Leishmania donovani: genetic diversity of isolates from Sudan characterized by PCR-based RAPD.

Drug unresponsiveness in patients with visceral leishmaniasis (VL) is a problem in many endemic areas. This study aimed to determine genetic diversity of Leishmania donovani isolates from a VL endemic area in Sudan as a possible explanation for drug unresponsiveness in some patients. Thirty clinically stibogluconate (SSG)-sensitive isolates were made SSG-unresponsive in vitro by gradually increasing SSG concentrations. The sensitive isolates and their SSG-unresponsive counterparts were typed using mini-circle kDNA and categorized using PCR-RAPD. All the isolates were typed as L. donovani, the resulting PCR-RAPD characterization of the SSG-sensitive isolates gave three distinct primary genotypes while, the SSG-unresponsive isolates showed only a single band. L. donovani isolates from eastern Sudan are diverse; this probably resulted from emergence of new L. donovani strains during epidemics due to the pressure of widespread use of antimonials. In this communication the possible role of isolates diversity in antimonial unresponsiveness and the in vitro changing PCR-RAPD band pattern in SSG-unresponsive strains were discussed.

Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan.

Eight patients with cutaneous ulcers were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected cutaneous leishmaniasis (CL). Diagnosis was confirmed parasitologically by both positive Giemsa-stained smears and successful culture of Leishmania promastigotes in NNN medium. The eight parasite isolates were shown to belong to the Leishmania donovani complex by kDNA PCR. Isoenzyme typing of three isolates revealed that they were identical to the L. donovani MON-82 reference strain, and the gp63 PCR-RFLP profile showed similar patterns to a reference strain of MON-82. CL is endemic in most regions of Sudan and has been reported previously as being caused by L. major MON-74. The results of this study suggest that L. donovani is also a cause of CL in Sudan and that further study of isolates from Sudanese patients with cutaneous ulcers is warranted to ascertain whether L. donovani or L. major is the causative agent.

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.

Haematogenous dissemination of tuberculous lymphadenitis.

OBJECTIVE: To determine whether Mycobacterium tuberculosis infection spreads through the blood to different lymph-node groups in patients with tuberculous lymphadenitis. DESIGN: Prospective analytical study. SETTING: The patients were recruited, managed and followed at the lymphodenopathy clinic, Central Police Hospital, Burr, Khartoum, Sudan. SUBJECTS: Fifty two sequential patients were enrolled. Thirty patients with FNAC diagnosis of tuberculous lymphadenitis and positive PCR for M. tuberculosis complex had a mean age of 26.9 +/- 11.2 years and similar male, female affection. Nine patients with FNAC tuberculous lymphadenitis, but negative PCR had a slightly higher mean age (32.6 +/- 18.2 years) with similar male: female proportions. Patients with reactive lymphadenopathy (9/52) were older than patients with tuberculous lymphadenitis with a mean age of 45 +/- 24.6 years. RESULTS: None of the patients were positive for HIV or had clinical or radiological evidence of pulmonary tuberculosis. M. tuberculosis DNA was detected in the blood samples of 30/39 (77%) patients with tuberculous lymphadenitis, but in none of the cases with reactive or malignant lymphadenopathy. The presence of M. tuberculosis DNA correlated strongly to multiple lymph-node involvement [OR (odds ratio) = 96.7, 95% confidence interval (CI) 9.0 - 1,039] and to caseating-granulomatous and predominantly necrotic cytomorphological categories [OR = 70, 95% confidence interval (CI) 7.0 - 703]. CONCLUSION: M. tuberculosis most probably disseminates through the blood from one node group to the other in patients with tuberculous lymphadenitis.

The pathogenesis of post kala-azar dermal leishmaniasis from the field to the molecule: does ultraviolet light (UVB) radiation play a role?

