Trypanosoma vivax, T. congolense "forest type" and T. simiae: prevalence in domestic animals of sleeping sickness foci of Cameroon.

In order to better understand the epidemiology of Human and Animal trypanosomiasis that occur together in sleeping sickness foci, a study of prevalences of animal parasites (Trypanosoma vivax, T. congolense "forest type", and T. simiae) infections was conducted on domestic animals to complete the previous work carried on T. brucei gambiense prevalence using the same animal sample. 875 domestic animals, including 307 pigs, 264 goats, 267 sheep and 37 dogs were sampled in the sleeping sickness foci of Bipindi, Campo, Doumé and Fontem in Cameroon. The polymerase chain reaction (PCR) based method was used to identify these trypanosome species. A total of 237 (27.08%) domestic animals were infected by at least one trypanosome species. The prevalence of T. vivax, T. congolense "forest type" and T. simiae were 20.91%, 11.42% and 0.34% respectively. The prevalences of 7 vivax and T. congolense "forest type" differed significantly between the animal species and between the foci (p < 0.0001); however, these two trypanosomes were found in all animal species as well as in all the foci subjected to the study. The high prevalences of 7 vivax and T congolense "forest type" in Bipindi and Fontem-Center indicate their intense transmission in these foci.

A new transmission risk index for human African trypanosomiasis and its application in the identification of sites of high transmission of sleeping sickness in the Fontem focus of southwest Cameroon.

A new index for the risk for transmission of human African trypanosomiasis was developed from an earlier index by adding terms for the proportion of tsetse infected with Trypanosoma brucei gambiense group 1 and the contribution of animals to tsetse diet. The validity of the new index was then assessed in the Fontem focus of southwest Cameroon. Averages of 0.66 and 4.85 Glossina palpalis palpalis (Diptera: Glossinidae) were caught per trap/day at the end of one rainy season (November) and the start of the next (April), respectively. Of 1596 tsetse flies examined, 4.7% were positive for Trypanosoma brucei s.l. midgut infections and 0.6% for T. b. gambiense group 1. Among 184 bloodmeals identified, 55.1% were from pigs, 25.2% from humans, 17.6% from wild animals and 1.2% from goats. Of the meals taken from humans, 81.5% were taken at sites distant from pigsties. At the end of the rainy season, catches were low and similar between biotopes distant from and close to pigsties, but the risk for transmission was greatest at sites distant from the sties, suggesting that the presence of pigs reduced the risk to humans. At the beginning of the rainy season, catches of tsetse and risk for transmission were greatest close to the sties. In all seasons, there was a strong correlation between the old and new indices, suggesting that both can be used to estimate the level of transmission, but as the new index is the more comprehensive, it may be more accurate.

Traditional medicine: past, present and future research and development prospects and integration in the National Health System of Cameroon.

Traditional medicine refers to health practices, approaches, knowledge and beliefs incorporating plant, animal and mineral based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination to treat, diagnose and prevent illnesses or maintain well-being. In the last decade traditional medicine has become very popular in Cameroon, partly due to the long unsustainable economic situation in the country. The high cost of drugs and increase in drug resistance to common diseases like malaria, bacteria infections and other sexually transmitted diseases has caused the therapeutic approach to alternative traditional medicine as an option for concerted search for new chemical entities (NCE). The World Health Organisation (WHO) in collaboration with the Cameroon Government has put in place a strategic platform for the practice and development of TM in Cameroon. This platform aims at harmonizing the traditional medicine practice in the country, create a synergy between TM and modern medicine and to institutionalize a more harmonized integrated TM practices by the year 2012 in Cameroon. An overview of the practice of TM past, present and future perspectives that underpins the role in sustainable poverty alleviation has been discussed. This study gives an insight into the strategic plan and road map set up by the Government of Cameroon for the organisational framework and research platform for the practice and development of TM, and the global partnership involving the management of TM in the country.

Domestic animals as potential reservoir hosts of Trypanosoma brucei gambiense in sleeping sickness foci in Cameroon.

