Specific antibody responses as indicators of treatment efficacy for visceral leishmaniasis.

Acute visceral leishmaniasis (VL) is caused by infection with parasites of the Leishmania donovani complex and may be fatal if not treated. Early diagnosis and efficacious treatment are the keys to effective VL management and control. Novel regimens are being developed to overcome limitations in VL treatment options, which are currently restricted by high costs, severe systemic side effects, and unresponsiveness. Although simple and accurate serological tests are available to help confirm VL, none are suitable to monitor treatment efficacy and cure. Here, we confirm that serum antibody responses to the diagnostic antigens rK39 and rK28 are unaltered by treatment, but demonstrate that antibodies produced against two antigens, rK26 and rK18, can be used as an indirect measure of parasite clearance. The levels of anti-rK18 and -rK26 antibodies were high in patients at initial diagnosis but declined in patients treated with either SSG (Ethiopia) or AmBisome (Bangladesh). Taken together, we propose that serological tests which measure antibodies to rK26 and rK18 merit consideration as potential markers of treatment success and cure.

Interleukin 10 production correlates with pathology in human Leishmania donovani infections.

We have found that an important Th2 cytokine, IL-10, is produced by tissues from patients acutely infected with Leishmania donovani. In all individuals tested, IL-10 mRNA production was increased in lymph nodes taken during acute disease over that observed in postacute samples. In contrast, both pre- and posttreatment lymph nodes had readily detected mRNA for IFN-gamma and IL-2. A down-regulating effect of IL-10 on leishmania-induced proliferative responses was demonstrated when Hu rIL-10 was added to cultures of PBMC from clinically cured individuals. PBMC from individuals with acute visceral leishmaniasis responded to stimulation with leishmania lysate by producing IL-10 mRNA. Simultaneously cultured PBMC collected from the same patients after successful chemotherapy produced no detectable IL-10 mRNA after leishmania antigen stimulation. Neutralizing anti-IL-10 mAb added to PBMC from patients with acute visceral leishmaniasis markedly increased the proliferative response to leishmania lysate. Finally, we observed mRNA for IL-10 and IFN-gamma concurrently in a lesion from a patient with post-kala-azar dermal leishmaniasis (PKDL). These results indicate the production of IL-10 during L. donovani infection, and suggest a role for this cytokine in the regulation of immune responsiveness during visceral leishmaniasis.

Cell-mediated immune responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects.

To determine the possible differences in the immune response to Plasmodium falciparum between sickle-cell trait (Hb AS) and normal haemoglobin (Hb AA) individuals, we examined 35 Hb AS and 24 Hb AA subjects matched for age and microenvironment. Their age was 2-55 years and all lived in a malaria endemic area 300 km south of Khartoum. Antibodies to ring-infected erythrocyte surface antigen (Pf155/RESA) and to circumsporozoite (CS) protein (anti-NANP40) indicated equal exposure to falciparum malaria. Peripheral blood mononuclear cells (BMNCs) from 20/35 (57%) Hb AS subjects compared with 10/24 (42%) Hb AA subjects, responded to affinity-purified P. falciparum soluble antigens (SPAg). Of those responding to SPAg, 9 (26%) Hb AS subjects and only two (8%) Hb AA subjects had high responses. The mean proliferative response to SPAg of BMNCs from Hb AS individuals was significantly higher than in Hb AA individuals (P less than 0.025). Responses of BMNCs to PPD and PHA were also higher among Hb AS individuals and correlated positively with responses to SPAg. These findings support the hypotheses that the sickle-cell trait protects individuals from P. falciparum infections, at least in part, by modulating the immune response.

Significance of eosinophiluria in urinary schistosomiasis. A study using Hansel's stain and electron microscopy.

