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. 1996 Oct;34(10):2401–2407. doi: 10.1128/jcm.34.10.2401-2407.1996

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique.

E Harris 1, J Detmer 1, J Dungan 1, F Doua 1, T White 1, J A Kolberg 1, M S Urdea 1, N Agabian 1
PMCID: PMC229279  PMID: 8880488

Abstract

We have developed a nonradioactive branched DNA (bDNA)-based assay for the diagnosis of the African trypanosomiases in simple buffy coat preparations of human blood. Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets of the bDNA assay, a technique which amplifies the signal from a target molecule rather than the target itself. Comparable sensitivities were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes, and bloodstream trypomastigotes. The results of bDNA analysis of human blood samples from Côte d'Ivoire (n = 50) showed excellent agreement with those of buffy coat microscopy. The bDNA technology offers certain advantages over alternative molecular biological techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from crosscontamination artifacts. We have successfully applied the bDNA technique to the detection of T. brucei in clinical samples from regions where T. brucei infection is endemic; to our knowledge, this is the first report of the molecular detection of T. brucei in human blood.

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Selected References

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  1. Bailey J. W., Smith D. H. The quantitative buffy coat for the diagnosis of trypanosomes. Trop Doct. 1994 Apr;24(2):54–56. doi: 10.1177/004947559402400204. [DOI] [PubMed] [Google Scholar]
  2. Barnes D. A., Mottram J., Selkirk M., Agabian N. Two variant surface glycoprotein genes distinguish between different substrains of Trypanosoma brucei gambiense. Mol Biochem Parasitol. 1989 May 1;34(2):135–146. doi: 10.1016/0166-6851(89)90005-4. [DOI] [PubMed] [Google Scholar]
  3. Belli A. A., Miles M. A., Kelly J. M. A putative Leishmania panamensis/Leishmania braziliensis hybrid is a causative agent of human cutaneous leishmaniasis in Nicaragua. Parasitology. 1994 Nov;109(Pt 4):435–442. doi: 10.1017/s0031182000080689. [DOI] [PubMed] [Google Scholar]
  4. Bienen E. J., Hammadi E., Hill G. C. Trypanosoma brucei: biochemical and morphological changes during in vitro transformation of bloodstream- to procyclic-trypomastigotes. Exp Parasitol. 1981 Jun;51(3):408–417. doi: 10.1016/0014-4894(81)90128-4. [DOI] [PubMed] [Google Scholar]
  5. Bromidge T., Gibson W., Hudson K., Dukes P. Identification of Trypanosoma brucei gambiense by PCR amplification of variant surface glycoprotein genes. Acta Trop. 1993 Apr;53(2):107–119. doi: 10.1016/0001-706x(93)90023-5. [DOI] [PubMed] [Google Scholar]
  6. Cattand P., de Raadt P. Laboratory diagnosis of trypanosomiasis. Clin Lab Med. 1991 Dec;11(4):899–908. [PubMed] [Google Scholar]
  7. Clayton C. E., Fueri J. P., Itzhaki J. E., Bellofatto V., Sherman D. R., Wisdom G. S., Vijayasarathy S., Mowatt M. R. Transcription of the procyclic acidic repetitive protein genes of Trypanosoma brucei. Mol Cell Biol. 1990 Jun;10(6):3036–3047. doi: 10.1128/mcb.10.6.3036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Lange T., Liu A. Y., Van der Ploeg L. H., Borst P., Tromp M. C., Van Boom J. H. Tandem repetition of the 5' mini-exon of variant surface glycoprotein genes: a multiple promoter for VSG gene transcription? Cell. 1983 Oct;34(3):891–900. doi: 10.1016/0092-8674(83)90546-9. [DOI] [PubMed] [Google Scholar]