An explanation of the endemic nature and/or the resurgence of Human African Trypanosomiasis (HAT) in the historic foci in West and Central Africa may be the existence of an animal reservoir. In some HAT foci, pigs were found infected by Trypanosoma brucei gambiense but the implication of the other domestic animals was not quite evaluated. This study aims to determine the prevalence of T. b. gambiense in domestic animal species (goat, sheep, pig and dog) commonly found in the four active HAT foci in Cameroon (Bipindi, Fontem, Campo and Doumé). Blood samples were collected from 307 pigs, 264 goats, 267 sheep and 37 dogs and used for parasitological (QBC), immunological (LiTat 1.3 CATT) and molecular (PCR) analyses. QBC detected trypanosomes in 3.88% domestic animals while 22.7% were sero-positive with LiTat 1.3 CATT tests. Of the 875 animals analysed, 174 (19.88%) harboured T. brucei s.l. DNA, found in each of the four types of animal and in the four localities. The infection rate significantly differed among the animal species (p < 0.0001) and localities (p < 0.0001). The PCR also revealed T. b. gambiense group 1 DNA in 27 (3.08 %) domestic animals. The specific infection rates were as follows: sheep (6.74%), goats (3.08%), pigs (0.32%) and dogs (O%). T. b. gambiense was found in 8 (3.92%) animals from Bipindi, 15 (4.83%) from Campo, 4 (2.59%) from FontemCenter and none from Doumé. The infection rates significantly differed between the localities, and correlated with the intensity of HAT transmission in the foci.

Serum lipid profile in highly active antiretroviral therapy-naïve HIV-infected patients in Cameroon: a case-control study.

BACKGROUND: HIV status has commonly been found to affect the serum lipid profile. OBJECTIVES: The aim of this study was to determine the effect of HIV infection on lipid metabolism; such information may be used to improve the management of HIV-infected patients. METHODS: Samples were collected from December 2005 to May 2006 at Yaounde University Teaching Hospital, Yaounde, Cameroon. Lipid parameters were obtained using colorimetric enzyme assays, while low-density lipoprotein cholesterol (LDLC) values were calculated using the formula of Friedewald et al. (1972) and atherogenicity index by total cholesterol (TC)/high-density lipoprotein cholesterol (HDLC) and LDLC/HDLC ratios. RESULTS: HIV infection was most prevalent in subjects aged 31 to 49 years. Most of the HIV-positive patients belonged to Centers for Disease Control and Prevention categories B (43.0%) and C (30.23%). Compared with control subjects, patients with CD4 counts<50 cells/microL had significantly lower TC (P<0.0001) and LDLC (P<0.0001) but significantly higher triglyceride (TG) values (P<0.001) and a higher atherogenicity index for TC/HDLC (P<0.01) and HDLC/LDLC (P=0.02); patients with CD4 counts of 50-199 cells/microL had significantly lower TC (P<0.001) and significantly higher TG values (P<0.001); patients with CD4 counts of 200-350 cells/microL had significantly higher TG (P=0.003) and a higher atherogenicity index for TC/HDLC (P<0.0002) and HDLC/LDLC (P=0.04); and those with CD4 counts >350 cells/microL had a higher atherogenicity index for TC/HDLC (P<0.0001) and HDLC/LDLC (P<0.001). HDLC was significantly lower in HIV-positive patients irrespective of the CD4 cell count. Lipid parameters were also influenced by the presence of opportunistic infections (OIs). CONCLUSION: HIV infection is associated with dyslipidaemia, and becomes increasingly debilitating as immunodeficiency progresses. HDLC was found to be lower than in controls in the early stages of HIV infection, while TG and the atherogenicity index increased and TC and LDLC decreased in the advanced stages of immunodeficiency.

Evaluation of oxidative stress and antioxidant status of pregnant women suffering from malaria in Cameroon.

