Universal PCR assays for the differential detection of all Old World Leishmania species.

For the epidemiological monitoring and clinical case management of leishmaniasis, determination of the causative Leishmania species gains importance. Current assays for the Old World often suffer from drawbacks in terms of validation on a geographically representative sample set and the ability to recognize all species complexes. We want to contribute to standardized species typing for Old World leishmaniasis. We determined the ribosomal DNA internal transcribed spacer 1 sequence of 24 strains or isolates, and validated four species-specific polymerase chain reactions (PCRs) amplifying this target. They discriminate L. aethiopica, L. tropica, L. major, and the L. donovani complex, use the same cycling conditions, and include an internal amplification control. Our PCRs amplify 0.1 pg of Leishmania DNA, while being 100% specific for species identification on an extensive panel of geographically representative strains and isolates. Similar results were obtained in an endemic reference laboratory in Kenya. Species could also be identified in clinical specimens. The presented PCRs require only agarose gel detection, and have several other advantages over many existing assays. We outline potential problems, suggest concrete solutions for transferring the technique to other settings, and deliver the proof-of-principle for analyzing clinical samples.

Diagnostic tests for kala-azar: a multi-centre study of the freeze-dried DAT, rK39 strip test and KAtex in East Africa and the Indian subcontinent.

Three diagnostic tests for visceral leishmaniasis (VL), the freeze-dried direct agglutination test (FD-DAT), the rK39 dipstick and a urine latex antigen test (KAtex), were evaluated for use in primary care in East Africa and the Indian subcontinent. Clinical suspects were prospectively recruited and tissue, blood and urine samples were taken. Direct microscopic examination of tissue smear, and FD-DAT, rK39 and KAtex were performed. Sensitivity and specificity with 95% credible intervals were estimated using Bayesian latent class analysis. On the Indian subcontinent both the FD-DAT and the rK39 strip test exceeded the 95% sensitivity and 90% specificity target, but not so in East Africa. Sensitivity of the FD-DAT was high in Ethiopia and Kenya but lower in Sudan, while its specificity was below 90% in Kenya. Sensitivity of the rK39 was below 80% in the three countries, and its specificity was only 70% in Ethiopia. KAtex showed moderate to very low sensitivity in all countries. FD-DAT and rK39 can be recommended for clinical practice on the Indian subcontinent. In East Africa, their clinical use should be carefully monitored. More work is needed to improve existing formats, and to develop better VL diagnostics.

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.

Operational validation of the direct agglutination test for diagnosis of visceral leishmaniasis.

The validity of the direct agglutination test (DAT) for visceral leishmaniasis (VL) was studied with a standardized field kit on 148 clinically suspected persons and 176 healthy controls recruited between 1993 and 1994 from an endemic area in Gedaref State, Sudan. A sensitivity of 95.9% and a specificity of 99.4% were found at a 1: 8,000 cut-off titer when parasitologically confirmed cases were compared with healthy controls. While corroborating previously reported sensitivity and specificity estimates of this serodiagnostic test, this study examined the bias generated by commonly used test validation procedures. The fundamental methodologic problem in VL test validation is the absence of a reliable gold standard. Moreover, any operational guideline on DAT use has to consider the critical dependency of the predictive values of the test on VL prevalence rates. The DAT diagnostic cut-off titer depends upon many external factors, among which the prevalence of disease in the area and the case mix seem the most important.

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.

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.

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.

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.

Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis.

This paper describes a new latex agglutination test ('KATEX') for the detection of leishmanial antigen in the urine of patients with visceral leishmaniasis. In preliminary laboratory trials, using urine collected from well-defined cases and controls from Brazil, Yemen and Nepal, the test had 100% specificity and a sensitivity between 68 and 100%. When used in a time-course experiment in cotton rats infected with Leishmania donovani, the test became positive 1 week after inoculation and antigen levels in urine declined rapidly after chemotherapy (the test was negative before the end of the course of treatment). Finally, in an integrated study performed in Sudan, KATEX was compared to microscopy and four different serological tests in a group of 73 patients having presented with clinical manifestations suggestive of visceral leishmaniasis. Compared to microscopy, KATEX performed better than any single serological test in predicting positivity and a particularly good result was obtained by combining KATEX and the direct agglutination test (DAT).

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.

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.