  9. Detmer J., Lagier R., Flynn J., Zayati C., Kolberg J., Collins M., Urdea M., Sánchez-Pescador R. Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology. J Clin Microbiol. 1996 Apr;34(4):901–907. doi: 10.1128/jcm.34.4.901-907.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dorfman D. M., Donelson J. E. Characterization of the 1.35 kilobase DNA repeat unit containing the conserved 35 nucleotides at the 5'-termini of variable surface glycoprotein mRNAs in Trypanosoma brucei. Nucleic Acids Res. 1984 Jun 25;12(12):4907–4920. doi: 10.1093/nar/12.12.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibson W. C., Borst P. Size-fractionation of the small chromosomes of Trypanozoon and Nannomonas trypanosomes by pulsed field gradient gel electrophoresis. Mol Biochem Parasitol. 1986 Feb;18(2):127–140. doi: 10.1016/0166-6851(86)90033-2. [DOI] [PubMed] [Google Scholar]
  12. Hasan G., Turner M. J., Cordingley J. S. Complete nucleotide sequence of an unusual mobile element from trypanosoma brucei. Cell. 1984 May;37(1):333–341. doi: 10.1016/0092-8674(84)90329-5. [DOI] [PubMed] [Google Scholar]
  13. Hide G., Tait A. The molecular epidemiology of parasites. Experientia. 1991 Feb 15;47(2):128–142. doi: 10.1007/BF01945413. [DOI] [PubMed] [Google Scholar]
  14. Hoff R. A method for counting and concentrating living Trypanosoma cruzi in blood lysed with ammonium chloride. J Parasitol. 1974 Jun;60(3):527–528. [PubMed] [Google Scholar]
  15. Horn T., Chang C. A., Fultz T. J., Ahle D., Hamren S. J., Urdea M. S. Branched oligodeoxyribonucleotides for use as amplification multimers in bioassays: chemical synthesis and characterization. Nucleic Acids Symp Ser. 1991;(24):201–202. [PubMed] [Google Scholar]
  16. Hunter C. A., Jennings F. W., Adams J. H., Murray M., Kennedy P. G. Subcurative chemotherapy and fatal post-treatment reactive encephalopathies in African trypanosomiasis. Lancet. 1992 Apr 18;339(8799):956–958. doi: 10.1016/0140-6736(92)91531-c. [DOI] [PubMed] [Google Scholar]
  17. Kimmel B. E., ole-MoiYoi O. K., Young J. R. Ingi, a 5.2-kb dispersed sequence element from Trypanosoma brucei that carries half of a smaller mobile element at either end and has homology with mammalian LINEs. Mol Cell Biol. 1987 Apr;7(4):1465–1475. doi: 10.1128/mcb.7.4.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kuzoe F. A. Current situation of African trypanosomiasis. Acta Trop. 1993 Sep;54(3-4):153–162. doi: 10.1016/0001-706x(93)90089-t. [DOI] [PubMed] [Google Scholar]
  19. Lumsden W. H., Kimber C. D., Dukes P., Haller L., Stanghellini A., Duvallet G. Field diagnosis of sleeping sickness in the Ivory Coast. I. Comparison of the miniature anion-exchange/centrifugation technique with other protozoological methods. Trans R Soc Trop Med Hyg. 1981;75(2):242–250. doi: 10.1016/0035-9203(81)90326-6. [DOI] [PubMed] [Google Scholar]
  20. Lumsden W. H., Kimber C. D., Evans D. A., Doig S. J. Trypanosoma brucei: Miniature anion-exchange centrifugation technique for detection of low parasitaemias: Adaptation for field use. Trans R Soc Trop Med Hyg. 1979;73(3):312–317. doi: 10.1016/0035-9203(79)90092-0. [DOI] [PubMed] [Google Scholar]
  21. Majiwa P. A., Otieno L. H. Recombinant DNA probes reveal simultaneous infection of tsetse flies with different trypanosome species. Mol Biochem Parasitol. 1990 May;40(2):245–253. doi: 10.1016/0166-6851(90)90046-o. [DOI] [PubMed] [Google Scholar]
  22. Majiwa P. A., Thatthi R., Moloo S. K., Nyeko J. H., Otieno L. H., Maloo S. Detection of trypanosome infections in the saliva of tsetse flies and buffy-coat samples from antigenaemic but aparasitaemic cattle. Parasitology. 1994 Apr;108(Pt 3):313–322. doi: 10.1017/s0031182000076150. [DOI] [PubMed] [Google Scholar]