Oxidative stress is thought to be involved in the pathophysiology of malaria, especially in pregnancy where natural resistance is markedly reduced. In the present study we investigated oxidative stress in 315 pregnant women out of which 159 had Plasmodium falciparum malaria and 154 controls. We evaluated the level of lipid peroxidation products (MDA level) in the plasma, the activity of erythrocyte antioxidant defense enzymes, superoxide dismutase (SOD, EC: 1.15.1.1) and catalase (Cat, EC: 1.11.1.6) as well as the ability to resist oxidative stress by the FRAP (Ferric Reducing Ability of Plasma) assay. Total erythrocyte protein levels were also examined. For the two groups of patients, several differences between the biochemical parameters tested were found. Median parasitaemia in women with malaria was 25,392 parasites/µl of blood (Range1200-82000), while in controls we had no parasites found in thin and thick smears. Levels of lipid peroxidation products (MDA) were significantly higher in patients with parasitemia than in healthy asymptomatic volunteers (mean: 0.844 ± 0.290 and 0.384 ± 0.129 respectively, p<0.001). This MDA level was higher in primigravidea and also correlates well with parasite density (p<0.001). Catalase activity in erythrocytes of women with malaria did not differ statistically from that of controls. In contrast, SOD activity of patients with malaria was found to be significantly higher than that of controls (mean: 0.7899 ± 0.2777 and 0.4263 ± 0.2629 respectively, p<0.05). FRAP values declined, from parasitemic patients (1.4619 ± 0.6565) compare to controls (2.4396 ± 0.8883, p<0.05), particularly in the first and third trimester of gestation (p<0.05 and p<0.01 respectively). Finally, total erythrocyte protein concentrations of women with malaria did not differ from that of the controls. Our results suggest an imbalance between oxidants and antioxidants in pregnant women suffering from malaria, a situation which could lead to severe damage for either the mother or the fetus. Therefore, further research should be done to assess the potential benefits of antioxidant supplementation for the pregnant women suffering from malaria.

Tsetse fly host preference from sleeping sickness foci in Cameroon: epidemiological implications.

To determine the tsetse fly host preferences in two sleeping sickness foci of southern Cameroon, four entomological surveys (two in each focus) were carried out. For the whole study, 4929 tsetse flies were caught: 3933 (79.8%) Glossina palpalis palpalis, 626 (12.7%) Glossina pallicera pallicera, 276 (5.6%) Glossina nigrofusca and 94 (1.9%) Glossina caliginea. One hundred and thirty-eight blood meals were collected and the origin of 118 (85.5%) meals was successfully identified: 38.4% from man, 23.9% from pig, 20.3% from sitatunga (Tragelaphus spekeii), 2.2% from sheep and 0.7% from golden cat (Profilis aurata). The number of Glossina palpalis palpalis with man blood meals is more important in the Human African Trypanosomiasis (HAT) focus showing endemic evolution (Campo) than in the focus (Bipindi) presenting a flare up of the disease. The consideration of both results of the prevalence of Trypanosoma brucei gambiense in vertebrate hosts and those of the tsetse fly host preferences indicates a wild animal reservoir of Gambian sleeping sickness and three transmission cycles (human, domestic and wild animals' cycles) in southern Cameroon HAT foci.

Proteinuria and onchocerciasis in an endemic area in Cameroon under community-based treatment with ivermectin.

The present study was aimed at determining the prevalence of onchocerciasis and proteinuria as well as the association between manifestations of heavy chronic onchocerciasis (HCO) and proteinuria among patients in Cameroon. Of the 482 (277: 57.5% females and 205: 42.5% males) subjects recruited from an area with an ivermectin treatment coverage rate of 77.8%, the average prevalence of microfilaridermia by skin snip (mf/ss) was 31.9%, the community microfilaria load was 9.3 mf/ss and the overall prevalence of proteinuria was 4.4%. There was no statistically significant difference in the prevalence of symptoms of HCO when subjects were matched in the presence and absence of proteinuria with regard to positive ss (P = 0.0860), presence of nodules (P = 0.5000), depigmentation (P = 0.1459), visual impairment (P = 0.5000) and recent ingestion of ivermectin (P = 0.6366). Fourteen (66.6%) of the 21 subjects with protein to creatinine ratios (P/CR) > or = 0.2 had HCO, while 15 (71.4%) of the 21 subjects with P/CR < 0.2 had HCO. This gives an odd ratio of 0.8 and a P value of 0.62. However, there is need to carry out studies with a larger sample size before firm conclusions can be drawn about the association between onchocerciasis and proteinuria.

