Comparison between the skin snip test and simple dot blot assay as potential rapid assessment tools for Onchocerciasis in the postcontrol era in Ghana.

Successful control of onchocerciasis through mass distribution of ivermectin needs to be coupled with reliable, sensitive, specific, yet affordable diagnostic methods to monitor and ensure the efficacy of such measures. The effort put into the development of diagnostic methods for onchocerciasis that can substitute for or work in combination with the present "gold standard," the skin snip test, has resulted in the discovery of a number of immunogenic proteins with potential use as diagnostic tools in the postcontrol era. Most of these proteins have now been produced through recombinant DNA techniques. However, when costs are not a trivial issue, none of them have yet found their way into the areas where the disease still exists. In the present study, we have evaluated the performance of a simple dot blot assay which uses a mixture of native proteins designated PakF as a serious contender in the quest for a less invasive and more sensitive method to detect Onchocerca volvulus infection in areas with diverse endemicities. Our results indicate that the assay we propose is more sensitive than the skin snip test and shows high specificity, both characteristics required for a suitable tool for the monitoring of onchocerciasis in the postcontrol era.

Evaluation of serological assays for diagnosis of onchocercosis.

Microscopic analysis of skin snips is the most widely used diagnostic technique for onchocercosis in endemic countries. The invasive nature and low sensitivity of that procedure has called for alternative diagnostic methods for this disease. Presently, serological assays detecting filariosis in general are available for routine analysis. However, serological assays specific for onchocercosis are still not universally available. We have evaluated the performance of a dot blot assay (DBA) as a potential method for specific detection of onchocercosis. The DBA, which detects IgG, as compared with 2 IgG4-immunoblot assays, all employing Onchocerca volvulus antigen. Furthermore, an enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence assay (IFA) based on antigens from Acanthocheilonema viteae and Brugia malayi, respectively, were included in the comparison. Samples from microfilariae-positive patients and negative controls from the onchocercosis-endemic country Ghana were analysed. The DBA was significantly more sensitive and specific than the IgG4-assays and the ELISA, respectively. Furthermore, the anti-filarial Ig was increased in patients 1 month post-ivermectin treatment. Sera from patients with suspected filariosis from different parts of the world were analysed using DBA, ELISA and IFA. Patients responding positively in the DBA (12%) had clinical symptoms compatible with onchocercosis whereas those positive in ELISA and IFA (53% and 48%, respectively) had various clinical symptoms. These results indicate that the DBA is more specific than and as sensitive as the ELISA and the IFA presently used for the diagnosis of onchocercosis.

Differential immune response to Onchocerca volvulus: IgG4 antibody responses differ in onchocerciasis patients from Guatemala and Ghana.

Geographical differences exist in the clinical features of onchocerciasis in Central America and West Africa, which could be due in part from variations in the antigenic composition of the infecting organism. In an attempt to address this question, adult female worms of Onchocerca volvulus derived from nodules of patients from Guatemala and Ghana were compared in terms of polypeptide composition and the IgG4 antibody responses induced in patients. It was shown that a Tris-buffer soluble extract from the worms obtained in the two regions differ in polypeptide composition. Furthermore, the diagnostic polypeptides were found to be in the 30 kDa region but the recognition of these antigens was less intense and less frequently observed in the sera of microfilaria (mf) positive patients from Ghana than equivalent age and sex matched patients from Guatemala.

Field diagnosis of onchocerciasis in an area of high versus low endemicity: evaluation of the Dot Blot Assay.

Parasitological examination of skin snips is the most widely used diagnostic method for onchocerciasis, but it is associated with inconvenience and low sensitivity. We describe an inexpensive antibody-based dot blot assay (DBA) for the detection of Onchocerca volvulus infection. A field evaluation of this method was performed in the onchocerciasis endemic country Ghana by testing 370 individuals living in a highly onchocerciasis endemic area and 122 in an area of low endemicity. Sera from individuals with other filarial infections were also tested. The DBA was able to detect 95% of the parasitologically confirmed infected individuals in the highly endemic area. Cross-reactivity occurred with a minority of the sera from individuals infected with other filarial worms. The DBA was as good as or superior to presently available diagnostic tests, and it also fulfilled the criteria for a good screening method.

Visceral leishmaniasis in Somalia: prevalence of markers of infection and disease manifestations in a village in an endemic area.