Urine deposit smears of 58 patients with urinary schistosomiasis, 20 with acute glomerulonephritis, and 14 with urinary stones were stained with Hansel's stain. Eosinophiluria was seen in all cases of urinary schistosomiasis (median 0.73 [73%] of urinary white blood cells [WBCs]); 13 (65%) of those with acute glomerulonephritis (median 0.03 [3%] of urinary WBCs); and 2 (14%) patients with urinary stones (0.01 [1%] and 0.05 [5%] of urinary WBCs). Other features included the presence of ova and activated macrophages and giant cells in urinary schistosomiasis, an increased number of mononuclear cells and stained cellular casts in acute glomerulonephritis, and an increased number of neutrophils in urinary stones. In schistosomiasis, urine differential leukocyte counts were unrelated to those of the blood. Moreover, urine eosinophil percentages were always higher than those in the blood. Thus, in urinary schistosomiasis, eosinophiluria could be explained by exudation, sloughing of granulomata, as well as bleeding from the urinary tract surface. Electron microscopic examination confirmed that most of the leukocytes were eosinophils. Some of these were adherent and degranulating over ova with variable miracidial damage. Urine eosinophil and egg counts were not correlated to each other. Staining urine deposit smears, with the use of Hansel's stain, is useful in the investigation of hematuria. Heavy eosinophiluria and few mononuclear cells favor the diagnosis of urinary schistosomiasis rather than acute glomerulonephritis or urinary stones. These results diminish the value of eosinophiluria as corroborative evidence of drug-induced interstitial nephritis in areas of endemic urinary schistosomiasis.

Endemic kala-azar in eastern Sudan: a longitudinal study on the incidence of clinical and subclinical infection and post-kala-azar dermal leishmaniasis.

Between April 1991 and April 1993, a longitudinal study was performed in the village of Um-Salala (1,430 inhabitants) in the endemic area of kala-azar (visceral leishmaniasis) in eastern Sudan. During the two years, a total of 92 kala-azar cases were diagnosed (male:female ratio = 1.8:1, mean age 6.6 years). The annual incidence rates were 38.4/1,000 and 38.5/1,000 person-years, respectively. The ratio of clinical to subclinical cases was 1.6:1 in the first year and 2.4:1 in the second year. Post-kala-azar dermal leishmaniasis occurred in 48 (56%) of 85 kala-azar cases that were followed-up for at least six months. Kala-azar occurred only in previously leishmanin-negative individuals. The majority of the population had a positive leishmanin skin test result, probably due to previous exposure to Leishmania major causing cutaneous leishmaniasis in their homeland in western Sudan from which they had migrated in the 1980s. It was thus postulated that previous cutaneous leishmaniasis might protect against kala-azar but this could not be proved.

A rapid and simple diagnostic test for active visceral leishmaniasis.

We have developed an immunodot assay for the serodiagnosis of active visceral leishmaniasis (AVL) which utilizes protein A colloidal gold as the visualizing agent. The test is simple, requires few reagents, and can be completed in two hours. It is sensitive and specific for active visceral leishmaniasis, and generally correlates with the ELISA. Either whole blood or sera in minute quantities may be used as test antibody. In addition, the use of the protein A gold immunodot is shown to detect anti-leishmania antibodies in infected dogs.

Congenital kala-azar and leishmaniasis in the placenta.

During an epidemic of visceral leishmaniasis in the Sudan, two cases of congenital kala-azar were seen. The first child, whose mother had contracted kala-azar in southern Sudan, was born in Khartoum, where no transmission of leishmaniasis is currently occurring. At seven months, the child had fever, lymphadenopathy, and hepatosplenomegaly; leishmania parasites were detected in the bone marrow. The child died and an autopsy showed leishmania parasites in all tissues including the lungs, kidneys, and thymus. In the second case, parasites were found in the placenta of a five-month-old fetus. These two cases demonstrate the importance of follow-up of infants born to mothers with leishmaniasis.

An improved serodiagnostic procedure for visceral leishmaniasis.

The enzyme-linked immunosorbent assay (ELISA) is a sensitive and specific serodiagnostic method for leishmaniasis. In this report, we describe how this versatile assay can be improved by the use of protein A or protein G conjugates for the specific detection of Leishmania antibody in the sera of patients with visceral leishmaniasis. In direct comparisons with anti-immunoglobulin conjugate, enzyme-linked protein A gave significantly higher absorbance values for positive sera without a corresponding increase in absorbance values for sera from normal individuals or from patients with other diseases known to cross-react with leishmaniasis. The effect was to increase the distance between positive and negative values, which aided in the interpretation of the results. This also permitted visual distinction between positive sera and negative or weakly reactive sera. The assay was effective using either blood or serum as the source of primary antibody. A further advantage of protein A over anti-Ig conjugate was its ability to detect specific antibody in dog as well as human sera. Finally, we demonstrated the usefulness of the protein A ELISA with a recombinant leishmania antigen, gp63.

