Ahmed M. Elhassan

Leishmaniasis in Sudan. 3. Visceral leishmaniasis

From the early 1900s, visceral leishmaniasis (VL; kala-azar) has been among the most important health problems in Sudan, particularly in the main endemic area in the eastern and central regions. Several major epidemics have occurred, the most recent--in Western Upper Nile province in southern Sudan, detected in 1988--claiming over 100,000 lives. The disease spread to other areas that were previously not known to be endemic for VL. A major upsurge in the number of cases was noted in the endemic area. These events triggered renewed interest in the disease. Epidemiological and entomological studies confirmed Phlebotomus orientalis as the vector in several parts of the country, typically associated with Acacia seyal and Balanites aegyptiaca vegetation. Infection rates with Leishmania were high, but subject to seasonal variation, as were the numbers of sand flies. Parasites isolated from humans and sand flies belonged to three zymodemes (MON-18, MON-30 and MON-82), which all belong to the L. donovani sensu lato cluster. Transmission dynamics have not been elucidated fully; heavy transmission in relatively scarcely populated areas such as Dinder national park suggested zoonotic transmission whereas the large numbers of patients with post kala-azar dermal leishmaniasis (PKDL) in heavily affected villages may indicate a human reservoir and anthroponotic transmission. Clinical presentation in adults and in children did not differ significantly, except that children were more anaemic. Fever, weight loss, hepato-splenomegaly and lymphadenopathy were the most common findings. PKDL was much more common than expected (56% of patients with VL developed PKDL), but other post-VL manifestations were also found affecting the eyes (uveitis, conjunctivitis, blepharitis), nasal and/or oral mucosa. Evaluation of diagnostic methods showed that parasitological diagnosis should still be the mainstay in diagnosis, with sensitivities for lymph node, bone marrow and spleen aspirates of 58%, 70% and 96%, respectively. Simple, cheap serological tests are needed. The direct agglutination test (DAT) had a sensitivity of 72%, specificity of 94%, positive predictive value of 78% and negative predictive value of 92%. As with other serological tests, the DAT cannot distinguish between active disease, subclinical infection or past infection. The introduction of freeze-dried antigen and control sera greatly improved the practicality and accuracy of the DAT in the field. An enzyme-linked immunosorbent assay using recombinant K39 antigen had higher sensitivity than DAT (93%). The polymerase chain reaction using peripheral blood gave a sensitivity of 70-93% and was more sensitive than microscopy of lymph node or bone marrow aspirates in patients with suspected VL. The leishmanin skin test (LST) was typically negative during active VL and converted to positive in c. 80% of patients 6 months after treatment. Immunological studies showed that both Th1 and Th2 cell responses could be demonstrated in lymph nodes from VL patients as evidenced by the presence of messenger ribonucleic acid for interleukin (IL)-10, interferon gamma and IL-2. Treatment of peripheral blood mononuclear cells from VL patients with IL-12 was found to drive the immune response toward a Th1 type response with the production of interferon gamma, indicating a potential therapeutic role for IL-12. VL responded well to treatment with sodium stibogluconate, which is still the first line drug at a dose of 20 mg/kg intravenously or intramuscularly per day for 15-30 d. Side effects and resistance were rare. Liposomal amphotericin B was effective, with few side effects. Control measures have not been implemented. Based on observations that VL does not occur in individuals who have a positive LST, probably because of previous cutaneous leishmaniasis, a vaccine containing heat-killed L. major promastigotes is currently undergoing a phase III trial.

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.

Diagnosing visceral leishmaniasis with the recombinant K39 strip test: Experience from the Sudan

We compared a strip test employing recombinant K39 (rK39) antigen and protein A/colloidal gold as read‐out agents with the rK39 ELISA for IgM and IgG antibodies and the direct agglutination test (DAT) using 55 sera from patients with parasitologically confirmed visceral leishmaniasis (VL). The rK39 strip test was positive in 37/55 (67%), the DAT in 50/55 (91%) at ≥ 1 : 1600 cut‐off value and in 47/55 (85%) at ≥ 1 : 6400 cut‐off value. The rK39‐ELISA gave positive IgG results for all sera; those who had a positive strip test had significantly higher IgG levels than those with a negative strip test (31.1 (SD=3.6) and 17.7 U/ml (SD=9.8), respectively, P < 0.0001). A total of 31/55 (56%) sera showed a positive IgM result; of these 27 (49%) had a positive strip test. We tested 115 apparently cured VL patients with the strip test during follow‐up; 68 were also tested with DAT. In the strip test, 25–43% of patients had a positive result at time points 3, 6, 9 and 12 months after treatment; for DAT (cut‐off ≥ 1 : 1600) these results were 67–83%. In neither test did a significant decrease in positivity rates occur over time (P=0.37 for the strip test, P=0.17 for the DAT). No correlation (P=0.33) was found between a positive strip test and a positive DAT result (cut‐off ≥ 1 : 1600), indicating that the strip test and DAT are complementary rather than interchangeable. Of 61 endemic controls two (3%) had a positive strip test result; both had a positive leishmanin skin test. The rK39 strip test has the ideal format for use in the field, but its sensitivity is limited; like DAT, but to a lesser extent, it remains positive after treatment.

