Conceived and designed the experiments: KW. Performed the experiments: HD LS XZ BS BL ZL. Analyzed the data: KW HD. Contributed reagents/materials/analysis tools: KW LS. Wrote the paper: KW HD.
The authors have declared that no competing interests exist.
Tuberculosis (TB) is a serious health problem in Tibet where Tibetans are the major ethnic group. Although genotyping of
In our study, a total of 576
The population structure of
Tuberculosis (TB) remains a major health problem in China. A 2000 national TB epidemiology survey conducted in China reported the average prevalence of TB amounts to 367 per 100,000 (0.0036%), with an estimated 4.5 million active pulmonary TB patients and 1.5 million new infections a year
Tibet Autonomous Region (Tibet) is located in the Qinghai-Tibet Plateau of western China and Tibetans account for more than 90% of this population. Based on the 1990 national TB epidemiology survey in China, the prevalence rate of TB in Tibet (1203.06/100,000) was higher than anywhere else in China. In 2005, 4291 active pulmonary TB cases were reported in Tibet, posing a serious threat to the public health in Tibet
Molecular typing of
Our study aimed to examine the strain diversity and the prevalence of Beijing genotype strains in Tibet. The power of the 24-locus MIRU-VNTR scheme to differentiate the Beijing genotype strains from Tibet was also investigated.
A total of 576
In terms of spoligotyping, we recognized a total of 22 distinct spoligotypes among the 576 isolates (
From left to right: 1) UPGMA dendrogram generated by 22 spoligotypes. 2) spoligotying patterns. 3) Spoligo-International-Type number. 4) Genetic lineage according to SITVIT2 database. 5) Strain numbers.
Clustering analysis revealed that 563 isolates were grouped into 9 clusters containing 2 to 512 isolates, while the other 13 isolates inhibited unique spoligotypes. The largest spoligotype lineage was Beijing genotype(90.63%, 522 isolates), most (512 strains) of which belonged to the classical Beijing genotype with a pattern that depicted the absence of the first 34 spacer oligonucleotides and the presence of spacers 35 to 43
Furthermore, there were no statistical significant associations between the Beijing lineage and age (
No. | No. of Beijing genotype | OR | P | 95%CI | |
|
576 | 522 | |||
|
|||||
Male | 329 | 298 | 1 | ||
Female | 247 | 224 | 0.983 | 1.000 | 0.558–1.731 |
|
|||||
0–19 | 57 | 49 | 1 | ||
20–39 | 340 | 312 | 0.561 | 0.211 | 0.242–1.300 |
40–59 | 143 | 129 | 0.678 | 0.457 | 0.268–1.716 |
60– | 36 | 32 | 0.781 | 0.763 | 0.217–2.809 |
|
|||||
New patient | 317 | 287 | 1 | ||
Treatment previously | 259 | 235 | 0.980 | 1.000 | 0.558–1.723 |
When using the 24-locus MIRU-VNTR method to typing the 576 isolates, the full set of results was obtained for 517 isolates. For 59 isolates, no PCR products were obtained at one or more loci. These observations remained consistent even after repeated testing. These findings might result from the chromosomal deletion, nucleotide polymorphism in the sequences complementary to PCR primers
In total, 24-locus MIRU-VNTR method differentiated 247 genotypes among the 517 isolates (
ETRA | ETRB | ETRC | MIRU2 | MIRU4 | MIRU10 | MIRU16 | MIRU20 | MIRU23 | MIRU24 | MIRU26 | MIRU27 | |
Whole sample | 0.169 | 0.109 | 0.110 | 0.003 | 0.097 | 0.193 | 0.222 | 0.413 | 0.031 | 0.000 | 0.482 | 0.078 |
Beijing genotype | 0.089 | 0.030 | 0.049 | 0.000 | 0.082 | 0.030 | 0.171 | 0.434 | 0.034 | 0.000 | 0.430 | 0.056 |
