Frequency of Mycobacterium tuberculosis AmongM. tuberculosis Complex Strains Isolated from Clinical Specimen

Hyunmi Cho; Jong-Bae Kim; Young Uh

doi:10.5145/ACM.2020.23.1.1

Journal List > Ann Clin Microbiol > v.23(1) > 1144292

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

Original Article

Ann Clin Microbiol 2020;23(1):21-12.

Published online: 12 March 2020

DOI: https://doi.org/10.5145/ACM.2020.23.1.1

Frequency of Mycobacterium tuberculosis AmongM. tuberculosis Complex Strains Isolated from Clinical Specimen

Hyunmi Cho¹, Jong-Bae Kim², Young Uh¹

^¹Department of Laboratory Medicine, Wonju Severance Christian Hospital, Yonsei University Wonju College of Medicine, Wonju

^²Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Korea

Corresponding author Young Uh E-mail: u931018@yonsei.ac.kr Tel: +82-33-741-1592 Fax: +82-33-731-0506

Received 12 June 2019 Revised 19 August 2019 Accepted 19 August 2019

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Mycobacterium tuberculosis complex, Sequence analysis, Spoligotyping

References

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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
Organisms (No.)	Rv0577	IS1561	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)
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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