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.

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.

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.

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.

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.

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%.

A new method for isolating Trypanosoma brucei gambiense from sleeping sickness patients.

Low infectivity to laboratory mammals and low virulence make Trypanosoma brucei gambiense difficult to isolate and grow in amounts sufficient for biochemical characterization. We report the isolation of T.b. gambiense by feeding cryopreserved primary isolates to laboratory-reared Glossina morsitans morsitans, followed by rapid cultivation in vitro of procyclic forms dissected from infected tsetse fly midguts. This technique allows the characterization of hitherto unsampled populations and avoids selection due to long-term subpassage. Of 16 primary isolates from trypanosomiasis patients of the Fontem focus in Cameroon, 12 (75%) produced infections in tsetse whereas only 4 (25%) infected rats. Ten isolates were subsequently cultivated as procyclic forms in vitro; 2 failed to grow owing to bacterial contamination. In addition, 2 primary isolates from Côte d'Ivoire patients and a stock of low virulence from the Congo Republic were similarly grown. Only one primary isolate produced tsetse salivary gland infections, an observation consistent with the hypothesis that some populations of T.b. gambiense are intrinsically incompatible with G.m. morsitans.

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.

Selective recovery of living microfilariae from Onchocerca volvulus nodules. Determination of optimal conditions for their culture in vitro for excretory/secretory products.

A method for the selective recovery of living microfilariae from Onchocerca volvulus nodules is described. The microfilariae migrate through solidified agar gel into overlayering Hanks balanced salt solution (HBSS). The highest recovery rates of the worms were obtained with 0.3 and 0.4% agar. Optimal conditions for in vitro cultures of the larvae in HBSS were established; pH range 7.0-7.5, glucose concentration 2-5 mg/ml for long term cultures, osmolality 200-309 mOsmoles/l, temperature 4 degrees C for prolonged cultures and 24-28 degrees C for overall best yield of excretory/secretory products (ESP). Subculturing of the larvae reduced contamination of ESP with human serum protein to minimal amounts after 9 recultures done within 96 h.

Absence of the LiTat 1.3 (CATT antigen) gene in Trypanosoma brucei gambiense stocks from Cameroon.

Antibodies to the variable antigen type (VAT) designated LiTat 1.3 are common in sera from parasitologically confirmed patients with gambian sleeping sickness. For this reason, LiTat 1.3 has been considered a suitable antigen for detecting Trypanosoma brucei gambiense in the Card Agglutination Test for Trypanosomiasis (CATT; Testryp-CATT, Smith Kline-RIT). However, surveys in the T.b. gambiense endemic focus of Fontem in Cameroon have suggested that expression of LiTat 1.3 might be rare or absent. We show here that the gene for LiTat 1.3 was indeed absent from some T.b. gambiense stocks isolated from this focus, and a LiTat 1.3-like gene was present in others. The divergent gene differed from the cloned version of LiTat 1.3. In addition, antibodies to LiTat 1.3 could not be detected in rabbits infected with either of the two kinds of T.b. gambiense from the Fontem area. We suggest that the absence of LiTat 1.3 expression in this focus may have important implications for the epidemiology and control of sleeping sickness, especially if heavy reliance is placed on the CATT.

Reactivation of an old sleeping sickness focus in Mamfe (Cameroon): epidemiological, immunological and parasitological findings.

We report the reactivation of an old sleeping sickness focus in Mamfe (Cameroon). Screening of 9827 people using the Testryp CATT (card agglutination test for trypanosomiasis) gave a total of 137 positive cases (1.4%). The prevalence of CATT positivity was significantly linked to sex, age, place of residence and type of occupation of the people. 26 of these immunological suspects were later confirmed as sleeping sickness patients, giving a morbidity index of 0.26%. Only 44% of 16 sera from these confirmed patients were CATT positive on serum while only 31% of the sera had a positive Indirect Immunofluorescent Antibody Test (IFAT) reaction, supporting the hypothesis of the existence of a new T.b gambiense serodeme in this region. The reasons for the reactivation of this old sleeping sickness focus are discussed.

[Absence of epidemiologic interrelations between retroviral infection by HIV and human African trypanosomiasis (HAT)]. / Absence d'inter-relations épidémiologiques entre l'infection rétrovirale à VIH et la trypanosomiase humaine africaine (THA). Analyse de trois enquêtes cas-témoins réalisées en 1989 et 1990 en Afrique centrale.

Authors are reporting results from 3 control-case surveys carried out in sleeping sickness foci in Cameroon, Central African Republic and Congo. HIV seroprevalence rates are comparable among sleeping sickness patients and trypanonegative control persons. These results lead towards the absence of inter-relationships between sleeping sickness and retroviral infection.

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.

An evaluation of the reactivity of the card agglutination test for trypanosomiasis (CATT) reagent in the Fontem sleeping sickness focus, Cameroon.

The TestrypR Card Agglutination Test for Trypanosomiasis (CATT) used for the serodiagnosis of gambiense trypanosomiasis is based on the variant antigen type (VAT) LiTat 1.3. This antigen is rarely expressed by trypanosomes in the Fontem focus of Cameroon, but the CATT has been used for serodiagnosis in the focus since 1985. We give here a summary of results obtained with the CATT in Fontem from 1985 to 1989. The CATT is specific for trypanosome antibodies since: (a) sera from persons with other parasitoses from areas non endemic for trypanosomiasis fail to react and (b) an enzyme-linked immunosorbent assay (ELISA) based on the detection of antibodies to somatic antigens of T.b gambiense from Fontem concorded with the CATT. CATT reactions in Fontem seem to be specific for the variant surface glycoprotein (VSG) since absorption of CATT reactive sera with formalin fixed bloodstream T. gambiense from Fontem and with culture produced procyclics of T. gambiense from Fontem failed to abrogate CATT reactivity. CATT on serum failed to confirm 37% of CATT positive cases on whole blood. Although immunoconglutinin (IK), anti-human red blood cell (RBC) antibodies and complement fixing immune complexes (ICs) were found in sera from Fontem, our results failed to incriminate immunoconglutination of RBCs, reactions of RBCs with their autoantibodies and immune adherence hemagglutination as contributory factors in this lack of agreement between CATT on serum and whole blood. Further, comparison of whole blood and serum CATT results of parasitologically confirmed patients leads to the conclusion that screening with the CATT in the Fontem focus should be done on whole blood, not serum or plasma. CATT reactions in Fontem are based on cross-reactions with as yet undefined VATs.

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.