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Utility and evaluation of new variable–number tandem-repeat systems for genotyping mycobacterial tuberculosis isolates
Journal of Microbiological Methods 77 (2009) 127–129
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j m i c m e t h
Note
Utility and evaluation of new variable–number tandem-repeat systems for genotyping mycobacterial tuberculosis isolates Horng-Yunn Dou a,1, Jang-Jih Lu b,c,1, Chih-Wei Lin a, Jia-Ru Chang a, Jun-Ren Sun b, Ih-Jen Su a,⁎ a b c
Division of Clinical Research, National Health Research Institutes, Zhunan, Taiwan. 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan, ROC Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, 325 Sec. 2 Chengkung Road, Taipei 114, Taiwan, ROC Department of Laboratory Medicine, China Medical University Hospital, 2, Yuh-Der Road, Taichung 404, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 29 August 2008 Received in revised form 24 December 2008 Accepted 12 January 2009 Available online 20 January 2009 Keywords: Mycobacterium tuberculosis MIRU–VNTR typing
a b s t r a c t We compared mycobacterial interspersed repetitive unit (MIRU)–variable number tandem repeat (VNTR) typing to traditional spoligotyping for discriminating Mycobacterium tuberculosis (MTB) strains. Our 17-loci MIRU–VNTR typing method was found to be superior to spoligotyping for non-Beijing family strains. To extend the method we also established PCR-based rapid genotyping protocols for Beijing, East-African-Indian and U lineages. © 2009 Elsevier B.V. All rights reserved.
Tuberculosis (TB) remains a worldwide human health problem. It is estimated that about one third of the world's population has been infected with Mycobacterium tuberculosis (MTB) bacilli, and 1.7 million persons died of the disease in 2006 (World Health Organization, 2008.). Globally, the tuberculosis epidemic consists of multiple genotype-specific sub-epidemics. Different MTB genotypes can be identified by variation in certain well-characterized repetitive sequences such as the IS6110 transposable element and the direct repeat (DR) region (Bhanu et al., 2002). The Beijing genotype family is well recognized as a distinct genetic lineage (Bifani et al., 2002) consisting of four monophyletic subgroups defined by large sequence polymorphisms (Brosch et al., 2002). It is dispersed worldwide yet predominates in certain geographic areas, particularly in parts of Asia (Cave et al., 1991; Chan et al., 2001) and Russia (Cowan et al., 2002). Molecular methods have gained increased acceptance as a tool in conjunction with conventional epidemiology to monitor the transmission of TB (Van Soolingen, 2001). Spoligotyping is a practical and reproducible PCR-based method which assays the presence or absence of a set of target sequences at the DR locus (Kamerbeek et al., 1997). Another molecular technique for strain typing of MTB is based on variable number tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRUs) (Mazars et al., 2001; Supply et al., 2000). This method makes use of the length variation at 12 independent minisatellite-like loci scattered throughout the MTB genome. A number of studies have shown that MIRU–VNTR typing is reliable and reproducible and enables a level of discrimination ⁎ Corresponding author. Tel.: +886 37 246 166, 35501; fax: +886 37 586 457. E-mail address: [email protected] (I.-J. Su). 1 Contributed equally to this manuscript. 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.01.007
between strains that is comparable to that of IS6110 typing (Mazars et al., 2001; Sola et al., 2003; Supply et al., 2001; Kolk et al., 1992). Here we further evaluated MIRU–VNTR typing on a set of 321 MTB complex clinical isolates from Taipei, Taiwan. Our aim was to establish the range of applicability of MIRU–VNTR typing in epidemiology and to evaluate its discriminatory power compared to spoligotyping. Three-hundred twenty-one samples were randomly collected from patients attending Tri-Service General Hospital between 2002 and 2005. All of the patients were sputum microscopy positive and culture positive for TB. Mycobacterial genomic DNA was extracted from cultured cells as described previously (Brudey et al., 2006; Kox