Follow-up of Card Agglutination Trypanosomiasis Test (CATT) positive but apparently aparasitaemic individuals in Côte d'Ivoire: evidence for a complex and heterogeneous population.

The aetiological diagnosis of human African trypanosomiasis (HAT) is based on the detection of the parasite, but currently available parasitological tests have low sensitivity and are hampered by fluctuating parasitaemia. The identification of seropositive individuals on whom to focus parasitological examination is based on antibody detection by means of the Card Agglutination Trypanosomiasis Test (CATT/T.b.gambiense). A complicating phenomenon is the occurrence of serologically positive but parasitologically unconfirmed results (isolated CATT positivity). This work presents a two-year longitudinal serological, parasitological and molecular follow-up of CATT-positive individuals including repeated examinations of each individual, to study the evolution over time of seropositivity at both the population and the individual levels. At the population level, the rate of seropositivity decreased during the first months of the survey, and afterwards showed remarkable stability. At the individual level, the results reveal the extreme heterogeneity of this population, with subjects showing fluctuating results, others with a short transient CATT positivity, and subjects that maintain their seropositivity over time. The stability of seropositivity and the pattern of results obtained with both immunological and parasitological examinations support the view that individual factors, such as immune response to infection, might be involved in the isolated CATT positivity phenomenon.

Comparative in vitro isolation of Trypanosoma theileri from cattle in Belgium.

Ten blood samples randomly collected from cows on a farm nearby Antwerp, Belgium, were inoculated into KIVI culture medium (Kit for In Vitro Isolation of trypanosomes) and RPMI 10%+feeder medium. Within 3 weeks of incubation all KIVI cultures and four RPMI 10%+feeder revealed presence of Trypanosoma theileri. Some practical implications regarding the use of KIVI for isolation of pathogenic African trypanosomes from cattle and other Bovidae are discussed.

Improved latex agglutination test for detection of antibodies in serum and cerebrospinal fluid of Trypanosoma brucei gambiense infected patients.

A rapid latex agglutination test (LATEX/T. b. gambiense) for detection of antibodies in patients infected with Trypanosoma brucei gambiense is presented. The reagent is coated with a mixture of three variable surface antigens of bloodstream form trypanosomes. Two hundred and forty sera and 79 CSF samples from patients with parasitologically confirmed trypanosome infection along with 173 sera and 38 CSF samples from non-trypanosomiasis patients have been tested. At 1:16 serum dilution, test specificity was 99%, while sensitivity ranged from 83.8 to 100% depending on the geographical origin of the samples. Undiluted CSF samples from non-trypanosomiasis and from first stage patients scored negative while 42 out of 66 CSF samples from second stage patients were positive. Stability and reproducibility of the lyophilized reagent were excellent.

A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense.

Infectivity of Trypanosoma brucei rhodesiense to humans is due to its resistance to a lytic factor present in human serum. In the ETat 1 strain this character was associated with antigenic variation, since expression of the ETat 1.10 variant surface glycoprotein was required to generate resistant (R) clones. In addition, in this strain transcription of a gene termed SRA was detected in R clones only. We show that the ETat 1.10 expression site is the one selectively transcribed in R variants. This expression site contains SRA as an expression site-associated gene (ESAG) and is characterized by the deletion of several ESAGs. Transfection of SRA into T.b. brucei was sufficient to confer resistance to human serum, identifying this gene as one of those responsible for T.b. rhodesiense adaptation to humans.

Diagnostic evaluation of PCR on dried blood samples from goats experimentally infected with Trypanosoma brucei brucei.

In seven goats experimentally infected with a pleomorphic clone of Trypanosoma brucei brucei, parasitaemia was monitored weekly for 6 weeks by wet blood film and microhaematocrit buffy coat examination. Dried blood samples on filter paper were concomitantly collected and tested by PCR using three different primer sets, putatively specific for Trypanozoon, T. vivax and T. congolense. With the originally designed ORPHON5J Trypanozoon primers, PCR tests became positive after 1 week (six animals) or 2 weeks (one animal) of infection and remained consistently positive until the end of the experiments, thus yielding an overall positivity rate of 97%, as compared with 74% for all parasitological tests together. The T. vivax and T. congolense primers yielded no positive PCR results.

Increased sensitivity of the card agglutination test CATT/Trypanosoma brucei gambiense by inhibition of complement.

CATT/Trypanosoma brucei (T.b.) gambiense is an antibody detection test currently used in field surveys on Gambian sleeping sickness. The screening test is usually performed on a drop of freshly collected heparinized blood, followed by a more specific confirmation test on diluted blood, plasma or serum. This approach may be biased by the occurrence of a complement-mediated prozone phenomenon causing lower test sensitivity at lower sample dilutions. A simple remedy is by addition of a Ca2+ chelating agent such as EDTA.

A semi-quantitative ELISA for detection of Trypanosoma brucei gambiense specific antibodies in serum and cerebrospinal fluid of sleeping sickness patients.