Prevalence of cutaneous leishmaniasis along the Nile River north of Khartoum (Sudan) in the aftermath of an epidemic in 1985.

Based on a pilot clinical study of the prevalence of cutaneous leishmaniasis (CL) among school children in villages on both banks of the Nile River north of Khartoum, Sudan in the aftermath of a 1985 epidemic, we studied a random sample (303 individuals) from one of these villages to determine the prevalence of infection and exposure to CL. Four percent of the population had active CL lesions, 47% had healed lesions, and another 43% reacted positively to sensitization with leishmanin in the absence of past or active CL lesions. Ninety-one percent of the total population reacted positively to leishmanin. The present status of CL in the area and possible reasons for the emergence of the epidemic are discussed, and gaps in our knowledge about the epidemic are identified.

Kala-azar in a high transmission focus: an ethnic and geographic dimension.

In 1994-1996, we studied a group of 58 game wardens stationed in an area known to be highly endemic for visceral leishmaniasis (kala-azar) for evidence of infection with Leishmania donovani. Leishmania DNA was detected by the polymerase chain reaction in the peripheral blood of cases of active kala-azar, former patients with visceral leishmaniasis, patients, and asymptomatic subjects. Using the cloned antigen rk39, antibodies were detected in 44.2% of the game wardens while leishmanin skin test result was positive in 77% of our sample. It was shown that certain tribes from northern Sudan were more likely to develop subclinical infections, while those of the Baria tribe from southern Sudan and those of the Nuba tribe from western Sudan were more likely to develop visceral leishmaniasis. Whether this is due to genetic factors or previous exposure to Leishmania parasites remains to be elucidated.

Kala-azar in western Upper Nile province in the southern Sudan and its spread to a nomadic tribe from the north.

Since the start in 1988 of the present epidemic of kala-azar (visceral leishmaniasis) in western Upper Nile state in southern Sudan, the epidemiology of the disease in all parts of the Sudan where kala-azar has been reported was reassessed by the Leishmaniasis Research Group in Khartoum. In this paper, the spread of the epidemic is described among a nomadic tribe originating from southern Kordofan state, who migrate every year with their cattle to the Bentiu area in western Upper Nile state where the epidemic is still raging. 200 cases from this tribe were seen in Khartoum; another 56 cases were found during a field trip to the area. In addition, the Bentiu area was visited, where 301 cases were under treatment and another 52 of 1120 individuals screened were confirmed parasitologically. 20 cases of post-kala-azar dermal leishmaniasis were found. Parasites isolated from the nomadic tribe were of the same zymodeme as parasites isolated previously from the Nuer in western Upper Nile. The epidemiological findings in each state are discussed in relation to the tribes that were affected and the ecology of the area.

Identification of Leishmania from mucosal leishmaniasis by recombinant DNA probes.

Three cases of mucosal leishmaniasis are described. Parasites isolated from mucosal lesions were identified by Southern blot analysis of their genomic deoxyribonucleic acids (DNAs) using recombinant DNA probe pDK20. Parasites from 2 patients were identified as Leishmania donovani s.l. One of the patients had pure mucosal lesions, while in the second patient there was dissemination of the parasite to other organs. The spectrum of the disease caused by L. donovani is discussed. The parasite from the third patient was identified as L. major.

The treatment of kala-azar in the Sudan with sodium stibogluconate: a randomized trial of three dosage regimens.