et al.,1994). Spoligotyping was carried out using a spoligotyping kit according to the manufacturer's instructions (Isogen Bioscience B.V.). Spoligotypes common to more than one strain were designated as shared types (ST) and assigned a shared international type number (SIT) according to the updated version of the international spoligotype database SpolDB4 (Brudey et al., 2006). Of the 321 isolates typed by spoligotyping, 258 (80%) fell into clusters; the maximum cluster size was 158. Among all of the isolates, 258 (80%) displayed one of 24 spoligotypes and 63 (20%) displayed unique spoligotypes (Table 1). The largest cluster was composed of the Beijing genotype (166/321; 51%), followed by Haarlem (H3) (43/321; 13%) and East African Indian (EAI; 35/321; 11%). MIRU–VNTR loci types ETR-A, B, C, D, E, F, MPTR-A, and MIRU-2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40 were individually amplified and analyzed as previously described by Supply et al. (2000). For MIRU– VNTR analysis, PCRs were carried out as follows. Five microliters of fivefold-diluted DNA solutions were added to a final volume of 50 µl containing the following reagents (Gibco-BRL): 0.2 µl of Herculase II Fusiog DNA polymerase (STRATAGENE,USA); 0.2 mM each of dATP, dCTP, dGTP, and dTTP; 5 µl of PCR buffer; 0.4 µM (2 µM for locus 7) of
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H.-Y. Dou et al. / Journal of Microbiological Methods 77 (2009) 127–129
Table 1 Comparison of the discriminatory power of spoligotyping and VNTR analyses Typing method
Spoligotyping 12-locus MIRU– VNTR 17-locus VNTR (12 MIRU and 5 ETRs) a
Total no. of type patterns
No. of unique types
No. of clusters
No. of clustered isolates (%)
Maximum no. of isolates in a cluster
HGDIa
87 140
63 109
24 31
258 (80) 212 (66)
158 84
0.751 0.919
165
138
37
183 (57)
80
0.962
Table 3 MIRU–VNTR loci used to subdivide each lineage into true clustered and true unique isolates Defined MIRU-VNTRa No. of Genotype No. of No. of clusters cluster unique loci repeat no./total isolates isolates isolates (%)
Beijing EAI U
HGDI was calculated as described by Hunter and Gaston (1998).
12 3
132 31
34 4
2
4
4
Combined MIRU– VNTR (n) No. of isolates with correct combined repeat no./total isolates (%)
26–≥6 96 31–≥ 5 95 93 D–≥6 100 4–≥ 5 100 95 24–2 98 26–2 98 A-5 100 10-8 88 88
a
primers; and 1 to 3.5 mM MgCl2. The primers and MgCl2 concentrations were as described by Mazars et al. (2001). The PCR fragments were analyzed by 1.5% agarose gel electrophoresis. Sizes of the amplicons were estimated by comparison with 50- and 100-bp ladders. MIRU–VNTR typing detected 140 different patterns, 212 of which were grouped into 31 clusters. The largest cluster comprised 84 from the Beijing family with an MIRU–VNTR profile of 223325173533. Thirty-five strains with unique patterns were identified in the Beijing family. The 166 Beijing strains clustered by spoligotyping were divided into 47 different MIRU genotypes. Of these 166 strains, 131 were grouped into 12 clusters. The Haarlem family was divided into 24 MIRU genotypes and five clusters; the largest cluster consisted of 8 genotypes with MIRU profile 222225153323. The T family was divided into 19 different MIRU genotypes. The EIA family, U family, and LAM were divided into 7, 6, and 3 different MIRU genotypes, respectively. The ‘other’ family (comprising MANU 2, Bovis 1, and unclassified) contains 34 different MIRU genotypes. The Hunter–Gaston equation (Hunter and Gaston, 1988) was used to calculate alleleic diversity (h) or the Hunter–Gaston discrimination index (HGDI) at each locus, according to Struelens (1996). Based on this index, three MIRU loci were designated as being “highly discriminating” (h N 0.6) (MIRU nos. 10, 26, and 31), three were designated as “moderately discriminating” (0.3 ≤h ≤ 0.6) (MIRU nos. 4, 39, and 40), and seven MIRUs (MIRU nos. 2, 6, 20, 23, 24, 27, and 40) were found to be poorly discriminating (h b 0.3) (Table 2). A similar calculation was also made for the five complementary VNTR loci (ETR-A, ETR-B, ETR-C, ETR-F,
Table 2 Allelic diversity of each MIRU locus MIRU
MIRU2 MIRU4, ETR-Db MIRU10 MIRU16 MIRU20 MIRU23 MIRU24 MIRU26 MIRU27 MIRU31, ETR-E MIRU39 MIRU40 ETR-A ETR-B ETR-C ETR-Fc MPTR-A a