Leishmaniasis in Sudan. 2. Mucosal leishmaniasis

Sudanese mucosal leishmaniasis is a chronic infection of the upper respiratory tract and/or oral mucosa caused mainly by Leishmania donovani. The disease occurs in areas of the country endemic for visceral leishmaniasis, particularly among Masalit and other closely related tribes in western Sudan. The condition may develop during or after an attack of visceral leishmaniasis, but in most cases it is a primary mucosal disease. Unlike South American mucocutaneous leishmaniasis, mucosal leishmaniasis in Sudan is not preceded or accompanied by a cutaneous lesion. Pathologically, the lesions show a mixture of macrophages, plasma cells and lymphocytes. An epithelioid granuloma may also be found. Parasites are scanty. Diagnosis is established by demonstration of parasites in smears or biopsies, by culture or animal inoculation, or with the aid of the polymerase chain reaction. Most patients give positive results in the direct agglutination test and leishmanin skin test. Patients respond well to treatment with pentavalent antimony compounds.

High levels of plasma IL-10 and expression of IL-10 by keratinocytes during visceral leishmaniasis predict subsequent development of post-kala-azar dermal leishmaniasis

Some patients develop post‐kala‐azar dermal leishmaniasis (PKDL) after they have been treated for the systemic infection kala‐azar (visceral leishmaniasis). It has been an enigma why the parasites cause skin symptoms after the patients have been successfully treated for the systemic disease. We report here that PKDL development can be predicted before treatment of visceral leishmaniasis, and that IL‐10 is involved in the pathogenesis. Before treatment of visceral leishmaniasis, Leishmania parasites were present in skin which appeared normal on all patients. However, IL‐10 was detected in the keratinocytes and/or sweat glands of all patients who later developed PKDL (group 1) and not in any of the patients who did not develop PKDL (group 2). Furthermore, the levels of IL‐10 in plasma as well as in peripheral blood mononuclear cell culture supernatants were higher in group 1 than in group 2.

Geographical Variation in the Response of Visceral Leishmaniasis to Paromomycin in East Africa: A Multicentre, Open-Label, Randomized Trial

Background Visceral leishmaniasis (VL) is a major health problem in developing countries. The untreated disease is fatal, available treatment is expensive and often toxic, and drug resistance is increasing. Improved treatment options are needed. Paromomycin was shown to be an efficacious first-line treatment with low toxicity in India. Methods This was a 3-arm multicentre, open-label, randomized, controlled clinical trial to compare three treatment regimens for VL in East Africa: paromomycin sulphate (PM) at 15 mg/kg/day for 21 days versus sodium stibogluconate (SSG) at 20 mg/kg/day for 30 days; and the combination of both dose regimens for 17 days. The primary efficacy endpoint was cure based on parasite-free tissue aspirates taken 6 months after treatment. Findings Overall, 135 patients per arm were enrolled at five centres in Sudan (2 sites), Kenya (1) and Ethiopia (2), when the PM arm had to be discontinued due to poor efficacy. The trial has continued with the higher dose of PM as well as the combination of PM and SSG arms. These results will be reported later. Baseline patient characteristics were similar among treatment arms. The overall cure with PM was significantly inferior to that with SSG (63.8% versus 92.2%; difference 28.5%, 95%CI 18.8% to 38.8%, p<0.001). The efficacy of PM varied among centres and was significantly lower in Sudan (14.3% and 46.7%) than in Kenya (80.0%) and Ethiopia (75.0% and 96.6%). No major safety issues with PM were identified. Conclusion The efficacy of PM at 15 mg/kg/day for 21 days was inadequate, particularly in Sudan. The efficacy of higher doses and the combination treatment warrant further studies.