In a randomized study in the Sudan, 3 different regimens of sodium stibogluconate were compared in patients with parasitologically confirmed kala-azar (visceral leishmaniasis): 10 mg/kg for 30 d (38 patients), 20 mg/kg for 30 d (29 patients), and 20 mg/kg for 15 d (37 patients). Treatment failures were defined as death, partial response, relapse, or the development of post-kala-azar dermal leishmaniasis. The hazard ratio for failure of 20 mg/kg for 30 d vs. 10 mg/kg for 30 d 2.1 (95% confidence interval [CI] = 0.6, 7.6) and for 20 mg/kg for 15 d vs. 10 mg/kg for 30 d it was 1.7 (95% CI = 0.5, 6.1). No significant difference was detected between the 3 regimens in the rate of return to normal of haematological criteria, regression of spleen size, or weight gain. After 15 d treatment parasite clearance with 20 mg/kg for 30 d and 20 mg/kg for 15 d was more profound than with 10 mg/kg for 30 d (P < 0.05), but the difference was no longer present at the end of treatment. Further investigation of the effectiveness of short, intensive treatment regimens in the treatment of kala-azar is warranted.

Post kala-azar dermal leishmaniasis in the Sudan: clinical features, pathology and treatment.

The clinical features, pathology, immune responses, diagnosis and treatment of post kala-azar dermal leishmaniasis (PKDL) in the Sudan are described and discussed. The disease is characterized by maculopapular or nodular lesions on the face, limbs or trunk. Lesions appear during or within months after the treatment of visceral leishmaniasis, but in 2 of 19 patients there was no previous history of kala-azar. PKDL may be confused with leprosy both clinically and pathologically. Similarities and differences between the 2 diseases are discussed. Unlike visceral leishmaniasis, the peripheral lymphoid cells of patients with PKDL respond to Leishmania antigen and some are leishmanin positive. The response to intravenous sodium stibogluconate (20 mg/kg for 30 d) was reasonably good but some patients required repeated or more prolonged treatment. Ketoconazole in a dose of 10 mg/kg daily for 4 weeks had no effect on PKDL.

Kala-azar: a comparative study of parasitological methods and the direct agglutination test in diagnosis.

In a comparative study 88 patients were diagnosed as suffering from kala-azar (visceral leishmaniasis) using 3 parasitological methods simultaneously. Splenomegaly was absent in 4 cases. In 84 patients with splenomegaly, splenic aspiration appeared to be the most sensitive method (96.4%), followed by bone marrow aspiration (70.2%) and lymph node aspiration (58.3%). There was no relation between titres in the direct agglutination test and parasite load as determined by the number of parasitological methods which were positive or parasite density in splenic aspirates. Splenic aspiration and bone marrow aspiration were compared as an assessment of cure in kala-azar. In 6 (13%) of 46 patients tested, parasites were found, all by splenic aspiration. Bone marrow showed parasites in one of these. The literature with regard to parasitological investigations before and after treatment is reviewed.

The direct agglutination test for diagnosis of visceral leishmaniasis under field conditions in Sudan: comparison of aqueous and freeze-dried antigens.

The performance of the direct agglutination test (DAT) was evaluated under field conditions in an endemic area of visceral leishmaniasis in eastern Sudan, using aqueous (Aq) antigen which has to be kept refrigerated and a newly developed freeze-dried (FD) antigen which is stable at ambient temperature. Both antigens compared well, with 92-98% of readings being identical or only with one dilution difference in titre. FD antigen gave titres that were identical with Aq antigen in 73% of samples, higher in 19%, and lower in 8%. Owing to high ambient temperatures and low humidity, microtitre plate wells dried out during the standard procedures for elution and incubation. However, shortening the elution time from 12 to 4 h proved possible for both antigens; incubation could be reduced from 24 to 10 h for Aq antigen, after which the plates could still be read. Incubation with FD antigen required 18 h and the plates needed to be kept cool because of evaporation. Despite the longer procedure with the FD antigen, the DAT can be completed in 24 h and the use of this stable antigen, that does not require refrigeration, is a major improvement in performing the DAT under unfavourable field conditions.

Kala-azar in displaced people from southern Sudan: epidemiological, clinical and therapeutic findings.