Allele Number of repeats no. (range) 4 11 8 4 2 6 2 10 5 5 4 6 6 8 5 6 3
1–4 0–7,9,11,3 1–5,7–9 1–4 2 1–6 1–2 1–10 0–4 2–6 1–4 1–4,6–7 2–7 1–8 2–6 1+1,2+1,0+2,1 +2,2+2,3+2 13,16
h indexa All Beijing Non-Beijing isolates isolates isolates 0.067 0.364 0.643 0.281 0.025 0.289 0.213 0.751 0.183 0.639 0.523 0.460 0.545 0.564 0.148 0.510 0.025
0 0.048 0.279 0.093 0.012 0.024 0.012 0.440 0.012 0.138 0.221 0.192 0.249 0.048 0.127 0 0
0.135 0.597 0.541 0.432 0.038 0.484 0.379 0.703 0.333 0.507 0.425 0.621 0.624 0.658 0.169 0.651 0.051
h index represents the allelic diversity of each locus, (see text). MIRU-4 contained 3 complete 77-bp repeats followed by an additional internal deletions of the VNTR sequence. c ETR-F is a complex locus composed of 55-bp and 79-bp repeat units of tandem repeats. b
Each code consists of the MIRU (number) or ETR (letter) designation followed by a dash and the number of copies. For example, 26 ≥ 6 indicates ≥ 6 copies of MIRU-26; A-5 indicates 5 copies of ETR-A.
and MPTR-A). ETR-B appears to be the most discriminative of these VNTR alleles (h = 0.564) and MPTRA the least discriminative (h = 0.025) (Table 2). There were important differences between Beijing family and non-Beijing isolates. For the Beijing family isolates, none of the 17 loci are highly discriminative; MIRU-26 is the only moderately discriminative locus; the others are poorly discriminative. Therefore, the proposed MIRU–VNTR typing method would not by itself be a useful molecular epidemiological tool in areas where Beijing strains are prevalent, such as in Taipai. However, for non-Beijing isolates, loci 26, 40, ETR-A, ETR-B, and ETR-F are highly discriminative; loci 4, 10, 16, 23, 24, 31, and 39 are moderately discriminative; and loci 2, 20, and ETR-C are poorly discriminative. About 82% of the 17 loci had an allelic diversity greater than 0.3. Remarkably, the order of magnitude of the diversity of the MIRU loci overall is the same as that previously observed in two collections of isolates from other geographic origins (Mazars et al., 2001; Supply et al., 2001). The present classification also agrees with that found by (Cowan et al., 2002) in a local collection from Michigan, USA, although the absolute values of allelic diversity are higher in our bacterial sample, which was selected for diversity. Allelic diversity was found to be low at certain loci and high at other loci. For example, in the Beijing lineage the majority of isolates contained more than six copies of MIRU-26 (96%) and five copies of MIRU-31 (95%) (Table 3). Taken together, 93% of the isolates in the lineage contain both polymorphisms. The same analysis was performed on the other lineages. The EAI lineage is highly conserved, with 95% of isolates containing more than five copies of MIRU-4, ≥6 copies of ETR-D, and two copies of MIRU-24 and MIRU-26. The U lineage also displays a high level of conservation, with 88% of isolates containing eight copies of MIRU-10 and 100% of isolates containing five copies of ETR-A. Therefore, these three strains lend themselves to PCR-based rapid and accurate genotyping. The present protocol, on the basis of its simplicity, specificity, and sensitivity, could provide an alternative genotyping method for clinical microbiology laboratories. Our results clearly demonstrate that MIRU typing is more discriminating than spoligotyping. The basic MIRU–VNTR system was shown to achieve good discrimination of these strains; therefore, we suggest that this MIRU–VNTR scheme could be used as the firstline screening method for routine epidemiological investigation of M. tuberculosis isolates in the Beijing area. However, secondary subtyping, either by IS6110-RFLP analysis or by testing hypervariable VNTR loci (Iwamoto et al., 2007), should be performed for clustered isolates. Acknowledgements This project was supported by grants from the National Health Research Institutes, National Science Council (NSC97-3112-B-400012), and the Department of Health (DOH97-DC-1501-01), and Department of Defense (DOD97-22-01), Taiwan. We thank the mycobacteriology laboratory of Tri-Service General Hospital for providing bacterial isolates. All participants of this consortium are acknowledged for valuable discussions.
