ABSTRACT

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on Löwenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.

Keywords: Tuberculosis; Mycobacterium bovis; Polymerase chain reaction.

RESUMO

Entre 2005 e 2006, 8.121 espécimes clínicos enviados ao Laboratório de Micobactérias do Hospital Universitário Clementino Fraga Filho/Instituto de Doenças do Tórax, no Rio de Janeiro, RJ, foram inoculados em meio Löwenstein-Jensen contendo glicerol e piruvato. Desses espécimes, 79 isolados de micobactérias tiveram crescimento somente em meio com piruvato, sendo selecionados para a identificação presuntiva de Mycobacterium bovis. Esses isolados foram submetidos à identificação por testes bioquímicos, amplificação por PCR com primers específicos (Rv0577 e Rv1510) e teste de suscetibilidade à pirazinamida. Todas as cepas apresentaram padrões fenotípicos e genotípicos de M. tuberculosis, não sendo detectado M. bovis.

Palavras-chave: Tuberculose; Mycobacterium bovis; Reação em cadeia da polimerase.

Historically, Mycobacterium bovis has been associated with extrapulmonary tuberculosis in children, usually due to the consumption of raw milk from infected cows.(1) This framework is still in place in developing countries, especially in Africa, where there are reports that M. bovis has been isolated in 10-20% of children with cervical lymphadenitis.(2) In addition, in middle- and high-income countries, the pattern of M. bovis infection has changed, causing disease in hunters,(3) as well as in immunosuppressed individuals, such as alcoholics and HIV-infected patients.(4) In the United States, M. bovis infection has been identified in immigrant communities, particularly in those with a history of consuming unpasteurized dairy products.(5) Although bovine tuberculosis has been identified in Brazil,(6) there are no data available on human tuberculosis caused by M. bovis in the country. The lack of reporting might be due to the fact that few mycobacteriology laboratories routinely use culture media containing pyruvate. However, there is considerable epidemiological interest on the prevalence of zoonotic tuberculosis in Brazil. In addition, the fact that M. bovis is naturally resistant to pyrazinamide, one of the three drugs used in the treatment of tuberculosis, gives the isolation and identification of M. bovis in human clinical specimens a practical purpose in that this knowledge can inform decisions regarding treatment. The Mycobacteriology Laboratory of the Hospital Universitário Clementino Fraga Filho/Instituto de Doenças do Tórax da Universidade Federal do Rio de Janeiro (HUCFF/IDT-UFRJ, Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, Federal University of Rio de Janeiro) receives approximately 5,500 samples annually. The hospital is a referral center for the diagnosis and treatment of severe forms of chronic and acute diseases, as well as of tuberculosis/HIV co-infection, in the state of Rio de Janeiro, Brazil. The objective of the present study was to investigate the prevalence of M. bovis in patients at the hospital.

Between 2005 and 2006, all clinical specimens referred to the Laboratory of Mycobacteriology of the HUCFF/IDT-UFRJ were decontaminated with the Kubica method,(7) and 0.2 mL were inoculated into two tubes of glycerol-containing Löwenstein-Jensen (LJ) culture medium and into one tube of pyruvate-containing LJ medium. The tubes were incubated at 37°C for up to 90 days.(7) The phenotypic identification of the M. tuberculosis complex was performed by means of biochemical tests (for niacin production, nitrate reduction, and thermal inactivation of catalase).(7,8) Specimens were also submitted to the pyrazinamide susceptibility test, as described by Canetti et al.(9): culture medium (pH = 5.0-5.2) + 100 mg/mL of pyrazinamide. In that test, the critical proportion of pyrazinamide is 10%.(7) For the molecular identification, DNA extraction was carried out in accordance with national guidelines.(10) Amplification was performed according to Huard et al.(11) with modifications. In brief, two PCR mixes were prepared, each containing 1.25 mL of DMSO, 2.5 mL of 10× buffer, 0.75 mL of 50 mM magnesium chloride, 0.5 mL of 10 mM dNTP (A, T, C, and G), 13.8 mL of purified water, 0.2 mL of platinum Taq polymerase (Invitrogen, Karlsruhe, Germany), and 5 mL of bacterial DNA, in a final volume of 25 mL. To each of the mixes, 0.5 mL of one of the primers used (Rv1510 and Rv0577) were added.(11) The PCR amplification was performed as follows: 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min; and a final extension step at 72°C for 10 min. The PCR product was run on a 1% agarose gel containing 0.5% ethidium bromide solution and observed under UV light.

