The Recent-Transmission of Mycobacterium tuberculosis Strains among Iranian and Afghan Relapse Cases: a DNA-fingerprinting using RFLP and spoligotyping

Restriction fragment length polymorphism (RFLP) using insertion sequence IS6110 is a well-established method of "DNA fingerprinting" that has been used to trace the transmission of particular strains of M. tuberculosis isolates [1, 2]. The method is based on the detection of differences in the numbers and locations of the insertion element IS6110 within the chromosomes of M. tuberculosis strains [2, 3]. Generally, the unrelated clinical isolates show a high degree of variation, whereas, epidemiologically related strains show identical or similar fingerprint patterns [4, 5]. By IS6110-RFLP, it was also possible to determine whether a new episode of disease is caused by reactivation of endogenous infection or by exogenous reinfection [6, 7]. The exogenous reinfection can either occur during therapy for the original infection or after therapy has been completed [8]. In overall, the frequency with which the patient reinfected with M. tuberculosis are reported to be variable. In South Africa, the frequency of exogenous reinfection reported to be high, whereas in other places it has been involved selected populations e.g., alcoholic residents of a homeless shelter or patients with advanced HIV infection[8, 9]. In Iran, the extent in which exogenous reinfection contributed to incidence of the diseases are not known. According to the World Health Organization, the estimated incidence of TB in Iran is 28 cases per 100000 populations [10]. The TB problem has become more serve because of an increase in MDR-TB strains. Based on national wide survey conducted in 1999, among all M. tuberculosis isolates tested for drug susceptibility, 10.9% were resistant to ≥ 1 anti-TB drug, and 6.7% were resistant to both isoniazid and rifampin (i.e., were MDR strains of M. tuberculosis)[11]. In further studies, the existence and transmission of XDR-TB strains (i.e., resistant to fluoroquinolones and to at least one of the three inject able second line drugs in addition to isoniazid and rifampin) in epidemiological related MDR-TB patients were demonstrated [12]. Considering the severity of diseases associated with spread and transmission of MDR or XDR-TB strains, we tried to determine the relative frequency of reactivation from recent transmission among relapse cases using IS6110-RFLP. However, since RFLP analysis with IS6110 alone may be inconclusive for strains carrying few copies of IS6110 [13], we also used an alternative PCR-technique called spoligotyping. The technique detects various non-repetitive spacer sequences located between small repetitive units (direct repeat DR) in the chromosome on M. tuberculosis complex [14]. In present study, the contribution of Afghan immigrants in maintaining the recurrent tuberculosis was also determined.

Using molecular typing, twenty-five percent of relapse cases (65; 25%) were clustered in 29 clusters. A retrospective studies revealed that patients in clusters developed their second episode of TB within the same period. Further analysis of spoligopatterns identified Haarlem I and Beijing types of M. tuberculosis strains in thirty eight percent of patients in clusters(38%)(table. 2). Therefore, the possibility of exogenous reinfection through transmission of particular M. tuberculosis strains were highlighted. Although, due to non-availability of previous culture genotyping results, we could not confirm the exogenous reinfection in them. Recently, investigators have suggested that the relative contribution of exogenous reinfection increases in parallel with the incidence of disease [6, 8, 18]. Most reported cases of exogenous reinfection observed among alcoholic residents of a homeless shelter or patients with advanced HIV infection [6, 9, 19]. We found no particular risk factors between patients in cluster and non-cluster cases. Retrospective analysis of cluster cases identified 41% of intra- community transmission between Iranian and Afghan TB patients. In our previous study, the impact of intra- community transmission was much lower (13.7%) than present result. Furthermore, we found that the Haarlem I and Beijing type of M. tuberculosis strains were the most frequent super families in intra-community transmission (figure. 1). The Haarlem I and Beijing strains have been reported in different geographical regions of the world and they thought to possess selective advantages in comparison to other M. tuberculosis super families [22, 23]. Previously, we demonstrated that 44% of MDR-TB patients in intra-community transmission were belonged to HaarlemI (73%) and Beijing (27%) superfamilies [21]. Therefore, based on previous and present reports, it is clear that both Haarlem I and Beijing strains can cause epidemic and from epidemiological point it is necessary to conduct more extensive surveillance of MDR-TB strains because they might cause serious outbreaks [12]. During the year 2000–2005, 32% of initial TB patients that referred to our unit were from Afghan born immigrants [20]. Majority of these patients (58%) had either resistant to any drug or drug combination including MDR-TB [20]. In present report also, the rate of resistance to any drug or drug combination in Afghan patients were more than two folds as compared to Iranian (table 1). These finding highlighted the need to reinforce the TB policy measures with regards to screening immigrants from neighboring countries, which is absent in the current system. Based on IS6110-RFLP, 74.8% of patients were grouped in non- cluster cases and it was assumed that reactivation of endogenous infection remains the more probable cause of active tuberculosis, in studied populations (table. 1). Today, it is known that sterilization of a pulmonary lesion is possible through effective treatment regimens. But, it is also accepted that subsequent episodes of TB are almost invariably caused by endogenous reactivation of resistant strains [7, 24]. In other words, majority of those who returned for treatment after default might develop resistant in comparison to those who returned after exogenous reinfection [7, 9, 25]. In this regards, we also found high number of MDR-TB strains among endogenous reactivation cases. The frequent superfamilies in MDR-TB cases of non-cluster cases were CASI (19; 31%) and EAI3 (12; 20%) (table. 2). Based on Sreevatsan et al, the EAI and CAS are belong to genetic group I organisms which are evolutionary older and they are the most frequent superfamilies in Central Asia and Middle East [26]. Our results also showed different susceptibility patterns for isolates in same clusters e.g., in the cluster numbered-27 (figure. 1); one strain was susceptible to all drugs tested and the other two strains were resistant to isoniazid. Mitchison [27] has already described the emergence of such drug resistance strains, solely due to irregularity in administration of drugs. That means the strains became acquired drug resistance isolates, as classically defined [24, 25]. In the studied population, no relation was found between the patterns of IS6110-RFLP and susceptibility results. In fact, the number and manner of IS6110 positioning along the genome of M. tuberculosis does not have any relation with drug-susceptibility results [2, 4]. Last but not the least, the major consideration of the usefulness of the IS6110-RFLP typing method is its specificity, which depends on the number of bands obtained. In the present study, 12% of collected strains had low copies of IS6110. Previously, we detected only 5.4% of strains with low IS6110 copy number [20]. Thereby, the prevalence of M. tuberculosis isolates with low or no IS6110 insert is not clear and further studies are required to show the real distribution of these strains within the country.

Previous studies showed that more than 90% of active cases of tuberculosis in Iran resulted from reactivation of infection-contracted years before, and that recently transmitted diseases had a minor role. In this study, we also showed the higher rate of reactivation (74.8%) versus recent transmission (25.2%). Although, the incidence of intra- community transmission had a significant rises from 13% in 2005 to 41% in 2007. Therefore, it is necessary to adapt new strategies for rapid diagnosis, and efficient treatment of TB patients.