A semi-quantitative ELISA, using variable surface glycoprotein of T.b. gambiense as antigen, was developed for the detection of antibodies of different immunoglobulin isotypes in serum and cerebrospinal fluid of sleeping sickness patients. Using the assay, the antibody profiles of paired serum and cerebrospinal fluid samples of 28 patients have been studied. Total concentrations of various Ig isotypes were determined as well. In serum and cerebrospinal fluid a drastic increase in IgG, basically IgG1, as well as in IgM levels was observed. The concentration of IgA remained relatively normal. The antitrypanosomal antibodies detected in serum and cerebrospinal fluid were mainly of the IgG (IgG1 and IgG3) and IgM isotypes. Measurement of immunoglobulin and trypanosome specific antibody concentrations in serum and CSF allows calculation of intrathecal antibody synthesis and is a possible tool for determining the clinical stage of sleeping sickness.

Human African trypanosomiasis: a latex agglutination field test for quantifying IgM in cerebrospinal fluid.

LATEX/IgM, a rapid agglutination test for the semi-quantitative detection of IgM in cerebrospinal fluid of patients with African trypanosomiasis, is described in this article. The lyophilized reagent has been designed for field use and remains stable at 45 degrees C for one year. The test has been evaluated on cerebrospinal fluid samples from trypanosome-infected and non-infected patients, by comparison with commercial latex agglutination, radial immunodiffusion, and nephelometry. All test systems yielded similar results.

Detection and characterization of autoantibodies directed against neurofilament proteins in human African trypanosomiasis.

In serum and in cerebrospinal fluid (CSF) from patients with human African trypanosomiasis (HAT) with central nervous system involvement, we detected autoantibodies directed to some proteins from these tissues. The characterization of antigenic proteins by Western blotting showed that the antibodies recognized the 200-kD and 160-kD proteins of neurofilament (NF). Serum anti-NF antibodies were more frequent in HAT patients than in control subjects (86% versus 24%; P < 10[-9]) and they belonged predominantly to the IgM class (anti-NF IgM = 86% versus anti-NF IgG = 4%; P < 10[-9]) in the patients with stage II (central nervous system involvement) HAT. The CSF antibodies to NF were IgM in 88% (22 of 25) of the cases and IgG in 32% (8 of 25) of the cases. Epitopes shared by NF and trypanosomes were detected by indirect immunofluorescence and this was confirmed by the disappearance of anti-NF reactivity after adsorption with trypanosome antigens (Trypanosoma brucei brucei or T. b. gambiense). Anti-NF antibodies were undetectable in the CSF from stage I HAT patients.

Diagnostic evaluation of PCR in goats experimentally infected with Trypanosoma vivax.

Six goats were experimentally infected with a stock of Trypanosoma vivax. Parasitaemia was weekly monitored by buffy coat and wet blood film examination during a period of 15 weeks and another 3 weeks following drug-treatment. Dried blood samples were tested by the polymerase chain reaction (PCR), using an extraction method with Chelex 100 (BioRad). PCR proved consistently more sensitive than the parasitological techniques.

Evaluation of variant specific trypanolysis tests for serodiagnosis of human infections with Trypanosoma brucei gambiense.

Twelve T.b. gambiense clone populations of distinct Variable Antigen Type (VAT) were combined in immune lysis tests with 340 sera of trypanosome infected patients from 8 different African countries and 267 non trypanosomiasis control sera. The diagnostic specificity of the test was 100%. At a serum dilution of 1:4 the overall test sensitivity with single VATs varied from 39.1 to 98.2% and from 12.1 to 86.8% at 1:32. At a serum dilution of 1:32 some combination tests with 2 VATs still scored above 96%. The VAT recognition patterns were clearly correlated with the geographical origin of the sera, reflecting a diversity in variable antigen repertoires.

[Evaluation of a direct serologic card agglutination test for the diagnosis of camel trypanosomiasis caused by Trypanosoma evansi]. / Evaluation d'un test sérologique d'agglutination directe sur carte dans le diagnostic de la trypanosomose caméline à Trypanosoma evansi.

The results of a novel direct serological card agglutination test for the diagnosis of camel trypanosomosis due to Trypanosoma evansi (CATT/T. evansi) were compared with those obtained by direct detection of parasites in a study using 1,093 sera from camels raised in northern Mali. A good correlation was revealed between the percentage of positive results obtained by CATT and the presence of trypanosomes (89%), as well as a good coincidence between the percentage of positive results obtained by CATT and low haematocrit values (packed cell volume). CATT revealed a global serological prevalence of 30.6%, whereas trypanosomes were found in only 5.85% of the corresponding animals. CATT/T. evansi is a quick and easy-to-read test, which merits further evaluation in camel-rearing countries.

