In vivo cytokine profiles in patients with kala-azar. Marked elevation of both interleukin-10 and interferon-gamma.

The immunological mechanisms underlying the susceptibility to disseminated visceral parasitism of mononuclear phagocytes in patients with kala-azar remain undefined. Resistance and susceptibility are correlated with distinct patterns of cytokine production in murine models of disseminated leishmanial disease. To assess lesional cytokine profiles in patients with kala-azar, bone marrow aspirates were analyzed using a quantitative reverse transcriptase PCR technique to amplify specific mRNA sequences of multiple Th1-, Th2-, and/or macrophage-associated cytokines. Transcript levels of IL-10 as well as IFN-gamma were significantly elevated in patients with active visceral leishmaniasis; IL-10 levels decreased markedly with resolution of disease. These findings suggest that IL-10, a potent, pleiotropic suppressor of all known microbicidal effector functions of macrophages, may contribute to the pathogenesis of kala-azar by inhibiting the cytokine-mediated activation of host macrophages that is necessary for the control of leishmanial infection.

Coeliac disease in Sudanese children with clinical features suggestive of the disease.

Our prospective hospital-based study examined frequency, clinical presentation and serological indicators of coeliac disease that correlated with intestinal biopsy among high-risk Sudanese children. From July 2001 to July 2002, 80 children aged 15 months-18 years presented with poor appetite, weight loss, pallor and proximal muscle wasting. We diagnosed coeliac disease in 18 (22.5%). Antigliadin antibodies (AGA-IgG, AGA-IgA or both) were high in 44; endomysial antibody retest was high in 30. Guardians of 12 children refused consent for biopsy. The other 18 were biopsied: 5 had total villous atrophy, 8 subtotal and 5 partial. All improved with gluten-free diet. Degree of villous atrophy did not correlate with diarrhoea duration or severity, anaemia severity or serological titres.

An ABC-ELISA for malaria serology in the field.

An avidin biotin peroxidase complex enzyme-linked immunosorbent assay (ABC-ELISA) was examined for the diagnosis of malaria in a controlled area in Sudan Gezira. The titers of the ABC-ELISA coincided with those of the IFAT. The method was more sensitive than the ordinary ELISA as the final enzyme reaction was amplified through the use of the ABC system. This allowed the resulting color spots on the dried plate wells to be read clearly with the naked eye. This test can be carried out without using major electrical equipment.

Studies on the leishmaniases in the Sudan. 1. Epidemic of cutaneous leishmaniasis in Khartoum.

A recent outbreak of cutaneous leishmaniasis (CL) in Khartoum province, the first ever reported, involved about 10,000 recorded cases. The peak incidence was in September 1986. It was widespread all over the province, but the most affected areas were along the banks of the River Nile. People of both sexes, all age groups, different ethnic origins, and all socio-economic classes were equally affected, suggesting that this epidemic was a new occurrence among a non-immune population. A leishmaniasis clinic was established and 736 cases were treated and studied. The control measures, responsible parasite, possible animal reservoir, and vector are discussed. Possible contributory factors were mass population movement from known endemic areas of CL in Khartoum, expanding towns and new settlements in previously uninhabited areas, high population densities of sandflies, and an increase in the rodent population, including Arvicanthis species.

Studies on the leishmaniases in the Sudan. 3. Clinical and parasitological studies on visceral and mucosal leishmaniasis.

The epidemiological, clinical and laboratory findings of 27 patients with visceral leishmaniasis (VL) and six with mucosal leishmaniasis (ML) in Sudan are described. The diagnosis was confirmed by the demonstration of amastigotes in bone-marrow in 20 cases with VL (80%) and impression smears in 2 cases with ML. The enzyme-linked immunosorbent assay and direct agglutination test were positive in all the sera tested. The leishmanin test was positive in all patients with ML, the diameter of the reaction ranging from 18 to 28 mm (mean 23 mm). Only one isolate (from a patient with VL) grew sufficiently well in culture to be typed and this was characterized by isoenzyme electrophoresis as Leishmania donovani zymodeme LON-46. The possibility that the ML isolate could have been L. major zymodeme LON-1 is discussed. The importance of further research to isolate and identify by biochemical procedures a more representative range of isolates of Leishmania from different clinical types in the Sudan, in order to elucidate questions such as the relationship between the vectors and possible animal reservoirs, is emphasized.