During the study period, our laboratory received 10,861 clinical samples for the routine diagnosis of mycobacterial infection, of which 8,121 were inoculated into tubes containing LJ medium + sodium pyruvate, yielding 1,793 positive cultures. Of those, 79 mycobacteria strains recovered from the clinical specimens presented growth only on LJ medium containing sodium pyruvate, and those isolates were selected for the presumptive identification of M. bovis. The screening using three biochemical tests-niacin production, nitrate reduction, and catalase inactivation-showed that 57 isolates belonged to the M. tuberculosis complex, 14 were nontuberculous mycobacteria, and 8 strains did not grow in a second culture in a new tube with LJ (Table 1).





All M. tuberculosis complex isolates showed the standard M. tuberculosis biochemical pattern: niacin production, nitrate reduction, and negativity for catalase. In order to confirm these data, a few isolates were randomly selected and submitted to the pyrazinamide susceptibility test. Scorpio et al.(12) cloned the pyrazinamidase gene (pncA) from M. tuberculosis and M. bovis and found that a mutation was only observed in strains of M. bovis and of several subspecies of BCG. This mutation makes M. bovis and BCG intrinsically resistant to pyrazinamide. Jong et al.(13) used the pyrazinamide test to identify M. bovis among 1,526 isolates and found that all 11 M. bovis isolates were resistant to pyrazinamide, compared with only 1.3% of the M. tuberculosis isolates. Of the 28 isolates identified as M. tuberculosis in the present study, only 3 were found to be resistant to pyrazinamide. In making the differential diagnosis between M. tuberculosis and M. bovis, it is advisable to use not only phenotypic tests but also genotypic markers,(14) and various molecular biology techniques have been used successfully in the characterization of M. tuberculosis isolates. Huard et al.(11) developed a PCR panel based on the deletions found in the complex in order to identify these species, using seven primers that amplified regions within the 16S rRNA loci. In the present study, we chose to perform PCR with two pairs of primers (Rv1510 and Rv0577) for better identification of M. tuberculosis isolates. The gene Rv0577 is present in all species of the M. tuberculosis complex, albeit absent in most nontuberculous mycobacteria, whereas the Rv1510 gene is known to be found in M. tuberculosis, M. africanum types I and II, and M. canettii, albeit absent in M. bovis and BCG. The 42 isolates that grew only on pyruvate and had a biochemical pattern consistent with M. tuberculosis were submitted to PCR, and all showed positive amplification for Rv1510, which is not found in M. bovis strains. Of the 42 isolates tested for Rv0577, 33 showed positive amplification, indicating that the isolates belonged to the M. tuberculosis complex (Table 2).




The results of the phenotypic and genotypic identification carried out in our study indicate that, during the study period, no M. bovis isolates were obtained. This result seems surprising because bovine tuberculosis is still prevalent in our country. In 2004, a study involving 454,108 animals showed a prevalence ranging from 0.37% in the southeast to 3.62% in the north.(6) In a study conducted in the state of Rio de Janeiro,(15) 12.7% of the dairy calves tested were found to be reactive to the skin prick test.