Post kala-azar dermal leishmaniasis (PKDL) is a dermatosis caused by persistence of Leishmania donovani parasites in the skin following apparently successful treatment of visceral leishmaniasis. The distribution of PKDL lesions in Sudanese patients often mirrors the clothing habits of those affected. It is most severe in or confined to the sun-exposed parts of the skin. It is well established that elimination of Leishmania parasites requires activation of parasitised macrophages by a Th1 immune response and that the latter is depressed by ultraviolet light (UVB). In this paper, we hypothesized that UVB light might be a key player in the pathogenesis of PKDL. This paper links observations made in the field with immunological data that are compatible with this hypothesis. We therefore investigated patients with PKDL immunologically for a possible role of UVB exposure in the pathogenesis of this condition. We marshal evidence that the changes in the tissues are compatible with the effects of UVB light and it is probable that UVB appears to be a key factor in the pathogenesis of PKDL. Immunopathologically the lesions were characterized by an influx of various inflammatory cells. The number of CD1a (Langerhans' cells) was decreased, they lost their dendrites, their HLA-DR and B7-1 expression was down regulated while B7-2 was expressed. Others have shown that Langerhans' cells with these features result from UVB exposure and that such cells are unable to present antigen to Th1 cells while retaining the capacity to present antigen to Th2 cells. Various cytokines known to be induced by UVB radiation could be demonstrated in PKDL lesions. Of these IL-10, TGF-beta, IL-12, IL-4 and TNF-alpha were found in different quantities. The Th-1 cytokine IFN-gamma was constantly present. The tissue origin of the Th-1 cells in PKDL is unknown. We believe that the antagonistic action of the different cytokines is the cause of the inflammation and chronicity of PKDL.

Rat testis as a radiobiological in vivo model for radionuclides.

The radiobiological effect of intracellularly localised radionuclides emitting low energy electrons (Auger electrons) has received much attention. Most in vivo studies reported have been performed in the mouse testis. We have investigated the rat testis as an in vivo radiobiological model, with sperm-head survival, testis weight loss and also alteration in the blood plasma hormone levels of FSH and LH as radiobiological endpoints. Validation of the rat testis model was evaluated by using mean absorbed doses of up to 10 Gy from intratesticularly (i.t.) injected (111)In oxine or local X-ray irradiation. Biokinetics of the i.t. injected radionuclide was analysed by scintillation camera imaging and used in the absorbed dose estimation. By the analysis of the autoradiographs, the activity distribution was revealed. Cell fractionation showed (111)In to be mainly associated with the cell nuclei. External irradiations were monitored by thermoluminescence dosimeters. The sperm-head survival was the most sensitive radiobiological parameter correlated to the mean absorbed dose, with a D(37) of 2.3 Gy for (111)In oxine and 1.3 Gy for X rays. The levels of plasma pituitary gonadal hormones FSH and LH were elevated for absorbed doses >7.7 Gy. This investigation shows that the radiobiological model based on the rat testis has several advantages compared with the previously commonly used mouse testis model. The model is appropriate for further investigations of basic phenomena such as radiation geometry, intracellular kinetics and heterogeneity, crucial for an understanding of the biological effect of low-energy electrons.

Uncommon clinical presentations of cutaneous leishmaniasis in Sudan.

Cutaneous leishmaniasis in Sudan is caused by Leishmania major zymodeme LON1. Self-healing usually occurs within 1 year but occasionally its duration is prolonged and treatment is required. The clinical forms are ulcers, nodules and noduloulcerative lesions. Here we describe seven patients with uncommon lesions that were difficult to recognize as Leishmania infections. These included mycetoma-like lesions, lesions that resembled L. tropica infection and others. One HIV/AIDS patient had Kaposi's sarcoma with Leishmania parasites in the Kaposi lesions. Most of these uncommon clinical forms were difficult to treat. The diagnosis depended on a high degree of suspicion and the demonstration of parasites in smears or culture. PCR was used to characterize parasites from the patients described here. Leishmania major was found by kDNA PCR in all patients, except one, who had a leishmanioma due to L. donovani. In three patients, including one with a L. tropica like-lesion, the parasites were confirmed as L. major by gp63 PCR-RFLP.

Coexistence of sickle cell nephropathy and lupus nephritis in a Sudanese child.

In spite of the wide distribution of sickle cell disease (SCD) in Africa, an association with systemic lupus erythromatosis (SLE) is seldom reported. This may be due to the poor association between the two diseases or the high prevalence of missed cases. Progressive renal injury is prominent in both SCD and SLE. In this communication, we are presenting a case of an 11-year-old male who presented with sickle cell nephropathy that manifested as nephrotic syndrome with no response to conservative therapy, alongside unexplained massive hemolysis. His renal biopsy proved SLE superimposed on sickle cell nephropathy. We are stressing the importance of considering alternate disease processes in patients with SCD when symptoms change or when there is an atypical clinical course.

