Memórias do Instituto Oswaldo Cruz On-line - Vol. 91(1) - Jan./Feb. 1996
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RESULTS AND DISCUSSION

Some preliminary results on the reactivity of monoclonal antibodies derived for selected species complexes of Leishmania (McMahon-Pratt et al. 1982, 1985, 1986, Jaffe & McMahon-Pratt 1983, Jaffe et al. 1984, Shaw et al. 1986, Pan & McMahon-Pratt 1988, Hanham et al. 1990) were confirmed in our recent studies using a large sample (1,500 Leishmania stocks, isolated from humans and a variety of mammalian and sandfly hosts) from different localities in the New World. Included are results of our work as well as data from other recently published studies (Grimaldi et al. 1987, 1989, 1991, Aguilar et al. 1989, Barral et al. 1991, Darce et al. 1991, Falqueto et al. 1991, Hashiguchi et al. 1991, Kreutzer et al. 1991, Ponce et al. 1991, Bonfante-Garrido et al. 1992).

Qualitatively, the reactivity of the monoclonal antibodies did not show any variation related to time in culture, culture media, or parasite virulence (Grimaldi et al. 1987). However, variation in the sensitivity of the test may occur due to the type of screening assay used (i.e., immunofluorescence, RIA or the ELISA technique). As an exemple, the L. donovani group-specific epitope recognized by monoclonal antibody D2, using RIA (Jaffe et al. 1984, Grimaldi et al. 1987) or ELISA, is weakly detected by IFA. However, as indicated in previous studies (McMahon-Pratt et al. 1986, Grimaldi et al. 1987, 1991, Barral et al. 1991), independent of the origin of Leishmania stock (i.e., host species involved or the clinical state of the infection) or of geographic area of isolation, some of the monoclonal antibodies showed a high and consistent qualitative specificity at a species level. In these analyses (see Table), the following monoclonals were the most specific: D2 (LXXVIII, 2E5-A8) for L. (L.) chagasi; B11 (VII-5G3-F3) for L. (V.) panamensis; B18 (XIV-2A5-A10) for L. (V.) braziliensis; M3 (IX-5H9-C10) for L. (L.) amazonensis; and V1 (CLXXVI-3C11-F14) for L. (L.) venezuelensis. However, significant differences between the reactivity patterns with specific monoclonal antibodies could be observed among stocks from certain species complexes of Leishmania from distinct endemic areas. These differences can be related with strain variation in the level of expression of certain antigenic determinants, as recognized by some of the monoclonal antibodies. For instance, Venezuelan isolates of L. (V.) braziliensis showed a distinct profile (Bonfante-Garrido et al. 1992) when they were compared with the same parasite species which circulates in Bolivia, Brazil or Colombia (Barral et al. 1991, Grimaldi et al. 1991, Grimaldi & McMahon-Pratt, unpublished data). Also, the species-specific epitope recognized by monoclonal antibody B19 (XLIV-5A2-B9) (Grimaldi et al. 1987) could not be detected in some variant strains of L. (V.) guyanensis (Grimaldi et al. 1991), indicating that they had lost the epitope. In addition, some L. (V.) braziliensis isolates from the Brazilian Amazon Region (Grimaldi et al. 1991), as well as other variant strains of this parasite from Bolivia and Peru (Grimaldi & McMahon-Pratt, unpublished data) did not react with the specific monoclonal antibody B16 (XIII-3E6-B11) that identify this species (Shaw et al. 1986, McMahon-Pratt et al. 1986, Grimaldi et al. 1987, Barral et al. 1991). Furthermore, although naturally occurring hybrid parasites (L. (V.) braziliensis x L. (V.) guyanensis) from Venezuela reacted with the specific monoclonal antibodies (B16 and B18) for L. (V.) braziliensis (Bonfante-Garrido et al. 1992, Grimaldi & McMahon-Pratt, unpublished data), conflicting results were obtained when the L. (V.) braziliensis x L. (V.) panamensis hybrids from Nicaragua were analyzed by the same method (Darce et al. 1991). Indeed, these monoclonals as well as the L. (V.) panamensis-specific monoclonal antibody (B11) did not react with the later hybrid isolates, indicating that they had lost the epitopes specific to both the parental species (Momen et al. 1993). On the other hand, numerical analysis of the enzymic profiles of L. (L.) venezuelensis isolates showed that this species was phenetically closely related to the WHO L. (L.) mexicana reference strain. However, these strains did not react with any of the monoclonal antibodies group-specific for L. mexicana complex parasites, other than the species-specific (V1) monoclonal antibody (Bonfante-Garrido et al. 1992).

