonotic tuberculosis in Korea.

References

1. Rodriguez-Campos S, Smith NH, Boniotti MB, Aranaz A. Overview and phylogeny of Mycobacterium tuberculosis complex organisms: Implications for diagnostics and legislation of bovine tuberculosis. Res Vet Sci. 2014; 97:S5–19.
crossref
2. Rastogi N, Legrand E, Sola C. The mycobacteria: An introduction to nomenclature and pathogenesis. Rev Sci Tech. 2001; 20:21–54.
crossref
3. Hwang S, Oh KJ, Jang IH, Uh Y, Yoon KJ, Kim HY, et al. Evaluation of the diagnostic performance of the AdvanSure TB/NTM realtime PCR kit for detection of mycobacteria. Korean J Clin Microbiol. 2011; 14:55–9.
crossref
4. Huard RC, Lazzarini LC, Butler WR, van Soolingen D, Ho JL. PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions. J Clin Microbiol. 2003; 41:1637–50.
5. Djelouadji Z, Raoult D, Daffé M, Drancourt M. A single-step sequencing method for the identification of Mycobacterium tuberculosis complex species. PLoS Negl Trop Dis. 2008; 2:e253.
crossref
6. Coitinho C, Greif G, Robello C, van Ingen J, Rivas C. Identification of Mycobacterium tuberculosis complex by polymerase chain reaction of exact tandem repeat-D fragment from mycobacterial cultures. Int J Mycobacteriol. 2012; 1:146–8.
crossref
7. World Health Organization. Global tuberculosis report 2018. Geneva: WHO press;2018.
8. World Health Organization, Food and Agriculture Organization of the United Nations (FAO) and World Organisation for Animal Health(OIE). Roadmap for zoonotic tuberculosis. Geneva: WHO press;2017.
9. Müller B, Dürr S, Alonso S, Hattendorf J, Laisse CJ, Parsons SD, et al. Zoonotic Mycobacterium bovis-induced tuberculosis in humans. Emerg Infect Dis. 2013; 19:899–908.
10. Dürr S, Müller B, Alonso S, Hattendorf J, Laisse CJ, van Helden PD, et al. Differences in primary sites of infection between zoonotic and human tuberculosis: results from a worldwide systematic review. PLoS Negl Trop Dis. 2013; 7:e2399.
crossref
11. Lim SK, Park JY, Park SD, Chang HK. Localized empyema due to Mycobacterium bovis. Korean J Med. 2012; 81:792–6.
12. Brown T, Nikolayevskyy V, Velji P, Drobniewski F. Associations between Mycobacterium tuberculosis strains and phenotypes. Emerg Infect Dis. 2010; 16:272–80.
13. Das S, Das SC, Verma R. Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis. Microbiol Immunol. 2007; 51:231–4.
14. Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol. 1997; 35:907–14.
crossref
15. Azadi D, Motallebirad T, Ghaffari K, Shojaei H. Mycobacteriosis and tuberculosis: laboratory diagnosis. Open Microbiol J. 2018; 12:41–58.
crossref
16. Nguyen VA, Choisy M, Nguyen DH, Tran TH, Pham KL, Dinh PT, et al. High prevalence of Beijing and EAI4-VNM genotypes among M. tuberculosis isolates in northern Vietnam: sampling effect, rural and urban disparities. PLoS One. 2012; 7:e45553.
crossref

Table 1.
Primers used in PCR-based MTB complex typing
Primer type and target locus Primer name Nucleotide sequence (5' → 3') Size, base pair
Rv0577 Rv0577F ATG CCC AAG AGA AGC GAA TAC AGG CAA 786
  Rv0577R CTA TTG CTG CGG TGC GGG CTT CAA  
IS1561 (Rv3349c) IS1561F G CT GGG TGG GCC CTG GAA TAC GTG AAC TC CT 943
  IS1561R A AAC TGC TCA CCC TGG CCA CCA CCA TTG AC CT
Rv1510 (RD4) Rv1510F GTG CGC TCC ACC CAA ATA GTT GC 1,033
  Rv1510R TGT CGA CCT GGG GCA CAA ATC AGT C  
Rv1970 (RD7) Rv1970F GCG CAG CTG CCG GAT GTC AAC 1,116
  Rv1970R CGC CGG CAG CCT CAC GAA ATG  
Rv3877/8 (RD1) Rv3877/8F CGA CGG GTC TGA CGG CCA AAC TCA TC 999
  Rv3877/8R CTT GCT CGG TGG CCG GTT TTT CAG C  
Rv3120 (RD12) Rv3120F G GTC GGC GAT AGA CCA TGA GTC CGT CTC CAT 404
  Rv3120R GCG AAA AGT GGG CGG ATG CCA GAA TAG T  
Rv2073c (RD9) Rv2073cF TCG CCG CTG CCA GAT GAG TC 600
  Rv2073cR TTT GGG AGC CGC CGG TGG TGA TGA  
Abbreviation: MTB, Mycobac cterium tuberculosis; IS, in sertion sequence; RD, region of difference.  
Table 2.
Primers used in spoligotyping and exact tandem repeat D sequencing
Primer type and target locus Primer name Nucleotide sequence (5' → 3') Size, base pair
Spoligotyping      
DR DRa GGT TTT GGG TCT GAC GAC Variable
  DRb CCG AGA GGG GAC GGA AAC  
ETR-D sequencing      
ETR-D ETRDF GTT GAT CGA GGC CTA TCA CG Variable
  ETRDR GAA TAG GGC TTG GTC ACG TA  

