First insights into the genetic diversity of Mycobacterium tuberculosis strains in Salvador , Bahia State , Brazil

This study constitutes a first attempt to describe the genetic population structure of Mycobacterium tuberculosis circulating in Salvador, Bahia State, Brazil. A total of 56 confirmed cases of pulmonary tuberculosis, identified between March and June 2008, were analyzed using restriction fragment length polymorphism (IS6110-RFLP). The study population was characterized by a predominance of males (71.43%) over 30 years of age (68.75%). Forty-one isolates were found to belong to a single pattern (73.2%), while 15 (26.7%) were found in group patterns, forming six clusters. The higher level of diversity observed is much more suggestive of endogenous reactivation than recent transmission. Tuberculosis; Molecular Epidemiology; Genetic Variation; Mycobacterium tuberculosis Introduction Tuberculosis (TB) is a global public health issue, with 9.27 million new cases and 1.3 million deaths worldwide in 2007 1. Brazil ranks among the 22 countries with the highest incidence rates of tuberculosis, with 100,000 new cases of TB being registered annually. In 2006, 6,500 cases of TB were registered in the State of Bahia, which is currently ranked as the third state in absolute numbers of cases in Brazil. Of these cases, 2,300 occurred in the city of Salvador 2. Recently, molecular techniques have been used to explain the epidemiological aspects of TB, contributing towards improving understanding of transmission dynamics that drive the disease 3. To date, no studies have been carried out on the epidemiologic and genetic diversity of circulating Mycobacterium tuberculosis in Salvador, a city in the Northeast Region of Brazil, where the incidence of this disease is high. The aim of the present study was to describe the genetic population structure of M. tuberculosis circulating in Salvador.

First insights into the genetic diversity of Mycobacterium tuberculosis strains in Salvador, Bahia State, Brazil Primeiro ensaio sobre diversidade genética das cepas de Mycobacterium tuberculosis em Salvador, Bahia, Brasil Introduction Tuberculosis (TB) is a global public health issue, with 9.27 million new cases and 1.3 million deaths worldwide in 2007 1 .Brazil ranks among the 22 countries with the highest incidence rates of tuberculosis, with 100,000 new cases of TB being registered annually.In 2006, 6,500 cases of TB were registered in the State of Bahia, which is currently ranked as the third state in absolute numbers of cases in Brazil.Of these cases, 2,300 occurred in the city of Salvador 2 .Recently, molecular techniques have been used to explain the epidemiological aspects of TB, contributing towards improving understanding of transmission dynamics that drive the disease 3 .
To date, no studies have been carried out on the epidemiologic and genetic diversity of circulating Mycobacterium tuberculosis in Salvador, a city in the Northeast Region of Brazil, where the incidence of this disease is high.The aim of the present study was to describe the genetic population structure of M. tuberculosis circulating in Salvador.
Otavio Mangabeira Specialist Hospital (HEOM/ SESAB), a referral unit for pneumopathies situated in the city of Salvador.The research project was approved by the National Health Surveillance Agency/Ministry of Health in Brasília, and by the ethics committee of the Collective Health Institute, Federal University of Bahia.

Bacterial isolates
The M. tuberculosis isolates were collected and processed from sputum samples in accordance with the protocol established by the Department of Mycobacteriology of the Central Laboratory of Public Health "Professor Gonçalo Muniz" (LACEN-BA) 4 .

IS6110-RFLP analysis and definitions
The clinical isolates of M. tuberculosis were typed using restriction fragment length polymorphism (IS6110-RFLP) analysis in accordance with the standardized protocol described by van Embden et al. 5 .The genetic profile of each strain was analyzed and visualized with GelCompar II version 4.0 (Applied Maths, Kortrijk, Belgium) using M. tuberculosis 14323 as the reference strain.All the strains with a similarity coefficient of > 90% were considered part of a "family".Within the molecular typing system, cluster strains were defined as being those which were found to have the same RFLP pattern (number of bands and position of the fragment) in two or more isolates of M. tuberculosis.Isolates which were found to have RFLP patterns distinctly different from the other patterns identified were considered to be non-clusters 3 .Isolates forming part of a cluster pattern were defined as representing a recently acquired infection and standard non-cluster isolates were defined as representing reactivation of an infection acquired in the past 3 .

