Brazilian Journal of Pulmonology

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Current Issue: 2004 - Volume 30 - Number 4 (July/August)

ORIGINAL ARTICLE

Molecular analysis of Mycobacterium tuberculosis strains from an outpatient clinic in Porto Alegre, (RS)

Análise molecular de cepas de Mycobacterium tuberculosis provenientes de um centro de saúde ambulatorial em Porto Alegre, (RS)

 

Michele Borges; Patrícia Izquierdo Cafrune; Lia Gonçalves Possuelo; Andréia Rosane de Moura Valim; Marta Osório Ribeiro; Maria Lucia Rosa Rossetti

 

Abstract

Background: Tuberculosis is an ancient disease, which still remains one of the major ills faced by mankind in the 21st century. In recent decades, new technologies employing the knowledge gained from molecular biology studies have allowed for more accurate detection of tuberculosis and increased investigation of the etiology and epidemiology of the disease.
Aim: Evaluating the degree of similarity among strains of Mycobacterium tuberculosis provided by the Phthisiology Sector of Centro de Saúde Navegantes (Navegantes Health Clinic) in Porto Alegre, RS, Brazil.
Method: A retrospective study was performed involving RFLP typing of 55 sputum samples from outpatients examined at the Centro de Saúde Navegantes. The results of the genotyping were correlated to the conventional epidemiology data.
Results: A single pattern was seen in 39 (70.9%) of the isolates, whereas 16 isolates (29.1%) showed clustering patterns and were grouped into 8 clusters of 2 patients each. An epidemiological link was found for 6 (37.5%) of the 16 patients in the clusters.
Conclusion: The appropriate combination of conventional epidemiology and genotyping of M. tuberculosis contributes to a better understanding of the dynamics of tuberculosis transmission even when such a study is performed in a single, isolated health clinic.

 

Resumo

INTRODUÇÃO: A tuberculose é uma doença antiga que ainda se mantém como um dos maiores males da humanidade no século XXI. Nas últimas décadas, o advento de novas tecnologias utilizando os conhecimentos de biologia molecular tem levado a um aumento na investigação da etiologia, detecção e epidemiologia da tuberculose.
OBJETIVO: Avaliar o grau de similaridade entre as cepas de Mycobacterium tuberculosis provenientes do setor de tisiologia do Centro de Saúde Navegantes, de Porto Alegre (RS).
MÉTODO: Foi realizado um estudo retrospectivo utilizando 55 amostras de escarro de pacientes atendidos ambulatorialmente no Centro de Saúde Navegantes para realização da técnica de RFLP. Os resultados obtidos pela genotipagem foram correlacionados com os dados gerados a partir da epidemiologia convencional.
RESULTADOS: Trinta e nove isolados (70,9%) apresentaram padrão único, enquanto dezesseis isolados (29,1%) apresentaram padrões agrupáveis e formaram 8 clusters, com 2 pacientes em cada. Foi encontrada relação epidemiológica em 6 (37,5%) dos 16 pacientes em cluster.
CONCLUSÃO: A associação adequada entre epidemiologia convencional e genotipagem de M. tuberculosis contribui para um melhor entendimento da dinâmica de transmissão da tuberculose mesmo quando o estudo é realizado em um único local.

 

 

Keywords: Mycobacterium tuberculosis. Transmission. Polymorphism, Restriction Fragment Length. Epidemiology.

 

Palavras-chave: Mycobacterium tuberculosis. Transmissão. RFLP. Epidemiologia.

 

 

Introduction

Tuberculosis (TB) has no flag, uniform or homeland. It has been a long-time companion of man, perhaps even since we became bipeds. Reports have shown evidence of TB in prehistoric human bones(1). Recent molecular studies show that the disease-causing agent, Mycobacterium tuberculosis (Mtb), has resided in the human organism for approximately 10,000 years, since the time at which humans first began to domesticate and herd animals(2). More than a hundred years after Robert Koch identified the disease-causing agent (in 1882), TB still has one of the highest rates of morbidity and mortality among all such chronic infections.

The increased incidence of TB over the last two decades has been attributed to the decline of public health systems, to the HIV pandemic and to the appearance of drug-resistant strains, as well as to the strong correlation between the disease and poverty(3).