As aforementioned, not all known species of Leishmania are recognized by a distinct/specific monoclonal antibody (Grimaldi et al. 1987, 1991, 1992, Kreutzer et al. 1991). However, the patterns observed with the less specific monoclonals (as defined by either qualitative or quantitative reactions with the expressed antigens) are indicative of these species. For example, monoclonal antibodies B3 (VI-4D10-D12) and B12 (XIII-3H6-A12) were found to be useful in the confirmation of L. (V.) braziliensis from Argentina, Bolivia, Brazil, Colombia, Nicaragua and Peru (Grimaldi et al. 1987, Barral et al. 1991, Grimaldi & McMahon-Pratt, unpublished data) or L. (V.) panamensis from Colombia, Costa Rica, Ecuador, Honduras and Nicaragua (Grimaldi et al., 1987 Grimaldi & McMahon-Pratt, unpublished data). In contrast, these monoclonals were not reactive in RIA tests with stocks of L. (V.) guyanensis, L. (V.) shawi, L. (V.) lainsoni, L. (V.) colombiensis or L. (V.) equatorensis regardless of geographic origin (Grimaldi et al., 1991, 1992, Kreutzer et al. 1991, Bonfante-Garrido et al. 1992, Grimaldi & McMahon-Pratt, unpublished data). In addition, although B4 (VI-2 A5-A4) crossreacted with L. (V.) panamensis, L. (V.) colombiensis and L. (V.) equatorensis (Kreutzer et al. 1991, Grimaldi et al. 1992), the former species could be easily distinguished using in conjunction the more specific monoclonal antibody (B11). Moreover, certain of the quantitative antigenic variations occurring betwen groups or species often exceed that detected within each of these taxonomic groups (Grimaldi et al. 1987).

There are other unusual features about leishmaniases of the New World. Although American cutaneous leishmaniasis is usually caused by parasite species belonging to the L. braziliensis or L. mexicana complex (Lainson & Shaw 1987, Grimaldi et al. 1989), a few cases of the disease from Brazil (Momen et al. 1985) and Ecuador (Hashiguchi et al. 1991) have been associated with a parasite similar to the Old World L. (L.) major. Interestingly, these L. major-like parasites cross-reacted with several monoclonal antibodies (T1, XLVI-5B8-A8; T2, XLVI-4H12-C2; T3, XLVI-5A5-D4; T4, LXVIII-1A4-G1; and T8, LXVII-3E12-F8) (Momen et al. 1985, Hashiguchi et al. 1991) produced against members of the L. major or L. tropica complex (Jaffe & McMahon-Pratt 1983). In addition, our experience would indicate the existence of a number of other leishmanial parasites circulating in the Americas [e.g., L. (V.) colombiensis; L. (L.) equatorensis; and L. (L.) venezuelensis] that also cross-reacted with the L. major species-specific monoclonals (Kreutzer et al. 1991, Bonfante-Garrido et al. 1992, Grimaldi et al. 1992). Work is now in progress to better define the phylogenetic relationship between these parasites and Old World L. (L.) major strains. Whatever the explanation for the existence of these L. major related parasites, the results point to caution for all researchers working with New World Leishmania isolates. We recommend that when classifying these parasites, reference strains of Old World species as well as the L. major-specific monoclonals (e.g., the monoclonal T1, XIX-2D8-D7) be included for comparison.

Several monoclonal antibodies (D-2, B-4, B-5, B-7, B-16, B-19, M-3, M-7, P-9, T-9) analyzed in this study were selected by the WHO Workshops and recommended for general use in the identification of Leishmania species. The analyses of strains brought by the participants of the Cali Workshop also pointed strongly to the need for the incorporation of additional monoclonals (e.g., B-3, B-11, B-12, B-18, V-1) in an expanded crosspanel. We should mention that a free "Monoclonal antibody kit" for diagnosis/identification of Leishmania species, consisting of lyophilized aliquots (100 µl) of the monoclonal antibodies (titers 10-4 to 10-6) will be available soon; as part of the kit, a description of methods (immunofluores-cence and the ELISA technique) will also be provided. Requests for the kit will be made to Dr F Modabber, World Health Organization, Geneva, Switzerland. A formal request form indicating the potential application and resources available for analyses will be requested by WHO.

In conclusion, problems related to the differentiation and identification of some leishmanial parasites were encountered using serodeme analysis with specific monoclonal antibodies, as well as when those samples were analyzed by isoenzyme characterization (Grimaldi et al. 1987, 1989, 1991). Some of these isolates represent additional new species (Grimaldi et al. 1991, 1992, Kreutzer et al. 1991) or hybrid parasites (Darce et al. 1991, Bonfante-Garrido et al. 1992) and further investigation with new monoclonal antibodies is recommended in these situations. A comparison of the discriminatory ability of the two typing methods for Leishmania using Simpson's index of diversity showed that serodeme analysis is more discriminating, even though the zymodeme analysis produced more groups (Cupolillo et al. 1993). The continual discovery of new leishmanial species in tropical America is, in part, a reflection of the increasingly sophisticated methods for parasite differentiation. However, it also indicates that there has been a much greater evolutionary divergence among this parasite group in the New World, compared to the Old World.

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