Abstract: The aim of present study was to determine the genotypes of isolates from East Azarbaijan province by this method. We performed (MIRU-VNTR) analysis of strains, isolated from 127 patients during a period of September 2002 to March 2003 in tuberculosis centers of the province. Among 127 isolates, we found 93 distinct MIRU-VNTR patterns, including in 21 clustered patterns and 72 unique patterns from isolated strains. The discriminatory power of MIRU-VNTR typing in present study was high (HGDI = 0.9932) for isolates. In clusters similar patterns of Nakhichevanees patients and Iranian patients was revealed in three clusters which showed Nakhichevanees patients referred to tuberculosis centers of province could be a source for transmission of tuberculosis. Tuberculosis in this province is relatively in good condition. The allelic diversity of our samples was lower than previous studies. These results indicate that MIRU-VNTR can be a useful and first line tool for studying genetic diversity of M. tuberculosis isolates in regional setting such as East Azarbaijan province of Iran.
INTRODUCTION
Tuberculosis is the leading cause of death in adults from a single infectious agent, killing about 3 million people every year. One-third of the human population is thought to be infected by the causative agent Mycobacterium tuberculosis (Cole et al., 1998; Burgos and Pym, 2002; Asgharzadeh et al., 2007a) and about 200 million additional people are at risk for developing the disease in the next 20 years if current trends continue (WHO, 1998; Grange and Zunmla, 1999). Therefore, efficient disease control can be achieved only by epidemiological surveillance systems to accurately monitor epidemic trends at a regional and global level.
Genotyping of M. tuberculosis has an important role in epidemiological studies (Asgharzadeh et al., 2007b). A large number of DNA fingerprinting methods for typing M. tuberculosis isolates have been developed in recent years, all have significant drawbacks and only a few have been adapted for widespread use. IS6110, spoligotyping and variable number tandem repeat-mycobacterial interspersed repetitive units are the most widely used methods (Kremer et al., 1999; Barow et al., 2001; Cowan et al., 2002).
Variable Number Tandem Repeat (VNTR) typing is an invaluable tool for genotyping and provides data in a simple and format based on the number of repetitive sequences in so-called polymorphic micro- or mini-satellite regions (Mazors et al., 2001). Despite some attempts to develop equivalent approaches for typing bacterial pathogens, only limited application for bacterial molecular epidemiology could be developed up to now (van Belkum et al., 1998; van Belkum, 1999). Mazors et al. (1998) introduced VNTR for M. tuberculosis which named Mycobacterial Interspersed Repetitive Unite-variable Number Tandem Repeat (MIRU-VNTR), each isolates is typed by the number of copies of repeated unites at 12 independent loci scattered throughout the genome (Supply et al., 1997). The repeated units are 52 to 77 nucleotides in length and the number of repeated unites can be determined by the size of the entire locus (Supply et al., 2000), previous studies demonstrated the importance of MIRU-VNTR method for tracking epidemiological key events, such as transmission or relapse and provide nonambiguous data which are highly portable between different laboratories (Mozars et al., 2001; Frothinghom et al., 1998; Supply et al., 1997).
The East Azarbaijan Province is located in the north west of Iran and in neighborhood of Nakhichevan state of Republic of Azerbaijan. The estimated population of the province is 3,500,000 (Asgharzadeh et al., 2006). The capital city of the province is Tabriz. The estimated rate of TB in Iran in 2004 was 27 in 100,000 (WHO, 2006) but in East Azarbaijan province the estimated rate was lower; it can be due to low case finding or low prevalence of TB in this province.
The objective of this study is to genotype M. tuberculosis isolates from 127 patients with tuberculosis in East Azarbaijan province and determine manner of transmission in this province.
MATERIALS AND METHODS
Patient population and bacterial isolates: The study population comprised 127 patients with TB confirmed by culture from September 2002 to March 2003 in TB centers of the province. Samples which were collected and submitted for culture in tuberculosis centers of province, clinical isolates were recovered from sputum (n = 99), bronchial fluids (n = 19), abscess aspirates (n = 2), urine (n = 2), cerebrospinal fluids (n = 2), pleural fluid (n = 1), endometrial biopsy (n = 1) and neck mass biopsy (n = 1). Fourteen strains were from Nakhichevanees patients which were compared with Iranian strains for detecting probable transmission of the infection. Information about age, sex, geographical origin and history of tuberculosis were recorded. The species identification of the isolates was based on Polymerase Chain Reaction (PCR) method and standard microbiological tests (Rieder et al., 1998).
