Molecular epidemiological approach to understand the emergence and spread of drug resistant Mycobacterium tuberculosis strains in Peru

Tuberculosis (TB) is a chronic infectious disease that remains a major health problem worldwide. It is more prevalent in underdeveloped and developing countries where it kills over 2 million people per year. One third ofthe world is thought to be infected by TB bacilli, and the World Health Organization estimates that nearly 1 billion additional people will become infected by M tuberculosis between 2000 and 2020 (36). In Latin America, Peru has the highest reported incidence rate of TB (160 per 100 000 population), with 18.5% of all cases occurring in the biggest district ofthe city ofUrna (San Juan de Lurigancho) (2). With the incorporation of the Directly Observed Therapy Shortcourse (DOTS) programme, Peru has become the model for TB control in Latin America. However, this DOTS success is threatened by the recent emergence of multidrug resistant (MDR) strains; defined as TB resistant to at least isoniazid (INH) and rifampicin (RMP), the two most powerful first-line anti-TB drugs. In 2005, the Technical Unit ofMDR-TB ofthe Peruvian Ministry ofHealth notified 1950 MDR-TB cases, among which 38% were from San Juan de Lurigancho (1). Drug resistance develops in the course of treatment and is the result of patients missing doses, doctors giving inappropriate treatment or patients failing to complete a course oftreatment. In addition, there is a new form ofTB caused by bacteria called extensively drug-resistant (XDR) TB, defined as MDR-TB being simultaneously resistant to one ofthe fluoroquinolones and one of the injectable second-line drugs (36). Diagnosis of drug-resistant TB can be achieved by in vitro drug-susceptibility testing after a positive culture. For some ofthe drugs molecular tools are available detecting mutations in relevant genes, such as the rpoB gene for detection ofRMPresistant TB. Previous sequencing studies revealed that the most common relevant rpoB mutations found in Peru were Ser531 followed by His526 mutations, and Ser315 as the most prevalent katG mutation (21,27). Nowadays studies are focusing on identifying specific mutations associated with second-line drugs resistance. Sequencing analysis has revealed two mutations in the rrs gene (A1401G and C1402T) associated with injectable drugs resistance (12) and two mutations in the gyrA gene (Ala-90 and Asp-94) associated to fluroquinolone resistance (34). Smear microscopy is the easiest, fastest and cheapest technique for laboratory-confirmed diagnosis of TB, but has a low detection rate. Cultivation followed by phenotypic identification and drug resistance testing is still the "gold standard", but has the disadvantage of taking a long time. Besides, biochemical characterization of isolates is not always conclusive and expensive. An alternative method for detection and/or identification comprises the amplification of specific loci by polymerase chain reaction (PCR). However, PCR-based systems lack sensitivity as compared to culture, partially due to the use of inadequate DNAextraction methods (26). So there still is a need for a rapid, simple and cost-effective diagnostic tool for detection of (drug-resistant) TB. The traditional view is that M tuberculosis strains are equally virulent. However, population-based genotyping has demonstrated that a small percentage of strains cause a disproportionately large number of cases, implying that some strains are spread more effectively than others. In the last two decades, epidemiological studies on TB have been assisted by the IS611O-based Restriction Fragment Length Polymorphism (RFLP) typing method (6,9, 13,15). Although IS611O-RFLP is considered as the gold standard typing method for TB, it also presents some disadvantages such as the labor-intensive manipulations and the complexity of comparing profiles (7, 16). Another typing technique described is based on the Variable Number ofTandem Repeats (VNTR) observed among TB-specific tandem repeats named Mycobacterial Interspersed Repetitive Units (MIRUs). These techniques have been also useful in studies on reinfection versus reactivation. Earlier, recurrent TB was systematically considered as a reactivation of the initial M tuberculosis strain, but DNA-fingerprinting revealed that reinfection with a new strain is possible as well. Despite their potential for genotyping and molecular epidemiology ofTB (29,30), these techniques are expensive for poor settings. So, for some specific purposes, it is better to do a first screening through the spoligotyping method which, in spite of a less discriminative power, is useful for the description of TB families. Also human factors playa role in the pathogenesis, evolution and outcome of disease. Involvement of human genetic heterogeneity in the susceptibility to TB has been suspected for many years (3). Although cytokines exhibit a low degree of genetic variations, an increasing number of association studies have implicated polymorphisms located on promoter regions or coding regions of cytokine genes as host factors influencing susceptibility to infectious diseases (4,14). The course ofM tuberculosis infection is regulated by two distinct T cell cytokine patterns. T helper 1 (Thl) cytokines, IL-2 and IFN-y, are associated with resistance to infection, whereas Th2 cytokines, IL-4 and IL-13, are associated with progressive disease (35). In addition, IL-l 0, one of the T-regulatory cytokines, seems to playa pivotal role during the chronic/latent stage of pulmonary TB, with increased production playing a potentially central role in promoting reactivation ofTB (33). Studies on TB patients with different ethnic backgrounds have shown conflicting evidence for 1NF-a (5,24,25) and IL-I0 -1082 (8, 18) polymorphisms association with TB. No association was found for TGF-~ (20). In contrast, published reports agree that IFN-y+874 allele A, associated with low IFN-y production, is associated with pulmonary TB (17). Finally there is also evidence that supports the association between polymorphism observed in IL12B (intron-2) and TB and between specific IL12B haplotypes and TB (32).

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