Concurrent infection with Leishmania donovani and Leishmania major in a Kenyan patient: clinical description and parasite characterization.

Leishmania isolates aspirated a few months apart from the spleen of an indigenous adult male kala-azar patient from Baringo District, Kenya, were biochemically characterized and compared. The patient lived within a dual focus of L. donovani kalazar and L. major cutaneous leishmaniasis. A primary Leishmania isolate from splenic aspirates was cryopreserved (NLB-294). The patient was treated with sodium stibogluconate for kala-azar and discharged. Three months later, he had clinical relapse and returned for retreatment. During his second visit, the patient participated in a diagnostic study in which urine and nasopharyngeal samples were cultured for leishmaniasis. Urine, nasopharyngeal, and splenic samples were positive for Leishmania. Secondary isolates from splenic (NLB-294-I) and urine (NLB-318) cultures were cryopreserved and characterized by cellulose acetate electrophoresis (CAE) using 20 enzymes. Whereas the urine isolate was typed as L. donovani, the splenic aspirate culture revealed a mixed infection with L. donovani and L. major. The primary isolate (NLB-294) was then characterized and also showed a mixed infection. To exclude the possibility of protein post-translational modifications in electrophoretic assays, the primary and secondary isolates were grown and processed under identical cultural and lysis conditions, and compared using CAE. The results were identical to the first electrophoretic assays showing mixed promastigote banding patterns. Stationary-phase promastigotes of the secondary splenic isolate (NLB-294-I) inoculated subcutaneously, intraperitoneally, and intracardially into Syrian hamsters and BALB/c mice produced both kala-azar and cutaneous leishmaniasis within 6.5 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Visceral leishmaniasis unresponsive to pentostam caused by Leishmania tropica in Kenya.

We report the characterization of 6 Leishmania tropica isolates from 2 patients with visceral leishmaniasis who were unresponsive to treatment with sodium stibogluconate. The Leishmania isolates, MHOM/KE/81/NLB-029A, -029XIB, and -029XIC and MHOM/KE/81/NLB-030I, -030B, and -030XXA, all from splenic aspirates, were characterized by cellulose acetate electrophoresis using 11 enzymes: malate dehydrogenase, malic enzyme, phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, superoxide dismutase, glutamate-oxaloacetate transaminase, adenylate kinase, nucleoside hydrolase, mannose phosphate isomerase, glucose phosphate isomerase, and phosphoglucomutase. Isozyme migration patterns were indistinguishable from those of 2 WHO reference strains of Leishmania tropica (MHOM/SU/60/LRC-L39, NLB-305 and MHOM/IQ/OO/LRC-L36, NLB-067). These are the first reported cases of visceral leishmaniasis (kala-azar) caused by L. tropica in Africa; these cases were refractory to sodium stibogluconate.

Cutaneous leishmaniasis caused by Leishmania major in Baringo District, Kenya.

Leishmania major was isolated from lesions of a patient suffering from cutaneous leishmaniasis in Baringo District of Kenya. Isoenzyme mobilities of this strain were compared with those of L. major, L. donovani, L. aethiopica and L. tropica reference strains and also L. major from a sand fly, Phlebotomus duboscqi, and a rodent, Arvicanthis niloticus, trapped in the same region. The patient's isolate had similar banding patterns to the L. major reference strain and also the rodent and the sand fly strains with the 9 enzymes examined. This is the first report in Kenya of an indigenous case with naturally acquired zoonotic cutaneous leishmaniasis.

A simple and economical direct agglutination test for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis.

A simple and economical direct agglutination test for the detection of visceral leishmaniasis is described. Trypsin-treated, Coomassie Brilliant Blue-stained, formalin-preserved promastigotes were used as antigen in re-usable V-well microtitre plates. In 21 patients with recent kala-azar, titres of 1:51200 or higher were found. Cured kala-azar patients treated 4 to 14 months before testing, showed titres in the range of 1:3,200 to greater than 1:51,200. Healthy and diseased controls had titres below 1:1,600 with the exception of African trypanosomiasis patients who showed titres of 1:200 to 1:12,800, overlapping with the titres of cured kala-azar patients. Where trypanosomiasis is not a consideration, a titre of 1:1,600 could be considered indicative of visceral leishmaniasis, the sensitivity and specificity were then 100%. The test was applied to sera of 280 inhabitants of Baringo District, a known focus of visceral leishmaniasis in Kenya. When treated cases were included, the test showed a sensitivity of 100% and specificity of 99.3%. This test could be used in district hospitals and health centres in endemic areas as an aid in diagnosis of kala-azar and in the field for sero-epidemiological studies.

Evaluation of a newly developed direct agglutination test (DAT) for serodiagnosis and sero-epidemiological studies of visceral leishmaniasis: comparison with IFAT and ELISA.

