Procyclic culture forms of Trypanosoma brucei species and antibodies to these parasites were used in developing antibody-detection and antigen-detection assays for diagnosis of African human sleeping sickness. An agglutination assay using live procyclic trypanosomes--the Procyclic Agglutination Trypanosomiasis Test (PATT) was developed for detecting anti-trypanosome antibodies in the sera of trypanosome-infected vervet monkeys and humans. Antibodies to procyclic surface antigens were detected by the PATT in sera of vervet monkeys as early as 7 days post-infection with T. b. rhodesiense. Positive agglutination titres were obtained with sera from monkeys with active, untreated infections and with sera taken soon after successful drug cure. Similar positive agglutination results were also observed using the PATT with sera from T. b. gambiense-infected patients from Cote d'Ivoire and Sudan and with documented sera from T. b. rhodesiense-infected patients from Kenya. No agglutination reactions were observed with preinfection sera from vervet monkeys, with sera from uninfected Canadians or with sera from Americans working in endemic areas. Together these results confirm the diagnostic value of using procyclic trypanosomes to detect anti-trypanosome antibodies in human African sleeping sickness.
A double antibody sandwich ELISA using monoclonal antibodies and polyclonal rabbit antibodies to the surface membrane antigens of procyclic trypanosomes was developed. This assay detected circulating trypanosomal antigens in the sera of trypanosome-infected mice and in the sera from parasite-infected patients. However, limited success was obtained with this sandwich ELISA when tested on a larger repertoire of sera from infected humans. Rabbit antibodies made against whole lysates of T. b. rhodesiense procyclics were then employed in an antigen-trapping sandwich ELISA. The
results demonstrated the effectiveness of this sandwich ELISA in revealing the infection
status of vervet monkeys or humans infected with either T. b. rhodesiense or T. h.
gambiense. Trypanosomal antigens were detected in the sera of parasitologically confirmed monkeys and patients but not in preinfection sera nor in control sera from uninfected North Americans.
The PATT and the sandwich ELISA exhibited higher sensitivities than the currently
employed diagnostic assay for human sleeping sickness, the Card Agglutination
Trypanosomiasis Test (CATT), when tested with sera of parasitologically-confirmed
humans. The sandwich ELISA was superior to the antibody-detecting PATT and CATT in
monitoring trypanocidal drug-treated patients. The overall sensitivity of the PATT and
sandwich ELISA was 94.3% and 97.4% and the specificity was 84.5% and 95.5%,
respectively. These results thus confirm the diagnostic value of these tests for the
diagnosis of human African sleeping sickness.
Identification of diagnostically useful antigens was attempted in order to facilitate the adaptation of these diagnostic assays to a simpler format for field application. Pooled sera obtained from trypanosome-infected patients was used as a probe to detect trypanosome antigens separated by high performance liquid chromatography, immunoaffinity and immunoblotting techniques. Most of the antigens were detected in the
higher molecular weight range (>62 Kd). Immunization of mice with the target antigens
yielded six trypanosome-specific monoclonal antibodies. In a double antibody sandwich
ELISA, these antibodies were successful in trapping circulating parasite antigens in sera
from trypanosome-infected mice as early as 3 days post-infection. Some of these antigens
have been partially biochemically characterized. Trypanosomal antigens were also detected
by these antibodies in the urine of infected mice. The antigen-capture sandwich ELISA
using either the selected monoclonal antibodies or the rabbit anti-procyclic whole lysate
antibodies gave similar results with sera from trypanosome-infected mice, human sleeping
sickness patients and uninfected humans from North America and Kenya. The results showed that these MAbs and their antigens were useful in the diagnosis of African human sleeping sickness. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9467 |
| Date | 18 June 2018 |
| Creators | Liu, Margaret Kim Mong |
| Contributors | Pearson, Terry W. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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