Prevalence and disease manifestations of visceral leishmaniasis (VL) were studied in a Somali village in an area which has long been known to be endemic for VL. Demographic data were collected from 102 households, comprising 438 inhabitants. Clinical examination was performed of 306 individuals, 72% of the 426 eligible persons. Of these, 276 (90%) agreed to give blood and 246 (80%) to be skin tested with leishmanin. Leishmanin reactions were positive; in 26% anti-Leishmania antibodies were detected in 11%, and splenomegaly was recorded in 14% (23% of those who were seropositive). Malaria was hypoendemic and therefore unlikely to be responsible for more than 10% of the cases with splenomegaly. Three of the seropositive villagers with splenomegaly complained of feeling ill. The remaining 91 sero- and/or leishmanin-positive individuals had no complaint regarding their health and had not experienced any long period of illness. There was a slight over-representation of males in the group of sero- and/or leishmanin-positive villagers, possibly due to a gender-associated difference in exposure to the parasite. Among the patients with clinical VL treated at Mogadishu hospitals during 1989 and 1990, the male/female ratio was 3.3:1, which may indicate a selection of male patients for hospital care. Most patients were < or = 15 years old, suggesting that the highest risk of becoming clinically ill was among children.

Visceral leishmaniasis in Somalia: prevalence of leishmanin-positive and seropositive inhabitants in an endemic area.

In an endemic area of Somalia both humoral and cell mediated immunity against Leishmania donovani was demonstrated in 246 inhabitants. In a study of 14 patients with active visceral leishmaniasis, we found that antibodies appear early in infection and that they are then demonstrable for a limited period only. Leishmanin positivity develops later and persists longer, but does not seem to be lifelong. The majority of the immunoreactive individuals were either sero- or leishmanin positive. This finding is in accord with the result obtained in recent experimental studies indicating a regulatory effect exerted on humoral and cell mediated immunity by different T lymphocyte subsets.

A simple dot blot assay adaptable for field use in the diagnosis of onchocerciasis: preparation of an adult worm antigen fraction which enhances sensitivity and specificity.

The lack of a convenient diagnostic method for onchocerciasis has motivated attempts to develop better detection techniques. Preliminary results indicate that a dot blot assay of the total immunoglobulin G (IgG) response to an easily available and simply prepared Tris buffer soluble fraction (TSF) of whole female worms may be applicable in the field as a first screening method. The specificity of the assay is improved by using a partially purified fraction of TSF of which the dominant component is a 23 kDa protein antigen, PakF. The IgG response to PakF was 100% sensitive and at least as specific as the IgG4 response to TSF. Sera from 189 individuals including onchocerciasis patients and apparently uninfected control subjects from an endemic area and from an urban area in Ghana were screened. The specificity of the assay with urban and endemic control sera was 93% and 68% respectively. The dot blot assay has advantages over the existing skin snip and enzyme-linked immunosorbent assay techniques in terms of simplicity, cost, consumption of antigen, risk of spreading other infections, and patient comfort.

Mechanisms of resistance to Leishmania aethiopica. I. Interferon-gamma in combination with a cytokine (not tumor necrosis factor-alpha) is required, but cannot act alone in the inhibition of intracellular forms of L. aethiopica in THP1 cells.

Following exposure to promastigotes of various Leishmania species, mononuclear cells from non-exposed as well as potentially exposed individuals produced a cytokine response which inhibited intracellular forms of Leishmania aethiopica in a permissive monocytic cell line (THP1). Interferon-gamma (IFN-gamma), was one of the cytokines responsible for this anti-leishmanial effect. IFN-gamma was necessary for inhibition but could not act on its own inhibiting L. aethiopica. Tumor necrosis factor-alpha seemed not to be involved in the anti-L. aethiopica effect. The observed effects were in the absence of endotoxin. The results suggest that the mechanisms of killing of L. aethiopica in human cells may differ from those responsible for inhibition of other Leishmania parasites (such as Leishmania major) in mouse macrophages. Furthermore, that potentially relevant responses to Leishmania antigens may exist in normal individuals.

Cytokine responses to parasite antigens: in vitro cytokine production to promastigotes of L. aethiopica by cells from non-Leishmania exposed donors may influence disease establishment.

The various cytokine responses associated with stimulation by parasites is discussed with emphasis on Leishmania parasites. Cells from normal individuals can respond to Leishmania antigens in vitro but the state of the antigen used for stimulation influences the outcome. We have used cells from non-Leishmania exposed donors and stimulated them in vitro with variously treated promastigotes of L. aethiopica. The levels of some cytokines released into the supernatant were measured. All the Leishmania preparations tested induced high levels of IL-6, whereas IFN-gamma production to the different stimuli was variable in the individual donors. The ability of these supernatants to inhibit intracellular forms of L. aethiopica was sometimes stronger in L. aethiopica-induced than in PHA-induced cultures. Such strong non-Leishmania specific responses, if they exist in vivo, may influence whether disease is established when the host encounters Leishmania parasites.

Cells from healthy non-exposed individuals produce cytokines to selected fractions of Leishmania promastigotes.