DNA used for the PCR analysis was extracted from cultured Mycobacterium’s. Two loops full of bacteria’s were suspended in 400 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and placed at 80°C for 20 min to kill the bacteria. DNA was extracted by lysozyme, SDS, proteinase K and CTAB. Extracted DNA after sedimentation with isopropanol and washing with ethanol 70% was redissolved in 100 μL TE buffer (van Soolingen et al., 1994).
MIRU-VNTR typing: PCR was performed in 20 μL volume that contained 5 to 50 ng of DNA, 0.5 μm of specific primers (Table 1) in the presence of 1.5 mM MgCl2, 100 μm of each dNTP, 50 mM KCl, 20 mM Tris_HCl, pH 8.4 and 1.25 U recombinant DNA polymerase (Cinnagen co. Iran). DNA was amplified by general PCR. All PCRs were initiated by a 7 min denaturizing step at 94°C and completed by a 7 min extension step at 72°C. The temperature cycles for different types of PCRs were as follow, 35 cycles of 45s at 94°C, annealing temperature for 45 sec and 72°C for 55 sec. Annealing temperatures were used as follow: 65, 63, 68, 65, 59, 67, 59, 65, 64, 63, 68 and 65 for MIRU loci 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39 and 40, respectively.
| Table 1: | Oligonucleotides used in MIRU-VNTR genotyping |
Negative controls consisted of the PCR components on reaction mixtures lacking mycobacterium DNA. PCR products were electrophoreses in 1.5% agarose gel and after staining with 0.5 μg mL-1 ethidium bromide visualized under UV light. The size of fragments was determined in comparing with 100 bp DNA ladder plus size marker (Fermentas) (Supply et al., 2001)
Statistical analysis: The MIRU-VNTR allelic diversity (h) at each of the loci was calculated by the equation.
h = 1-Σχi2 |
where, χi is the frequency of the ith allele at the locus (Sun et al., 2004).
The Hunter-Gaston Discriminatory Index (HGDI) described by Hunter and Gaston (Hunter and Gaston., 2003) was used as a numerical index for MIRU-VNTR discriminatory power. HGDI was calculated by using the following formula:
RESULTS
The age of the patients ranged from 12 to 90 years which young patients (≤45 years old) represented 43% of study population. The male to female ratio was 1.3:1. Using MIRU-VNTR typing; 93 distinct were identified, including 21 cluster patterns and 72 unique patterns (Table 2). Fifty five isolates (43.3%) included in clusters. And the discriminatory power of MIRU-VNTR typing was high (HGDI = 0.9932) for our isolates. The minimum estimate for the proportion of tuberculosis that was due to transmission in East Azarbaijan Province is 26.8% (55-21)/127).
In 21 clusters (Fig. 1), the largest cluster comprised six patients, four of these patients were female and two were male, which this transmission can be happen by contact or by frequenting in the same place. The rest were residents from different parts of the city and no epidemiological linking was detected; however all were suffering from poor living condition. The second cluster was made up of four patients from Tabriz and Maraghe, a city about 200 km away from Tabriz and no connection between the patients were detected. Seven clusters were composed three patients and twelve clusters composed two patients.
Three cluster has been defined that shared MIRU-VNTR pattern between Iranian and Nakhichevanees patients. The first cluster composed one Nakhichevanees patients and two Iranian patients, the second cluster composed one Nakhichevanees and one Iranian patient and equally the last cluster composed one Nakhichevanees and one Iranian patient. One Cluster composed three Nakhichevanees patients with the same pattern, which were one female and two male.
The allelic diversities of the 12 MIRU-VNTR locus based on this study isolates. Locus 26 and 40 were highly discriminative (≥0.6), locus 16, 23, 24 and 31 were moderately discriminative (≥0.3) and locus 2, 4, 10, 20, 27, 39 were poorly discriminate (<0.3) (Table 3).