Data analysis
Exploratory data management was performed using Stata version 7.0 (Stata Corp., College Station, USA).Prevalence ratios (PR) and their respective 95% confidence intervals (95%CI) were calculated.

Findings Analysis of patient characteristics and associated factors
Fifty six patients whose sputum cultures tested positive for M. tuberculosis were enrolled in this study between March and June 2008,.Of this total, the majority (71.43%) were male and over 30 years of age (68.75%) (Table 1).Regarding ethnic background, most of the patients (80%) classified themselves as non-white.With respect to education, 63.89% of the patients were illiterate or had failed to complete primary school.A quarter of the patients did not reside in Salvador and 23.4% had been submitted to a category III treatment regimen [indicated in Brazil for the treatment of cases in which previous treatment with regimens I, IR (recurrence after cure) and II, has failed].

RFLP analysis
Of the 56 patients analyzed, 41 of the isolates (73.21%) had single patterns and 15 (26.78%) had group patterns.Overall, all the isolates had 2-16 copies of IS6110-RFLP and 10.7% of all samples analyzed had six or fewer copies of this repetitive element.Fifteen isolates formed 6 distinct clusters denominated CL I, CL II, CL III, CL IV, CL V and CL VI.Each cluster was composed of 2 isolates except for CL V, which was composed of 5 isolates (Figure 1).

Discussion
This is the first exploratory study conducted in Bahia with the aim of describing the genetic population structure of M. tuberculosis, as regards recent infection or reactivation.The principal finding refers to the fact that 26.78% of isolates formed cluster patterns, suggesting recent transmission of TB in this group of patients.This proportion is slightly higher than the findings of studies conducted in Rio de Janeiro using similar methodology (19%) 6 and similar to the results of studies carried out in isolates in Porto Alegre (State of Rio Grande do Sul) and São Paulo 3,7 which amounted to 29.1% and 33%, respectively.In the present study, the percentage of cluster strains found was slightly lower than those reported in studies conducted in France (35%) 8 and San Francisco (40%) 9 .
Cad. Saúde Pública, Rio de Janeiro, 27(9):1859-1863, set, 2011 The proportion of non-cluster strains was 73.21%, suggesting the need to improve TB control, promoting preventive actions against reactivation.In Brazil, recent studies 3,7 show similar findings.Around 70% of cases, result from old infections (non-cluster strains), showing that the occurrence of new cases in the country originates predominantly from endogenous reactivation.
In this study, patients were from a heterogeneous group of people concentrated in the capital city, the Greater Metropolitan Region of Salvador and other towns in the state, in addition to two patients living in another state (São Paulo).This finding shows the mobility of the population-genetic characteristics of tuberculosis between different regions, so providing a contribution to implementing adequate measures in cases of recent infection as well as reactivation of an old infection.
One of the limitations of this study is that, due to the small sample size, it was not possible to obtain a statistically significant association between the formation of cluster patterns and the factors analyzed.Another limitation refers to the fact that the findings on the proportion of cluster patterns may be affected by the incidence of TB, by the heterogeneous nature of the population and by the sampling fraction included in the study.An analysis of these aspects would contribute towards obtaining a more accurate calculation of recent infection in a given community 6,10 .
Despite these limitations, a wide genetic diversity was found in the strains of M. tuberculosis analyzed in TB patients in this referral hospital in the city of Salvador.Further studies should be carried out to acquire more data on this subject in Salvador and enable the investigation into associations between the various co-variables and the proportion of cluster strains in this city.

Table 1
Bivariate analysis of the association between the co-variables and restriction fragment length polymorphism (RFLP) pattern (cluster or non-cluster) of Mycobacterium tuberculosis isolates from patients infected with tuberculosis (TB).Salvador, Bahia State, Brazil.Dendrogram representing the different RFLP patterns found in isolates of M. tuberculosis from patients receiving care at a referral hospital, according to the identifi cation number of the sample, date of isolation, sex, age, place of residence and treatment regimen.Salvador, Bahia State, Brazil.ContributorsA.C. N. Silva, J. N. Reis, S. M. Pereira, E. L. A. Mota, and M. G. Reis participated in the article's conception, data analysis and interpretation, writing of the article, relevant critical review of the intellectual content, and final approval of the version for publication.L. Ferrazoli collaborated in the conception, data analysis and interpretation, writing of the article, and critical review of the intellectual content.V. Simonsen and T. M. A. Carmo contributed to the article's conception and data interpretation.