H.-Y. Dou et al. / Journal of Microbiological Methods 77 (2009) 127–129
References Bhanu, N.V., van Soolingen, D., van Embden, J.D., Dar, L., Pandey, R.M., Seth, P., 2002. Predominace of a novel Mycobacterium tuberculosis genotype in the Delhi region of India. Tuberculosis 82, 105–112 (Edinb). Bifani, P.J., Mathema, B., Kurepina, N.E., Kreiswirth, B.N., 2002. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol. 10, 45–52. Brosch, R., Gordon, S.V., Marmiesse, M., Brodin, P., Buchrieser, C., Eiglmeier, K., et al., 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl. Acad. Sci. U S A. 99, 3684–3689. Brudey, K., Driscoll, J.R., Rigouts, L., Prodinger, W.M., Gori, A., Al-Hajoj, S.A., et al., 2006. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6, 23. Cave, M.D., Eisenach, K.D., McDermott, P.F., Bates, J.H., Crawford, J.T., 1991. IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting. Mol. Cell. Probes 5, 73–80. Chan, M.Y., Borgdorff, M., Yip, C.W., de Haas, P.E., Wong, W.S., Kam, K.M., et al., 2001. Seventy percent of the Mycobacterium tuberculosis isolates in Hong Kong represent the Beijing genotype. Epidemiol. Infect. 127, 169–171. Cowan, L.S., Mosher, L., Diem, L., Massey, J.P., Crawford, J.T., 2002. Variable–number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units. J. Clin. Microbiol. 40, 1592–1602. Hunter, P.R., Gaston, M.A., 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26, 2465–2466. Iwamoto, T., Yoshida, S., Suzuki, K., Tomita, M., Fujiyama, R., Tanaka, N., et al., 2007. Hypervariable loci that enhance the discriminatory ability of newly proposed 15loci and 24-loci variable–number tandem repeat typing method on Mycobacterium tuberculosis strains predominated by the Beijing family. FEMS Microbiol. Lett. 270, 67–74.
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Kamerbeek, J., Schouls, L., Kolk, A., van Agterveld, M., van Soolingen, D., Kuijper, S., et al., 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35, 907–914. Kolk, A.H., Schuitema, A.R., Kuijper, S., van Leeuwen, J., Hermans, P.W., van Embden, J.D., et al., 1992. Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system. J. Clin. Microbiol. 30, 2567–2575. Kox, L.F., Rhienthong, D., Miranda, A.M., Udomsantisuk, N., Ellis, K., van Leeuwen, J., et al., 1994. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J. Clin. Microbiol. 32, 672–678. Mazars, E., Lesjean, S., Banuls, A.L., Gilbert, M., Vincent, V., Gicquel, B., et al., 2001. Highresolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc. Natl. Acad. Sci U S A. 98, 1901–1906. Sola, C., Filliol, I., Legrand, E., Lesjean, S., Locht, C., Supply, P., et al., 2003. Genotyping of the Mycobacterium tuberculosis complex using MIRUs: association with VNTR and spoligotyping for molecular epidemiology and evolutionary genetics. Infect. Genet. Evol. 3, 125–133. Struelens, M.J., 1996. Consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems. Clin. Microbiol. Infect. 2, 2–11. Supply, P., Mazars, E., Lesjean, S., Vincent, V., Gicquel, B., Locht, C., 2000. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol. Microbiol. 36, 762–771. Supply, P., Lesjean, S., Savine, E., Kremer, K., van Soolingen, D., Locht, C., 2001. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J. Clin. Microbiol. 39, 3563–3571. Van Soolingen, D., 2001. Molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements. J. Intern. Med. 249, 1–26. World Health Organization, 2008. http://www.who.int/tb/publications/global_report/ 2008/key_points/en/index.html. Global tuberculosis control: surveillance, planning, financing.