| Table 2: | MIRU-VNTR patterns of strains from East Azarbaijan Province of Iran |
| *: IPs are Iranian patients number and NPs are Nakhichevanees patients number | |
| Table 3: | Allelic diversity of each MIRU-VNTR locus |
| Allelic diversity (h) was defined as h = 1 - Σχi2, where χi is the frequency of the ith allele at the locus | |
| Fig. 1: | Dendrogram of isolates in this study. It revealed twenty one clusters and similarity of our isolates |
DISCUSSION
MIRU-VNTR is a PCR-based method for typing M. tuberculosis isolates that require amplification followed by size analysis of 12 independent loci; it can be highly effective in detecting the sources of infection and providing data for improving the control programs of tuberculosis. In this study we used MIRU-VNTR to investigate tuberculosis transmission in East Azarbaijan province of Iran. From one hundred and twenty seven isolates, fifty five isolates (43.3%) included in one of twenty one clusters, which show high transmission of tuberculosis in our region. But it was lower than Singapore with 55.3% (Sola et al., 2003) and Spain with 71% (de Viedgma et al., 2005) also the minimum estimate of transmission of tuberculosis in East Azarbaijan by MIRU-VNTR was 26.8% which was higher than San Francisco with 24% (Borgdorff et al., 2000) or London with 14.4% (Magurie et al., 2002) but it was lower than South Africa with 50% (Godfrey-Fausset et al., 2000). This rate of clustering among M. tuberculosis strains, suggesting that the majority of the tuberculosis cases in East Azarbaijan are due to reactivation.
It is known that in mixed populations the degree of DNA polymorphism is greater (van Embden et al., 1993). Although immigration from other parts of the country is almost in existent in East Azarbaijan. There is considerable number of patients from Nakhichevan (during our study, 11% of initial strains were isolated from nakhichevanees patients). In addition, the percentage of infection in Nakhichevan and Republic of Azerbaijan is high (WHO, 2006; Pfyffer et al., 2001). We often face with traveling from this region and in this study fourteen isolates that were affected by tuberculosis were from Nakhichevan. Six Nakhichevanees patients (42.85%) included in one of the four clusters, which one of the clusters composed three nakhichevanees patients that show internal transmission of tuberculosis between them but in next three clusters we had Nakhichevanees patients and Iranian patients with the same MIRU-VNTR pattern. Therefore, we can conclude these Nakhichevanees patients were the source of spread infection in East Azarbaijan by traveling and residing in this region, similar transmission introduced previously in San Francisco (Casperm et al., 1996), Denmark (Troels et al., 2001) and Spain (de Viedgma et al., 2005). In the other hand, multidrug resistance in Republic of Azerbaijan strains is high (Pffyfer et al., 2001). Treatment of tuberculosis in Iran is entirely free, so these patients refer to our province for treatment and it cause new transmission and more expenses for country. Furthermore it should be noted that the study was performed during a period of six month only and because of the characteristics of disease transmission and development a study using a large time frame should be performed to evaluate the real clustering rate.
In a study on tuberculosis transmission in Paris, 70% of the patients were male sex and male sex was a risk factor for clustering (Gutierrez et al., 1998). In agreement with mentioned study, in our study, however not significant, prevalence of tuberculosis was higher for male (58.27%) than female (41.73%). In our study, no difference was observed between the younger or older age categories; unemployment and generalized poor living condition could be the reason for this.
In our study allelic diversity of the 12 MIRU-VNTR was low, especially in loci 2, 4, 10, 20, 27 and 39. Low diversity of MIRU-VNTR loci apparently allow to the available data for a philogenetic analysis (Mokrousov et al., 2004). The allelic diversity of our samples was lower than previous studies (Sun et al., 2004; Sola et al., 2003).
The result of this study showed that sanitation of tuberculosis in East Azarbaijan is relatively in good condition and both of reactivation of latent infection and transmission of infection are effective in prevalence of tuberculosis in our province. In order to prevent transmission of tuberculosis from Nakhichevan, new tuberculosis centers should be establish near the border to prohibit traveling of Nakhichevanees patients and long staying of them.
In conclusion MIRU-VNTR as a simple, automation, a short turnaround time, high throughput, perfect and reproducible technique, can be use for study manner of transmission and epidemiological links in regional studies and patterns allow comparing results with other laboratories as a global investigation. MIRU-VNTR typing will be likely prevailing as a first line method for genotyping of M. tuberculosis.
ACKNOWLEDGMENTS
We gratefully thank Davood Habibzadeh for assistance in obtaining patients information and entire staff of Tabriz Tuberculosis and Lung Disease Research Center for their generous cooperation. This study was supported by the Molecular Biology Laboratory, Faculty of Medicine and Biotechnology Research Center, Tabriz University of Medical Sciences.