MIRU31 | MIRU39 | MIRU40 | Mtub04 | Mtub21 | Mtub29 | Mtub30 | Mtub34 | Mtub39 | QUB11b | QUB26 | QUB4156c | |
Whole sample | 0.663 | 0.243 | 0.251 | 0.278 | 0.539 | 0.014 | 0.151 | 0.034 | 0.257 | 0.75 | 0.583 | 0.560 |
Beijing genotype | 0.611 | 0.141 | 0.211 | 0.224 | 0.488 | 0.015 | 0.034 | 0.030 | 0.165 | 0.714 | 0.558 | 0.554 |
To identify a minimal set of MIRU-VNTR loci for differentiating Beijing genotype strains in Tibet, the allelic diversity of each MIRU-VNTR locus were calculated separately (
Based on the allelic diversity of each MIRU-VNTR locus among Beijing genotype strains, the cumulative HGDI of the MIRU-VNTR locus combination was calculated and compared (
Locus combination | VNTR alias | VNTR locus | No. of patterns | No. of clusters | No. of clustered isolates | No. of isolates in each cluster | Clustering rate (%) | HGDI (cumulative) |
1 | VNTR2163 | QUB11b | ||||||
2 | VNTR3192 | MIRU31 | 24 | 17 | 466 | 2–135 | 94.9 | 0.8590 |
3 | VNTR4052 | QUB26 | 69 | 35 | 439 | 2–71 | 85.4 | 0.9332 |
4 | VNTR4156 | QUB4156 | 96 | 44 | 421 | 2–65 | 79.7 | 0.9506 |
5 | VNTR1955 | Mtub21 | 119 | 50 | 404 | 2–64 | 74.8 | 0.9582 |
6 | VNTR2059 | MIRU20 | 125 | 50 | 398 | 2–63 | 73.57 | 0.9598 |
7 | VNTR2996 | MIRU26 | 158 | 57 | 372 | 2–52 | 66.59 | 0.9713 |
8 | VNTR0424 | Mtub04 | 175 | 62 | 360 | 2–48 | 63.00 | 0.9751 |
9 | VNTR0802 | MIRU40 | 189 | 60 | 344 | 2–48 | 60.04 | 0.9784 |
10 | VNTR3690 | Mtub39 | 196 | 60 | 337 | 2–44 | 58.56 | 0.9805 |
11 | VNTR1644 | MIRU16 | 207 | 62 | 328 | 2–41 | 56.24 | 0.9830 |
12 | VNTR4348 | MIRU39 | 222 | 61 | 312 | 2–39 | 53.06 | 0.9850 |
13 | VNTR2165 | ETR A | 228 | 63 | 308 | 2–39 | 51.80 | 0.9855 |
14 | VNTR0580 | MIRU4 | 233 | 63 | 303 | 2–38 | 50.74 | 0.9860 |
15 | VNTR3007 | MIRU27 | 235 | 64 | 302 | 2–38 | 50.32 | 0.9863 |
16 | VNTR0577 | ETR C | 239 | 62 | 296 | 2–37 | 49.47 | 0.9867 |
17 | VNTR2401 | Mtub30 | 241 | 61 | 293 | 2–37 | 49.05 | 0.9869 |
18 | VNTR2531 | MIRU23 | 242 | 61 | 292 | 2–37 | 48.84 | 0.9870 |
19 | VNTR3171 | Mtub34 | 244 | 61 | 290 | 2–37 | 48.41 | 0.9872 |
20 | VNTR2461 | ETR-B | 245 | 62 | 290 | 2–37 | 48.20 | 0.9875 |
21 | VNTR0960 | MIRU10 | 246 | 62 | 289 | 2–37 | 47.99 | 0.9876 |
22 | VNTR2347 | Mtub29 | 247 | 62 | 288 | 2–37 | 47.78 | 0.9877 |
23 | VNTR0154 | MIRU2 | 247 | 62 | 288 | 2–37 | 47.78 | 0.9877 |
24 | VNTR2387 | MIRU24 | 247 | 62 | 288 | 2–37 | 47.78 | 0.9877 |
Our results demonstrated that the population structure of
Our results showed that there was no association between the prevalence of Beijing genotype and sex, age, and treatment status. This indicated that maybe there were other factors that contributed to the spread of Beijing genotype strains. Demographic factors may be responsible for the dominance of Beijing genotype strains based on a co-evolution between the host and the pathogen
We found that the spoligotyping method could not effective distinguish Beijing genotype strains. Therefore in our study, these strains were further subjected to the newly proposed 24-locus MIRU-VNTR method and we found that the allelic diversity of the VNTR loci varied significantly at each locus. Among the 24 loci we investigated, QUB11b and MIRU31 were highly discriminative (h≥0.6), QUB4156, Mtub21, MIRU20 and MIRU26 were moderately discriminative, and other loci were poorly discriminative. When scrutinizing the 24-locus scheme, we found that MIRU24 and MIRU2 remained monomorphic in every published setting in China, Japan and Russia