Contents lists available at ScienceDirect
Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j m i c m e t h
Note
Utility and evaluation of new variable–number tandem-repeat systems for genotyping mycobacterial tuberculosis isolates Horng-Yunn Dou a,1, Jang-Jih Lu b,c,1, Chih-Wei Lin a, Jia-Ru Chang a, Jun-Ren Sun b, Ih-Jen Su a,⁎ a b c
Division of Clinical Research, National Health Research Institutes, Zhunan, Taiwan. 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan, ROC Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, 325 Sec. 2 Chengkung Road, Taipei 114, Taiwan, ROC Department of Laboratory Medicine, China Medical University Hospital, 2, Yuh-Der Road, Taichung 404, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 29 August 2008 Received in revised form 24 December 2008 Accepted 12 January 2009 Available online 20 January 2009 Keywords: Mycobacterium tuberculosis MIRU–VNTR typing
a b s t r a c t We compared mycobacterial interspersed repetitive unit (MIRU)–variable number tandem repeat (VNTR) typing to traditional spoligotyping for discriminating Mycobacterium tuberculosis (MTB) strains. Our 17-loci MIRU–VNTR typing method was found to be superior to spoligotyping for non-Beijing family strains. To extend the method we also established PCR-based rapid genotyping protocols for Beijing, East-African-Indian and U lineages. © 2009 Elsevier B.V. All rights reserved.
Tuberculosis (TB) remains a worldwide human health problem. It is estimated that about one third of the world's population has been infected with Mycobacterium tuberculosis (MTB) bacilli, and 1.7 million persons died of the disease in 2006 (World Health Organization, 2008.). Globally, the tuberculosis epidemic consists of multiple genotype-specific sub-epidemics. Different MTB genotypes can be identified by variation in certain well-characterized repetitive sequences such as the IS6110 transposable element and the direct repeat (DR) region (Bhanu et al., 2002). The Beijing genotype family is well recognized as a distinct genetic lineage (Bifani et al., 2002) consisting of four monophyletic subgroups defined by large sequence polymorphisms (Brosch et al., 2002). It is dispersed worldwide yet predominates in certain geographic areas, particularly in parts of Asia (Cave et al., 1991; Chan et al., 2001) and Russia (Cowan et al., 2002). Molecular methods have gained increased acceptance as a tool in conjunction with conventional epidemiology to monitor the transmission of TB (Van Soolingen, 2001). Spoligotyping is a practical and reproducible PCR-based method which assays the presence or absence of a set of target sequences at the DR locus (Kamerbeek et al., 1997). Another molecular technique for strain typing of MTB is based on variable number tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRUs) (Mazars et al., 2001; Supply et al., 2000). This method makes use of the length variation at 12 independent minisatellite-like loci scattered throughout the MTB genome. A number of studies have shown that MIRU–VNTR typing is reliable and reproducible and enables a level of discrimination ⁎ Corresponding author. Tel.: +886 37 246 166, 35501; fax: +886 37 586 457. E-mail address: [email protected] (I.-J. Su). 1 Contributed equally to this manuscript. 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.01.007
between strains that is comparable to that of IS6110 typing (Mazars et al., 2001; Sola et al., 2003; Supply et al., 2001; Kolk et al., 1992). Here we further evaluated MIRU–VNTR typing on a set of 321 MTB complex clinical isolates from Taipei, Taiwan. Our aim was to establish the range of applicability of MIRU–VNTR typing in epidemiology and to evaluate its discriminatory power compared to spoligotyping. Three-hundred twenty-one samples were randomly collected from patients attending Tri-Service General Hospital between 2002 and 2005. All of the patients were sputum microscopy positive and culture positive for TB. Mycobacterial genomic DNA was extracted from cultured cells as described previously (Brudey et al., 2006; Kox et al.,1994). Spoligotyping was carried out using a spoligotyping kit according to the manufacturer's instructions (Isogen Bioscience