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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.

The HIV/AIDS pandemic is spreading at an alarmingly high rate in Africa. Leishmaniasis is also highly prevalent in the continent. Despite the emergence of Leishmania/HIV co-infection in Africa, the numbers reported are disproportionately low. Moreover, the number of cases of co-infection is expected to rise in Africa owing to the simultaneous spread of the two infectious diseases and their increasingly overlapping geographical distribution.

SUMMARYThe most effective protection against human leishmaniasis has been achieved following vaccination with live promastigotes. Killed promastigotes + BCG can protect, albeit to a lower degree. To explore what mechanisms may be involved in these differences, the ability of live and dead promastigotes to induce immune responses were evaluated in vitro. The data showed that live and dead promastigotes differ in their ability to induce proliferation and cytokine production. Cytokine gene expression of Th1 related cytokines (IL-12, IFNγ and TNFα) in adult PBMC was more evident to live than to heat killed promastigotes. This was coupled with significantly higher number of IFNγ secreting cells induced by live than killed promastigotes. However, α-IL-12 antibodies did not block the IFNγ response induced by live promastigotes. Proliferative responses were variable. In contrast to adult PBMC no IFNγ secreting MNC could be detected in cord blood. However, in these cells the live promastigotes consistently induced higher proliferative response compared to dead. Implications of these findings are discussed.

SUMMARYWe have previously shown that natural killer (NK) cells play a role in protection against leishmaniasis. Furthermore, we have shown that NK cells in mononuclear cells derived from unexposed donors are induced to proliferate in vitro in response to leishmanial antigens. Since interleukin (IL)-12, a strong inducer of NK cells, acts on the early events in NK cells and T-cells, and is considered as an adjuvant for use in a potential antileishmaniasis antigen, we wished to investigate how this cytokine influences the in vitro Leishmania induced proliferative and cytokine response in healthy donors. We demonstrate that in an innate response to Leishmania antigen involving NK cells, a critical level of IL-12 is required to induce interferon (IFN)-γ secretion below which, IL-10 is released in amounts which apparently inhibit IFN-γ secretion and cellular proliferation. However, at higher IL-12 levels, there is simultaneous secretion of IFN-γ and IL-10 as well as proliferation of cells. In a similar vein, exogenous IL-10 in turn inhibited IFN-γ secretion as well as proliferation when used at low/medium concentrations, but at high concentrations this effect was abolished and replaced by the simultaneous detection of IFN-γ, IL-10 and proliferation. The contribution of NK cells in cross regulation of these two very important immuneregulatory cytokines and the effect of exogenous IL-12 in a Leishmania driven response are discussed.

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.

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.

The immune response of normal human peripheral blood mononuclear cells (PBMC) after stimulation with human immunodeficiency virus-1 (HIV-1) antigens plus Leishmania donovani promastigotes in vitro was investigated. HIV-1-antigen stimulation of PBMC did not induce the intracellular accumulation of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma). However, cells stimulated with L. donovani antigens exhibited the production of IL-6 and TNF-alpha, but not IFN-gamma. Furthermore, co-stimulation of PBMC with HIV-1 antigen plus L. donovani resulted in the intracellular accumulation of IL-6 and TNF-alpha comparable to that of cells that were activated with L. donovani antigen alone. Heat-inactivated HIV-1 antigen did not appear to induce or suppress cytokine production by PBMC. However, the same HIV antigens did suppress L. donovani-induced proliferation as well as PPD-induced proliferation in a dose-dependent fashion. Elevated levels of serum cytokines have been demonstrated in patients with HIV infection indicating their role in the pathogenesis of HIV-associated immunosuppression. The results may partially support the idea that the abnormally increased cytokine levels in the sera of HIV-infected subjects is due to the various opportunistic pathogens that these patients contract, rather than a response to HIV antigens. As cytokines have been shown to up-regulate HIV replication, the data suggest a role for opportunistic infections in cytokine-induced transactivation of HIV-1 and disease progression.

