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Molecular approaches to DNA diagnosis

Published online by Cambridge University Press:  06 April 2009

D. C. Barker
D. C. Barker
Affiliation:
MRC Outstation of NIMR, Molteno Laboratories, Department of Pathology, Cambridge CB2 1QP
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Summary

The DNA of a parasite is the ultimate blueprint of that parasite, the one characteristic which normally remains unchanged during every stage of the life-cycle. All the DNA sequence in the egg of a species of parasite are also in the larvae and adults of the same species. The same DNA is present in the parasite whether it is in a free-living stage, in an invertebrate vector or in a vertebrate host such as man. The molecular basis for DNA diagnosis is to allow labelled single-stranded species or strain-specific DNA sequences, selected from well-characterized reference species, to find and hybridize with homologous DNA from, or in, the unknown isolates of parasites. DNA probes are now available for most vector borne parasitic diseases. Parasitological identification problems are mostly concerned with distinguishing closely related strains or subspecies, for example detecting Taenia solium eggs as opposed to T. saginata eggs, or finding which of the 15 man-infecting subspecies of Leishmania is present in a single cutaneous lesion, the commonest clinical sign of the disease, or in a sandfly. For efficient hybridization by the present methods there has to be enough of a particular sequence present in a parasite's genome to make a feasible target. Therefore, DNA probes for parasites have been selected from repetitive, reiterated or multicopy DNA with intrinsic extensive sequence variation. DNA, which is free of coding restraint, can evolve rapidly to give differences between species, so that introns, ribosome gene spacers, variant genes, pseudo-genes and non-conserved DNA have all been used for DNA diagnosis. The major problems of sequence selection have been greatly aided by the use of recombinant DNA methods, which have the added advantage of economical production of DNA probes. The unique characteristics of kinetoplast mini-circle DNA in Leishmania has allowed the selection of a complex species, subspecies, strain and even isolate-specific DNA probes. These have been used successfully for Southern filter endonuclease fragment DNA identification, for dot-blot recognition of less than 200 parasites and non-radioactive detection of DNA sequence homology by ‘in situ’ hybridization and light microscopy in a single Leishmania cell. The adaptation of the forensic human genetic fingerprinting technique has allowed identification of L. braziliensis DNA in human biopsy material, even the presence of a vast excess of human DNA. Fingerprinting with ribosomal spacer-derived recombinant DNA probes has been used to discriminate Echinococcus species. The identification of Taenia species has been accomplished using probes from a genomic library of size-selected DNA fragments, and synthetic oligonucleotides are available for Onchocerca subspecies detection. The new technologies combining repeated genomic sequence probes with pulse field separation of the chromosomes of parasites, has opened up new avenues of research. Double probes for the simultaneous identification of the insect vector and the carried parasite have recently been reported. Lastly, the polymerase chain reaction technique has given us the opportunity of amplifying even single copy genes in one parasite to give sufficient DNA for positive identification. DNA diagnosis in parasitology is now on a par with the DNA diagnosis of viruses and human genetic disorders. The last ten years have seen many exciting developments in DNA diagnosis; the next years should see DNA diagnosis routinely used in epidemiological and clinical situations in countries where parasitic diseases are a major public health problem.