Post-kala-azar dermal leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL) is a complication of visceral leishmaniasis (VL); it is characterised by a macular, maculopapular, and nodular rash in a patient who has recovered from VL and who is otherwise well. The rash usually starts around the mouth from where it spreads to other parts of the body depending on severity. It is mainly seen in Sudan and India where it follows treated VL in 50% and 5-10% of cases, respectively. Thus, it is largely restricted to areas where Leishmania donovani is the causative parasite. The interval at which PKDL follows VL is 0-6 months in Sudan and 2-3 years in India. PKDL probably has an important role in interepidemic periods of VL, acting as a reservoir for parasites. There is increasing evidence that the pathogenesis is largely immunologically mediated; high concentrations of interleukin 10 in the peripheral blood of VL patients predict the development of PKDL. During VL, interferon gamma is not produced by peripheral blood mononuclear cells (PBMC). After treatment of VL, PBMC start producing interferon gamma, which coincides with the appearance of PKDL lesions due to interferon-gamma-producing cells causing skin inflammation as a reaction to persisting parasites in the skin. Diagnosis is mainly clinical, but parasites can be seen by microscopy in smears with limited sensitivity. PCR and monoclonal antibodies may detect parasites in more than 80% of cases. Serological tests and the leishmanin skin test are of limited value. Treatment is always needed in Indian PKDL; in Sudan most cases will self cure but severe and chronic cases are treated. Sodium stibogluconate is given at 20 mg/kg for 2 months in Sudan and for 4 months in India. Liposomal amphotericine B seems effective; newer compounds such as miltefosine that can be administered orally or topically are of major potential interest. Although research has brought many new insights in pathogenesis and management of PKDL, several issues in particular in relation to control remain unsolved and deserve urgent attention.

T-cell response in human leishmaniasis.

In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered from cutaneous leishmaniasis (CL) responded by IFN-gamma production following stimulation with Leishmania antigens whereas cells from patients recovered from visceral leishmaniasis (VL) showed a mixed pattern of IFN-gamma and IL-4 responses. The cells producing these cytokines were predominantly CD4+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans.

Liver morphology and function in visceral leishmaniasis (Kala-azar).

AIM: To study the morphology and function of the liver in visceral leishmaniasis (Kala-azar). METHODS: Percutaneous liver biopsy specimens from 18 patients with confirmed visceral leishmaniasis were examined under light and electron microscopy before and after treatment with pentovalent antimony. The tissue was also examined for hepatitis B surface and core antigens using immunoperoxidase staining. Liver function was investigated in nine patients before and after treatment. RESULTS: Specimens before treatment showed Kupffer cells and macrophages colonised by leishmania parasites in 40% of cases. A chronic mononuclear cell infiltrate had affected the portal tracts and lobules. Ballooning degeneration of the hepatocytes, fibrosis of the terminal hepatic venules, and pericellular fibrosis were common findings. The fibrosis was related to Ito cells transforming to fibroblast-like cells. None of the patients had hepatitis B infection. All patients had biochemical evidence of liver dysfunction before treatment. Liver function improved after treatment. CONCLUSION: Visceral leishmaniasis causes morphological and functional disturbance in the liver. Focal fibrosis rather than cirrhosis occurs. The exact aetiology of hepatic damage is unclear but may have an immunological basis.

Endemic kala-azar in eastern Sudan: post-kala-azar dermal leishmaniasis.

In a longitudinal study between 1991 and 1993 in an endemic area in eastern Sudan, 85 cases of kala-azar (visceral leishmaniasis) were diagnosed, of whom 48 (56%) developed post-kala-azar dermal leishmaniasis (PKDL). Another four cases of PKDL had no clinical history of kala-azar. In children, PKDL was more frequent in the very young; seven of nine kala-azar cases (78%) in the group 0-1 years of age and 13 of 16 (81%) in the group 2-3 years of age developed PKDL. On the average, PKDL occurred 56 days (mean; range 0-180) after the end of treatment of kala-azar. To assess the severity of PKDL, a classification was developed using three grades of severity based on differences in density and distribution of lesions. In young children, PKDL was more severe. Incomplete treatment of kala-azar may be important in the pathogenesis of PKDL. Thirty-one patients were followed-up for at least six months; of these, 20 were not treated (17 healed, two improved, and in one, the condition was unchanged), three healed after incomplete treatment with sodium stibogluconate, and eight were cured after treatment but two required two courses. Considerable morbidity was caused by PKDL and should be taken into consideration in the management and follow-up of kala-azar patients. The high incidence of PKDL may have important implications in transmission.