SUMMARYCells from normal non-Leishmania-exposed individuals could respond in vitro by proliferation and interferon-gamma (IFN-γ) production to Leishmania aethiopica stimulation. The main cell type that appeared to be activated following such stimulation was CD3−, CD16+/56+. i.e. NK cells. Of the few CD3+ cells responding, an involvement of CD8+ cells was evident in the absence of activation of CD4+ cells in normal individuals, while a different feature was observed when patients' cells were investigated. Cells from patients with L. aethiopica infection did not show this NK response, but rather the CD4+ cells were the prominent responding cells. No evidence of the involvement of superantigens or cells utilizing the γδ T cell receptor (γδcells) in the response of unexposed individuals was noted.

Cutaneous leishmaniasis (CL) is caused by Leishmania infection of dermal macrophages and is associated with chronic inflammation of the skin. L. aethiopica infection displays two clinical manifestations, firstly ulcerative disease, correlated to a relatively low parasite load in the skin, and secondly non-ulcerative disease in which massive parasite infiltration of the dermis occurs in the absence of ulceration of epidermis. Skin ulceration is linked to a vigorous local inflammatory response within the skin towards infected macrophages. Fas ligand (FasL) and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expressing cells are present in dermis in ulcerative CL and both death ligands cause apoptosis of keratinocytes in the context of Leishmania infection. In the present report we show a differential expression of FasL and TRAIL in ulcerative and non-ulcerative disease caused by L. aethiopica. In vitro experiments confirmed direct FasL- and TRAIL-induced killing of human keratinocytes in the context of Leishmania-induced inflammatory microenvironment. Systemic neutralisation of FasL and TRAIL reduced ulceration in a model of murine Leishmania infection with no effect on parasitic loads or dissemination. Interestingly, FasL neutralisation reduced neutrophil infiltration into the skin during established infection, suggesting an additional proinflammatory role of FasL in addition to direct keratinocyte killing in the context of parasite-induced skin inflammation. FasL signalling resulting in recruitment of activated neutrophils into dermis may lead to destruction of the basal membrane and thus allow direct FasL mediated killing of exposed keratinocytes in vivo. Based on our results we suggest that therapeutic inhibition of FasL and TRAIL could limit skin pathology during CL., Author Summary Cutaneous leishmaniases are associated with parasite-induced inflammatory lesions of the skin. The degree of clinical pathology is not associated with parasitic burden; on the contrary, ulcerative lesions are associated with low infectious load, and non-ulcerative lesions are associated with an abundant parasite infiltration. Leishmania are intracellular parasites in mammalian hosts and reside in macrophages in the deep layers of the skin, the dermis. The exact mechanism of ulceration in CL is not known and Leishmania parasites do not directly induce destruction of keratinocytes in the most superficial layer of the skin, the epidermis. In this study we investigated if ulcerated lesions were associated with higher expression of FasL- and TRAIL-induced cell-death of keratinocytes. We found a higher expression of FasL and TRAIL in human skin samples from ulcerative as compared to non-ulcerative leishmaniasis. In a mouse model of ulcerative leishmaniasis neutralisation of FasL and TRAIL reduced ulceration. We suggest that FasL and TRAIL participate in the ulcer formation during leishmaniasis both as a chemoattractant of activated neutrophils leading to tissue destruction and through direct killing of keratinocytes. Possible approaches to use this concept in therapeutical interventions with the aim to reduce immunopathology associated with leishmaniasis are discussed.

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.

The sensitivity and specificity of parasite demonstration methods (smear, culture and histology) and serological assays (enzyme-linked immunosorbent assay [ elisa ], direct agglutination test and immunoblot) were compared in the diagnosis of leishmaniasis in Ethiopia. Culture was found to be the most sensitive diagnostic method, followed by elisa , for the diagnosis of cutaneous leishmaniasis (CL). When the clinical type of CL was taken into consideration, serological and parasitological methods were equally good for the diagnosis of diffuse cutaneous leishmaniasis. Overall, the serological assays were not sensitive enough to diagnose all the parasitologically confirmed cases of localized cutaneous leishmaniasis. Both groups of diagnostic methods performed equally well in the diagnosis of visceral leishmaniasis patients. In cases of CL where clinical diagnosis was a problem and histology could not give a definitive diagnosis due to the absence of demonstrable parasites, one of the serological assays, preferably elisa , was very useful in establishing the final diagnosis.