High prevalence of Trypanosoma brucei gambiense group 1 in pigs from the Fontem sleeping sickness focus in Cameroon.

To understand the importance of domestic pigs in the epidemiology of human trypanosomiasis, PCR was used to identify trypanosome populations in 133 pigs from the Fontem sleeping sickness focus of Cameroon. The results from this study show that 73.7% (98/133) of pigs from the Fontem area carry at least one trypanosome species. Trypanosoma vivax, T. brucei s.l. and T. congolense forest were found in 34.6% (46/133), 40.0% (53/133) and 46.0% (61/133) of the pigs respectively. T. simiae and T. congolense savannah were not identified in these animals. The use of repeated DNA sequences detected T. b. gambiense group 1 in 14.8% (15/101) of the pigs. Such pigs can be possible reservoir hosts for T. b. gambiense group 1 and contribute to the maintenance of the disease in the area. Mixed infections were revealed in 35.3% (47/133) of the pigs. Furthermore, we observed that under natural conditions, 52.4% (11/21) of the pigs from the Fontem focus carry mixed infections with T. b. gambiense group 1. No significant difference was observed between the percentage of T. b. gambiense group 1 single and mixed infections, and between the prevalence of this trypanosome in pigs from villages with and without sleeping sickness patients.

[Development of an antigen detection dot blot assay for the diagnosis of human onchocerciasis based on the biotin-avidin binding system]. / Développement d'un test dot blot de détection d'antigène, basé sur le système de fixation biotine- avidine, pour le diagnostic de l'onchocercose humaine.

The development of a highly sensitive and specific diagnostic test is of urgent need for the field assessment of human onchocerciasis and for monitoring the success of control programs. We report here the development and evaluation of a Dot blot Immunobinding Assay (DIA-BA) based on the biotin-avidin binding system, for the detection of O. volvulus specific antigens in body fluids. Specific antibodies were produced by immunizing rabbits with the O. volvulus recombinant antigen Oncho-C71 and labelled with biotin. The biotinylated probes were then used to detect O. volvulus specific antigens initially blotted onto a nitrocellulose membrane. The smallest amount of blotted antigens detectable by the new test is 0.5ng, 1ng, 1ng and 2ng respectively in urine, dermal fluid, tears and serum samples. Out of 456 onchocerciasis endemic subjects examined, 98.4%, 96.5%, 90.8% and 75.0% were positive by the DIA-BA test on urine, dermal fluid, tears and serum respectively The test was most sensitive (100%) when used on urine and least (54.76%) when used on serum from skin snip positive subjects. The specificity of the test, determined amongst non-exposed individuals, was 100% on all but for dermal fluid samples (97.5%). Also, the color intensities on the blot were observed to positively correlate (r = 0.8 on urine) with the skin microfilaria loads on the individuals. We conclude that DIA-BA test could be very useful for mass diagnosis of prepatent, of low and high level infections due to O. volvulus.

Trypanosoma brucei s.l.: characterisation of stocks from Central Africa by PCR analysis of mobile genetic elements.

To better understand the epidemiology of sleeping sickness in the Central African sub-region, notably the heterogeneity of Human African Trypanosomiasis (HAT) foci, the mobile genetic element PCR (MGE-PCR) technique was used to genotype Trypanosoma brucei s.l. (T. brucei s.l.) isolates from this sub-region. Using a single primer REV B, which detects positional variation of the mobile genetic element RIME, via amplification of flanking regions, MGE-PCR revealed a micro genetic variability between Trypanosoma brucei gambiense (T. b. gambiense) isolates from Central Africa. The technique also revealed the presence of several T. b. gambiense genotypes and allowed the identification of minor and major ubiquitous genotypes in HAT foci. The presence of several T. b. gambiense genotypes in HAT foci may explain the persistence and the resurgence phenomena of the disease and also the epidemic and the endemic status of some Central African sleeping sickness foci. The MGE-PCR technique represents a simple, rapid, and specific method to differentiate Central African T. brucei s.l. isolates.