When comparing the HGDI of this locus set with the VNTR loci reported in other areas (
VNTR alias | Tibet, China(this study) | Beijing, China | Heilongjiang China | Shanghai, China | Wuhan, China | Hong Kong,China | Hong Kong, China | Kobe, Japan | St. Petersburg, Russia |
QUB11b | 0.694 | 0.651 | 0.704 | 0.655 | 0.669 | 0.618 | 0.772 | 0.205 | |
MIRU31 | 0.617 | 0.169 | 0.395 | 0.246 | 0.23 | 0.156 | 0.200 | 0.322 | 0.160 |
QUB26 | 0.525 | 0.518 | 0.607 | 0.595 | 0.314 | 0.299 | 0.741 | 0.636 | |
QUB4156 | 0.519 | 0.395 | 0.182 | 0.492 | 0.167 | 0.611 | 0.082 | ||
Mtub21 | 0.491 | 0.556 | 0.396 | 0.523 | 0.393 | 0.330 | |||
MIRU20 | 0.440 | 0.014 | 0.061 | 0.25 | 0.022 | 0.120 | |||
MIRU26 | 0.429 | 0.353 | 0.596 | 0.612 | 0.60 | 0.200 | 0.383 | 0.520 | |
Mtub04 | 0.224 | 0.306 | 0.391 | 0.297 | 0.459 | 0 | |||
MIRU40 | 0.221 | 0.194 | 0.292 | 0.147 | 0.23 | 0.196 | 0.327 | 0.122 | |
Mtub39 | 0.166 | 0.171 | 0.174 | 0.061 | 0.186 | 0 | |||
MIRU16 | 0.158 | 0.068 | 0.200 | 0.242 | 0.55 | 0.058 | 0.310 | 0.082 | |
MIRU39 | 0.147 | 0.119 | 0.290 | 0.286 | 0.03 | 0.320 | 0.221 | 0 | |
ETR A | 0.090 | 0.232 | 0.238 | 0.031 | 0.188 | 0.201 | 0.147 | 0.158 | |
MIRU4 | 0.066 | 0.120 | 0.212 | 0.061 | 0.08 | 0.072 | 0.019 | 0.086 | 0 |
MIRU27 | 0.058 | 0.014 | 0.031 | 0.08 | 0.115 | 0 | |||
ETR C | 0.054 | 0.094 | 0.057 | 0.165 | 0.022 | 0.042 | |||
Mtub30 | 0.033 | 0.068 | 0.133 | 0.091 | 0.403 | 0.042 | |||
MIRU23 | 0.033 | 0.014 | 0.061 | 0.25 | 0.176 | 0 | |||
Mtub34 | 0.029 | 0.014 | 0.089 | 0.065 | 0 | ||||
ETR B | 0.029 | 0.014 | 0 | 0.064 | 0 | 0 | 0 | ||
MIRU10 | 0.025 | 0.144 | 0.154 | 0.195 | 0.52 | 0.377 | 0.419 | 0.082 | |
Mtub29 | 0.013 | 0.119 | 0.123 | 0.061 | 0.043 | 0.087 | |||
MIRU2 | 0 | 0 | 0 | 0 | 0 | 0 | |||
MIRU24 | 0 | 0 | 0 | 0 | 0 | 0 |
The 24-locus MIRU-VNTR scheme has some technical limitations: in China, MIRU-VNTR can be only performed manually and, consequently, was very time-consuming and tedious for this study. Some reports demonstrated that combination of 24-locus MIRU-VNTR and spoligotyping could improve the discriminatory power of
In conclusion, Beijing genotype strains appeared to be widely disseminated across Tibet. The analysis of MIRU-VNTR data might be useful to select appropriate VNTR loci for the genotyping of
The study was approved by the Ethics Committee of National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. All patients in the study signed informed consent form.Clinical isolates and DNA samples.
We randomly collected a total of 590
We performed spoligotyping according to a standard protocol as described by Kamerbeek et al
In terms of the 24-locus MIRU-VNTR typing, we used PCR primers flanking each of the 24 loci that were described by Supply et al
To minimize the risk of laboratory cross-contamination, DNA extraction and PCR amplification were conducted in separate rooms. The PCR laboratory has four disconnected rooms for preparation of the PCR mixtures, addition of the DNA, PCR amplification, and electrophoretic fractionation. Negative controls (sterile water) were included to control for cross contamination.
Bionumerics software version 5.0 and MIRU-VNTRplus (
The chi square test was used to assess association of Beijing genotye with sex, age, and treatment status by using SPSS 11.5 (SPSS Inc., Chicago, IL, USA).
(XLSX)
We thank the staffs of the respective institutes in Tibet for their contribution to this study.