B.V.). Spoligotypes common to more than one strain were designated as shared types (ST) and assigned a shared international type number (SIT) according to the updated version of the international spoligotype database SpolDB4 (Brudey et al., 2006). Of the 321 isolates typed by spoligotyping, 258 (80%) fell into clusters; the maximum cluster size was 158. Among all of the isolates, 258 (80%) displayed one of 24 spoligotypes and 63 (20%) displayed unique spoligotypes (Table 1). The largest cluster was composed of the Beijing genotype (166/321; 51%), followed by Haarlem (H3) (43/321; 13%) and East African Indian (EAI; 35/321; 11%). MIRU–VNTR loci types ETR-A, B, C, D, E, F, MPTR-A, and MIRU-2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40 were individually amplified and analyzed as previously described by Supply et al. (2000). For MIRU– VNTR analysis, PCRs were carried out as follows. Five microliters of fivefold-diluted DNA solutions were added to a final volume of 50 µl containing the following reagents (Gibco-BRL): 0.2 µl of Herculase II Fusiog DNA polymerase (STRATAGENE,USA); 0.2 mM each of dATP, dCTP, dGTP, and dTTP; 5 µl of PCR buffer; 0.4 µM (2 µM for locus 7) of
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H.-Y. Dou et al. / Journal of Microbiological Methods 77 (2009) 127–129
Table 1 Comparison of the discriminatory power of spoligotyping and VNTR analyses Typing method
Spoligotyping 12-locus MIRU– VNTR 17-locus VNTR (12 MIRU and 5 ETRs) a
Total no. of type patterns
No. of unique types
No. of clusters
No. of clustered isolates (%)
Maximum no. of isolates in a cluster
HGDIa
87 140
63 109
24 31
258 (80) 212 (66)
158 84
0.751 0.919
165
138
37
183 (57)
80
0.962
Table 3 MIRU–VNTR loci used to subdivide each lineage into true clustered and true unique isolates Defined MIRU-VNTRa No. of Genotype No. of No. of clusters cluster unique loci repeat no./total isolates isolates isolates (%)
Beijing EAI U
HGDI was calculated as described by Hunter and Gaston (1998).
12 3
132 31
34 4
2
4
4
Combined MIRU– VNTR (n) No. of isolates with correct combined repeat no./total isolates (%)
26–≥6 96 31–≥ 5 95 93 D–≥6 100 4–≥ 5 100 95 24–2 98 26–2 98 A-5 100 10-8 88 88
a
primers; and 1 to 3.5 mM MgCl2. The primers and MgCl2 concentrations were as described by Mazars et al. (2001). The PCR fragments were analyzed by 1.5% agarose gel electrophoresis. Sizes of the amplicons were estimated by comparison with 50- and 100-bp ladders. MIRU–VNTR typing detected 140 different patterns, 212 of which were grouped into 31 clusters. The largest cluster comprised 84 from the Beijing family with an MIRU–VNTR profile of 223325173533. Thirty-five strains with unique patterns were identified in the Beijing family. The 166 Beijing strains clustered by spoligotyping were divided into 47 different MIRU genotypes. Of these 166 strains, 131 were grouped into 12 clusters. The Haarlem family was divided into 24 MIRU genotypes and five clusters; the largest cluster consisted of 8 genotypes with MIRU profile 222225153323. The T family was divided into 19 different MIRU genotypes. The EIA family, U family, and LAM were divided into 7, 6, and 3 different MIRU genotypes, respectively. The ‘other’ family (comprising MANU 2, Bovis 1, and unclassified) contains 34 different MIRU genotypes. The Hunter–Gaston equation (Hunter and Gaston, 1988) was used to calculate alleleic diversity (h) or the Hunter–Gaston discrimination index (HGDI) at each locus, according to Struelens (1996). Based on this index, three MIRU loci were designated as being “highly discriminating” (h N 0.6) (MIRU nos. 10, 26, and 31), three were designated as “moderately discriminating” (0.3 ≤h ≤ 0.6) (MIRU nos. 4, 39, and 40), and seven MIRUs (MIRU nos. 2, 6, 20, 23, 24, 27, and 40) were found to be poorly discriminating (h b 0.3) (Table 2). A similar calculation was also made for the five complementary VNTR loci (ETR-A, ETR-B, ETR-C, ETR-F,
Table 2 Allelic diversity of each MIRU locus MIRU
MIRU2 MIRU4, ETR-Db MIRU10 MIRU16 MIRU20 MIRU23 MIRU24 MIRU26 MIRU27 MIRU31, ETR-E MIRU39 MIRU40 ETR-A ETR-B ETR-C ETR-Fc MPTR-A a
Allele Number of repeats no. (range) 4 11 8 4 2 6 2 10 5 5 4 6 6 8 5 6 3