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.

We have examined by immunoblotting the antigen profiles of Leishmania parasites which have undergone upward shifts in ambient temperature during culture. Parasites in the promastigote insect vector stage were grown to stationary growth phase at 25 degrees C, and then further cultured at the 37 degrees C temperature experienced in the mammalian host. Changes in the immunoblot profiles of the parasites occurred within one day of culture at mammalian ambient temperature. Serum antibodies from patients with active Leishmania infections showed reactivity with antigenic determinants of greater than Mr 38,000 that were expressed by parasites at 37 degrees C, and which were not comparably observed on immunoblots of 25 degrees C cultured organisms. The promastigotes of Leishmania species which cause either cutaneous or visceral leishmaniasis express differing forms of the 37 degrees C induced high molecular weight determinants, however, these molecules express cross-reactive epitopes. Previous studies have suggested that temperature may play a role in the differentiation process between the insect and host life cycle stages of Leishmania. Our results suggest that the antigenic profile of Leishmania parasites may also be affected by the expression of products from temperature sensitive biosynthetic pathways.

The usefulness and sensitivity of a direct agglutination test (DAT) in the diagnosis of cutaneous leishmaniasis due to Leishmania aethiopica infection has been investigated. Formalin-fixed, trypsin-treated and Coomassie blue-stained Leishmania promastigotes of various origins were used as antigens. L. Major, L. Donovani, L. aethiopica but not L. tropica antigen preparations were able to distinguish sera from individuals infected with Leishmania from sera of uninfected controls, although the titres of sera from patients with localized cutaneous leishmaniasis were low. Comparable results were obtained when the same sera were tested using freshly prepared antigen or antigen stored for 10 months at 4 °C. The assay was also used to monitor improvement of disease status following treatment of diffuse cutaneous leishmaniasis patients, and it was found to correlate well with the changing clinical status of the patients.

Leishmania aethiopica infection results in two main clinical entities, diffuse disease (DCL) and localized ulcers (LCL). The lack of reactivity to leishmanial antigens has been attributed, among other things, to some inherent immunological defect of the host or considered as a consequence of the initial site of infection. Properties unique to the infecting parasite have been said to contribute little if anything to the differences between DCL and LCL found in the same areas of Ethiopia. Data are given to show that infected individuals respond by higher production of IL-2 to antigens from LCL isolates (lcl antigen), than to antigens derived from DCL isolates (dcl antigen). Furthermore, dcl antigen induced less gamma interferon from lymphocytes of all individuals tested than did lcl antigen. Lymphocytic proliferation of cells from control individuals working in the endemic area was higher in response to lcl isolates than to dcl isolates. These findings suggest that some differences in the parasites may contribute to the clinical outcome of infection with L. aethiopica.

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.

SummarySeveral studies in murine systems have suggested a role of apoptosis in the pathogenesis of leishmaniasis. However, the role of apoptosis in visceral leishmaniasis in man has not been explored. In this study, we show that patients with visceral leishmaniasis demonstrate significant dysregulation of Fas and Fas ligand. Levels of soluble Fas (sFas) and soluble Fas ligand (sFasL) were elevated in plasma of patients with active visceral leishmaniasis (VL) and individuals co-infected with VL-HIV-1 compared to healthy controls. The levels of sFas and sFasL were normalized 6 months after successful treatment. In VL patients, the expression of membrane bound Fas, and to a lower extent FasL, were up-regulated on Leishmania donovani-infected spleen cells, the site of parasite multiplication. Expression of Fas and FasL on peripheral blood mononuclear cells was within normal range, probably reflecting that the blood is not a normal site of L. donovani infection. Furthermore, this is suggested by the finding that in vitro infection of macrophages with L. donovani up-regulated Fas expression on the surface of infected cells and enhanced the levels of sFasL in supernatants from infected cultures. How this dysregulation may affect the pathogenesis of human visceral leishmaniasis is discussed.

