E. A. G. Khalil

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.

Diagnosis of tuberculous lymphadenitis by FNAC, microbiological methods and PCR: a comparative study

Despite its usefulness in the diagnosis of tuberculous lymphadenitis, fine needle aspiration cytology (FNAC) faces several limitations, and its sensitivity and specificity are not well established. The diagnostic accuracy and limitations of FNAC were studied in comparison with conventional microbiological methods and polymerase chain reaction (PCR). Sixty patients with lymphadenopathy and a clinical diagnosis of tuberculous lymphadenitis were subjected to FNA. The aspirate was used for cytological examination, Ziehl‐Neelsen staining, mycobacterial culture and PCR. PCR was performed using two sets of oligonucleotide primers for Mycobacterium tuberculosis and a single primer for M. bovis species. The results of FNAC, microbiological methods and PCR correlated with the clinical outcome after follow‐up for an average period of 24 months. Twenty‐five cases (41.6%) were treated and responded well to anti‐tuberculosis therapy, among them 17 were correctly diagnosed by FNAC (68%), eight by microbiological methods (32%) and 24 by PCR (96%). When PCR is considered the gold standard, FNAC predicted the correct diagnosis in 62% of cases with a high false negative rate (38%) due to the absence of granuloma/necrosis in smears from cases of early tuberculosis. In the latter group PCR proved to be the most valuable and a diagnostic success of 100% was achieved when FNAC and PCR were combined. In addition, PCR allowed immediate characterization of M. tuberculosis in the vast majority (96.2%) of cases in the study population.

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.

Genetic susceptibility to visceral leishmaniasis in The Sudan: linkage and association with IL4 and IFNGR1

Longitudinal studies in Sudan show ethnic differences in incidence and clinical phenotypes associated with Leishmania donovani. Immunologically, bias in type 1 vs type 2 cytokine responses is important. To determine whether polymorphisms at IL4/IL9 or IFNGR1 contribute to susceptibility, we examined 59 multicase families of visceral leishmaniasis (VL) with/without post Kala-azar dermal leishmaniasis (PKDL). Multipoint nonparametric analysis (Allegro) linked IL4/IL9 to VL per se (P=0.002). Transmission disequilibrium testing with robust variance estimates confirmed association in the presence of linkage between VL per se and IL4 (P=0.008) but not IL9. Stepwise logistic regression analysis showed both IL4RP2 and IL4RP1 markers contributed significantly to the association, suggesting a common disease-associated haplotype. In contrast, IFNGR1 was linked (P=0.031) and associated (P=0.007) to PKDL but not VL or VL per se. Hence, polymorphism in a type 2 cytokine gene influences underlying susceptibility to VL, whereas IFNGR1 is specifically related to susceptibility to PKDL.

Epidemiology and clinical manifestations of Leishmania donovani infection in two villages in an endemic area in eastern Sudan

We conducted a longitudinal study in an endemic area for visceral leishmaniasis (VL) in eastern Sudan to compare the epidemiology and clinical spectrum of Leishmania donovani infection in two populations differing in ethnic background and duration of residence in the area. The study took place in two villages from April 1994 to April 1996. In Um‐Salala village, which is inhabited by members of the Masaleet tribe, half of the villagers had previous exposure to cutaneous leishmaniasis (Leishmaria major) before moving there. The population of the second village, Mushrau Koka, belong to the Hausa tribe and most were born there. The incidence of VL was 20.4/1000 person‐years in 1994/1995 and increased sharply to 38.3/1000 person‐years in 1995/1996 in Um‐Salala. A rise in the incidence of VL was also observed in Mushrau Koka but with a lower incidence, 3.3/1000 person‐years to 4.6/1000 person‐years. The incidence rate of confirmed VL reflects only a limited part of the total infection rate which includes various forms of subclinical infection. The ratio of clinical to subclinical infection in Um‐Salala was 1.2 : 1 in 1994/1995 compared with 2.6 : 1 in 1995/1996. This ratio was 1 : 11 in 1994/1995 and 1 : 2.5 in 1995/1996 in Mushrau Koka. In both villages the mean age of subclinical cases was higher, but in Mushrau Koka the mean age of subclinical cases also was higher than that of subclinical cases in Um‐Salala. The leishmanin skin test (LST) was positive in 56% of individuals in Um‐Salala and in 33% in Mushrau Koka. VL only occurred in leishmanin‐negative individuals. Post kala‐azar dermal leishmaniasis (PKDL) followed in 58% of confirmed VL patients in Um‐Salala; the low incidence of VL for Mushrau Koka did not permit to estimate a PKDL rate. The clinical manifestations resulting from exposure to L. donovani range from subclinical infection to VL and PKDL. No firm conclusion as to the difference in incidence of VL between the two villages could be reached but differences in exposure to VL and cutaneous leishmaniasis (CL) as well as other factors such as ethnic background and differences in nutritional status may play a role.

Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Guérrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers

In a previous efficacy study, autoclaved Leishmania major (ALM) + bacille Calmette-Guérrin (BCG) vaccine was shown to be safe, but not superior to BCG alone, in protecting against visceral leishmaniasis. From June 1999 to June 2000, we studied the safety and immunogenicity of different doses of alum-precipitated ALM + BCG vaccine mixture administered intradermally to evaluate whether the addition of alum improved the immunogenicity of ALM. Twenty-four healthy adult volunteers were recruited and sequentially allocated to receive either 10 microg, 100 microg, 200 microg, or 400 microg of leishmanial protein in the alum-precipitated ALM + BCG vaccine mixture. Side effects were minimal for all doses and confined to the site of injection. All volunteers in the 10 microg, 100 microg, and 400 microg groups had a leishmanin skin test (LST) reaction of > or = 5 mm by day 42 and this response was maintained when tested after 90 d. Only 1 volunteer out of 5 in the 200 microg group had a LST reaction of > or = 5 mm by day 42 and the reasons for the different LST responses in this group are unclear. This is the first time that an alum adjuvant with ALM has been in used in humans and the vaccine mixture was safe and induced a strong delayed type hypersensitivity (DTH) reaction in the study volunteers. On the basis of this study we suggest that 100 1 microg of leishmanial protein in the vaccine mixture is a suitable dose for future efficacy studies, as it induced the strongest DTH reaction following vaccination.

Treatment of visceral leishmaniasis with sodium stibogluconate in Sudan: management of those who do not respond

Almost all (98%) of 1593 visceral leishmaniasis (VL) patients treated with sodium stibogluconate (Pentostam; Wellcome) in Sudan between 1989 and 1995 and follow-up responded well to treatment. However, the other 33 patients, all of whom were seronegative for HIV, showed partial or no response. The two main causes of unresponsiveness were primary drug resistance (39.3%) and low drug dosages given at peripheral dispensaries (30.3%). All of those who had been sub-optimal doses were cured when adequate doses of the drug were given. A third cause was concurrent disease, particularly pulmonary tuberculosis (18%). With treatment of the concurrent disease, patients responded well to Pentostam. Eight patients who failed to respond to repeated courses of Pentostam did not benefit from pentamidine or sterol inhibitors. Three of these patients responded to liposomal amphotericin B, two responded to splenectomy in association with Pentostam therapy, and three died. Pentostam, given in adequate doses, still appears to be the drug of choice for the treatment of VL in the Sudan Liposomal amphotericin B is a suitable second-line drug.

The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition of Leishmania reactivity by peripheral blood mononuclear cells (PBMC)

PKDL develops in about 50% of Sudanese patients treated for visceral leishmaniasis (kala‐azar). Patients with kala‐azar were entered into this study and followed for a period of up to 2 years. During follow up 12 patients developed PKDL and eight did not. Proliferative responses and cytokine production to Leishmania donovani and control antigens were measured in vitro using PBMC isolated at the time of diagnosis of kala‐azar, after treatment of visceral leishmaniasis, during follow up, and at the time of diagnosis of PKDL. Proliferative responses and interferon‐gamma (IFN‐γ) production were low at diagnosis and increased after treatment of kala‐azar in both patients who developed (group 1) and those who did not develop PKDL later (group 2). In group 1, development of PKDL was always associated by an increased PBMC response to Leishmania antigen in proliferation and IFN‐γ production assays. There were no differences in Leishmania antigen‐induced production of IL‐4, IL‐5 and IL‐10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack on parasites, which have survived in the skin despite the drug treatment. The finding that PKDL develops after treatment of kala‐azar as Leishmania‐reactive T cells start to circulate in peripheral blood in sufficient numbers to be detected in in vitro assays supports this hypothesis.