Studies on the leishmaniases in the Sudan. 2. Clinical and parasitological studies on cutaneous leishmaniasis.

In the Omdurman Hospital for Tropical Diseases, Sudan, from 6 October to 1 December 1986, 736 patients with cutaneous leishmaniasis (CL) were studied. The duration of the lesions varied from a few days to 4 months, usually (56%) 1-3 months. Multiple lesions ranging from one to 50 (mean = 4) were present in 80% of cases. Ulcerative lesions were found in 44%, nodulo-ulcerative in 31%, nodular in 31% and others, including infiltrated, fungating and warty lesions, in 5% of patients. The lower limbs were involved in 66%, upper limbs in 50%, face in 6%, back in 4%, chest in 2%, abdomen in 1% and buttocks in 0.1%. Lesions were also found on the ear, scalp, genitalia and mucocutaneous junctions. Lymphatic involvement was present in 11% of the patients, secondary infection in 18%, fever in 17%, pain in 38% and itching in 61%; 3% of the patients were diabetics. Features of diffuse cutaneous leishmaniasis were noted in one patient and three cases presented with an 'id' reaction. A slit-skin smear was positive in 88% of the cases. Using thin-layer starch gel electrophoresis, 23 stocks from man and one from Arvicanthis niloticus were identified by the characterization of 12 enzymes as Leishmania major zymodeme LON-1. Various therapeutic regimens are discussed. Only patients with severe lesions (approximately 15%) were given 3 to 4 weeks' treatment with intravenous Pentostam, to which all except 2 responded satisfactorily with minimal side effects.

A comparison of the direct agglutination test and enzyme-linked immunosorbent assay in the sero-diagnosis of leishmaniasis in the Sudan.

The sensitivity and specificity of the direct agglutination test (DAT) and the enzyme-linked immunosorbent assay (ELISA) were compared for the sero-diagnosis of visceral (VL) and cutaneous (CL) leishmaniasis in Sudanese patients. All the sera from parasitologically confirmed cases of VL were positive in both ELISA and DAT. Some minor discrepancies were apparent between the two tests in patients with clinical signs of VL, but in whom VL was not confirmed parasitologically. In parasitologically confirmed CL both tests performed equally badly, with the DAT detecting 67% of cases and ELISA 60%. For the sero-diagnosis of VL, ELISA and DAT performed equally well, but on grounds of simplicity and low cost the DAT was preferred.

Antileishmanial antibodies in an outbreak of visceral leishmaniasis in eastern Sudan: high antibody responses occur in resistant subjects and are not predictive of disease.

A 3-year longitudinal survey was carried out from 1998 to 2000 in a village in eastern Sudan where a visceral leishmaniasis (VL) outbreak occurred. Leishmania-specific antibodies were analysed by enzyme-linked immunosorbent assay and immunoblotting. Immunoblot analysis detected antibodies to Leishmania in 80% of the healthy subjects and half of them harboured high immunoglobulin (Ig) G antibody levels, similar to those of VL patients. These antibodies belonged to the IgG1 and IgG3 subclasses but neither their respective levels nor the immunoblot recognition patterns were predictive of VL. During this epidemic, a large proportion of subjects had a high antileishmanial antibody response, indicating that they were infected by Leishmania though most of them remained healthy during the whole study period. These results obtained in the context of an outbreak contrast with those obtained from studies performed in endemic areas characterized by lower parasite transmission levels. Furthermore, the clinical and serological follow-up of our study subjects showed that VL occurred mainly in subjects who had been serologically positive for 5-24 months rather than resulting from primo infection by the parasite.