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.

Summary Natural killer (NK) cells have been implicated in the natural protection and healing of leishmaniasis by their ability to secrete the macrophage activating cytokine interferon (IFN)γ. Previous studies have demonstrated that early production of interleukin (IL)-12 triggers IFNγ secretion by NK cells. Here we report that live Leishmania promastigotes (the form that is injected by the vector) can directly induce human peripheral blood NK cells from healthy donors to IFNγ secretion in the absence of IL-12 and professional antigen presenting cells. Killing of promastigotes abolishes this property. This novel mechanism of activation of the innate immune response may be relevant for establishment of infection and thus also the design of vaccines against leishmaniasis.

Objective: To investigate whether clearance of Leishmania parasites from tissue aspirate smears in patients with HIV and visceral leishmaniasis (VL) co-infection treated with pentavalent antimonials is influenced by initial HIV viral load and to assess the effect of active VL on HIV viral load and replication in vivo. Methods: Leishmania parasites were identified in Giemsa-stained smears prepared from tissue aspirates. Parasite index was determined by quantifying Leishmania donovani bodies in smears. HIV-1 RNA was quantitated by using the nucleic acid sequence-based amplification technique with a limit of detection of 500 copies/ml. All patients were treated with pentavalent antimonials at 20 mg pentavalent antimony (Sb v )/kg daily for a total of 28 days. None of the patients received specific anti-retroviral therapy. Results: Seventeen patients (73.9%) showed good initial response to anti-leishmanial treatment and the remaining six (26.1%) had very poor response. Among the good responders, 11 (64.7%) had no demonstrable Leishmania donovani bodies in post-therapy tissue aspirate smear preparations, and in the remaining six (35.3%) their parasite loads were reduced to very low levels. Patients with poor response had persistently high parasite index despite completion of anti-leishmanial chemotherapy. Poor responders had pre-treatment median HIV viral load that was > 160-fold higher than responders to anti-leishmanial chemotherapy; [410 000 copies/ml (quartile range, 33 000-530 000) and 2500 copies/ml (quartile range 500-297 500), respectively]. Furthermore, compared with pre-treatment viral concentrations, patients with good response showed marked reduction in post-treatment viral load. In contrast, post-treatment HIV viral concentrations were markedly increased among patients with poor response to anti-leishmanial therapy. Conclusions: The results suggest that pre-treatment HIV viral load influences response to anti-leishmanial chemotherapy and active VL is associated with increased viral replication in vivo, supporting the notion that dual infection plays an important role in the pathogenesis and disease progression of either infection.