A newly developed direct agglutination test (DAT) for visceral leishmaniasis, IFAT and ELISA were applied to sera of patients with visceral leishmaniasis, African and American trypanosomiasis, other parasitic infections and healthy controls. The sensitivities of the 3 tests were comparable (96.3% to 100%); excluding patients with African and American trypanosomiasis, the specificities of DAT and IFAT were 100% and ELISA 87.3%. When trypanosomiasis sera were included, the specificities were 72.6%, 94.3% and 79.4% in DAT, IFAT and ELISA respectively. In 273 sera from a leishmaniasis endemic area (Baringo District, Kenya), the sensitivity was 80% in DAT and IFAT and 60% in ELISA, specificities being 99.6% (DAT), 98.5% (IFAT) and 62.5% (ELISA). As the new DAT is economical and easy to perform, it is recommended for sero-epidemiological field work on visceral leishmaniasis.

Acute phase protein concentrations predict parasite clearance rate during therapy for visceral leishmaniasis.

Visceral leishmaniasis (VL) remains a major health problem in Kenya and other parts of Africa, Central America and Asia. Currently, splenic aspirate smear and culture are the standard methods of monitoring therapy and relapse. Acute phase reactant markers, C-reactive protein (CRP), serum amyloid A protein (SAA) and alpha 1-acid glycoprotein (AGP) were evaluated as less invasive techniques for monitoring therapy in 59 patients with VL before, during and after therapy. CRP, SAA and AGP were elevated in VL patients at admission and the concentrations decreased with effective therapy to reach normal levels by the end of therapy (SAA and AGP) or by 3 months follow-up (CRP). Two groups of patients were selected on the basis of rate of parasite clearance. The acute phase protein concentrations were significantly raised in those slower to clear parasites. Analysis of sensitivity and specificity of acute phase proteins as predictors of parasite clearance suggested that they might represent useful non-invasive markers for monitoring disease activity, response to therapy and relapse in VL.

Visceral leishmaniasis unresponsive to antimonial drugs. I. Clinical and immunological studies.

Ten Kenyan patients with visceral leishmaniasis, unresponsive to sodium stibogluconate at a dose of 16 to 20 mg Sb/kg/day given for 30 to 98 days, have been studied clinically and immunologically and compared with 57 antimony-responsive patients. Pulmonary tuberculosis and previous treatment with antimonial drugs were the only factors which were more common in unresponsive patients. The degree of immunosuppression and rate of recovery of immunoreactivity did not differ between antimony-responsive and -unresponsive patients. Only one patient had never been treated before (primary unresponsiveness). In the other nine patients secondary unresponsiveness occurred after one or more treatment courses, suggesting that the parasite developed resistance to antimony. Antimony-unresponsiveness in visceral leishmaniasis is a serious problem numerically, clinically and economically. A plea is made that the initial treatment of visceral leishmaniasis should be adequate in dose and duration.

Visceral leishmaniasis unresponsive to antimonial drugs. III. Successful treatment using a combination of sodium stibogluconate plus allopurinol.

Five patients with long-standing visceral leishmaniasis who were unresponsive to sodium stibogluconate, 20 mg antimony/kg body-weight once or twice daily, were treated for 14 to 54 days with a combination of sodium stibogluconate at the same dose plus allopurinol at a dose of 20 mg/kg body-weight per day in three divided doses. This combination was safe and effective. Negative splenic aspirate smears were obtained from all patients within 19 days, and none has relapsed in at least 12 months of follow-up.

Visceral leishmaniasis unresponsive to antimonial drugs. II. Response to high dosage sodium stibogluconate or prolonged treatment with pentamidine.

Ten Kenyan patients with visceral leishmaniasis unresponsive to sodium stibogluconate, at a dose of 16 to 20 mg Sb/kg body-weight/day given for 30 to 98 days, were treated with 20 mg Sb/kg bw given every eight hours. This regimen was modified or abandoned in six patients because of suspected toxicity, although toxicity was difficult to assess because of intercurrent illness. Toxic effects included lethargy, anorexia, vomiting, electrocardiographic changes, fall in haemoglobin and rise in liver enzymes. One patient died, probably from a cardiac arrhythmia. Two patients were cured, four responded partially and four showed no response. Pentamidine, at a dose of 4 mg/kg body-weight given one to 3 times per week for 5 to 39 weeks, was given as initial treatment in one patient and after failure of sodium stibogluconate in seven. Toxic effects included nephritis, hepatitis, transient diabetes and subcutaneous abscesses. Two patients were cured, two responded partially, three showed no response and one, after apparent cure, relapsed and was unresponsive to additional pentamidine treatment. Low-frequency, long-duration pentamidine was often useful in maintaining any improvement made during treatment with the less well tolerated high-dose, high frequency sodium stibogluconate. We observed the step-wise development of resistance to both sodium stibogluconate and pentamidine. The problems of managing patients with visceral leishmaniasis which is unresponsive to conventional doses of pentavalent antimonials are discussed and some tentative suggestions put forward.

Schistosomiasis caused by Schistosoma mansoni in Baringo District, Kenya: case report.