Field evaluation of latex agglutination test for detecting urinary antigens in visceral leishmaniasis in Sudan.

A latex agglutination test to detect urinary antigens for visceral leishmaniasis (VL) was studied. In 204 patients with suspected VL, KAtex had a sensitivity of 95.2% with good agreement with microscopy smears but poor agreement with 4 different serology tests. It was also positive in 2 confirmed VL cases co-infected with HIV. In all K4tex-positive confirmed cases actively followed up after treatment, the test became negative 1 month after completion of treatment. While IC4tex had a specificity of 100% in healthy endemic and non-endemic controls, the direct agglutination test (DAT) was positive in 14% of the KAtex-negative healthy endemic controls. KAtex is a simple addition to the diagnostics of VL particularly at field level and as a complementary test for the diagnosis of VL in smear-negative cases with positive DAT results.

Identification of a novel G245R polymorphism in the IL-2 receptor beta membrane proximal domain associated with human visceral leishmaniasis.

Binding of the interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) triggers a series of intracellular events culminating in lymphocyte proliferation and differentiation. We report here the identification of a novel G245R polymorphism in the membrane proximal domain of the IL-2 receptor beta chain (IL-2Rbeta). Present at a frequency of 7.2%, the IL-2-Rbeta G245R was identified in a population of Eastern Sudan exposed to a severe outbreak of visceral leishmaniasis (VL), a disease associated with a marked depression of T-cell antigen-specific responses. The location of the G245R polymorphism next to the box1/box2 proximal cytokine receptor homology segment and suggestive genetic association with the development of disease (P=0.043), suggest that it may affect Janus kinase (JAK) association and impair growth signal transduction. However, additional genetic association with a synonymous single nucleotide polymorphism (IL2RB+8777T) suggests that other variations of IL2RB or nearby genes participate in the highly significant linkage with VL at 22q12 previously reported for this population.

Genetic control of visceral leishmaniasis in a Sudanese population: candidate gene testing indicates a linkage to the NRAMP1 region.

There is some evidence showing that genetic factors are involved in human susceptibility to parasitic diseases such as schistosomiasis and malaria. Studies have shown that the Nramp1 and H-2 genes are implicated in the control of Leishmania donovani infection in mice. We sought genetic loci involved in the control of susceptibility to visceral disease caused by L. donovani in humans. We studied 37 families with at least two affected sibs living in a village in eastern Sudan, where an outbreak of visceral leishmaniasis occurred between 1995 and 2000. The genetic markers located in five chromosomal regions containing candidate genes were typed: 2q35 (NRAMP1), 5q31-q33 (Th2 cytokine cluster), 6p21 (HLA/TNF-alpha), 6q23 (INFGRI) and 12q15 (INF-gamma). Linkage (multipoint lod-score=1.08; P=0.01) was observed for the 5'(CA) repeat polymorphism in the NRAMP1 promoter. This suggests that genetic variations of this gene affect susceptibility to visceral leishmaniasis in this population.

The sandfly fauna in the visceral-leishmaniasis focus of Gedaref, in the Atbara-River area of eastern Sudan.

Visceral leishmaniasis (VL) is an acute public-health problem in Sudan. Between 1997 and 2000, four, brief entomological surveys were carried out in Barbar El Fugarra, a village in the state of Gedaref, in the Atbara-River area of eastern Sudan. Between 1996 and 1999, 658 cases of VL occurred among the village's population of about 4000. CDC miniature light-traps set inside and outside human dwellings were used to collect a total of 12,745 sandflies, including five species of the genus Phlebotomus and 19 of Sergentomyia. Phlebotomus papatasi and P. orientalis made up 7% and 5% of the collected sandflies, respectively. Seasonal variation was observed in the numbers of P. orientalis, P. papatasi, S. schwetzi and S. magna caught. Almost all (88%) of the sandflies collected were caught inside houses or granaries and there appeared to be particularly large indoor populations of P. orientalis, P. papatasi, S. schwetzi, S. magna and S. clydei. Phlebotomus orientalis could be responsible for the indoor transmission of the parasites causing the local VL, between humans and between humans and local dogs (which have been found infected by some of the Leishmania zymodemes found in humans). The co-occurrence in this focus of P. papatasi and Arvicanthis niloticus, which are known vectors and reservoir hosts, respectively, of L. major, indicates the possibility that outbreaks of human cutaneous leishmaniasis might occur in the area.