Development and evaluation of an antigen detection dipstick assay for the diagnosis of human onchocerciasis.

To improve on the diagnosis of onchocerciasis, especially light infections, we developed and evaluated an oncho-dipstick test based on the detection of Onchocerca volvulus specific antigens in urine and tears. The test was able to detect as little as 25 ng/ml of parasite specific antigens in samples and took as little as 3 h. Evaluation of the assay on 456 residents of an onchocerciasis hyperendermic area in Cameroon resulted in 408 (89.5%) positives in urine and 374 (82%) positives in tears. The prevalence of onchocerciasis in the study area, as determined by Rapid Epidemiological Mapping of Onchocerciasis (REMO) and skin snip methods, was 52 and 36.8%, respectively. The sensitivity of the oncho-dipstick assay was 100% in urine and 92% in tears; its specificity was 100% in both. Concordance between urine and tear test results from the same individuals was 87%. The test strips were sufficiently reactive when left at room temperature for up to 8 months. The test would be useful for laboratory diagnosis of onchocerciasis in low transmission zones and to ascertain successful treatment of patients in experimental drug studies.

Novel, sensitive and low-cost diagnostic tests for 'river blindness'--detection of specific antigens in tears, urine and dermal fluid.

Sensitive, specific and low-cost diagnostic tests for onchocerciasis are indispensable for monitoring the efficacy of control programs, as well as for preventing blindness (when the tests are combined with efficacious chemotherapy. Three new tests to detect Onchocerca-specific antigens in tears, dermal fluid and urine employ antibodies to O. volvulus-specific recombinant proteins, Oncho-C27 and OvD3B, encoded by genes within the immunodominant Onchocerca OV 33-3 gene family, and expressed in yeast and in E. coli, respectively. In these assays, Onchocerca-specific antigens in test samples are bound onto a solid surface and revealed using appropriate enzyme-labelled antibodies. Proteins in the samples are first transferred to Hybond-N + membrane disks or nitrocellulose paper using either a transblot or a dotblot machine, and then reacted with specific O. volvulus antibodies. Bound antibodies are revealed with species-specific peroxidase-labelled antibodies and peroxidase substrate. Positive tests give a brown colour. In one of the two assays developed to detect Onchocerca antigens in tears, the sensitivity was enhanced by first adsorbing the specific antibodies onto the membrane surface in order to immobilize and concentrate the Onchocerca-specific antigen molecules on the membrane. The specificity of the recombinant proteins for Onchocerca volvulus had been verified by ELISA, classical Western blot and modified DSIA. The tests are a dipstick immunobinding assay for ocular microfilariae (DSIA), a transblot immunobinding assay for the detection of skin microfilariae (TADA) and a dot-blot immunobinding assay for detecting urinary microfilariae and their antigens (DIA). Their specificity and sensitivity were evaluated in the field on 110 subjects with proven ocular microfilariae, 130 subjects with clinical and parasitological evidence of onchocerciasis, 25 subjects infected with other helminths and 120 normal controls. The minimal detection limits of Oncho-C27 protein by DSIA, TADA and DIA were 500 ng/ml, 154 ng/ml and 508 ng/ml, respectively By contrast, their sensitivities were: 100% for DSIA and 82.5% for TADA employed on samples of tears; 97% for TADA skin test and 96% for DIA used on urine samples.

Influence of a past history of Gambian sleeping sickness on physical growth, sexual maturity and academic performance of children in Fontem, Cameroon.

Little has been published on the long-term complications of Gambian sleeping sickness (GSS) following treatment. A case-control study to compare physical growth, sexual maturity and academic performance of children with and without a past history of GSS was therefore conducted. The study took place over a period of 6 months, in the 10 villages of the Fontem GSS focus, which is known to be very endemic for the disease. Overall, 100 young subjects (aged 6-20 years) with a past history of GSS were pair-matched for age (+/- 5 months), sex, place of residence, and socio-economic and cultural backgrounds with 100 other, control subjects who had no history of GSS and who were sero-negative for GSS when checked with a card agglutination test (Testryp-CATT). On average, the cases weighed 4.25 kg less, were 3 cm shorter and had 1.15-cm smaller mid-upper-arm circumferences than the controls (P < 0.05 for each). The mean sexual-maturity rating of the two groups was similar but the controls tended to have attained puberty earlier than the cases. When the cases were subdivided into those treated with melarsoprol and those given pentamidine, only the melarsoprol-treated sub-group was significantly different from the corresponding controls in terms of physical growth and sexual maturity.