Schistosomiasis caused by Schistosoma mansoni has not been reported in Baringo District of Rift Valley Province. The intermediate host (Biomphalaria species) though has been reported to occur along the shores of the lakes in this region. Three children from Baringo District were diagnosed to have schistosomiasis caused by S. mansoni by finding ova in their stools. They gave no history of visiting an endemic area. There are many dams being built for land reclamation, creating favourable conditions for the spread of the disease, in presence of the intermediate and definitive host. Studies on the current status of the disease and malacology should be undertaken in order to control the spread of the disease at an early stage.

Use of beef extract as a diluent in direct agglutination test for visceral leishmaniasis.

Direct Agglutination Tests (DAT) have been developed for sero-diagnosis of visceral leishmaniasis (VL). The objective of this study was to find a more suitable diluent for serum dilution in a Direct Agglutination Test. 1% of beef extract (BEX) was used as a diluent in the Direct Agglutination Test; 1% foetal bovine serum (FBS) and 0.2% gelatine (Gel) were included for comparison as alternative diluents. Serum from VL patients, individuals suffering from hydatidosis, syphilis and from healthy adult Kenyans was used as control. Beef extract was found to be easy to handle, less expensive and results comparable to those obtained from the other diluents.

Relationship between direct agglutination test and splenic aspirate smear parasite load in visceral leishmaniasis at Baringo District, Kenya.

Direct agglutination test was carried out in Baringo District on 100 persons presenting with signs and symptoms suggestive of visceral leishmaniasis. Splenic aspirate smears and cultures were done on these 100 persons in order to parasitologically confirm the findings of the direct agglutination test. It was found that the direct agglutination test positively detected all 79 (79%) patients parasitologically confirmed to have visceral leishmaniasis. Irrespective of the splenic aspirate smear parasite rate, whether 1+ or 6+ on a logarithmic scale, direct agglutination test was positive. There were 21% false positives, two of whom had Schistosoma mansoni in their stools. It was not immediately known about the cause of the other false positives. It was concluded that the direct agglutination test is a good provisional serodiagnostic test for visceral leishmaniasis and should be considered for wider field application.

Post kala-azar dermal leishmaniasis: the Kenyan experience.

Post kala-azar dermal leishmaniasis (PKDL) occurs occasionally after successful cure of visceral leishmaniasis. Twelve patients with diagnosis consistent with PKDL were seen at Clinical Research Centre from 1981 to 1985. Clinical presentation ranged from macular hypopigmented lesion to generalized nodular lesions. All lesions cleared either by self-cure or by treatment with sodium stibogluconate.

Problems in the treatment of kala-azar: case report.

Frequent relapses after treatment for visceral leishmaniasis and apparent parasitological cure is not commonly reported. Seven year old boy who relapsed four times with clinical and parasitological evidence of the disease at each two months follow-up period is presented. He had Leishimania donovani Kenya strain. After treatment, review would be after two months, six months and twelve months periods. Splenic aspirates were routinely done weekly and on the last day of each treatment. The drugs administered for varying periods included intravenous sodium stibogluconate 20 mg/kg daily, P20 in combination with allopurinol 21 mg/kg three times daily, and Pentamidine 4 mg/kg three times weekly and antituberculous drugs. The presence of abundant extra cellular leishmania donovani bodies in the bone marrow and possible pulmonary tuberculosis might have precipitated the frequent relapses. It is not clear which of the drugs effected the cure. It was observed that inspite of prolonged antileishmanial drug administration no side effects were noted.

Jejunal function and pathology in visceral Leishmaniasis.

Small-intestinal function was studied in 10 patients with visceral leishmaniasis. Vitamin-A absorption was impaired in 7 and d-xylose in 1. In 5 of the 10 patients Leishmania were demonstrated in biopsy specimens of intestinal mucosa. Parasitised macrophages were present in villous tips and less commonly in the lamina propria and submucosa; a moderate inflammatory infiltrate was composed of lymphocytes and plasma cells. 2 patients had partial villous atrophy. There was no correlation between intensity of parasitisation and severity of malabsorption. After treatment with sodium stibogluconate there was a significant improvement in absorption of vitamin A and d-xylose, and biopsy specimens became normal. In 1 patient visceral leishmaniasis was thought to be the cause of chronic diarrhoea.

Improvement of a direct agglutination test for field studies of visceral leishmaniasis.

To increase the potential for the wide-scale application of our direct agglutination test for visceral leishmaniasis, modifications in the components and procedures were introduced. Supplementation with 0.056 M citrate of the suspension medium stabilized the antigen for 9 weeks at 37 degrees C. To circumvent the need for cooling systems in the field, 0.2% (wt/vol) gelatin was added to the serum diluent instead of fetal bovine serum, with reliable results. Specificity and sensitivity were improved by the incorporation of 0.1 M 2-mercaptoethanol in samples with borderline titers. The test could be performed on samples of whole blood; thus the difficulties of preparation and storage of serum, plasma, or filter paper blood are avoided. For mass screening programs, a single serum dilution of 1:6,400 could be employed, contributing to a further reduction in test expenses. Sera from different geographical areas showed equal reactivities in this direct agglutination test despite the nonhomologous Leishmania donovani antigens used.