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.

[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.

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.

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%.

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.

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.

Value of whole-cell antigen extracts for serologic detection of Helicobacter pylori.

Whole-cell protein extracts of Helicobacter pylori strains were evaluated by enzyme-linked immunosorbent assay to detect immunoglobulin G antibody against H. pylori in 113 patients with upper gastrointestinal complaints. These antigen preparations were of value for detecting infection by H. pylori in patients with high antibody titers (> or = 12,800), whereas for patients with lower titers, the results were inconclusive.

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.

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.

Enhancement of drug susceptibility in Plasmodium falciparum in vitro and Plasmodium berghei in vivo by mixed-function oxidase inhibitors.

A number of compounds, as exemplified by verapamil and desipramine, have been shown to enhance the susceptibility of resistant malaria parasites to chloroquine. The mechanism by which these agents reverse resistance is still controversial but is though to involve alterations in drug transport causing an increase in steady-state drug concentrations. We have proposed that an alternative resistance mechanism may involve the metabolic deactivation of the drug in some resistant parasites via cytochrome P-450 mixed-function oxidases. If the hypothesis is true, it should be possible to alter drug susceptibility in malaria parasites by the use of agents known to inhibit or induce cytochrome P-450 activities. We have assessed the ability of known inhibitors of cytochrome P-450 enzymes (cimetidine, metyrapone, and alpha-naphthoflavone) to enhance chloroquine susceptibility in Plasmodium falciparum culture-adapted and wild-type isolates in vitro and P. berghei in vivo. In all three systems, the inhibitor cimetidine enhanced parasite susceptibility to chloroquine, and this increase in susceptibility was unrelated to changes in chloroquine steady-state concentrations in vitro or to alterations in host pharmacokinetics in vivo. Additionally, the cytochrome P-450 inducer phenobarbital produced slight decreases in P. falciparum drug susceptibility in vitro. We have compared the ability of the cytochrome P-450 inhibitors cimetidine and metyrapone to enhance drug susceptibility with that of verapamil by using wild-type malaria isolates obtained from Cameroon. Verapamil completely reversed resistance, i.e., to below the cutoff point of 70 nM, in all the resistant isolates. Cimetidine enhanced chloroquine susceptibility in 60% of the isolates and reduced 50% inhibitory concentrations by at least 43% in all the resistant isolates. The compounds tested had little or no effect on the 50% inhibitory concentrations for the susceptible isolates. The data support a possible role for detoxification in chloroquine resistance, and even in the absence of such a process we have observed apparent chemosensitization by agents whose common biological feature is the inhibition of cytochrome P-450 enzymes. Additionally, sensitization has been observed in wild-type isolates of P. falciparum obtained form immune residents of an area of endemicity as well as culture-adapted parasites.

[Renal puncture biopsy in nephrotic syndrome in Cameroonian children, adolescent and adults: histopathologic profile according to age]. / Ponctions-biopsies rénales au cours du syndrome néphrotique de l'enfant, du jeune et de l'adulte camerounais: profile histopathologique selon l'âge.

Eighty-six renal biopsies were under-taken in the Nephrology Service of the Yaounde Central Hospital (Cameroon, Central Africa) during a 4 year period (1st January 1986 to 31st of December 1989). Fifty of them (58%) were done in case of nephrotic syndrome associated or not with hypertension, renal failure. Only 42 of these 50 biopsies were valid and concerned 9 patients aged 8 to 15 years, 22 aged 16 to 30 years and 11 above 30 years. On the whole, 3 main lesions were noted: minimal changes (16 cases: 38.4%), membranous glomerulonephritis (13 cases: 31.2%) and proliferative glomerulonephritis (10 cases: 24%). From the analysis of lesions observed in each age group, it was found that the histopathologic profile of renal biopsies during nephrotic syndrome is practically identical in cameroonian children, young and adults with a relative predominance of minimal change and various glomerular lesions regardless of the age group.

Comparative studies of clinical groups of patients in an onchocerciasis endemic area for evidence of immune-mediated protection.

Four clinical groups of persons from an area endemic for onchocerciasis were compared using certain immunological parameters. The groups were: generalised onchocerciasis, patients with restricted distribution of onchocercal skin lesions, microfilaredermia patients with no clinical manifestations, and a group which clinically, had successfully resisted the infection. Specific serum antibodies to O. volvulus antigens were found in all groups. The IgG specific antibodies were highest in patients with generalised onchocerciasis and lowest in the group who had apparently contained the infection. The sera of persons from the latter group mediated leukocyte adherence to and immobilised microfilariae of O. volvulus. Using the Western blot technique, there were no onchocercal protein antigens that reacted exclusively with sera from the "protected group". However, the staining of the reaction bands was most intense when sera from this patient group reacted with low molecular weight specific onchocercal antigens (M. W. 10-57 KD).

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.

Immunity to Onchocerca volvulus. Serum mediated leucocyte adherence to infective larvae in vitro.

Leucocyte adherence to infective larvae of Onchocerca volvulus in the presence of serum was evaluated using sera from four clinically distinct groups of patients with Onchocerciasis from an area hyperendemic for the disease. Significant cellular adherence to infective larvae occurred for the most part in the presence of sera obtained from subjects with either no microfilaridemia and few or no palpable nodules. These patients had, as well, the highest serum titres of specific anti-O. volvulus IgG antibodies. In contrast, sera from subjects with many palpable nodules and heavy skin infiltration with microfilariae (generalised disease) did not mediate significant adherence of leucocytes to infective larvae targets. Further, this group had the lowest serum levels of specific anti-O. volvulus antibodies of the IgG isotype. The findings are in keeping with the hypothesis that specific protective immunity may occur in O. volvulus infections.

[An echographic study of the urologic lesions of urinary bilharziasis in children in Barombi Kotto (Cameroon)]. / Etude échographique des lésions urologiques de la bilharziose urinaire chez les enfants de Barombi Kotto (Cameroun).

Ultrasonographic examination was performed on 212 school age children (152 proven Schistosomiasis, 60 normal) living in the endemic area of Barombi Kotto (Cameroon). 86.8% of urologic lesions were observed in the diseased group. 73 renal lesions were detected: these consisted of various degrees of dilatations. Urinary bladder lesions were present in 132 cases: they consisted of wall thickenings (84.9%), wall irregularities (79.6%) and localized hypertrophies (24.3%). 11 normal (18.4%) children presented with urologic complications. The prevalence of urologic lesions increased with the intensity of infection as measured by urinary egg output, but there seemed to be no direct relationship between the parasitic load and the severity of morbidity in the study. The authors recommend the use of ultrasonography as a public health tool for the diagnosis and control of disease complications in endemic areas.