A multi-centre evaluation of the card indirect agglutination test for trypanosomiasis (TrypTect CIATT).

A version of the card indirect agglutination test for trypanosomiasis, the TrypTect CIATT, was evaluated for the diagnosis of Trypanosoma brucei gambiense and T. b. rhodesiense sleeping sickness. The results of this antigen-detection test indicated high relative sensitivity (99.3%) and specificity (99.4%), and also much higher prevalences of infection in the general population of endemic foci (27.9% for T. b. gambiense and 21.8% for T. b. rhodesiense) than detected by parasitological diagnosis (1.6% and 1.1%, respectively). TrypTect CIATT detected (and could therefore be used for the diagnosis of) non-patent infections. Among the suspected cases (i.e. those initially found to be parasite-negative but to be antigen-positive), trypanosomes were detected in 29 (4.2%) of those checked at a 3-month follow-up, and 17 more such suspects when they were followed up at 6-18 months. Moreover, a high proportion of blood samples from a random sample of the rest of the suspects tested positive for trypanosome-specific DNA by PCR (79.9% for T. b. gambiense and 13.9% for T. b. rhodesiense). ELISA also demonstrated the presence of anti-trypanosome antibodies in many of the suspects tested (63%, 38%, 24% and 66.9% of those in Cameroon, Côte d'Ivoire, Tanzania, and Malawi, respectively). A follow-up of 164 patients treated with melarsoprol revealed that, by 9 months post-treatment, 113 (69.0%) had no detectable trypanosome antigens in their peripheral blood. The test could therefore be used for evaluating chemotherapeutic cure, as well as for diagnosis.

Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast.

A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33. Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O. volvulus worms was screened. Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O. volvulus. These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S. cerevisiae. Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae. The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin. Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae. The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients. The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively. The combined sensitivity using both recombinant proteins was 94%.

Detection of Trypanosoma brucei gambiense, in serologically positive but aparasitaemic sleeping-sickness suspects in Cameroon, by PCR.

Diagnosis of Gambian sleeping sickness is problematic because of the very low levels of parasitaemia encountered in the field. A PCR method developed for the sensitive detection of Trypanosoma brucei was used to diagnose parasitologically negative suspects in a recent survey in Cameroon. Individuals were screened in two foci (Mbam and Fontem), firstly with the card agglutination test for trypanosomiasis (CATT) as a primary serological test, together with palpation and puncture of enlarged cervical lymph glands. Any suspects found positive by CATT (CATT+) and any clinical suspects were then subjected to several parasitological tests (examination of thick blood films and use of hematocrit centrifugation, mini-anion-exchange chromatography and a commercial kit for in-vitro isolation). Overall, 43 of the 1703 subjects screened in the Mbam focus were CATT+ and three (two of whom were CATT+) had enlarged glands. In Fontem, 56 of the 1210 subjects screened were CATT+, 78 (24 of whom were CATT+) had enlarged glands and two (both CATT+) had trypanosomes in their gland juice. However, all the suspected cases of sleeping sickness, including the two gland-positives, gave negative results in the secondary, parasitological tests. Blood samples from 28 suspects from Mbam and 30 from Fontem were selected for PCR analysis on the basis of high CATT response or clinical grounds. For each suspect, DNA was prepared from 0.5 ml blood by phenol extraction or differential lysis and then amplified by PCR using specific primers for T. brucei ssp. Four samples from Mbam and nine from Fontem, including the two gland-positives, were found positive by PCR. Compared with the other parasitological techniques, therefore, PCR was the most sensitive diagnostic method in this study, with an estimated sensitivity of 25 trypanosomes/ml blood. Although PCR analysis is too expensive for routine diagnosis, it could be very useful in determining which sleeping-suspects should be closely followed up.