Six hundred and ninety-three patients with kala-azar were seen in Khartoum, Sudan, from January 1989 to February 1990. They were almost exclusively from the Nuer tribe, originating from the western Upper Nile province in southern Sudan, an area not known previously to be endemic for kala-azar. Because of the civil war in southern Sudan no treatment was available locally and massive migration to northern Sudan occurred; many died on the way. All age groups were affected; there was a slight male preponderance (56%). In the clinical presentation, marked generalized lymphadenopathy was prominent (84%). Splenomegaly was absent in 4% of cases. Patients usually showed anaemia, leucopenia and/or thrombocytopenia. 623 patients were treated with sodium stibogluconate, 10 mg/kg for 30 d; relapse occurred in 4% and death in 12%. Latterly, 70 patients were treated with sodium stibogluconate at 2 x 10 mg/kg for 15 d, with relapse in 6% and death in 6%. The difference between the 2 regimens in the number of relapses and deaths was not significant. The outbreak may have been caused by a combination of factors: the introduction of the parasite from an endemic area to a non-immune population, the presence of malnutrition caused by loss of cattle and unavailability of other food sources, and possibly an ecological change in favour of the sandfly vector.

Direct agglutination test for diagnosis and sero-epidemiological survey of kala-azar in the Sudan.

132 patients with suspected kala-azar (visceral leishmaniasis) were included in a prospective study to compare the performance of the direct agglutination test (DAT) with smears of lymph node, bone marrow and splenic aspiration. A titre greater than 1:3200 was considered positive. 67 patients provided positive smears and 65 were smear-negative. Compared with the results obtained from smears, the sensitivity of the DAT was 94% and its specificity 72%. Of 18 patients who were negative by smears but positive by DAT, 3 were treated on strong clinical suspicion of kala-azar and responded well to therapy. One other patient had post-kala-azar dermal leishmaniasis. Of the remaining 14, 8 were tested with the leishmanin skin test; 6 gave a positive result, suggesting past or sub-clinical infection. In 4 smear-positive patients, the DAT was negative; in 2 of these, the test remained negative during 6 months' follow-up. In a sero-epidemiological survey in a camp for displaced people, 30 of 600 sampled individuals gave a positive DAT. Of these, 11 had been treated for kala-azar; 4 others were leishmanin positive. The DAT is a useful screening test (sensitivity 94%; predictive value of a negative test 92%), but it does not differentiate between past kala-azar, sub-clinical infection and active disease.

Sudanese mucosal leishmaniasis: epidemiology, clinical features, diagnosis, immune responses and treatment.

The epidemiology, clinical features, pathology, immune responses, diagnosis and treatment of 14 patients with mucosal leishmaniasis in the Sudan are described. The condition occurred mainly in adult males, particularly in certain closely related tribes from the western Sudan. It affected the mucosa of the upper respiratory tract and/or the oral mucosa and sometimes followed treated kala azar. The parasites were sometimes confined to the mucosa, sometimes spread to the lymph nodes, and rarely infected the bone marrow and spleen. One of the 2 patients with both visceral and mucosal leishmaniasis differed from classical kala azar cases; his infection was longer lasting, he was leishmanin positive, and his peripheral mononuclear cells proliferated in response to leishmanial antigens. Mucosal leishmaniasis following treated kala azar is a similar phenomenon to post-kala azar dermal leishmaniasis and post-kala azar uveitis. Post-kala azar mucosal leishmaniasis can therefore be added to the other post-kala azar leishmanial infections. Using the polymerase chain reaction, Southern blot analysis with specific probes, and isoenzyme characterization, the causative parasite was identified as Leishmania donovani in 4 patients and as L. major in one. Unlike American mucocutaneous leishmaniasis, mucosal leishmaniasis in the Sudan was not preceded or accompanied by cutaneous lesions and the response to pentavalent antimony or ketoconazole was good.

Biochemical and molecular characterization of Leishmania parasites isolated from an endemic focus in eastern Sudan.

Twelve Leishmania isolates from visceral leishmaniasis patients in eastern Sudan were characterized using isoenzyme analysis, Southern blotting and polymerase chain reaction (PCR) 'fingerprinting'. Isoenzyme analysis revealed the presence of 3 zymodemes: MON-18, MON-30 and MON-82, corresponding to Leishmania donovani sensu stricto, L. infantum and L. archibaldi (still of uncertain taxonomic status), respectively. Southern blotting and PCR 'fingerprinting' revealed identical patterns for all 3 zymodemes, which were indistinguishable from those of L. donovani s.s.