In spite of having a nationwide TB control program, Brazil reports approximately 90,000 new cases of the disease every year, although it is estimated that the actual number is 129,000(4). In the state of Rio Grande do Sul, the rate of TB incidence in the year 2000 was 55.7/100,000 inhabitants, and the number of new cases reported was 6,152, 65% of which occurred in the metropolitan area of Porto Alegre(5).

Genotyping techniques used to differentiate among strains of Mtb have improved understanding of the dynamics of TB transmission. The active form of the disease develops as commonly after a recently acquired infection as it does after reactivation of an infection acquired long before(3,6). In a population-based study, it was postulated that patients with TB presenting single strains should be considered cases of TB by reactivation, and those presenting isolates sharing indistinguishable patterns be designated as belonging to clusters, representing the development of the disease from a recently acquired infection(6,7).

The restriction fragment length polymorphism (RFLP) technique uses the 6110 insertion sequence (IS6110) to analyze polymorphism. The RFLP is the genotyping method most widely used to differentiate among Mtb strains and has been useful in carrying out various types of epidemiologic studies(6,8).

Because a better understanding of the dynamics of TB transmission is necessary, the present study aimed to assess the degree of similarity among strains of Mtb provided by the Phthisiology Sector of the Centro de Saúde Navegantes (CSN, Navegantes Health Clinic) in Porto Alegre. Molecular biology techniques were combined with conventional epidemiology.

Methods

Patients and Samples

The Mtb isolates used in the study were provided by the Tuberculosis Laboratory of the IPB-LACEN/RS, which receives patient samples from the Phthisiology Sector of the Navegantes Health Clinic, an outpatient clinic in the northern part of Porto Alegre. We analyzed 55 sputum samples obtained from patients examined at the CSN between 1999 and 2000. Apart from primary culture, the samples were also tested for resistance to anti-TB drugs. Clinical and demographic data were obtained from patient files, as well as through review of laboratory records and data in the Sistema de Informação de Agravos de Notificação (SINAN, National Case Registry) database. The present study was approved by the Ethics Reasearch Committee of the Fundação Estadual de Produção e Pesquisa em Saúde do Rio Grande do Sul (Rio Grande do Sul State Foundation for Health Research).

Microorganism Culture and Susceptibility Test

The Kudoh & Kudoh method was used to cultivate clinic samples(9). Drug-susceptibility tests were performed according to the proportion method. As described by Canetti et al. (10), the following drugs were used: 40 mg/mL of rifampin; 0.2 mg/mL of isoniazid; 4 mg/mL of streptomycin 2 mg/mL of ethambutol; 100 mg/mL of pyrazinamide; and 20 mg/mL of ethionamide. Strains resistant to at least the combination of isoniazid and rifampin were considered multidrug-resistant.

The Restriction Fragment Length Polymorphism (RFLP)/IS6110 Technique

Genomic DNA was extracted using cetyltrimethylammonium bromide method and the IS6110 RFLP was carried out according to the protocol devised by Van Embden et al. (11).

Data Analysis

GelCompar software, version 4.2 (Applied Maths, Kortrijk, Belguim) and visual analysis were used to analyze digitized RFLP images. EpiInfo software, version 6.04d (Centers for Disease Control and Prevention, USA) was used to analyze the clinical and demographic data of patients.

Results

Among the 55 Mtb isolates analyzed using the RFLP technique, 47 different band patterns were identified. Thirty-nine isolates (70.9%) presented a single pattern, whereas 16 isolates (29.1%) presented clustering patterns. Isolates having 6 or less copies of IS6110 represented 5.4% of the total number of samples analyzed. The remaining 94.6% had 7 or more copies of the insertion element. The number of bands ranged from 2 to 8. The average and the trend were 10 copies of IS6110 per isolate.

Isolates presenting 100% similarity were considered clusters. Out of the 55 isolates studied, 16 were grouped into 8 different clusters designated CL I, CL II, CL III, CL IV, CL V, CL VI, CL VII and CL VIII. Each cluster consisted of 2 isolates. Among the strain patterns identified through RFLP, three patterns belonged to the "A" family found in São Paulo(8) (Figure 1). Those patterns were visually compared with each other and then with the Mtb reference strain 14323, which was used as an external marker in each gel.

Cluster patients sharing the same household, living in the same neighborhood or belonging to the same family were considered to have an epidemiological link. Such an epidemiological link was observed for 6 patients (37.5%) in 3 of the 8 clusters identified. The links connecting those patients were based on residential proximity or family relation.