Characterization of Trypanosoma brucei gambiense isolates using restriction fragment length polymorphisms in 5 variant surface glycoprotein genes.

Fifty-eight Type I Trypanosoma brucei gambiense (G) stocks, including 16 from 3 sleeping sickness foci in Cameroon, were compared by Restriction Fragment Length Polymorphism (RFLP) analysis with 14 T.b. brucei and T.b. rhodesiense stocks from various endemic areas of Africa. Loci examined were for 5 variant surface glycoprotein (VSG) genes: the LiTat 1.3, AnTat 11.17 and 2K genes were present as single copy genes, while the VSG 117 and U2 gene probes hybridised with a family of related genes. The RFLP data were subjected to cluster analysis to produce a dendrogram constructed from similarity coefficients. The LiTat 1.3 and AnTat 11.17 genes are considered to be characteristic of G stocks, and neither gene was found in the non-G stocks; however, the LiTat 1.3 gene was absent from 6 of the 58 G stocks, while the AnTat 11.17 gene was absent from 8. Supplementation of the LiTat 1.3 antigen in the Card Agglutination Test for Trypanosomiasis with the AnTat 11.17 antigen might thus improve performance of the test, particularly in Cameroon. The U2 VSG gene probe gave a characteristic RFLP pattern for G stocks, as did the VSG 117 gene; the latter is an isogene of AnTat 1.8 previously used extensively to characterise G stocks by other workers. The 2K gene was absent in some G stocks, while present in some non-G stocks, and was not therefore useful for characterisation of G stocks. In cluster analysis, the T.b. gambiense stocks formed a large homogeneous group, subdivided into 5 subgroups, with the non-gambiense stocks as a heterogeneous outgroup.

Genetic heterogeneity in the Trypanosoma brucei gambiense genome analysed by random amplification of polymorphic DNA.

The random amplification of polymorphic DNA (RAPD) technique has the potential to produce large amounts of characterisation data very quickly and simply, using far less DNA than conventional restriction-fragment-length polymorphism (RFLP) analysis. In the present study we assessed genetic heterogeneity among 34 Trypanosoma brucei gambiense isolates from various endemic areas in Africa by the RAPD technique using 8 arbitrary primers and compared the results with those obtained previously from RFLP analysis of polymorphisms in 5 variant surface glycoprotein (VSG) genes. The isolates were compared both among themselves and with 3 T. b. non-gambiense isolates. Most of the primers produced RAPD profiles specific for T. b. gambiense, with 4 primers showing marked polymorphisms between T. b. gambiense and non-gambiense stocks. These primers also showed minor variations between the T. b. gambiense stocks, and 2 revealed differences between Cameroonian stocks. These results were comparable with those produced by RFLP analysis, where certain polymorphisms are characteristic of T. b. gambiense. Numerical analysis showed a high correlation between the RAPD and RFLP data, with genetic variation being detected at a finer level by RAPD analysis. We conclude that RAPD analysis provides a simple and accurate method for the characterisation of T. b. gambiense.

Glycolipid antigens of adult O. volvulus reactive with patients sera from onchocerciasis endemic areas in Cameroon.

Lipid extraction of adult O. volvulus worms using a chloroform/methanol/water mixture yielded 10 lipid fractions of which 8 were demonstrated by the orcinol reagent to be glycolipids. In TLC, two of these lipid fractions had mobilities similar to cholesterol and cholesterol ester (Rf.: 0.95, 0.86) whereas two others migrated as sphingomyelin and lecithin (Rf.: 0.40, 0.35) respectively. Other components migrated at intermediate positions. The glycolipids were immunologically active and reacted with sera from onchocerciasis patients. The highest reaction was obtained with the IgG antibody class, followed by IgM while no appreciable reactivity was observed with IgE. Sera from patients infected with other filariae such as Loa-loa and Dipetalonema perstans did not show any significant reaction with these antigens. The significance of these results is discussed.