Keywords

DNA diagnosisleishmaniasishybridizationkinetoplast DNA
Type
Research Article
Information
Parasitology , Volume 99 , supplement S1: Symposium of the British Society for Parasitoiogy and the Linnean Society Special Issue: Research developments in the study of parasitic infections , January 1989 , pp. S125 - S146
Copyright
Copyright © Cambridge University Press 1989

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References

REFERENCES

Alvar, J. & Garcia-Saiz, A. (1989). Leishmaniasis–AIDS Association: New epidemiological perspectives: In Leishmaniasis: The First Centenary (1885–1985). New Strategies for Control (ed. Hart, D. T.). NATO/ASI Series A: Life Sciences, London: Plenum Publishing Co. Ltd. 161, 761–3.Google Scholar
Anon. (1984). The Leishmaniases. Report of a WHO Expert Committee. WHO Technical Report Series 701, 1–140.Google Scholar
Arnot, D. E. & Barker, D. C. (1981). Biochemical identification of cutaneous Leishmania by analysis of kinetoplast DNA. II Sequence homologies in Leishmania kDNA. Molecular and Biochemical Parasitology 3, 47–56.CrossRefGoogle ScholarPubMed
Bain, O. (1981). Le genre Onchocerca: hypothesis sur son évolution et clé dichorotique des espèces. Annals of Tropical Medicine and Parasitology 56, 503–26.Google Scholar
Barker, D. C. (1980). The ultrastructure of kinetoplast DNA with particular reference to the interpretation of darkfield electron microscopy images of isolated purified networks. Micron 11, 21–62.Google Scholar
Barker, D. C. (1987). DNA diagnosis of human leishmaniasis. Parasitology Today 3, 177–84.Google Scholar
Barker, D. C. (1989). Characterization by light microscope ‘in situ’ hybridization within 12 hours of live Leishmania observation. In Leishmaniasis: The First Centenary (1885–1985). New Strategies for Control (ed. Hart, D. T.). NATO/ASI Series A: Life Sciences, London: Plenum Publishing Co. Ltd. 161, 869–77.Google Scholar
Barker, D. C. & Arnot, D. E. (1981). Biochemical identification of cutaneous Leishmania by analysis of kinetoplast DNA. I. Ultrastructure and buoyant density analysis. Molecular and Biochemical Parasitology 3, 33–46.Google Scholar
Barker, D. C., Arnot, D. E. & Butcher, J. (1982). DNA characterization as a taxonomic tool for identification of kinetoplastic flagellate protozoans. In Proceedings of the Workshop of Pan American Health Organization. Biochemical Characterization of Leishmania. Washington D. C. (1980), (ed. Chance, M. L. & Walton, B. C.), pp. 139–180. Geneva: UNDP/World Bank/WHO.Google Scholar
Barker, D. C. & Butcher, J. (1983). The use of DNA probes in the identification of leishmaniasis: discrimination between isolates of the Leishmania mexicana and L. braziliensis complexes. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 285–97.Google Scholar
Barker, D. C., Butcher, J., Gibson, L. J. & Williams, R. H. (1986 c). Characterization of Leishmania sp. by DNA hybridization probes. UNDP/World Bank/WHO/TDR. Laboratory Manual 57 pp.Google Scholar
Barker, D. C., Butcher, J., Gibson, L. J., Kennedy, W. P. K. & Williams, R. H., Cuba Cuba, C. A., Marsden, P. D., Lainson, R. & Shaw, J. J. (1986 b). Sequence homology of kinetoplast DNA in Leishmania studied by filter hybridization of endonuclease digested fragments and ‘in situ’ hybridization of individual organisms. In Leishmania, Taxonomie et Phylogenese. Imeee, Montpellier, pp. 41–55.Google Scholar
Barker, D. C., Gibson, L. J. & Williams, R. H. (1989). Assessment of the use in the diagnosis of leishmaniasis of biotinylated mini-circle derived DNA sequences. In Leishmaniasis: The First Centenary (1885–1985). New Strategies for Control (ed. Hart, D. T.). NATO/ASI Series A: Life Sciences, London: Plenum Publishing Co. Ltd. 161, 481–93.Google Scholar
Barker, D. C., Gibson, L. J., Kennedy, W. P. K., Nasser, A. A. A. & Williams, R. H. (1986 a). The potential of using recombinant DNA species-specific probes for the identification of tropical Leishmania. Parasitology, 91 (Suppl.), S139–S174.CrossRefGoogle Scholar