The average age was 42 among patients who developed TB after recent transmission and 43 among those who had reactivation TB. In addition, no significant gender-related age difference was found between cases resulting from recent transmission and those resulting from reactivation.

Between 1999 and 2000, the CSN reported 837 new TB cases. In 55 (6.57%) of those cases, samples were obtained and sent to the Tuberculosis Laboratory of the IPB-LACEN/RS. Only samples from patients not responding to Regime I were sent for culture, which explains the high proportion of previously treated patients (90.9%) in the study (Table 1).




Figure 1. Dendrogram representing the various RFLP patterns found in Mycobacterium tuberculosis isolates obtained from patients treated at the Centro de Saúde Navegantes. Clusters were designated CL I, CL II, CL III, CL IV, CL V, CL VI, CL VII, and CL VIII.
*Patterns of strains beloging to the "A" family found in São Paulo(8) ** M. tuberculosis reference strain 14323


TABLE 1 - Clínical and demographic characteristics of the patients evaluated



Forty isolates (72.7%) had different resistance profiles and only 15 (27.3%) were sensitive to all drugs used in the treatment of TB. Twenty-three isolates (41.8%) were resistant to one or more drug and 17 (30.9%) were multidrug-resistant.

Concerning the factors that may be associated with the development of TB, no significant correlation was found between clinical/demographic data and cluster isolates (Table 1). Despite what has been mentioned, data obtained from patients infected with resistant strains show the significance of these patients as links in the chain of recent transmission.

Discussion

Not so long ago, it was virtually impossible to identify individual Mtb strains. Therefore, tracking the transmission of a given strain in a region or institution was equally difficult. In recent years, genotyping techniques have been used to complement traditional methods of collecting epidemiological data.

Through RFLP analysis of Mtb isolates, 29.1% of patients evaluated in the present study were found to be in cluster, similar to proportions reported in studies involving isolates obtained from patients in Rio de Janeiro and São Paulo (22% and 23%, respectively)(12,8). In a study conducted in the metropolitan area of Porto Alegre, 34.5% of patients were found to be in cluster(13). Therefore, efforts aimed at control of the disease should focus not only on interrupting the chain of transmission but also on preventing reactivation TB. Our results are very similar to those of other studies carried out in Brazil (Table 2). As described in various other studies(14-16), patterns with an average of 10 bands per isolate were observed.

Even though 81.8% of patients were male, the rate of recent transmission among males did not differ from that found for female patients. This indicates that, although male gender may be a risk factor for the development of TB, it is not necessarily correlated with higher rates of recent transmission(6,8,17,18). In addition, no significant race-related difference was found in the percentage of cluster patients, although there is a greater tendency among black patients for developing the disease from recent transmission. In the present study, the small number of black patients involved probably accounted for the lack of any significant difference, since blacks represent only 5.2% of the total Rio Grande do Sul population, whereas whites represent 86.3%(19). It has been shown that blacks are more likely to develop the disease from recent transmission(6). Although this is probably related to socioeconomic status, there have been studies investigating the possibility that genetic factors influence susceptibility(20).

In the present study, 14.3% of HIV-positive patients developed TB from recent transmission, which was not significantly related the proportion of HIV-negative patients (34.6%) who developed the disease under the same conditions . Probably due to the small sample size, our data differ from what has been reported in the literature, in which HIV-positive patients have been shown to be more likely to develop TB - both from reactivation and from recently acquired infections(3).

Through analysis of the clinical data, an epidemiological link was identified for 6 patients (37.5%) who developed the disease from recent transmission. The percentage found in the present study was similar to that found in studies performed in Hong Kong (30.2%) and in the USA (Missouri: 46%; Arkansas: 42%)(21,22,23).

The high level of resistance to at least one drug (72.7%) observed in the present study is directly related to sample selection. A history of previous treatment is a predisposing factor for resistant bacilli selection(24), as evidenced by the fact that 90.9% of the patients evaluated in the present study had previously undergone treatment.


TABLE 2 - Comparison with results from other molecular epidemiological studies conducted in Brazil



The correlation between recent transmission and resistance was not significant, although 35% of patients presenting resistant strains developed TB after a recent transmission. Other studies have also shown that patients with resistant strains contribute significantly to cluster formation(6,8,15,25,29), which could be related to the difficulties encountered in detecting resistance and in treating these patients. In comparison to patients infected with sensitive strains, patients with drug-resistant strains require treatment that is more protracted, toxic and expensive, present lower cure rates, and tend to remain infected for longer periods(26).

