RESUMO

Os bacilos que causam a tuberculose estão agrupados no complexo Mycobacterium tuberculosis, composto por: M. tuberculosis, M. bovis subsp. bovis, M. africanum e M. microti, além da cepa vacinal M. bovis BCG. Tem sido proposta a inclusão de novas espécies neste complexo, como M. canettii, uma variante de M. tuberculosis encontrada na região da Somália(1), M. caprae, agente etiológico de tuberculose em caprinos(2), e M. pinnipedii, que causa tuberculose em leões marinhos, mas que também pode infectar o homem(3).

The bacilli that cause tuberculosis (TB) belong to the Mycobacterium tuberculosis complex, which is composed of M. tuberculosis, M. bovis subsp. bovis, M. africanum and M. microti, as well as the M.bovis BCG strain used for vaccination. It has been proposed that newlydiscovered species should also be included in this complex. These new speciesinclude M. canettii, a variant of M. tuberculosis found in the Somaliaregion(1), M. bovissubsp. caprae, the etiologicagent of TB in goats(2) and M. pinnipedii, which causes TB in sealions and may also infect humans(3).

Studiesinvolving DNA-DNA hybridization and sequence analysis of the 16S rDNA sequence,the 16S-23S intergenic spacer sequence and the gene encoding the hsp65 heatshock protein(4) have shown thatthis complex, in fact, constitutes a single species. For reasons essentiallyrelated to the medical and veterinary significance of this group of bacteria,as well as to its pathogenic power and the wide spectrum of hosts receptive toeach species, the nomenclature remains unchanged.

Withinthis complex of bacteria, M. tuberculosisis the principal pathogen in humans. However, cases of human TB resulting frominfection with M. africanum and M. canettii have been reported, mainlyin Africa(1). In addition, M. bovis, the etiologic agent of bovineTB, may also infect humans and other animals. Studies conducted in Argentinaand England showed that M. bovis isresponsible for 0.4% to 1% of human TB cases(5,6). According to the Pan American Health Organization(7), 7000 new cases of TB appear annually in SouthAmerica. There are no data available regarding human cases of TB resulting fromM. bovis infection in Brazil. Thisspecies is naturally resistant to pyrazinamide (PZA), a drug used in thetreatment of TB in humans, which, in certain cases, makes the differentiationbetween species relevant. Such cases include those in which epidemiologicalevidence suggests M. bovisinvolvement and those in which the patient fails to respond to treatmentregimes that include PZA.

Adiagnosis of TB caused by M. tuberculosisor by M. bovis can be made throughanalysis of clinical data and radiological evidence. However, bacteriologicaldiagnosis is necessary in order to confirm the diagnosis and identify thespecies involved. M. tuberculosis andM. bovis can be differentiated usingphenotyping techniques such as tests for niacin production and nitratereductase, as well as cultures to assess bacterial growth in the presence ofthiophene-2-carboxylic acid hydrazide and PZA(4). The polymerase chain reaction (PCR) method has been incorporated intothe routine of many laboratories as a diagnostic alternative due to its greaterspeed, sensitivity and specificity. This technique allows the distinctionbetween M. tuberculosis and M. bovis to be made through differentialamplification of the pncA and oxyR gene sequences(8), amplification and analysis of enzymatic restriction within the gyrBsequence(9), multipleamplifications (multiplex PCR) of the DR fragment regions, the insertionsequence 6110 (IS6110) and the hsp65gene(10), as well asamplification of the mtp40 fragment, which is exclusive to M. tuberculosis and therefore absent from M. bovis(11).

Themtp40 fragment, contained within the plcA gene sequence, which encodes thephospholipase C enzyme of M. tuberculosis,has been widely used for specific diagnosis of M. tuberculosis in (uncultured) clinical samples(11) and in cultures of isolated strains(12). The absence of mtp40 from M. bovis has been verified by various authors, although the claimthat mtp40 is present in all clinically isolated M. tuberculosis strains has been challenged(13).

Inour laboratory, we analyzed the results from the amplification of the mtp40fragment in 790 M. tuberculosisstrains isolated at the Rijksinstituutvoor Volksgezondheid en Milieu (RIVM, National Institute for Public Healthand the Environment) reference laboratory (Bilthoven, the Netherlands). Theprimers PT1 (CAACGCGCCGTCGGTGG) and PT2 (CCCCCCACGGCACCGC) were employed. Wealso evaluated the results of such amplification in 105 M. africanum strains and 10 M.canettii strains isolated at the CentreNational de Référence des Méningocoques, Institut Pasteur (NationalReference Center for Meningococci, Pasteur Institute, Paris France). Positiveamplification results were obtained in 94.6%, 54.6% and 70%, respectively, ofthe isolated strains of each species (data from the RIVM)(14).

Westudied, in detail, a set of 32 strains belonging to these two collections.These strains were chosen because they presented varying PCR results inrelation to the mtp40 fragment. In 17 strains, a fragment of the expected size(396 bp) was amplified, no amplification occurred in 13, and a fragment largerthan expected (1700 bp) was amplified in 2 of the strains (both M. tuberculosis). The presence ofgenetic polymorphisms in the phospholipase C gene, which would explain theabsence of the mtp40 fragment from the isolates, was evaluated. The resultsshow that, in most cases, the lack of mtp40 fragment amplification results fromcomplete deletion of the plcA gene, as well as of the adjacent genes. Inaddition, insertion of a copy of the IS6110element into the mtp40 fragment was observed in 2 of the isolates, therebyimpeding amplification of the fragment of the correct size (Figure 1)(15).





Thereare no available data regarding the relevance of using this marker in Brazilianstrains. However, the existence of M.tuberculosis, M. africanum and M.canettii isolates presenting genetic polymorphisms serves as an indicatorthat we should be choosing molecular markers that are capable of definitivelyidentifying M. tuberculosis anddifferentiating it from other members of the complex. Curretly, it is advisableto use a combinaton of phenotypic and genotypic markers in the differentialdiagnosis between M. tuberculosis andM. bovis.

Cristina Viana-Niero; Sylvia Cardoso Leão

Department of Microbiology, Immunology and Parasitology, UNIFESP-EPM
e-mail: cviana@ecb.epm.br

References

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Financial support:
FAPESP (grant no. 00/02525-3);
CABBIO-CNPq (grant no. 480382/01-8);
Rede Brasileira de Pesquisa em TB (Rede-TB, Brazilian Tuberculosis Research Network)/grant no. 62.0055/01-4-PACDT-Milenio