The food of the larvivorous fish Gambusia affinis (Baird and Girard) and Oreochromis (formerly Tilapia) niloticus (Linnaeus) in Gezira irrigation canals.

The food of both Gambusia affinis and Oreochromis niloticus was studied. Organisms eaten by both species of fish are tabulated, together with the amounts eaten during the various months of the year. G. affinis larger than 25 mm are carnivorous and become more so with age. Food selection by G. affinis depends on the availability of food items rather than choice. It showed a marked preference for mosquito larvae. O. niloticus smaller than 150 mm were markedly carnivorous, but this decreased with age. Only small fish of this species are useful for the biological control of mosquitoes. Fish larger than 150 mm showed a marked preference for higher aquatic macrophytes.

Survival, growth and reproduction of the imported ampullarid snail Marisa cornuarietis in Central Sudan.

The ampullarid snail Marisa cornuarietis was imported to the Sudan in 1981 for evaluation as a biological control agent against the planorbid snails which transmit human schistosomes. In initial field studies in small protected ponds the generation time of M. cornuarietis was 4 months, as in Puerto Rico. The snails reached 4 cm in diameter after 1 year, compared to 3 cm in Puerto Rico. Their population density varied from 60 to 175 snails per metre of shore-line, compared to a similar pond in Puerto Rico where the stable density was about 115 snails per metre. The proportion surviving after 1 year was 0.03, less than the annual proportion surviving of 0.10 in Puerto Rico. Thus the preliminary results indicated that the ampullarid snails could establish strong populations in permanent habitats in central Sudan where there was adequate food, although it might take longer than it does in Puerto Rico.

The impact of the exotic fish Gambusia affinis (Baird and Girard) on some natural predators of immature mosquitoes.

The effects of Gambusia affinis on some suspected predators of mosquitoes were studied for a period of 12 months. A list of such predators was given. The most abundant invertebrate predators were Odonata, followed by Hemiptera and Ephemeroptera and the least common were Coleoptera. The population density of mosquito predators, with exception of Notonectidae and the fish Oreochromis niloticus, was markedly reduced in the presence of G. affinis. Application of aerial pesticides reduced the population of both G. affinis and O. niloticus fish. However it had no effect on the numbers of all invertebrate predators.

Mapping of a visceral leishmaniasis-specific immunodominant B-cell epitope of Leishmania donovani Hsp70.

We have shown that a member of the 70-kDa heat shock protein (Hsp70) family is a major target of the humoral immune response during Leishmania donovani infection. A recombinant fusion protein was recognized by sera from 92% (35 of 38) of patients with visceral leishmaniasis, including representatives from each of the major regions where it is endemic. Serological analysis of recombinant Hsp70, expressed by a series of deletion constructs, identified the carboxy-terminal region as the immunodominant site. This region, which is the most evolutionarily divergent part of the molecule, was recognized by all sera from patients with visceral leishmaniasis which exhibited an anti-Hsp70 response. Purified recombinant L. donovani Hsp70 was not recognized by sera from patients with cutaneous leishmaniasis, Chagas' disease, leprosy, malaria, or schistosomiasis. To determine the regions involved in antibody recognition, a series of overlapping peptides were synthesized on polyethylene pins by the Pepscan method, and a hexamer, EADDRA, was identified by the visceral leishmaniasis serum samples as an immunodominant B-cell epitope.

Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing.

Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.