498
Limitations of the use of the mtp40 fragment as a marker
of differentiation between
Mycobacterium tuberculosis
and
M. bovis
CRISTINA VIANA- NIERO, SYLVIA CARDOSO LEÃO
The bacilli t hat cau se t u bercu losis (TB) belon g to the Mycobacterium tuberculosis complex, which is composed of M. t u bercu losis, M. bovis su bsp. bovis, M. african u m an d M. microt i, as well as t he M. bovis BCG st rain u sed for vaccin at ion . It has b een p ro p o sed t h a t n ewly d isco vered sp ecies shou ld also be in clu ded in t his complex. These n ew species in clu de M. can et t ii, a varian t of M. tuberculosis found in the Somalia region(1), M. bovis
su bsp. caprae, the etiologic agent of TB in goats(2)
an d M. pin n ipedii, which cau ses TB in sea lion s an d may also in fect hu man s(3 ).
Studies involving DNA- DNA hybridization and sequence analysis of the 16S rDNA sequence, the 16S- 23S intergenic spacer sequence and the gene en codin g t he hsp65 heat shock prot ein(4 ) have
shown that this complex, in fact, constitutes a single sp ecies. Fo r reaso n s essen t ially relat ed t o t h e medical and veterinary significance of this group of bacteria, as well as to its pathogenic power and the wide spectrum of hosts receptive to each species, the nomenclature remains unchanged.
Within this complex of bacteria, M. tuberculosis is t he prin cipal pat hogen in hu man s. However, cases of hu man TB resu lt in g from in fect ion wit h M. african u m an d M. can et t ii have been report ed, m a in ly in Africa(1 ). In a d d it io n , M. b o vis, t h e
et iologic agen t of bovin e TB, m ay also in fect hu man s an d ot her an imals. St u dies con du ct ed in Argen t in a an d En glan d showed t hat M. bovis is respon sible for 0.4% t o 1% of hu man TB cases(5,6).
Ac c o r d in g t o t h e P a n Am e r ic a n He a lt h Org an izat io n(7 ), 7 0 0 0 n ew cases o f TB ap p ear
an n u ally in Sou t h Am erica. There are n o dat a available regardin g hu man cases of TB resu lt in g from M. bovis in fect ion in Brazil. This species is n at u rally resist an t t o pyrazin amide (PZA), a dru g u sed in t he t reat men t of TB in hu man s, which, in cert ain cases, makes t he differen t iat ion bet ween species relevant. Such cases include those in which
e p id e m io lo g ica l e vid e n ce su g g e st s M. b o vis in volvemen t an d t hose in which t he pat ien t fails to respond to treatment regimes that include PZA. A diagnosis of TB caused by M. tuberculosis or by M. bovis can be made through analysis of clinical d a t a a n d ra d io lo g ic a l e vid e n c e . Ho w e ve r, bacteriological diagnosis is necessary in order to con firm t he diagn osis an d iden t ify t he species in volved. M. t u bercu losis an d M. bovis can be differentiated using phenotyping techniques such as tests for niacin production and nitrate reductase, as well as cultures to assess bacterial growth in the presence of thiophene- 2- carboxylic acid hydrazide and PZA(4). The polymerase chain reaction (PCR)
method has been incorporated into the routine of many laboratories as a diagnostic alternative due to its greater speed, sensitivity and specificity. This t ech n iq u e a llo ws t h e d ist in ct io n b et ween M. t u bercu losis and M. bovis t o be made t hrou gh differential amplification of the pncA and oxyR gene sequences(8), amplification and analysis of enzymatic
rest rict ion wit hin t he gyrB sequ en ce(9), mu lt iple
amplifications (multiplex PCR) of the DR fragment regions, the insertion sequence 6110 (IS6110) and the hsp65 gene(10), as well as amplification of the
m t p 4 0 f ra g m e n t , w h ic h is e xc lu sive t o M. tuberculosis and therefore absent from M. bovis(11).
The mtp40 fragment, contained within the plcA gene sequence, which encodes the phospholipase 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 claim that mtp40 is present in all clinically isolated M. tuberculosis strains has been challenged(13).
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an d Mycobact eriu m bovis as a cau se of hu man disease in Argen t in a. Trop Geogr Méd 1987; 39:222- 7. 6 . Yat es MD, Gran ge J M. In ciden ce an d n at u re of hu man
t u b ercu lo sis d u e t o b o vin e t u b ercle b acilli in So u t h -Ea st En g la n d : 1 9 7 7 - 1 9 8 7 . Ep id e m io l In fe ct 1 9 8 8 ; 101 : 2 2 5 - 9 .
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Fig ure 1. Location of the mtp40 fragment in the Mycobacterium tuberculo genome: (A) M. tuberculosis H37Rv; (B) insertion of a copy of the IS6 element into the mtp40 fragment; (C) deletion of the 8.6- kilobase fragm in clu din g t he mt p40 fragmen t . The black squ are wit hin t he plcA ge represents the mtp40 fragment and the hashed rectangle represents the insert sequence 6110. This region does not exist within the M. bovis genome.
for Public Health and the Environment) reference laboratory (Bilthoven, the Netherlands). The primers PT1 (CAACGCGCCGTCGGTGG) a n d PT2 (CCCCCCACGGCACCGC) were employed. We also evaluated the results of such amplification in 105 M. africanum strains and 10 M. canettii strains isolated at the Centre National de Référence des Méningocoques, In st it u t Past eu r (Nat ion al Referen ce Cen t er for Meningococci, Pasteur Institute, Paris France). Positive amplification results were obtained in 94.6%, 54.6% and 70%, respectively, of the isolated strains of each species (data from the RIVM)(14).
We studied, in detail, a set of 32 strains belonging to these two collections. These strains were chosen because they presented varying PCR results in relation to the mtp40 fragment. In 17 strains, a fragment of t h e exp ect ed size (3 9 6 b p ) was am p lified , n o amplification occurred in 13, and a fragment larger than expected (1700 bp) was amplified in 2 of the st rain s (bot h M. t u bercu losis). The presen ce of genetic polymorphisms in the phospholipase C gene, which would explain the absence of the mtp40 fragment from the isolates, was evaluated. The results show that, in most cases, the lack of mtp40 fragment amplification results from complete deletion of the plcA gene, as well as of the adjacent genes. In addition, insertion of a copy of the IS6110 element into the mtp40 fragment was observed in 2 of the isolat es, t hereby impedin g amplificat ion of t he fragment of the correct size (Figure 1)(15).
Th ere a re n o a va ila b le d a t a reg a rd in g t h e relevance of using this marker in Brazilian strains. However, t he exist en ce of M. t u bercu losis, M. africanum and M. canettii isolates presenting genetic polymorphisms serves as an indicator that we should be choosing molecular markers that are capable of d e fin it ive ly id e n t ifyin g M. t u b e rcu lo sis a n d differentiating it from other members of the complex. Curretly, it is advisable to use a combinaton of phenotypic and genotypic markers in the differential diagnosis between M. tuberculosis and M. bovis.
Cristina Viana-Niero, Sylvia Cardoso Leão. Department of Microbiology, Immunology and
Parasitology, UNIFESP-EPM e-mail: cviana@ ecb.epm.br
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.
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