We are interested in cellular responses to antigens of parasites to which the cell donor has not been previously exposed and how such responses may influence parasite establishment. In order to characterize such responses we have used cells from unexposed healthy donors and analysed the lymphoproliferative response to various Leishmania aethiopica antigen preparations and the cytokines produced in the process. Peripheral blood lymphocytes were stimulated with SDS-PAGE separated L. aethiopica antigen coupled to nitrocellulose particles. Fifteen of the 16 unexposed individuals tested had proliferative responses to either the whole or/and the antigen-bearing nitrocellulose fractions (NC fractions). Although the degree of response to the fractionated antigen varied in individuals, major stimulatory fractions were found in the high molecular weight region of 110-80 kDa (fractions 3-6) and low molecular weight region of 46-18 kDa (fractions 12-16). Substantial amounts of interferon gamma (IFN-gamma) and interleukin 2 (IL-2) were present in the supernatants of cells stimulated with the whole unfractionated antigen. The potential relevance of such responses in resistance to Leishmania infection is discussed.

Contribution of non-Leishmania-specific immunity to resistance to Leishmania infection in humans.

Lymphocytes of individuals from a country non-endemic for Leishmania (Sweden), responded with a vigorous interferon-gamma (IFN-gamma) and IL-6 response when exposed to live or dead promastigotes of Leishmania aethiopica. This response was sometimes as strong as when the same cells were exposed to the mitogen (phytohaemagglutinin (PHA)). Furthermore, supernatants of cells exposed to Leishmania promastigotes were able to inhibit the amastigote form of the same parasite. In some few instances there was no such reactivity to Leishmania parasites. It is proposed that most individuals have such a first line cytokine response which is enough to prevent further spread and growth of the parasites. In exposed individuals who display disease symptoms, this non-Leishmania-specific response is overcome (by dose) or is weak (for genetic reasons). In the latter instances curbing of parasite growth would depend on acquired immunity.

Serum antibody specificities to Leishmania aethiopica antigens in patients with localized and diffuse cutaneous leishmaniasis.

In order to characterize the antigenic determinants of Leishmania aethiopica, we have analysed by immunoblotting the antibody reactivity of leishmaniasis patients with either the localized (LCL) or diffuse (DCL) clinical forms of disease. In this study we have compared the reactivity of antibodies from eight LCL and DCL patients to parasites isolated from each individual, or the parasite isolates of the other LCL and DCL patients studied. The immunoblot profiles of antibodies from LCL patients differed from the antibody profiles of DCL patients. Serum antibodies from LCL patients showed limited recognition of somatic antigens of less than Mr 50,000 which were recognized by antibodies present in DCL patients. A direct comparison of individual LCL and DCL patient derived promastigotes determined that the lack of antibody to these antigens in LCL patients was not due to the differential expression of these determinants by the LCL and DCL derived promastigotes. The results of this study suggest that although either LCL or DCL derived promastigotes express a wide variety of antigenic moieties which are potentially reactive with antibodies, only a subset of antibodies against these specificities develop in any individual patient, during active infection.

The pathogenesis of Leishmania aethiopica infection in BALB/c mice.

A mouse model for L. aethiopica infection is described. BALB/c mice were unable to clear an infection with 1 x 10(7) promastigotes injected into the hind footpad. However, there was no ulceration of the lesion and no development of overt clinical symptoms after 203 days of infection. Spread of viable organisms was evident in the draining lymph node but not in the spleen or liver. The control of the infection was associated with the development of classical delayed hypersensitivity responses to phenolized promastigotes and appeared as a localized granulomtaous infiltration. The infiltration had features of classical tuberculoid granulomas, but superimposed on it was a strong eosinophilic infiltration. The relevance of such cells though unclear is discussed.

Differential recognition of Leishmania aethiopica antigens by lymphocytes from patients with local and diffuse cutaneous leishmaniasis. Evidence for antigen-induced immune suppression.

Data are presented to suggest that differential Ag expression by parasites derived from diffuse (DCL) vs local (LCL) cutaneous leishmaniasis patients may be responsible for the Ag-specific anergy seen in DCL patients. The evidence suggests that promastigotes derived from DCL patients express epitopes which preferentially stimulate suppressor activities in DCL patients. These determinants appear to be expressed less, if at all by promastigotes derived from LCL patients. The Ag-specific suppression or nonresponsiveness which dominates the immune response in DCL patients during an active infection can be abrogated by drug treatment or removal of live DCL parasites, which suggests that Ag-induced regulatory cells, probably of T cell lineage, are most likely responsible for the nonresponsiveness seen in untreated DCL patients. Thus the mechanisms of immune regulation operating in this disease differ from that of lepromatous leprosy where the specific unresponsiveness (anergy) is irreversible even after successful treatment.