Correlation between serum levels of antibodies to the 96-kD antigen of Plasmodium falciparum and protective immunity in Cameroon: a longitudinal study.

A longitudinal study was conducted in the Yaounde area of Cameroon that involved 211 individuals in June 1990, and 70 individuals for the follow-up study in December 1990. Sera from these subjects were tested against the recombinant 96-thermoresistant antigen of Plasmodium falciparum and the kinetics of antibody production to this protein show that titers tend to increase with age and are also related to antigen exposure. The increase in antibody titers with age correlates positively with the ability of the individual to prevent development of a high parasitemia. Adults who maintained stable high titers generally did not experience clinical attacks during the study period. The data suggest that antibodies against the 96-kD antigen participate in conferring some immunity to falciparum malaria.

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%.

Immunological studies on onchocerciasis. Varying skin hypersensitivity and leucocyte migration inhibition in a clinical spectrum of the disease.

Soluble antigen was prepared from adult worms and microfilariae of Onchocerca volvulus. In 27 patients clinically characterized as having generalized type (17) and localized reaction type (10) onchocerciasis, the response to this antigen was assessed in vivo by skin testing and in vitro by leucocyte migration inhibition assay. Three varieties of skin hypersensitivity were observed: Immediate, Arthus-type and delayed or type IV hypersensitivity. The first 2 occurred in all patients, but type IV hypersensitivity was noted only in 10, eight of whom had localized reactive type onchocerciasis, whilst the other 2 had mild generalized lesions which had for the most part healed with treatment. The leucocyte migration inhibition assay showed a spectrum of responsiveness with significant inhibition in the presence of antigen in 8 patients with localized lesions and in 2 others with mild generalized disease which had largely healed.--The specificity of the skin reaction and leucocyte migration inhibition to the antigen used was confirmed by the results obtained when a control group of 18 subjects, with no evidence of previous filarial infection, was similarly tested. 16 of them showed no skin hypersensitivity and 2 had a doubtful immediate reaction. No significant inhibition of leucocyte migration in the presence of antigen was noted in this group.

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.

Nephropathy in Cameroon: evidence for filarial derived immune-complex pathogenesis in some cases.

This study was carried out in two parts. Part 1 consisted of an epidemiological survey of 1011 subjects aged 3-65 yrs from 2 adjoining villages hyper-endemic for Onchocerciasis and 890 subjects in a control area, relatively free from this infection but otherwise with a similar parasitological profile. There was a significantly higher prevalence of proteinuria in subjects from the onchocercal zone than in controls (observed difference greater than 5 1/2 times its standard error). Part 2 comprised detailed investigations, including renal biopsy, of 63 consecutive patients admitted into hospital with severe proteinuria and/or renal failure from a caption area extending into the onchocercal zone. There were a variety of causative factors, but in 9 cases filarial antigen was demonstrable in the immune-complex deposits in the kidney. A plenum of renal histopathological changes were seen in patients with onchocerciasis. The significance of these findings is discussed.

Adult response to initial treatment with ampicillin in community acquired pneumonia in Yaoundé, Cameroon.

A prospective study was conducted to evaluate the efficacy of ampicillin as an initial therapy in 60 adult patients with community acquired pneumonia. Bacteriological etiology was obtained only in 24 (40pc) patients by microscopic examination, culture and antigen detection. Streptococcus pneumoniae was the leading causative agent identified in 15 cases. Other etiologies were Klebsiella pneumoniae (3), Streptococcus pyogenes (2) Staphylococcus aureus (2), Haemophilus influenzae (1) and Moraxella catarrhails (1). Patients were started on ampicillin one gram twice daily, empirically, and treated for 10 days. There were 52(86.7pc) patients cured, two(3.3pc) patients improved and six (10pc) were therapeutic failures. The causative agents in patients with failures were: Klebsiella pneumoniae (1), Staphylococcus aureus (1), Streptococcus pneumoniae (1) and unknown in three cases. Only six of 60 patients still febrile after three days were switched to amoxycillin/clavulanic acid or cefaclor according to culture results and susceptibilities or to roxythromycin because no organisms were isolated. Side effects were observed in only one (1.7pc) patient who developed a mild skin rash. Due to its low cost and its high efficacy, ampicillin still appears to be the drug of choice in adult community acquired pneumonia in our region.

[Variations under genetic control of onchocerca infection as a function of clinical profile in the endemic center of Cameroon]. / Variations sous contrôle génétique de l'infection onchocerquienne en fonction du profil clinique dans les foyers endémiques du Cameroun.

Onchocerciasis, also known as "river blindness", presents a plenum of clinical manifestations which vary from one individual to another, and from one area to another. This large spectrum of clinical manifestations of the disease is an indication of the complexity of the pathogenesis of onchocerciasis and suggests that many interacting factors might influence the clinical features of the disease. The present study has focused on the heterogenicity of the host immune response as a plausible explanation for differences in clinical manifestations of the infection. Host genetic factors, namely HLA genes, might play an important role in determining the nature of the immune response mounted against the parasite Onchocerca volvulus, and thus the development of different manifestations of the infection. Genetic diversity of onchocerciasis was assessed in different endemic foci in Cameroon. In order to investigate the possibility that the Major Histocompatibility Complex (MHC) genes might be associated with the different clinical types of onchocerciasis, 146 subjects living in three endemic areas of Cameroon were studied. They were classified in four groups: A (asymptomatic subjects), P (putatively immune subjects) L (patients with localised disease) and G (patients with generalised disease). The four groups differed in the distribution of HLA class II alleles as determined by Direct Heteroduplex Analysis. On the one hand, allele HLA-DQA1*0501 appeared to be associated with protection against severe onchocerciasis; on the other, allele HLA-DQB1*0201 might play an important role in the severe form of the disease.