Abbreviation: DR, direct repeat; ETRDF, exact tandem repeat D forward; ETRDR, exact tandem repeat D reverse.

Table 3.
Characteristics of 310 Mycobacterium tuberculosis complex cases in clinical specimen
Variables M. tuberculosis complex No. (%)
Age, years  
0–9 1 (0.3)
10–19 5 (1.6)
20–29 11 (3.6)
30–39 13 (4.2)
40–49 36 (11.6)
50–59 53 (17.1)
60–69 31 (10.0)
≥70 160 (51.6)
Sex  
Male 182 (58.7)
Female 128 (41.3)
Specimen  
Sputum 141 (45.5)
Bronchial washing 140 (45.2)
Pleural fluid 9 (2.9)
Urine 3 (1.0)
Cerebrospinal fluid 2 (0.6)
Colon 2 (0.6)
Lymph node 2 (0.6)
Others 11 (3.6)
Pyrazinamide susceptibility  
Resistant 7 (2.3)
Susceptible 303 (97.7)
Table 4.
PCR-based MTB complex typing
Organisms (No.) Rv0577 IS1561 PCR-based MTB complex typing
Rv1510 (RD4) Rv1970 (RD7) Rv3877/8 (RD1) Rv3120 (RD12) Rv2073c (RD9)
MTB H37Rv + + + + + + +
M. bovis AN5 + + +
M. bovis* + + +
M. bovis BCG + +
M. africanum + + + + +
Isolates (3) + + + + + + +
Isolates (7) + + + + + + +

* Isolate from cattle.

† Isolates were positive in Advansure Mycobacterium Genoblot Assay but negative in M. tuberculosis specific real-time PCR.

‡ Pyrazinamide-resistant strains.

Abbreviation: MTB, Mycobacterium tuberculosis; IS, insertion sequence; RD, region of difference; BCG, Bacillus Calmette–Guérin.

Table 5.
Spoligotyping-based MTB complex differentiation
Species/ Specimen No. Octal value SIT Clade (strain) Spoligotype pattern
MTB H37Rv 777777477760771 451 T-H37Rv  
M. bovis AN5 676773777777600 482 BOV_1  
M. bovis 664073777777600 683 BOV_2  
M. bovis BCG 676773777777600 482 BOV_1  
M. africanum 770777777777671 181 AFRI_1  
Y1* 777777777760571 520 T1  
Y2* 777737774413771 564 EAI4-VNM  
Y3* 777737774413771 564 EAI4-VNM  
P1 777737777760771 37 T3  
P2 000000000003771 1 Beijing  
P3 000000000003771 1 Beijing  
P4 000000000003771 1 Beijing  
P5 000000000003771 1 Beijing  
P6 000000000003771 1 Beijing  
P7 000000000003771 1 Beijing  
S1 777777777763771 54 Manu2  
S2 000000000003771 1 Beijing  

* Y1–3, Mycobacteriu um tuberculosis-negaative strains for real-time PCR

† P1–7, pyrazinamide-resistant strains.

‡ S1–2, M. tuberculosi is-positive strains for real-time PCR.

Abbreviation: MTB, M. tuberculosis; SIT, shared international type; EAI4-VNM, East-African-Indian 14-Vietnam; BCG, Bacillus Calmette–Guérin.

Table 6.
Sequencing based MTB complex differentiation for exact tandem repeat D
Organisms Total No. of ETR-D
MTB H37Rv 3 (3 × 77 bp)
M. bovis AN5 4 (3 × 77 bp, 1 × 53 bp)
M. bovis* 4 (3 × 77 bp, 1 × 53 bp)
M. bovis BCG 2 (2 × 77 bp)
M. africanum 4 (3 × 77 bp, 1 × 53 bp)
Isolate No. 1 3 (2 × 77 bp, 1 × 53 bp)
Isolate No. 2 10 (9 × 77 bp, 1 × 53 bp)
Isolate No. 3 8 (7 × 77 bp, 1 × 53 bp)
* Isolate from cattle.  

Isolate was positive in Advansure Mycobacterium Genoblot Assay but negative in Mycobacterium tuberculosis specific real-time PCR. Abbreviation: MTB, M. tuberculosis; ETR-D, exact tandem repeat-D; bp, base pair; BCG, Bacillus Calmette–Guérin.

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