Immunogenicity and safety of autoclaved Leishmania major plus BCG vaccine in healthy Sudanese volunteers

In a longitudinal study in the epidemiology of Leishmania donovani infection in an endemic focus in eastern Sudan, we observed that previous exposure or infection with Leishmania major appeared to protect against visceral leishmaniasis caused by L. donovani. We therefore conducted a study to test the safety and immunogenicity of a vaccine consisting of autoclaved L. major (ALM) plus BCG in inducing protection in vaccinated individuals. Leishmanin-negative healthy Sudanese volunteers were enrolled in the study and were divided into three groups: group (A) received ALM+BCG, group (B) received BCG alone, and group (C) received the vaccine diluent. The subjects were examined for their clinical and immunological responses before intervention, following intervention and 6-8 weeks after vaccination. Vaccinated subjects (group A) developed localized reactions at the sites of vaccine inoculation that ulcerated and healed within 4-6 weeks; 61.6% of them converted to leishmanin reactive following vaccination. Only one subject in group (C) became leishmanin-positive. A total 76.9% of the vaccinated volunteers in group (A) produced significant levels of interferon-gamma in response to L. major antigen. The vaccine produced significant cellular immune responses that may protect against natural challenge. None of the groups had systemic reactions and all the reactions observed in the vaccinated group were comparable with the BCG-vaccinated group.

Efficacy of liposomal amphotericin B (AmBisome®) in the treatment of persistent post-kala-azar dermal leishmaniasis (PKDL)

Abstract A dermatosis commonly known as post-kala-azar dermal leishmaniasis (PKDL) may develop following the treatment of human visceral leishmaniasis (VL). In about 15% of PKDL cases the disfiguring lesions persist, sometimes for many years. Such persistent lesions currently require daily injections of sodium stibogluconate (SSG) for 2–4 months and even then treatment may not be successful. Alternative, quicker and cheaper treatment options that cause less toxicity are being explored. Immuno–chemotherapeutic regimens (based on leishmaniasis candidate vaccines/BCG with SSG) are still experimental but treatment with liposomal amphotericin B (AmBisome®) has already been found effective, albeit in a small number of patients. AmBisome is considered less nephrotoxic than non-liposomal amphotericin B because it specifically targets the macrophages in which the Leishmania parasites develop. The aim of the present study was to evaluate further the usefulness of AmBisome in the treatment of persistent PKDL, in Sudan. The 12 subjects, all of whom gave their informed consent, had each had PKDL lesions for >6 months and shown no improvement after repeated injections of SSG. During the study period, they were hospitalized and regularly screened, haematologically and biochemically, for adverse effects. The AmBisome, given intravenously at 2.5 mg/kg.day for 20 days, completely cleared the skin rash of 10 (83%) of the patients and caused no detectable adverse effects. In the 10 patients who responded well to the treatment, the papular lesions regressed and became flat while the hypopigmented lesions darkened (continuing to do so even after the last AmBisome injections). Treatment outcome appeared to be unaffected by the age or gender of the patient (P= 0.7 for each) but the time taken for the PKDL lesions to heal was correlated with the age of the lesions (P= 0.009). The macular lesions healed more slowly than the papular (P= 0.02). In conclusion, Ambisome appears suitable for the treatment of persistent PKDL lesions in Sudan. Once certain favourable clinical signs (the regression and/or darkening of the PKDL lesions) have been noted, the lesions will probably continue to clear without the need for more injections.