Even though our study was limited to patients treated at the CSN, considerable diversity was seen among Mtb strain patterns, and a high rate of epidemiologic links was found among patients belonging to clusters. This can be attributed to the appropriate combination of conventional epidemiology techniques and genotyping, which provided an overview of the transmission dynamics of the disease at this location.

Acknowledgments

We would like to thank Nurse Mara Pessini of the Phthisiology Sector of the CSN, who was kind enough to provide us with the data necessary for the completion of the study. We are also grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council for Scientific and Technological Development) for providing financial support.

References

1. Conde MB, Souza GM, Kritski AL. Tuberculose sem medo.Editora Atheneu. 1ª ed. São Paulo: 2002.
2. Young DB. Blueprint For The White Plague. Nature 1998; 393.
3. Sepkowitz KA, Raffalli J, Riley L, Kiehn TE, Armstrong D. Tuberculosis in the AIDS era. Clin Microbiol 1995; 8:180-99.
4. Brasil. Ministério da Saúde. Manual técnico para o controle da Tuberculose: Cadernos de atenção básica. Brasília; 2002.
5. SES-RS. Secretaria da Saúde do Estado do Rio Grande do Sul. Programa de Controle da Tuberculose; 2003.
6. Small PM, Hopewell PC, Singh SP, Paz A, Parsonnet J, Ruston DC, et al. The epidemiology of tuberculosis in San Francisco; A population-based study using conventional and molecular methods. N Engl J Med 1994; 330:1703-9.
7. Tenover FC, Arbeit RD, Goering RV. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists. Infect Control Hosp Epidemiol 1997; 18:426-39.
8. Ferrazoli L, Palaci M, Marques LRM, Jamal LF, Afiune JB, Chimara E, et al. Transmission of tuberculosis in an endemic urban setting in Brazil. Int J Tuberc Lung Dis 2000; 4:1-8.
9. Kudoh S, Kudoh TA. A simple technique for culturing tubercle bacilli. Bulletin of the WHO 1974; 51:71-82.
10. Canetti GW, Fox A, Khomenko HT, Mahler NK, Menon D A, Mitchison N, et al. Advances in techniques of testing mycobacterial drug sensivity, and the use of sensitivity tests in tuberculosis control programmes. Bull.WHO 1969; 41:21-43.
11. van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, et al. Strain identification of M. tuberculosis by DNA fingerprinting: recommendations for a standadized methodology. J Clin Microbiol 1993; 31:406-9.
12. Suffys PN, Ivens de Araújo ME, Rossetti ML, Zaha A, Barroso EW, Barreto AMW, et al. Usefulness of IS6110-restriction fragment length polymorphism typing of Brazilian strains of Mycobacterium tuberculosis and comparison with an international fingerprint database. Res Microbiol 2000; 151:343-51.
13. Possuelo LG. Epidemiologia Molecular da Tuberculose na Região Metropolitana de Porto Alegre. Dissertação apresentada à Universidade Federal do Rio Grande do Sul para obtenção do título de Mestre em Ciências Biológicas: Bioquímica, 2003.
14. Niemann S, Rüsch-Gerdes S, Richter, E. IS6110 fingerprinting of Mycobacterium tuberculosis strains isolated in Germany during 1995. J Clin Microbiol 1997; 35:3015-20.
15. Fandinho FCO, Kritski AL, Hofer C, Conde HR, Ferreira RMC, Saad MHF, et al. RFLP patterns and risk factors for recent tuberculosis transmission among hospitalized tuberculosis patients in Rio de Janeiro, Brazil. Trans Royal Soc Trop Med and Hygiene 2000; 94:271-5.
16. Calusni ALR, Roscani GN, Villares MC, Soini H, Graviss E, Ramos MC. IS6110 Restriction Fragment Polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas, Brazil; Evidence of International Distribution of Strains. Mem Inst Oswaldo Cruz 2003; 98:655-8.