Bettini, S., Gramiccia, M., Gradoni, L., Pozio, E., Mugnai, S. & Maroli, M. (1983). Leishmaniasis in Tuscany (Italy) VIII–Human population response to leishmanin in the focus of Monte Argentario (Grosseto) and epidemiological evaluation. Annals de Parasitologic 58, 539–47.Google Scholar
Beverley, S. M., Ismach, R. B. & McMahon, Pratt D. (1987). Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns. Proceedings of the National Academy of Sciences, USA 84, 484–8.Google Scholar
Bishop, R. P. & Miles, M. A. (1987). Chromosome size polymorphisms of Leishmania donovani. Molecular and Biochemical Parasitology 24, 263–72.Google Scholar
Blaxter, M. L., Miles, M. A. & Kelly, J. M. (1988). Specific serodiagnosis of visceral leishmaniasis using a Leishmania donovani antigen identified by expression cloning. Molecular and Biochemical Parasitology 30, 259–70.CrossRefGoogle ScholarPubMed
Bryceson, A. D. M. (1972). Immunological aspects of cutaneous leishmaniasis. In Essays on Tropical Dermatology (ed. Marshall, J.), pp. 230–41. Amsterdam: Excerpta Medica.Google Scholar
Borst, P. & Hoeijmakers, J. H. J. (1979). Kinetoplast DNA (Review). Plasmid 2, 20–40.CrossRefGoogle Scholar
Chapman, C. J. (1988). The development of rapid identification techniques for ‘Old World’ leishmaniasis using monoclonal antibodies and kinetoplast DNA methodologies. Ph.D. thesis, University of London, London School of Hygiene and Tropical Medicine.Google Scholar
Comeau, A. M., Miller, S. I. & Wirth, D. F. (1986). Chromosome location of four genes in Leishmania. Molecular and Biochemical Parasitology 21, 161–9.Google Scholar
De Bruijn, M. H. L. (1988). Diagnostic DNA amplification: no respite for the elusive parasite. Parasitology Today 4, 293–5.CrossRefGoogle ScholarPubMed
Ellis, J. & Crampton, J. (1988). Characterization of a simple, highly repetitive DNA sequence from the parasite Leishmania donovani. Molecular and Biochemical Parasitology 29, 9–18.CrossRefGoogle ScholarPubMed
Englund, P. T. (1979). Kinetoplast DNA. Journal of Biological Chemistry 254, 4895–4900.Google Scholar
Erlich, H. A., Gelfand, D. H. & Saiki, R. K. (1988). Specific DNA amplification. Nature, London 461–2.Google Scholar
Erttmann, K. D., Unnasch, T. R., Greene, B. M., Albeiz, E. J., Boateng, J., Denke, A. M., Ferraroni, J. J., Karam, M., Schulz-Key, H. & Williams, P. N. (1987). A DNA sequence specific for forest form Onchocerca volvulus. Nature, London 327, 415–17.Google Scholar
Giannini, S. H., Schittini, M., Keithly, J. S., Warburton, P. W., Cantor, C. R. & Van Den Ploeg, L. H. T. (1986). Karyotype analysis of Leishmania species and its use in classification and clinical diagnosis. Science 232, 762–5.CrossRefGoogle ScholarPubMed
Giannini, S. H. (1988). Chromosome size homologies in Leishmania major determined by molecular karyotyping. In Leishmaniasis: The First Centenary (1885–1985). New Strategies for Control (ed. Hart, D. T.). NATO/ASI Series A: Life Sciences, London: Plenum Publishing Co. 161, 883–9.Google Scholar
Gibson, W. C., Dukes, P. & Gashumba, J. K. (1988). Species-specific DNA probes for the identification of African trypanosomes in tsetse flies. Parasitology 97, 63–73.CrossRefGoogle ScholarPubMed
Gill, P., Jeffreys, A. J. & Werrett, D. J. (1985). Forensic application of DNA fingerprints. Nature, London 318, 577–9.Google Scholar
Harnett, W., Chambers, A. E., Renz, A. & Parkhouse, R. M. E. (1988). The use of synthetic oligonucleotide probes in the DNA identification of Onchocerca volvulus. Molecular and Biochemical Parasitology 35, 119–26.CrossRefGoogle Scholar
Harrison, L. J. S., Delgado, J. & Parkhouse, R. M. E. (1988). Differentiation of Taenia saginata and Taenia solium by the use of cloned DNA fragments. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 174.Google Scholar
Harrison, L. J. S., Delgado, J. & Parkhouse, R. M. E. (1988). DNA probes in the differentiation of human Taenia saginata and Taenia solium infection. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 939.Google Scholar
