UNIFESP-EPM Departamento de Microbiologia, Imunologia e ParasitologiaUNIFESP, EPM, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Leao, Sylvia Cardoso

Viana-Niero, Cristina

Jornal Brasileiro de Pneumologia

Cristina Viana-Niero

Sylvia Cardoso Leão

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Limitações do uso do fragmento mtp40 como marcador de diferenciação entre Mycobacterium tuberculosis e M. bovis Limitations of the use of the mtp40 fragment as a marker of differentiation between Mycobacterium tuberculosis and M. bovis

Viana-Niero, Cristina [UNIFESP]

Leao, Sylvia Cardoso [UNIFESP]

Repositório Institucional UNIFESP

498Limitations of the use of the mtp40 fragment as a markerof differentiation between Mycobacterium tuberculosisand M. bovisCRISTINA VIANA-NIERO, SYLVIA CARDOSO LEÃOThe bacilli that cause tuberculosis (TB) belongto the Mycobacterium tuberculosis complex, whichis composed of M. tuberculosis, M. bovis subsp.bovis, M. africanum and M. microti, as well as theM. bovis BCG strain used for vaccination. It hasbeen proposed that newly discovered speciesshould also be included in this complex. Thesenew species include M. canettii, a variant of M.tuberculosis found in the Somalia region(1), M. bovissubsp. caprae, the etiologic agent of TB in goats(2)and M. pinnipedii, which causes TB in sea lionsand may also infect humans(3).Studies involving DNA-DNA hybridization andsequence analysis of the 16S rDNA sequence, the16S-23S intergenic spacer sequence and the geneencoding the hsp65 heat shock protein(4) haveshown that this complex, in fact, constitutes a singlespecies. For reasons essentially related to themedical and veterinary significance of this groupof bacteria, as well as to its pathogenic power andthe wide spectrum of hosts receptive to each species,the nomenclature remains unchanged.Within this complex of bacteria, M. tuberculosisis the principal pathogen in humans. However,cases of human TB resulting from infection withM. africanum and M. canettii have been reported,mainly in Africa(1). In addition, M. bovis, theetiologic agent of bovine TB, may also infecthumans and other animals. Studies conducted inArgentina and England showed that M. bovis isresponsible for 0.4% to 1% of human TB cases(5,6).According to the Pan American HealthOrganization(7), 7000 new cases of TB appearannually in South America. There are no dataavailable regarding human cases of TB resultingfrom M. bovis infection in Brazil. This species isnaturally resistant to pyrazinamide (PZA), a drugused in the treatment of TB in humans, which, incertain cases, makes the differentiation betweenspecies relevant. Such cases include those in whichepidemiological evidence suggests M. bovisinvolvement and those in which the patient failsto respond to treatment regimes that include PZA.A diagnosis of TB caused by M. tuberculosis orby M. bovis can be made through analysis of clinicaldata and radiological evidence. However,bacteriological diagnosis is necessary in order toconfirm the diagnosis and identify the speciesinvolved. M. tuberculosis and M. bovis can bedifferentiated using phenotyping techniques suchas tests for niacin production and nitrate reductase,as well as cultures to assess bacterial growth in thepresence of thiophene-2-carboxylic acid hydrazideand PZA(4). The polymerase chain reaction (PCR)method has been incorporated into the routine ofmany laboratories as a diagnostic alternative dueto its greater speed, sensitivity and specificity. Thistechnique allows the distinction between M.tuberculosis and M. bovis to be made throughdifferential amplification of the pncA and oxyR genesequences(8), amplification and analysis of enzymaticrestriction within the gyrB sequence(9), multipleamplifications (multiplex PCR) of the DR fragmentregions, the insertion sequence 6110 (IS6110) andthe hsp65 gene(10), as well as amplification of themtp40 fragment, which is exclusive to M.tuberculosis and therefore absent from M. bovis(11).The mtp40 fragment, contained within the plcAgene sequence, which encodes the phospholipase Cenzyme of M. tuberculosis, has been widely used forspecific 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 verifiedby various authors, although the claim that mtp40 ispresent in all clinically isolated M. tuberculosis strainshas been challenged(13).In our laboratory, we analyzed the results fromthe amplification of the mtp40 fragment in 790 M.tuberculosis strains isolated at the Rijksinstituut voorVolksgezondheid en Milieu (RIVM, National Institute499REFERENCES1. van Soolingen D, Hoogenboezem T, de Haas PEW,Hermans PWM, Koedam MA, Teppema KS, et al. A novelpathogenic taxon of the Mycobacterium tuberculosiscomplex, Canettii: Characterization of an exceptionali so late f rom Afr ica .  Int  J  Syst  Bacter io l  1997;47(4):1236-45.2. Aranaz A, Cousins D, Mateos A, Domínguez L. Elevationof Mycobacterium tuberculosis subsp. caprae Aranazet al. 1999 to species rank as Mycobacterium capraecomb. nov., sp. nov. Int J Syst Evol Microbiol 2003;53:1785-9.3. Cousins D, Bastida R, Cataldi A, Quse V, Redrobe S,Dow S, et al. A Tuberculosis in seals caused by a novelmember of the Mycobacterium tuberculosis complex:Mycobacterium pinnipedii sp. nov. Int J Syst EvolMicrobiol 2003; 53:1305-14.4. Euzéby JP. List of Bacterial names with Standing inNomenclature – Societé de Bactériologie Systématiqueet Vétérinaire – France. Disponível em (URL: http://www.bacterio.cict.fr). Acesso em 20 out 2003.5. Barrera L, De Kantor IN. Nontuberculous mycobacteriaand Mycobacterium bovis as a cause of human diseasein Argentina. Trop Geogr Méd 1987; 39:222-7.6. Yates MD, Grange JM. Incidence and nature of humantuberculosis due to bovine tubercle bacilli in South-East England: 1977-1987. Epidemiol Infect 1988;101:225-9.7. Pan American Health Organization. 