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.

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.

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.

The pathology of cutaneous leishmaniasis due to Leishmania major in Sudan.

The pathology of cutaneous leishmaniasis in Sudan, where the disease is caused by Leishmania major, was studied by light and electron microscopy. Lesions were classified into four distinct groups based on the ratio of different cell types, especially lymphocytes, macrophages, and plasma cells in the inflammatory infiltrate, and the formation of compact epithelioid granulomas or the presence of necrosis. In the lesions, there was a positive correlation between the number of lymphocytes and the number of activated macrophages and epithelioid cells. We suggest that the parasites are eliminated from the lesion by two processes: 1) a lytic mechanism in which parasites are lysed within activated macrophages and 2) necrosis of parasitized macrophages. Morphologic evidence for these two mechanisms of parasite elimination was detected by both light and electron microscopy. The evolution of the pathology of the lesions was followed by rebiopsy when the lesion had regressed in size under antileishmanial therapy.

Oronasal leishmaniasis caused by a parasite with an unusual isoenzyme profile.

A 45-year-old Sudanese man from western Sudan presented with oronasal leishmaniasis of three years duration. He had no history of previous kala-azar or cutaneous leishmaniasis. The parasite isolated from the oral mucosa was characterized by isoenzymes using 12 enzymes and by polymerase chain reaction amplification of kinetoplast DNA using species-specific primers. The specific primers gave products indistinguishable from those of the Leishmania donovani complex. However, the isoenzyme profile showed a zymodeme pattern that was significantly different from the zymodemes previously reported in the Sudan and the Ethiopian region.

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.

Granulomatous mammary disease: ten years' experience with fine needle aspiration cytology.

OBJECTIVE: To determine the aetiological types of granulomatous disease of the breast in women presenting with mammary complaints in the Sudan. METHODS: Clinical history and physical examination, complete blood counts, Mantoux test, histopathology and fine needle aspiration cytology (FNAC). RESULTS: Granulomatous mastitis was seen in 11/2500 (0.44%) patients with mammary disease over a 10-year period. All were of childbearing age (mean 26.0 +/- 5.9 years). Common presentations were diffuse swelling, well-circumscribed masses, nipple retraction, multiple sinuses and superficial skin ulcers. Lymphadenopathy was seen in more than 60% of the patients. Diagnosis was based on cytomorphological features in 10/11 cases and histopathology in one. Nine were diagnosed with tuberculous mastitis and two with idiopathic granulomatous mastitis. Acid-fast bacilli (AFB) could not be demonstrated in any of the cytology smears. Tuberculous mastitis responded to empirical anti-tuberculosis treatment, with a minimum follow-up of 2 years in seven women. CONCLUSION: Tuberculous mastitis is a rare entity in women with mammary disease in the Sudan. Alternative diagnoses such as idiopathic granulomatous mastitis should be made only after failure of an adequate trial of anti-tuberculosis treatment. FNAC is a useful diagnostic tool even if AFB cannot be demonstrated.

Leishmanin and tuberculin sensitivity in leishmaniasis in the Sudan, with special reference to kala-azar.

The application of leishmanin and tuberculin skin tests was studied in patients with leishmaniasis in the Sudan. 35 cases of active kala-azar and 3 relapse cases were leishmanin negative. 81% of patients treated for kala-azar showed a positive reaction after 6 months. 17 of 29 patients with post kala-azar dermal leishmaniasis (PKDL) were leishmanin positive. 2 of 16 patients with kala-azar tested with tuberculin were positive; one was diagnosed as tuberculosis. In 7 initially tuberculin negative patients, the tuberculin test became positive after treatment. A new Leishmania major skin test antigen (Pasteur Institute, Iran) was more reactive than other antigens in patients with cutaneous leishmaniasis, mucocutaneous leishmaniasis and PKDL, but not in treated kala-azar cases. In a field study in an area of endemic kala-azar, the new L. major antigen proved more reactive both in individuals previously exposed to L. major (causing cutaneous leishmaniasis) and in those with past exposure to L. donovani. The literature concerning skin testing with leishmanin and tuberculin in kala-azar is reviewed.