Harrison, L. J. S. & Parkhouse, R. M. E. (1988). Taenia saginata and Taenia solium: reciprocal models. In Cysticercosis – Now; Satellite Symposium on Human Neurocysticercosis. Acta Leidensia (in the Press).Google Scholar
Harrison, L. J. S. & Sewell, M. M. H. (1988). The zoonotic Taeniae of Africa. In Parasitic Worms, Zoonosis and Human Health in Africa. (ed. MacPherson, C. N. L. & Craig, P. S.). London: Unwin Hyman. (in the Press).Google Scholar
Heath, S., Chance, M. L., Hommel, M. & Crampton, J. M. (1987). Cloning of a gene encoding the immunodominant surface antigen of Leishmania donovani promastigotes. Molecular and Biochemical Parasitology 23, 211–22.Google Scholar
Hommel, M. (1978). The genus Leishmania: biology of the parasites and clinical aspects. Bulletin Institute Pasteur 75, 5–102.Google Scholar
Jackson, P. R., Lawrie, J. M., Stiteler, J. M., Hawkins, D. N., Wohlhieter, J. A. & Rowton, E. D. (1986). Detection and characterization of Leishmania species and strains from mammals and vectors by hybridization and restriction endonuclease digestion of kinetoplast DNA. Veterinary Parasitology 20, 195–215.Google Scholar
Jackson, P. R., Wohlhieter, J. A., Jackson, J. E., Sayles, P., Diggs, C. L. & Hockmeyer, W. T. (1984). Restriction endonuclease analysis of Leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease. American Journal of Tropical Medicine and Hygiene 33, 808–19.Google Scholar
Jeffreys, A. J., Wilson, V. & Thein, S. L. (1985). Hypervariable ‘minisatellite’ regions in human DNA. Nature, London 314, 67–73.Google Scholar
Kelly, J. M., Blaxter, M. L., Kellet, E. & Miles, M. A. (1988). Cloning and characterization of Leishmania donovani genes coding for immunodominant antigens. In Leishmaniasis: The First Centenary (1885–1985). New Strategies for Control (ed. Hart, D. T.). NATO/AS I Series A: Life Sciences, London: Plenum Publishing Co. Ltd. 161, 579–85.Google Scholar
Kennedy, W. P. K. (1984). Novel identification of differences in the kinetoplast DNA of Leishmania isolates by recombinant DNA techniques and ‘in situ’ hybridization. Molecular and Biochemical Parasitology 12, 313–25.Google Scholar
Kidane, G. Z., Hughes, D. & Simpson, L. (1984). Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast mini-circle DNA from Leishmania tarentolae. Gene 27, 265–77.Google Scholar
Kitchen, P. A., Klein, V. A., Fein, B. I. & Englund, P. T. (1984). Gapped mini-circles: A novel replication intermediate of kinetoplast DNA. Journal of Biological Chemistry 259, 15532–9.CrossRefGoogle Scholar
Lainson, R. (1962). Leishmaniasis parasites of mammals in relation to human diseases. In Animal Disease in Relation to Animal Conservation. Symposium of the Zoological Society of London 50, 137–79.Google Scholar
Laison, R. & Shaw, J. J. (1987). Evolution, classification and geographical distribution. In The leishmaniases in Biology and Medicine (ed. Peters, W. & Killick-Kendrick, W.). London: Academic Press.Google Scholar
Langer, P. R., Waldrop, A. A. & Ward, D. C. (1981). Enzymatic-synthesis of biotin-labelled polynucleotides: novel nucleic acid affinity probes. Proceedings of the National Academy of Sciences, USA 78, 6633–7.Google Scholar
Lawrie, J. M., Jackson, P. R., Stiteler, J. M. & Hockmeyer, W. T. (1985). Identification of pathogenic Leishmania promastigotes by DNA:DNA hybridization with kinetoplast DNA cloned to E. coli plasmids. American Journal of Tropical Medicine and Hygiene 34, 257–65.CrossRefGoogle ScholarPubMed
Le Blancq, S. M., Lanham, S. M. & Evans, D. A. Comparative isoenzyme profiles of Old and New World Leishmania. In Leishmaniases in Biology and Medicine (ed. Peters, W. & Killick-Kendrick, R.). London: Academic Press.Google Scholar
Le Riche, P. D. & Sewell, M. M. H. (1977). Differentiation of Taenia saginata and Taenia solium by enzyme electrophoresis. Transations of the Royal Society of Tropical Medicine and Hygiene 71, 237–8.Google ScholarPubMed
La Riche, P. D. & Sewell, M. M. H. (1978). Differentiation of taeniid cestodes by enzyme electrophoresis. International Journal for Parasitology 8, 479–83.Google Scholar