1–4 0–7,9,11,3 1–5,7–9 1–4 2 1–6 1–2 1–10 0–4 2–6 1–4 1–4,6–7 2–7 1–8 2–6 1+1,2+1,0+2,1 +2,2+2,3+2 13,16
h indexa All Beijing Non-Beijing isolates isolates isolates 0.067 0.364 0.643 0.281 0.025 0.289 0.213 0.751 0.183 0.639 0.523 0.460 0.545 0.564 0.148 0.510 0.025
0 0.048 0.279 0.093 0.012 0.024 0.012 0.440 0.012 0.138 0.221 0.192 0.249 0.048 0.127 0 0
0.135 0.597 0.541 0.432 0.038 0.484 0.379 0.703 0.333 0.507 0.425 0.621 0.624 0.658 0.169 0.651 0.051
h index represents the allelic diversity of each locus, (see text). MIRU-4 contained 3 complete 77-bp repeats followed by an additional internal deletions of the VNTR sequence. c ETR-F is a complex locus composed of 55-bp and 79-bp repeat units of tandem repeats. b
Each code consists of the MIRU (number) or ETR (letter) designation followed by a dash and the number of copies. For example, 26 ≥ 6 indicates ≥ 6 copies of MIRU-26; A-5 indicates 5 copies of ETR-A.
and MPTR-A). ETR-B appears to be the most discriminative of these VNTR alleles (h = 0.564) and MPTRA the least discriminative (h = 0.025) (Table 2). There were important differences between Beijing family and non-Beijing isolates. For the Beijing family isolates, none of the 17 loci are highly discriminative; MIRU-26 is the only moderately discriminative locus; the others are poorly discriminative. Therefore, the proposed MIRU–VNTR typing method would not by itself be a useful molecular epidemiological tool in areas where Beijing strains are prevalent, such as in Taipai. However, for non-Beijing isolates, loci 26, 40, ETR-A, ETR-B, and ETR-F are highly discriminative; loci 4, 10, 16, 23, 24, 31, and 39 are moderately discriminative; and loci 2, 20, and ETR-C are poorly discriminative. About 82% of the 17 loci had an allelic diversity greater than 0.3. Remarkably, the order of magnitude of the diversity of the MIRU loci overall is the same as that previously observed in two collections of isolates from other geographic origins (Mazars et al., 2001; Supply et al., 2001). The present classification also agrees with that found by (Cowan et al., 2002) in a local collection from Michigan, USA, although the absolute values of allelic diversity are higher in our bacterial sample, which was selected for diversity. Allelic diversity was found to be low at certain loci and high at other loci. For example, in the Beijing lineage the majority of isolates contained more than six copies of MIRU-26 (96%) and five copies of MIRU-31 (95%) (Table 3). Taken together, 93% of the isolates in the lineage contain both polymorphisms. The same analysis was performed on the other lineages. The EAI lineage is highly conserved, with 95% of isolates containing more than five copies of MIRU-4, ≥6 copies of ETR-D, and two copies of MIRU-24 and MIRU-26. The U lineage also displays a high level of conservation, with 88% of isolates containing eight copies of MIRU-10 and 100% of isolates containing five copies of ETR-A. Therefore, these three strains lend themselves to PCR-based rapid and accurate genotyping. The present protocol, on the basis of its simplicity, specificity, and sensitivity, could provide an alternative genotyping method for clinical microbiology laboratories. Our results clearly demonstrate that MIRU typing is more discriminating than spoligotyping. The basic MIRU–VNTR system was shown to achieve good discrimination of these strains; therefore, we suggest that this MIRU–VNTR scheme could be used as the firstline screening method for routine epidemiological investigation of M. tuberculosis isolates in the Beijing area. However, secondary subtyping, either by IS6110-RFLP analysis or by testing hypervariable VNTR loci (Iwamoto et al., 2007), should be performed for clustered isolates. Acknowledgements This project was supported by grants from the National Health Research Institutes, National Science Council (NSC97-3112-B-400012), and the Department of Health (DOH97-DC-1501-01), and Department of Defense (DOD97-22-01), Taiwan. We thank the mycobacteriology laboratory of Tri-Service General Hospital for providing bacterial isolates. All participants of this consortium are acknowledged for valuable discussions.
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