  23. Masiga D. K., Smyth A. J., Hayes P., Bromidge T. J., Gibson W. C. Sensitive detection of trypanosomes in tsetse flies by DNA amplification. Int J Parasitol. 1992 Nov;22(7):909–918. doi: 10.1016/0020-7519(92)90047-o. [DOI] [PubMed] [Google Scholar]
  24. Mathieu-Daude F., Bicart-See A., Bosseno M. F., Breniere S. F., Tibayrenc M. Identification of Trypanosoma brucei gambiense group I by a specific kinetoplast DNA probe. Am J Trop Med Hyg. 1994 Jan;50(1):13–19. doi: 10.4269/ajtmh.1994.50.13. [DOI] [PubMed] [Google Scholar]
  25. Miezan T. W., Meda A. H., Doua F., Cattand P. Evaluation des techniques parasitologiques utilisées dans le diagnostic de la trypanosomose humaine a Trypanosoma gambiense en Côte-d'Ivoire. Bull Soc Pathol Exot. 1994;87(2):101–104. [PubMed] [Google Scholar]
  26. Moser D. R., Cook G. A., Ochs D. E., Bailey C. P., McKane M. R., Donelson J. E. Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction. Parasitology. 1989 Aug;99(Pt 1):57–66. doi: 10.1017/s0031182000061023. [DOI] [PubMed] [Google Scholar]
  27. Murphy N. B., Pays A., Tebabi P., Coquelet H., Guyaux M., Steinert M., Pays E. Trypanosoma brucei repeated element with unusual structural and transcriptional properties. J Mol Biol. 1987 Jun 20;195(4):855–871. doi: 10.1016/0022-2836(87)90490-6. [DOI] [PubMed] [Google Scholar]
  28. Myler P. J., Glick D., Feagin J. E., Morales T. H., Stuart K. D. Structural organization of the maxicircle variable region of Trypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites. Nucleic Acids Res. 1993 Feb 11;21(3):687–694. doi: 10.1093/nar/21.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nelson R. G., Parsons M., Barr P. J., Stuart K., Selkirk M., Agabian N. Sequences homologous to the variant antigen mRNA spliced leader are located in tandem repeats and variable orphons in trypanosoma brucei. Cell. 1983 Oct;34(3):901–909. doi: 10.1016/0092-8674(83)90547-0. [DOI] [PubMed] [Google Scholar]
  30. Pachl C., Todd J. A., Kern D. G., Sheridan P. J., Fong S. J., Stempien M., Hoo B., Besemer D., Yeghiazarian T., Irvine B. Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay. J Acquir Immune Defic Syndr Hum Retrovirol. 1995 Apr 15;8(5):446–454. doi: 10.1097/00042560-199504120-00003. [DOI] [PubMed] [Google Scholar]
  31. Perry K. L., Watkins K. P., Agabian N. Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8190–8194. doi: 10.1073/pnas.84.23.8190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Running J. A., Urdea M. S. A procedure for productive coupling of synthetic oligonucleotides to polystyrene microtiter wells for hybridization capture. Biotechniques. 1990 Mar;8(3):276–279. [PubMed] [Google Scholar]
  33. Sloof P., Bos J. L., Konings A. F., Menke H. H., Borst P., Gutteridge W. E., Leon W. Characterization of satellite DNA in Trypanosoma brucei and Trypanosoma cruzi. J Mol Biol. 1983 Jun 15;167(1):1–21. doi: 10.1016/s0022-2836(83)80031-x. [DOI] [PubMed] [Google Scholar]
  34. Sloof P., de Haan A., Eier W., van Iersel M., Boel E., van Steeg H., Benne R. The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA. Mol Biochem Parasitol. 1992 Dec;56(2):289–299. doi: 10.1016/0166-6851(92)90178-m. [DOI] [PubMed] [Google Scholar]
  35. Smith J. L., Chapman A. B., Agabian N. Trypanosoma vivax: evidence for only one RNA polymerase II largest subunit gene in a trypanosome which undergoes antigenic variation. Exp Parasitol. 1993 May;76(3):242–246. doi: 10.1006/expr.1993.1029. [DOI] [PubMed] [Google Scholar]
  36. Stuart K., Gobright E., Jenni L., Milhausen M., Thomashow L., Agabian N. The IsTaR 1 serodeme of Trypanosoma brucei: development of a new serodeme. J Parasitol. 1984 Oct;70(5):747–754. [PubMed] [Google Scholar]

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