Lopes, U. G., Momen, H., Grimaldi, G. Jr. Marzochi, M. C. A., Pacheco, R. S. & Morel, C. M. (1984). Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis. Journal of Parasitology 70, 89–98.Google Scholar
Lopes, U. G. & Wirth, D. F. (1986). Identification of visceral Leishmania species with cloned sequences of kinetoplast DNA. Molecular and Biochemical Parasitology 20, 77–84.Google Scholar
Lopez, M., Montoya, Y., Arana, M., Cruzalegui, F., Braga, J., Llanos-Cuentas, A., Romero, G. & Arevalo, J. (1988). The use of non-radioactive DNA probes for the characterization of Leishmania isolates from Peru. American Journal of Tropical Medicine and Hygiene 38, 308–14.Google Scholar
Marsden, P. D. (1985). Clinical presentations of Leishmania braziliensis braziliensis. Parasitology Today 1, 129–33.Google Scholar
Massamba, N. N. & Williams, R. O. (1984). Distinction of African trypanosoma species using nucleic acid hybridization. Parasitology 88, 55–65.CrossRefGoogle ScholarPubMed
Mauel, J. & Behin, R. (1982). Leishmaniasis. In Immunology of Parasite Infections, 2nd Edn. (ed. Cohen, S. & Warren, K. S.), pp. 299–355. Oxford: Blackwell Scientific Publications.Google Scholar
McGee, J. O'D. & Fleming, K. A. (1988). Analysis of disease by NISH. In Modern Approaches to ‘in situ’ Hybridization Histochemistry. Proceedings of The Royal Microscopical Society 23 (In the Press).Google Scholar
McManus, D. P. & Simpson, A. J. G. (1985). Identification of the Echinococcus (hydatid disease) organisms using cloned DNA markers. Molecular and Biochemical Parasitology 17, 171–8.Google Scholar
Morel, C. & Simpson, L. (1980). Characterization of pathogenic trypanosomatidae by restriction endonuclease fingerprinting of kinetoplast DNA mini-circles. American Journal of Tropical Medicine and Hygiene 29, Suppl. 1070–4.Google Scholar
Ntambi, J. M. & Englund, P. T. (1985). A gap at unique location in newly replicated kinetoplast DNA mini-circles from Trypanosoma equiperdum. Proceedings of the VII International Congress of Protozoology, Nairobi, Kenya 128, Abstract 299.Google Scholar
Omar, M. S., Denke, A. M. & Raybould, J. N. (1979). The development of Onchocerca ochengi (Nematoda: Filarioidea) to the infective stage in Similium damnosum s.l. with a note on the histochemical straining of the parasite. Tropenmedizin und Parasitologic 30, 157–62.Google Scholar
Paindavoine, P., Pays, E., Laurent, M., Geltmeyer, Y., Le Ray, D., Mehlitz, D. & Steinert, M. (1986). The Use of DNA hybridization and numerical taxonomy in determining relationship between Trypanosoma brucei stocks and subspecies. Parasitology 92, 31–50.Google Scholar
Perler, F. B. & Karam, M. (1986). Cloning and characterization of two Onchocerca volvulus repeated DNA sequences. Molecular and Biochemical Parasitology 21, 171–8.Google Scholar
Peters, W., Elbihari, S. & Evans, D. A. (1986). Leishmania infecting man and wild animals in Saudi Arabia 2 Leishmania arabica n. sp. Transactions of the Royal Society of Tropical Medicine and Hygiene 80, 497–502.Google Scholar
Post, R. J. & Crampton, J. M. (1987). Probing the unknown. Parasitology Today 3, 380–3.Google Scholar
Ramirez, J. L. & Guevara, P. (1987). The ribosomal gene spacer as a tool for taxonomy of Leishmania. Molecular and Biochemical Parasitology 22, 177–83.Google Scholar
Ready, P. D., Smith, D. F. & Killick-Kendrick, R. (1988). DNA hybridizations on squash-blotted sandflies to identify both Phlebotomus papatasi and infecting Leishmania major. Medical and Veterinary Entomology 2, 109–16.CrossRefGoogle ScholarPubMed
Ridley, D. C. (1980). A histological classification of cutaneous leishmaniasis and its geographical expression. Transactions of the Royal Society of Tropical Medicine and Hygiene 74, 515–21.Google Scholar
Rishi, A. K. (1987). Molecular characterization of taeniid cestodes. Ph.D. thesis, University of London.Google Scholar
Rishi, A. K. & McManus, D. P. (1987 a). Genomic cloning of human Echinococcus granulosus DNA: isolation of recombinant plasmids and their use as genetic markers in strain characterization. Parasitology 94, 369–83.Google Scholar