SUMMARY Onchocerciasis is associated with blindness and gross skin changes, believed to be a consequence of the immune response to antigens released from the offspring of the female worm of Onchocerca volvulus, the microfilariae (mf). An effective microfilaricidal drug is now available which quickly reduces the mf burden without affecting the adult worm. There exist foci in onchocerciasis endemic areas where some of the patients have many mf in their skin but relatively few clinical symptoms. This state of hyposensitivity is believed to be due to immunosuppression. The aim of this study was to address the question of the basis of, and the effect of ivermectin treatment on this immunosuppression. Female adult worms of O. volvulus were used as whole or fractionated antigens to stimulate peripheral blood mononuclear cells. Microfilariae are found in the reproduction tract of the female worms, and thus an antigen preparation of the female adult O. volvulus contains both exclusive adult antigens as well as antigens from microfilariae. Cells were obtained from onchocerciasis patients, individuals of similar socio-economic status living in the same Ghanaian village, but who showed no parasitological or clinical evidence of onchocerciasis (exposed endemic controls), healthy Ghanaians living in areas where transmission of onchocerciasis does not seem to occur (non-exposed endemic controls) and unexposed healthy Swedish donors. As a group, cells from onchocerciasis patients proliferated to a lesser degree than cells from the exposed endemic control and the non-exposed endemic control groups to the whole worm antigen, whereas the phytohaemagglutinin (PHA) response was strongest in the patients. Proliferative responses of above 1000 ct/min to fractions of the worm extract were only evident in the cells from a few individuals in each of the various groups. However, 28 days following ivermectin treatment, cells from all onchocerciasis patients were able to mount significantly enhanced proliferation to a fraction of approximately 96 kD (fraction 3), while only four of nine of this group showed an increased response to the whole worm antigen. The proportional increase in the response to the whole organism in these individuals was of a much lower magnitude than the increased response to fraction 3. The O. volvulus antigen-specific immunosuppression observed in these onchocerciasis patients appears to be due to suppressive antigens which have the capacity to mask the potential response to selected antigens of O. volvulus, and ivermectin treatment possibly modulates the immune response, allowing for stepwise recognition of such antigens. Since ivermectin treatment kills only the microfilariae and not the adult worm, the putative suppressive antigens would be expected to be from the microfilariae.

The role of natural versus acquired immunity to Leishmania aethiopica infection in humans is the focus of our studies. We found in previous studies that mononuclear cells from nonexposed healthy Swedish donors responded to Leishmania antigen stimulation by proliferation and gamma interferon production. The main cell type responding was CD3 − CD16/56 + natural killer (NK) cells. These findings led us to suggest that the potential to produce a rapid, nonacquired NK cell response may be a protective phenotype. In order to test this hypothesis, an area in Ethiopia where Leishmania is endemic was selected, and peripheral blood mononuclear cells were obtained from individuals who had lived in the area most of their lives but had no evidence of past or present leishmaniasis. Their responses were compared with those of confirmed leishmaniasis patients from the same region with active lesions or cured leishmaniasis lesions. Cells from these donors were stimulated in vitro with L. aethiopica antigen. Responses were measured by proliferation, cytokine production, and phenotype analysis by fluorescence-activated cell sorting. The association of NRAMP1 alleles with the studied phenotype and susceptibility to L. aethiopica -induced leishmaniasis was also evaluated. The results show that Leishmania antigens can induce NK cell and CD8 + -T-cell responses in vitro. This is clearly seen in proliferating cells from the cured (immune) individuals and the apparently protected controls from the area of endemicity. It contrasted with the reactivity of the patients, where some NK proliferation was coupled with enhanced CD4 + -T-cell proliferation. We conclude from these observations that NK cells and CD8 + cells proliferating in response to Leishmania stimulation are involved in protection from and healing of (Ethiopian) cutaneous leishmaniasis; however, such mechanisms appear to be unrelated to the NRAMP1 host resistance gene.

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.

We coincubated killed or live human immunodeficiency virus type-1 (HIV-1) with human monocyte-derived cells infected with Leishmania donovani and examined the effect of the virus preparations on the intracellular growth of the parasite. We found that there was significant enhancement (by a mean of 53%, p0.001) of intracellular L. donovani growth in the human monocytic leukaemia THP-1 cell line coincubated with killed HIV-1. Infection of peripheral blood monocyte-derived macrophages with live HIV-1 initiated after L. donovani infection led to an increase in intracellular parasites by an overall mean of 2.8% vs 4.9% (p0.01) at 2 and 5 d after HIV infection in L. donovani and L. donovani plus HIV-1 infected, respectively, and by an overall mean of 5.0% vs 13.3% (p0.001) at 5, 12 and 15 d after HIV-1 infection in L. donovani and L. donovani + HIV-1 infected, respectively. Further, L. donovani infection 2 d after infection with HIV-1 led to enhanced parasite growth (34.5%, p0.001) compared with cells infected with L. donovani alone (5.5%), and those where HIV-1 was added after L. donovani (18.1%). In all cases, HIV-1 from live and killed virus preparations led to decreased anti-leishmanial activity of the macrophages as evidenced by decreased control of intracellular multiplication. The findings may suggest a mechanism not requiring live virus to explain how HIV-1 coinfection may impair the control of intracellular Leishmania growth in individuals with pre-existing asymptomatic infection leading to the reactivation of the parasite. Moreover, patients with HIV-1 infection might be at increased risk of developing Leishmania infection.