The present study was conducted at a tertiary hospital that serves as a referral center for transplant assessment, as well as for AIDS, cancer, and other diseases, which would facilitate the externalization of the M. bovis tuberculosis. Kantor et al.(16) extensively reviewed the international literature on zoonotic tuberculosis in Latin America, finding a prevalence ranging from 0.0% to 2.5%. According to the authors, human tuberculosis caused by M. bovis has been reported only in Argentina, Brazil, Ecuador, and Venezuela. The same authors showed that, during a 20-year period (1987-2006), the Professor Hélio Fraga Referral Center, located in the city of Rio de Janeiro and operating under the auspices of the Brazilian National Ministry of Health, identified only one case of tuberculosis caused by M. bovis, and that strain was isolated from the blood of an HIV-positive patient. From 2001 to 2005, the referral laboratories of the Adolfo Lutz Institute, located in the city of São Paulo, tested approximately 355,000 cultures using a pyruvate medium and isolated only two M. bovis strains, one from a lymph node sample and one from a liquor sample. In Rio Grande do Sul, a state with a tradition of animal husbandry, M. bovis was not identified among the 5,000 M. tuberculosis isolates tested. We were unable to identify any strain with an M. bovis profile among 1,793 mycobacteria isolates recovered during the study period.

One limitation of the present study is that the HUCFF/IDT-UFRJ primarily serves patients from urban areas. Although our data and those obtained by the other authors cited above showed no isolation of M. bovis, the presence of M. bovis in raw milk was demonstrated by two groups of authors in São Paulo.(17,18) Given that there is significant consumption of raw milk among the Brazilian population, these studies demonstrated that there is a potential risk of contamination by M. bovis that might lead to infection and illness. However, there have been very few reports of tuberculosis caused by M. bovis in the country. Zoonotic tuberculosis in Brazil is definitely underreported. Cultures for mycobacteria are performed, in regional and national laboratories, only in special cases or for drug resistance surveys and only on glycerol-containing media, on which M. bovis grows poorly. It is also possible that the lack of isolation of M. bovis is the result of improved control activities of bovine tuberculosis. Supporting this hypothesis is the fact that, in 2001, the Brazilian National Program for the Control and Eradication of Brucellosis and Animal Tuberculosis made it mandatory to carry out skin prick tests in animals owned by milk production cooperatives.


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* Study carried out in the Mycobacteriology Laboratory of the Hospital Universitário Clementino Fraga Filho/Instituto de Doenças do Tórax da Universidade Federal do Rio de Janeiro - HUCFF/IDT-UFRJ, Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil - and at Professor Paulo de Góes Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Correspondence to: Luciana Fonseca Sobral. Laboratório de Bacteriologia Veterinária, Universidade Federal Fluminense, CEP  24210‑-130, Niterói, RJ, Brazil.
Tel. 55 21 2629-2435. Fax: 55 21 2629-2432. E-mail: lucianafonsecas@gmail.com
Financial support: This study received financial support from the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council for Scientific and Technological Development), Institutos Nacionais de Ciência e Tecnologia (INCT, National Institutes of Science and Technology), and the International Clinical Operational and Health Services Research and Training Award (ICOHRTA) Mandate (AIDS/TB Grant no. 5 U2R TW006883-03). Luciana F. Sobral is the recipient of a grant from Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ, Rio de Janeiro Research Foundation).
Submitted: 8 December 2010. Accepted, after review: 2 May 2011.
** A versão completa em português deste artigo está disponível em www.jornaldepneumologia.com.br




About the authors

Luciana Fonseca Sobral
Biologist. Universidade Federal Fluminense, Niterói, Brazil.

Rafael Silva Duarte
Associate Professor. Prof. Paulo de Góes Institute of Microbiology, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Gisele Betzler de Oliveira Vieira
Pharmacist. Mycobacteriology Laboratory of Hospital Universitário Clementino Fraga Filho/Instituto de Doenças do Tórax da Universidade Federal do Rio de Janeiro - HUCFF/IDT-UFRJ, Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, Federal University of Rio de Janeiro - Rio de Janeiro, Brazil.

Marlei Gomes da Silva
Biologist. Professor Paulo de Góes Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.

Neio Boechat
Associate Professor. Federal University of Rio de Janeiro School of Medicine, Rio de Janeiro, Brazil.

Leila de Souza Fonseca
Full Professor. Professor Paulo de Góes Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.