1991. Health conditionsin the Americas, vol I. Scientific publication nº524. PanAmerican Health Organization, Washington, DC.8. Espinosa de los Monteros LE, Galan JC, Gutierrez M,Figure 1. Location of the mtp40 fragment in the Mycobacterium tuberculogenome: (A) M. tuberculosis H37Rv; (B) insertion of a copy of the IS6element into the mtp40 fragment; (C) deletion of the 8.6-kilobase fragmincluding the mtp40 fragment. The black square within the plcA gerepresents the mtp40 fragment and the hashed rectangle represents the insertsequence 6110. This region does not exist within the M. bovis genome.for Public Health and the Environment) referencelaboratory (Bilthoven, the Netherlands). The primersPT1 (CAACGCGCCGTCGGTGG) and PT2(CCCCCCACGGCACCGC) were employed. We alsoevaluated the results of such amplification in 105 M.africanum strains and 10 M. canettii strains isolated atthe Centre National de Référence des Méningocoques,Institut Pasteur (National Reference Center forMeningococci, Pasteur Institute, Paris France). Positiveamplification results were obtained in 94.6%, 54.6%and 70%, respectively, of the isolated strains of eachspecies (data from the RIVM)(14).We studied, in detail, a set of 32 strains belongingto these two collections. These strains were chosenbecause they presented varying PCR results in relationto the mtp40 fragment. In 17 strains, a fragment ofthe expected size (396 bp) was amplified, noamplification occurred in 13, and a fragment largerthan expected (1700 bp) was amplified in 2 of thestrains (both M. tuberculosis). The presence ofgenetic polymorphisms in the phospholipase C gene,which would explain the absence of the mtp40fragment from the isolates, was evaluated. The resultsshow that, in most cases, the lack of mtp40 fragmentamplification results from complete deletion of theplcA gene, as well as of the adjacent genes. Inaddition, insertion of a copy of the IS6110 elementinto the mtp40 fragment was observed in 2 of theisolates, thereby impeding amplification of thefragment of the correct size (Figure 1)(15).There are no available data regarding therelevance of using this marker in Brazilian strains.However, the existence of M. tuberculosis, M.africanum and M. canettii isolates presenting geneticpolymorphisms serves as an indicator that we shouldbe choosing molecular markers that are capable ofdefinitively identifying M. tuberculosis anddifferentiating it from other members of the complex.Curretly, it is advisable to use a combinaton ofphenotypic and genotypic markers in the differentialdiagnosis between M. tuberculosis and M. bovis.Cristina Viana-Niero, Sylvia Cardoso Leão.Department of Microbiology, Immunology andParasitology, UNIFESP-EPMe-mail: cviana@ecb.epm.brFINANCIAL SUPPORT:FAPESP (grant no. 00/02525-3);CABBIO-CNPq (grant no. 480382/01-8);Rede Brasileira de Pesquisa em TB (Rede-TB, BrazilianTuberculosis Research Network)/grant no. 62.0055/01-4-PACDT-Milenio500Samper S, Garcia Marin JF, Martin C, Dominguez L, etal. Allele-specific PCR method based on pncA and oxyRsequences for distinguishing Mycobacterium bovisfrom Mycobacterium tuberculosis: Intraspecific M.bovis pncA sequence polymorphism. J Clin Microbiol1998; 36:239-42.9. Chimara E, Ferrazoli L, Leão SC. Mycobacteriumtuberculosis complex differentiation using gyrB-restrictionfragment length polymorphism (gyrB-RFLP) analysis.Submetido a Memórias do Instituto Oswaldo Cruz. 2004.10 . Yeboah-Manu D, Yates MD, Wilson SM. Application ofa Simple Multiplex PCR To Aid in Routine Work of theMycobacterium Reference Laboratory. J Clin Microbiol2001; 39(11): 4166-8.11 . Del Portillo P, Murillo LA, Patarroyo ME. Amplificationof a species-specific DNA fragment Mycobacteriumtuberculosis and its possible use in diagnosis. J ClinMicrobiol 1991; 29(10):2163-8.12 . Liébana E, Aranaz A, Francis B, Cousins D. Assessmentof genetic markers for species differentiation withinthe Mycobacter ium tuberculosis complex. J Cl inMicrobiol 1996; 34(4):933-8.13 . Vera-Cabrera L, Hoard ST, Laszlo A, Johnson WM.Analysis of genetic polymorfism in the phospholipaseregion of Mycobacterium tuberculosis. J Clin Microbiol1997; 35(5):1190-5.14 . Viana-Niero C, Vincent V. 1999. Étude moléculaire desbac i l les  de la  tuberculose d ’or ig ine af r ica ine:Mycobacterium africanum et souches “canetti”. Tesede mestrado apresentada à Université Paris V etUniversité Paris XI.15 . Viana-Niero C, de Haas PE, van Soolingen D, Leão S C.Analysis of genetic polymorphisms affecting the fourphosphol ipase C (p lc )  genes in Mycobacter iumtuberculosis complex clinical isolates. Microbiol 2004;150:967-78.

Limitations of the use of the mtp40 fragment as a marker of differentiation between Mycobacterium tuberculosis and M. bovis

http://dx.doi.org/10.1590/S1806-37132004000400018

Limitations of the use of the mtp40 fragment as a marker of differentiation between Mycobacterium tuberculosis and M. bovis

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