17. Diel R, Schneider S, Meywald-Walter K, Ruf CM, Rüsch-Gerdes S, Niemann S. Epidemiology of tuberculosis in Hamburg: long-term population-based analysis alpplying classical and molecular epidemiological techniques. J Clin Microbiol 2002; 40:532-9.
18. Gutiérrez MC, Vincent V, Aubert D, Bizet J, Gaillot O, Lebrun L, et al. Molecular fingerprinting of Mycobacterium tuberculosis and risk factors for tuberculosis transmission in Paris, France, and surroundig area. J Clin Microbiol 1998; 36:486-92.
19. IBGE - Instituto Brasileiro de Geografia e Estatística. 2000; Tabela 136. http//www.ibge.gov.br/SIDRA.
20. Stead WW. Variation in vulnerability to tuberculosis in America today: random, or legacies of different ancestral epidemics? Int Tuberc Lung Dis 2001; 5:807-14.
21. McConkey SJ, Willians M, Weiss D, Adams H, Cave MD, Yang Z, et al. Prospective use of molecular typing of Mycobacterium tuberculosis by use of restriction fragment-length polymorphism in a public tuberculosis-control program. Clin Infec Dis 2002; 34:612-9.
22. Chan-Yeung M, Tam CM, Wong H, Leung CC, Wang J, Yew WW. Molecular and Conventional Epidemiology of Tuberculosis in Hong Kong: a Population-Based Prospective Study. J Clin Microbiol 2003; 41:2706-8.
23. Braden CR, Templeton GL, Cave MD, Valway S, Onorato IM, Castro KG. Interpretation of restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from a state with a large rural population. J Infec Dis 1997; 175:1446-52.
24. Natal S, Valente JG, Sanches AR, Penna MLF. Isoniazid and rifampicin resistance and prior treatment for tuberculosis. Cad Saúde Pública 2003; 19(5):1277-81.
25. Alland D, Kalkut GE, Moss AR, McAdam RA, Hahn JA, Bosworth W, et al. Transmission of tuberculosis in New York City an analysis by DNA fingerprint and conventional epidemiologic methods. N Engl J Med 1994; 17:1710-6.
26. Sepkowitz KA, Raffalli J, Riley L, Kiehn TE, Armstrong D. Tuberculosis in the AIDS era. Clin Microbiol 1995; 8:180-99.
27. Ivens de Araújo ME, Fandinho FC, Werneck Barreto AM, Gonçalves Veloso V, Grinstejn B, Lourenço MK, et al. DNA fingerprint of Mycobacterium tuberculosis patients with and without AIDS in Rio de Janeiro. Braz J Med Res 1998; 31:369-72.
28. Lourenço MCS, Grinsztejn B, Fandinho Montes FCO, Silva MG, Saad MHF, Fonseca LS. Genotypic patterns of multiple isolates of M. tuberculosis from tuberculous HIV patients. Tropical Med and Intern Health 2000; 5:488-94.
29. Baptista IMFD, Oelemann MC, Opromolla DVA, Suffys, PN. Drug Resistance and Genotypes of Strais of Mycobacterium tuberculosis isolated from Human Immunodeficiency Virus-infected and Non-infected Tuberculosis Patients in Bauru, São Paulo, Brazil. Mem Inst Oswaldo Cruz 2002; 97:1147-52.

Abbreviations used in this paper:
CSN - Centro de Saúde Navegantes (Navegantes Health Clinic)
EMB - Ethambutol
ETH - Ethionamide
HIV - Human immunodeficiency virus
INH - Isoniazid
IS - Insertion sequence
Mtb - Mycobacterium tuberculosis
PZA - Pyrazinamide
RMP - Rifampicin
RFLP - Restriction Fragment Length Polymorphism
TB - Tuberculosis

Correspondence
Michele Borges
Av. Ipiranga, 5400, Bairro Jardim Botânico
CEP:90610-000, Porto Alegre, RS, Brasil
Email: micheborges@yahoo.com.br

Submitted: 20 February 2004.
Accepted, after review: 25 May 2004.
Financial support provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council for Scientific and Technological Development) and the Rede Brasileira de Pesquisa em TB (Rede-TB, Brazilian Tuberculosis Research Network)/Grant no. 62.0055/01-4-PACDT-Milenio.

* Study carried out at the Centro de Desenvolvimento Científico e Tecnológico (Center for Scientific and Technological Development) of the Fundação Estadual de Produção e Pesquisa em Saúde (State Foundation for Health Research), Porto Alegre, RS, Brazil

 

 


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