Rishi, A. K. & McManus, D. P. (1987 b). DNA probes which unambiguously distinguish Taenia solium from Taenia saginata. Lancet 1988, 1275.Google Scholar
Rishi, A. K. & McManus, D. P. (1988). Molecular cloning of Taenia solium genomic DNA and characterization of taeniid cestodes by DNA analysis. Parasitology 97, 161–76.Google Scholar
Rogers, W. O. & Wirth, D. F. (1987). Kinetoplast DNA mini-circles: regions of extensive sequence divergence. Proceedings of the National Academy of Sciences, USA 84, 565–9.CrossRefGoogle Scholar
Rogers, W. O. & Wirth, D. F. (1988). Gneration of sequence diversity in the kinetoplast DNA mini-circles of Leishmania mexicana amazonensis. Molecular and Biochemical Parasitology 30, 1–8.CrossRefGoogle Scholar
Sanchez, D. O., Madrid, R., Engel, J. C. & Frasch, A. C. C. (1984). Rapid identification of Trypanosoma cruzi isolate by ‘dot-spot’ hybridization. Federation of European Biochemical Societies Letters 168, 139–42.CrossRefGoogle ScholarPubMed
Shah, J. S., Karam, M., Piessens, W. F. & Wirth, D. F. (1987). Characterization of an Onchocerca-specific DNA clone from Onchocerca volvulus. American Journal of Tropical Medicine and Hygiene 37, 376–84.CrossRefGoogle ScholarPubMed
Shaw, J. J., Lainson, R., Ryan, L., Braga, R. R., McMahon-Pratt, D. & David, J. R. (1987). Leishmaniasis in Brazil XXIII. The identification of Leishmania braziliensis braziliensis in wild caught neotropical sandflies using monoclonal antibodies. Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 69–72.Google Scholar
Sheppard, H. W., & Dwyer, D. M. (1986). Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis. Molecular and Biochemical Parasitology 19, 34–43.Google Scholar
Silviera, F. T., Shaw, J. J., Braga, R. R. & Ishikawa, E. (1987). Dermal leishmaniasis in the Amazon region of Brazil: Leishmania (Viannaia) laisonsi sp.n. A new parasite from the state of Pava. Memorias do Instituto Oswaldo Cruz 82, 289–92.Google Scholar
Smith, G. E. & Summers, M. D. (1980). The biosectional transfer of DNA and RNA to nitrocellulose on diazobenzyloxymethyl paper. Analytical Biochemistry 109, 123–9.Google Scholar
Southern, E. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 503–17.Google Scholar
Spithill, T. W. & Grumont, R. J. (1984). Identification of species, strains and clones of Leishmania by characterization of kinetoplast DNA mini-circles. Molecular and Biochemical Parasitology 12, 217–36.Google Scholar
Spithill, T. W. & Samaras, N. (1985). The molecular karyotype of L. major and mapping of α- and β-tubulin gene families to multiple unlimited chromosomal loci. Nucleic Acids Research 13, 4155–69.Google Scholar
Van Eys, G. J. J. M., Schoone, G. J., Lighthart, G. S., Laarman, J. J. & Terpstra, W. J. (1987). Detection of Leishmania parasites by DNA ‘in situ’ hybridization with non-radioactive probes. Parasitology Research 73, 199–202.Google Scholar
Van Eys, G. J. J. M., Schoone, G. J., Ligthart, G. S., Alvar, J., Evans, D. A. & Terpstra, W. J. (1989). Identification of ‘Old World’ Leishmania by DNA recombinant probes. Molecular and Biochemical Parasitology 34, 53–62.Google Scholar
Voller, A. & De Savigny, D. (1981). Diagnostic serology of tropical parasitic diseases. Journal of Immunological Methods 46, 1–29.Google Scholar
Wirth, D. F. & McMahon-Pratt, D. (1982). Rapid identification of Leishmania species by specific hybridization of kinetoplast DNA in cutaneous lesions. Proceedings of the National Academy Sciences, USA 79, 6999–7003.CrossRefGoogle ScholarPubMed
Wirth, D. F. & Rogers, W. O. (1985). Rapid identification of Leishmania species using specific hybridization of kinetoplast DNA sequences. In Rapid Detection and Identification of Infectious Agents, (ed. Kingsburg, D. T. & Falbow, S.), pp. 127–137. New York: Academic Press.Google Scholar
Wirth, D. F., Rogers, W. O., Barker, R., Dourado, H., Suesebang, L. & Albuquerque, B. (1986). Leishmaniasis and Malaria: New tools for epidemiologic analysis. Science 234, 975–9.Google Scholar