1. Cutaneous leishmaniasis

Abstract Cutaneous leishmaniasis (CL) in Sudan is caused by Leishmania major, zymodeme LON-1. The disease is endemic in many parts of the country. The vector is Phlebotomus papatasi and the animal reservoir is probably the Nile rat Arvicanthis niloticus. Clinically, patients usually present with papules, nodules, or noduloulcerative lesions, mainly on the exposed parts of the skin. In 20% of cases the parasite disseminates through the lymphatics, producing sporotrichoid-like lesions. The pathology of the lesion is described. Langerhans cells are the main antigen-presenting cells in CL. They pickup antigen from the dermis and migrate to regional lymph nodes where they present it to T cells. Antigen-specific activated T cells home to the dermis where they stimulate macrophages to eliminate the parasite. Peripheral blood mononuclear cells (PBMC) proliferate in response to Leishmania antigen in vitro and produce cytokines. PBMC of patients with mild and severe disease produce Th1- and Th2- like cytokine patterns, respectively. The criteria for the clinical diagnosis of CL are described. The diagnosis is confirmed by the demonstration of parasites in slit smears in 50–70% of cases and in histological sections in 70%. With primers specific for L. major, the polymerase chain reaction is positive in 86% of cases. Since CL is a self-limiting disease, treatment is confined to patients with severe disease.

Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major: a comparison with direct microscopy of smears and sections from lesions

We have compared the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool against conventional microscopical diagnostic techniques in patients with cutaneous leishmaniasis from the Sudan. Twenty-eight patients were diagnosed according to clinical criteria followed by microscopical examination of histological sections and slit or impression smears. The PCR had a sensitivity of 86% when used alone, and 93% when combined with Southern blotting. In contrast, microscopy of histological sections had a sensitivity of 76% and slit and impression smears of only 55% and 48%, respectively. The PCR should be considered as a valuable and sensitive diagnostic tool in the diagnosis of cutaneous leishmaniasis; it has the added advantage of identification of the species of Leishmania causing the lesion.

Post-kala-azar dermal leishmaniasis in the Sudan: clinical presentation and differential diagnosis.

Post‐kala‐azar dermal leishmaniasis (PKDL) is a common complication following kala‐azar (visceral leishmaniasis). In a prospective study in a village in the endemic area for kala‐azar in the Sudan, 105 of 183 (57%) kala‐azar patients developed PKDL. There was a significantly higher PKDL rate (69%) in those who received inadequate and irregular treatment of kala‐azar than in those who were treated with stibogluconate 20 mg kg−1 daily for 15 days (35%). The group of patients who developed PKDL did not differ from those who did not develop PKDL with regard to age and sex distribution, reduction in spleen size, and conversion in the leishmanin skin test (LST). In a clinical study, 416 PKDL patients were analysed and divided according to grade of severity. Severe PKDL was more frequent in younger age groups (P < 0·001); there was an inverse correlation between grade and conversion in the LST (P < 0·01). In 16% of patients tested, parasites were demonstrated in inguinal lymph node or bone marrow aspirates, indicating still visceral disease (para‐kala‐azar dermal leishmaniasis); there was no correlation between the presence of parasites and grade of severity. Conversion rates in the LST were lower than in those who did not have demonstrable parasites (11% and 37%, respectively; P < 0·01). In the absence of reliable and practical diagnostic tests, PKDL may be diagnosed on clinical grounds and differentiated from other conditions, of which miliaria rubra was the most common. Differentiation from leprosy was most difficult.

Sodium Stibogluconate (SSG) & Paromomycin Combination Compared to SSG for Visceral Leishmaniasis in East Africa: A Randomised Controlled Trial

Background Alternative treatments for visceral leishmaniasis (VL) are required in East Africa. Paromomycin sulphate (PM) has been shown to be efficacious for VL treatment in India. Methods A multi-centre randomized-controlled trial (RCT) to compare efficacy and safety of PM (20 mg/kg/day for 21 days) and PM plus sodium stibogluconate (SSG) combination (PM, 15 mg/kg/day and SSG, 20 mg/kg/day for 17 days) with SSG (20 mg/kg/day for 30 days) for treatment of VL in East Africa. Patients aged 4–60 years with parasitologically confirmed VL were enrolled, excluding patients with contraindications. Primary and secondary efficacy outcomes were parasite clearance at 6-months follow-up and end of treatment, respectively. Safety was assessed mainly using adverse event (AE) data. Findings The PM versus SSG comparison enrolled 205 patients per arm with primary efficacy data available for 198 and 200 patients respectively. The SSG & PM versus SSG comparison enrolled 381 and 386 patients per arm respectively, with primary efficacy data available for 359 patients per arm. In Intention-to-Treat complete-case analyses, the efficacy of PM was significantly lower than SSG (84.3% versus 94.1%, difference = 9.7%, 95% confidence interval, CI: 3.6 to 15.7%, p = 0.002). The efficacy of SSG & PM was comparable to SSG (91.4% versus 93.9%, difference = 2.5%, 95% CI: −1.3 to 6.3%, p = 0.198). End of treatment efficacy results were very similar. There were no apparent differences in the safety profile of the three treatment regimens. Conclusion The 17 day SSG & PM combination treatment had a good safety profile and was similar in efficacy to the standard 30 day SSG treatment, suggesting suitability for VL treatment in East Africa. Clinical Trials Registration www.clinicaltrials.gov NCT00255567