[Serological evidence of the existence of a wild reservoir of Trypanosoma brucei gambiense in the Pendjari biosphere reservation in the Republic of Benin]. / Indications sérologiques de l'existence d'un réservoir sauvage du Trypanosoma brucei gambiense dans la réserve de la biosphère de la Pendjari en République du Bénin.

In the national park of Pendjari, situated in the North-West of Benin, 91 wild animals, belonging to seven species, were darted. Thick and thin blood smears were examined for trypanosomes and plasma for trypanolytic antibodies against 6 antigenic variants of Trypanosoma brucei gambiense. Parasites were found in 13.92% and trypanolytic antibodies in 20.88% of the samples. A total of 28.57% of animals were positive by at least one of the two test systems used. Morphologically Trypanosoma congolense, T. vivax and T. brucei were identified. Overall prevalence was 40% in Adenota kob (n: 50), 13.63% in Alcelaphus buselaphus (n: 22), 10% in Hippotragus equinus (n: 10), 33% in Kobus defassa (n: 3), 0% in Phacochoerus aethiopicus (n: 3) and in Syncerus caffer (n: 2). The only lion (Panthera leo) examined was serologically positive. The results indicate that the wild animals are reservoirs of animal trypanosomes and suggest that among them Adenota kob and Panthera leo are carriers of T. brucei gambiense, one of the etiological aspects of human trypanosomiasis.

An experimental latex agglutination test for antibody detection in human African trypanosomiasis.

A latex card agglutination test for detection of antibodies in human African trypanosomiasis is presented. The latex was covalently coated with semipurified surface glycoprotein of Variable Antigen Type LiTat 1.6 of Trypanosoma brucei gambiense. Sera from 100 patients infected with T.b. gambiense, 26 patients infected with T.b. rhodesiense and 707 individuals without trypanosomiasis, including 132 malaria seropositives, have been tested. At serum dilution 1:16, sensitivity of the test was 91% for the T.b. gambiense and 42.3% for the T.b. rhodesiense group. Specificity was over 99%. The reagent remained stable at +/- 6 degrees C for at least 3 months. Reagent kept at 37 degrees C for 3 months retained its sensitivity and showed a slight decrease in specificity.

Variant-specific trypanolytic antibodies in sera from patients infected with Trypanosoma brucei rhodesiense.

Infections with salivarian trypanosomes are characterized by the successive development of parasite populations of distinct variable antigen types (VATs), the corresponding antibodies accumulating in the blood of the host. Sixty VATs had been cloned during previous studies on variable antigen repertoires of Trypanosoma brucei rhodesiense; 12 of these were selected for immunolysis tests against 85 sera from T.b. rhodesiense patients in Busoga (Uganda). One variant, ETat 1/1, reacted with 59 out of 65 sera that contained detectable lytic antibodies. ETat 1/1 in combination with two other variants ETat 1/14 and Utat 1/1, covered all the seropositive sera; other VATs showed varying degrees of reactivity. The results suggest that sera from T.b. rhodesiense patients contain easily detectable VAT-specific antibodies and that their corresponding antigens might be used in the preparation of serodiagnostic reagents for the disease.

A gene expressed only in serum-resistant variants of Trypanosoma brucei rhodesiense.

The human infective African trypanosomes are host range variants of Trypanosoma brucei which are resistant to a lytic component in primate serum. T. b. rhodesiense occurs both as a form sensitive to lysis by normal human serum and as a form resistant to this lysis. Switching from one phenotype to the other has been observed in both directions. In the cloned T. b. rhodesiense ETAR1-repertoire we have detected 1.5-kb mRNAs only present in the resistant forms. In T. b. gambiense, which always occurs as a normal human serum-resistant form, no such transcript could be detected, indicating that another mechanism of resistance is involved here. Starting from an independent non-cloned T. b. rhodesiense population isolated from an infected patient, both resistant and sensitive trypanosomes have been prepared. Northern blot analysis of the total RNA prepared from these populations has revealed again the differential occurrence of the resistance-specific transcript, indicating that we are dealing with a general phenomenon associated with serum resistance in T. b. rhodesiense. As expected, Southern blot analyses have demonstrated that both serum-resistant and serum-sensitive forms of T. b. rhodesiense contain the gene coding for this transcript.

Spread of Trypanosoma brucei to the nervous system: early attack on circumventricular organs and sensory ganglia.

The distribution of Trypanosoma brucei brucei in the nervous system of experimentally infected Sprague-Dawley rats and BALB/c and deer mice was examined with immunohistochemical techniques. The trypanosomes showed an early invasion in areas lacking a so-called blood-brain or blood-nerve barrier, i.e., in sensory ganglia and circumventricular organs including the area postrema, pineal gland, and median eminence. This distribution of trypanosomes may relate to the origin of cardinal symptoms of the disease, e.g., sensory disturbances, nausea, disturbed circadian rhythm, and neuroendocrinological dysfunctions. Trypanosome infections in rodents may provide a model for studies of how an infectious agent or factors released by the immune response may relatively selectively interfere with these functionally defined regions of the nervous system.