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.

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.

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.

Mengistu G, AkuiTo H. Fehniger TE. Immunobloi Analysis of Sera Trom Ethiopian CutaneousLeishmaniasis by Antibody Class. Scand J Immunol 1992;36(Suppl.l I):l49-52We have determined the specificity of the classes of anti-leishmanial antibodies which aredelectable in serum from patients with active cutaneous leishmaniasis. On immunobtots.differences exist in the patterns of antigen recognition by IgG. IgM. IgA and IgE antibodiespresent in localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL)patient serum. Each class of antibody showed differing patterns of banding to the variousmolecular species of parasite antigens. There was signiticant variation in the specificities of theIgG antibody reactivities between individual patients. The patterns of IgM binding were generallyhomogeneous and restricted to antigens with M, >40 kDa. The only class o^ AnX'i-Leishnuiniaantibodies which showed shared patterns of common antigen recognition by all of the patientsstudied were IgA antibodies. The reactivities of IgE antibodies encompassed two antigens of Mr36 and 46-48 kDa which were not recognized by any of the other isotypes. Such antibody classassociated reactivity may be useful in the design of" serodiagnostic assays for the detection ofLeishmania infection or other infectious agents.Genncne Mengisiu. Department of Infectious DLsease.v, Karolinska Institute. Huddinge Hospital.14186 Huddinge. SwedenThere is supportive evidence that antibodies playsome functional role in the pathogenesis ofhuman leishmaniasis. Plasma cells (IgG, IgA orIgM) account for 10-50';'i> ofthe infiltrating cellsin the lesions of patients with American cuta-neous leishmaniasis [ I ]. and are commonly foundin the lesions of Ethiopian patients with diffusecutaneous leishmaniasis (DCL) [2]. Recentstudies have further shown that plasma cells areinduced in the lesions of patients with activecutaneous leishmaniasis following the local ad-ministration of intranodular injections of rIL-2[3]. Serum IgG antibodies reactive v^ith Leishma-nia aerhiopiia are observed in high titres inpatients with localized cutaneous leishmaniasis(LCL) and DCL patients [4]. At the level ofantigen-specific recognition, we have recentlyshown that the immunoblot profiles of serumantibodies ofany given LCL or DCL patient werelimited to only a subset ofthe potential antigeniccomponents expressed by the parasite [5]. How-ever, significant heterogeneity was observed in theantibody repertoire of individual LCL and DCLpatients.In this study we reported the general distribu-tion patterns and specificity of the classes ofserum antibody which developed in patients withcutaneous leishmaniasis.MATERIALS AND METHODSSubjecf.s. Serum samples were obtained from eightDCL and seven LcL parasitologically confirmedpatients attending the dermatology clinics of the AilAfrica Leprosy and Rehabilitation Training Center(ALERT) in Addis Ababa. The duration of" diseasesvaried from 9 months to > 26 years. Despite themultiple drug therapy in some of the DCL patients.there was only temporary recovery from the signs andsymptoms of the disease. Ten healthy controls (fiveEuropeans living in leishmania non-endemic country,and five Ethiopians living in lj;ixhmania endemic andnon-endemic areas) oT both se.ites were also studied.Serum was stored at —20 C until used.Antigen preparation. Promasligotcs were grown onNNN medium from biopsies of patient lesions aspreviously described [5]. Parasites were stored at a totalprotein concentration of 1-2 mg/ml at —70 C untilused.SDS-PAGE and immiinohloi analysis. Whole celllysates of organisms (100 }ig protein/cm gel surface)149