Resistant hypertension in Yaounde.

Sixty six consecutive patients with resistant hypertension were studied over a period of 12 months. During that same period 565 patients were seen in all for hypertension given an average prevalence of 11.7% of resistant hypertension. Men were more frequently affected, however mean systolic and diastolic blood pressure levels were higher in women. Past history of poor compliance, overweight, renal impairment and cardiac abnormalities were important predictors of poor response to therapy. Resistance to therapy was not related to the class or the type of medication taken. Side effects were more numerous in the resistant group. Biochemically, apart from abnormal renal function tests, the other baseline tests were similar in both groups of resistant and non resistant hypertensives.

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.

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.

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.

Immunological studies in congestive cardiomyopathy in Cameroon.

Significantly higher levels of immunoconglutinin were found in the sera of 40 Cameroonian patients aged between 15 and 80 years with congestive cardiomyopathy than in a matched group of normal Cameroonian controls. C3 values were also abnormal in the group with congestive cardiomyopathy, and a negative correlation was found between the C3 and immunoconglutinin values, indicating complement incorporation into immune complexes. Further, the levels of immunoglobulins G, M, and A were all raised, the most striking increase being of IgM, which was greater than in the normal controls and in a group with 'other cardiac disease' studied for comparison. Complement-fixing autoantibodies to cardiac muscle of the class IgG and IgM were found by immunofluorescence techniques in a significant proportion of the patients with congestive cardiomyopathy. Finally, evidence of trypanosomiasis was found by immunofluorescence tests in 27-5 per cent of patients with congestive cardiomyopathy, compared with 8-5 per cent of patients with other cardiac disease, and 1-9 per cent of normal Cameroonian controls selected randomly. We think that some cases of congestive cardiomyopathy in Cameroon may be caused by untreated subclinical attacks of African trypanosomiasis which produce immune complexes that damage the cardiac tissue where the parasite is lodged. Secondary autoimmune carditis modifies the course of the disease, with the resulting end-stage picture.

Immunological studies in onchocerciasis in Cameroon.

Immunological studies were undertaken in a group of twenty patients with varying clinical manifestations of onchocerciasis. Significantly higher levels of immunoconglutinin, IgG and IgM, and alterations in C3 levels were found in the patient group than in controls. IgG and IgM complement-fixing antibodies to microfilariae of O.volvulus were detected in the sera of these patients. The lymphocyte transformation rate following PHA stimulation was high in patients with localised skin lesions in which the microfilariae were difficult to demonstrate in contrast to the low transformation rate obtained in those with diffuse lesions and in whom microfilariae were readily detected in skin biopsies. The significance of these findings is discussed.

Serum immunoglobulin E levels in onchocerciasis: the development of a radioallergosorbent test for Onchocerca volvulus infection.

The effects of Onchocerca volvulus infection on immunoglobulin levels have been studied in subjects from three localities with different onchocerciasis prevalence rates. Infestation by O. volvulus provoked a 200-500% rise in total serum IgE levels. Immunoglobulins G, M. and A were increased less dramatically. To study further the IgE response, radioallergosorbent test (RAST) systems were developed using total extract of O. volvulus and Onchocerca supernatant (excretory-secretory) antigens. Both antigen preparations reacted with more than 80% of the onchocerciasis sera from the hyperendemic village, but the reactions were not proportionate to skin microfilarial density. There were also positive reactions with sera of some individuals whose skin biopsies showed no microfilariae, indicating prior exposure to the parasite. European control sera tested under similar conditions reacted only slightly in the RAST system. The developed RAST systems may thus be used for determining prior exposure to O. volvulus.

Serum antibodies against human brain myelin proteins in Gambian trypanosomiasis.

Human brain myelin was obtained by flotation centrifugation on 0.88 M sucrose. The myelin proteins were extracted with 0.1% SDS-0.01 M Tris, pH 7.4 from the myelin floating on 0.88 M sucrose following centrifugation. Using ELISA and immunoblotting, we have shown that antibodies against the myelin proteins exist in sera collected from Gambian trypanosomiasis patients. The antibodies were more prevalent in patients with CNS involvement since of 21 sera from patients with more than 4 cells/ml cerebrospinal fluid (CSF), 15 (71.4%) had antimyelin antibodies, as compared to 3 of 13 (23.1%) from patients with less than 4 cells/ml CSF. Thus, of 18 sera that had antimyelin antibodies, 15 (83.3%) were from patients with CNS involvement. Using the immunofluorescence test, selected sera detected antigens in cryocuts of human brain.

[Subpopulations of lymphocytes in onchocercal nodules]. / Sous-populations de lymphocytes dans les nodules onchocerquiens.

Fourty onchocercal nodules were taken from patients living in forest areas where onchocerciasis is highly endemic, in order to identify the various types of lymphocytes present in these nodules. For this purpose, each nodule was submitted to two histological procedures: one after fixation in formalin of part of the nodule and hematoxylin-eosin stain; the other after freezing part of the nodule in liquid nitrogen, cryostat cutting and staining by immuno-peroxidase method using avidin-biotin and the following monoclonal antibodies: Pan B, OK T3, OK T4 and OK T8. Fifty-five of the 40 nodules show lymphocytes. All the 25 nodules with lymphocytes present T-lymphocytes. Only 20 of these 25 nodules have B lymphocytes and therefore only 20 nodules with lymphocytes have both T and B lymphocytes. Of the 25 nodules with T lymphocytes: 19 present T4 lymphocytes, 25 show T8 lymphocytes and therefore 19 have both T4 and T8 lymphocytes. These results are analysed and discussed in this study which notices that there is an intense cellular and humoral immune reaction in onchocercal nodules, an insufficient reaction as parasitic antigens persist and are not destroyed.