Subtotal laparoscopic cholecystectomy influences the rate of conversion in patients with difficult laparoscopic cholecystectomy: Case series.

OBJECTIVES: This study aimed to show that subtotal laparoscopic cholecystectomy (SLC) is a safe procedure that reduces the rate of conversion in patients with difficult laporoscopic cholecystectomies in resource-meagre settings. PATIENTS AND METHODS: Following informed consent, patients with gallstones reporting to Atbara Medical Centre, Atbara, Northern Sudan from February 2012 to July 2013 were managed laparoscopically except those with choledocholithiasis. SLC was done for patients with difficult cholecystectomy and obscured Callot's triangle. Clinical presentation, duration of symptoms, ultrasound findings, frequency of conversion to open operation, frequency of difficult cholecystectomy, operation duration and numbers/types of complications were recorded. Statistical analysis was carried out using SPSS. RESULTS: One hundred and nine patients with a median age of 48 years, F:M ratio of 9 and mean duration of symptoms of 14.8 ± 12.9 months were enrolled. A quarter (29/109, 26.6%) had acute choleycystitis, 13% had difficult laparoscopic cholecystectomy. SLC was done for 16.2%. Retained stones were statistically significant in patients who underwent subtotal laparoscopic cholecystectomy (p = 0.02) with a conversion rate of 5.5%. CONCLUSION: SLC is feasible, safe and can reduce the rate of conversion for patients with difficult laporoscopic cholecystectomy. Sub-total laparoscopic cholecystectomy is not a substitute to conversion and in difficult conditions it is not a failure for the surgeon but a wisdom.

Treatment-based strategy for the management of post-kala-azar dermal leishmaniasis patients in the Sudan.

UNLABELLED: Post-kala-azar dermal leishmaniasis (PKDL) is a dermatosis that affects more than 50% of successfully treated visceral leishmaniasis (VL) patients in Sudan. PKDL is considered an important reservoir for the parasite and its treatment may help in the control of VL. Currently, treatment is mainly with sodium stibogluconate (SSG), an expensive and fairly toxic drug and without universally in treatment protocols used. A literature review, a consensus of a panel of experts, and unpublished data formed the basis for the development of guidelines for the treatment of PKDL in the Sudan. Six treatment modalities were evaluated. Experts were asked to justify their choices based on their experience regarding of drug safety, efficacy, availability, and cost. The consensus was defined by assigning a categorical rank (first line, second line, third line) to each option. Regarding the use of AmBisome the presence of the drug in the skin was confirmed in smears from PKDL lesions. RECOMMENDATIONS: AmBisome at 2.5 mg/kg/day/20 days or SSG at 20 mg/kg/day/40 days plus four/weekly intradermal injection of alum-precipitated autoclave L. major vaccine are suggested as first- and second-treatment options for PKDL in the Sudan, respectively. SSG at 20 mg/Kg/day/60 or more days can be used if other options are not available.

Blindness following visceral leishmaniasis: a neglected post-kala-azar complication.

Visceral leishmaniasis (VL) is an important cause of morbidity and mortality that affects multiple organs. Post-kala-azar ocular involvement is a serious complication that can manifest as blepharo-conjuctivitis or pan-uveitis. Failure of prompt diagnosis and treatment can result in blindness. We report five cases with pan-uveitis that followed the successful treatment of VL and consequent post-kala-azar dermal leishmaniasis were presented. Two patients lost their sight permanently but the rest were successfully treated. A high index of suspicion and prompt treatment are of paramount importance in order to avoid blindness following post-kala-azar ocular uveitis.

Surrogate markers of subtle renal injury in patients with visceral leishmaniasis.

Sudanese visceral leishmaniasis (VL) is a disease of children that is characterized by fever, hepatosplenomegaly, lymphadenopathy, pancytopenia, and renal injury. Microalbuminuria (MA) and urinary retinol binding protein (urRBP) are useful markers for glomerular and tubular dysfunctions, respectively. We report the prevalence of subtle renal injury in 88 parasitologically confirmed VL patients in a cross-sectional and hospital-based study. Blood and urine were collected before treatment for hematological, biochemical profiles in addition to MA and urRBP measurement using competitive solid phase, sandwich enzyme-linked immune sorbent assay (ELISA), and immunoturbidometry. All the patients had normal serum urea and creatinine levels and no detectable urRBP. However, 40% of the patients had MA detected by ELISA, and 42% were reactive with turbidometry. The sensitivity, specificity, positive and negative predictive values for MA turbidometric technique were calculated as 100%; 96%; 95% and 100%, respectively. In conclusion; subtle renal injury in VL is mainly glomerular. Turbidometry for MA measurement is a simple, inexpensive, sensitive, and specific technique with high predictive values.

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.

Immunological stimulation for the treatment of leishmaniasis: a modality worthy of serious consideration.

Instead of relying on drugs to reduce the parasite burden of leishmaniasis, and waiting for the effector immune response to develop in time to control the parasites, immunotherapy in conjunction with chemotherapy can rapidly induce the effector immune response. With a safe and potent drug plus an affordable therapeutic vaccine (immunostimulant), which remains to be developed, a single visit by patients with visceral or cutaneous leishmaniasis might be sufficient to induce a quick and lasting recovery. Drug toxicity and the emergence of resistance could also be dramatically reduced compared with present long-term monotherapy. Immunotherapy could be an effective addition to chemotherapy for leishmaniasis.

Efficacy of killed whole-parasite vaccines in the prevention of leishmaniasis: a meta-analysis.

Despite decades of investigation in countries on three continents, an efficacious vaccine against Leishmania infections has not been developed. Although some indication of protection was observed in some of the controlled trials conducted with "first-generation" whole, inactivated Leishmania parasite vaccines, convincing evidence of protection was lacking. After reviewing all previously published or unpublished randomized, controlled field efficacy clinical trials of prophylactic candidate vaccines, a meta-analysis of qualified trials was conducted to evaluate whether there was some evidence of protection revealed by considering the results of all trials together. The findings indicate that the whole-parasite vaccine candidates tested do not confer significant protection against human 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.

IFNG and IFNGR1 gene polymorphisms and susceptibility to post-kala-azar dermal leishmaniasis in Sudan.

Post-kala-azar dermal leishmanaisis (PKDL) in Sudan is associated with elevated interferon-gamma (IFN-gamma). To study interferon-gamma pathways in PKDL, we genotyped 80 trios from the Masalit ethnic group for polymorphisms at -470 ins/delTT, -270T/C, -56T/C and +95T/C in IFNGR1 and at -179G/A and +874T/A in IFNG. No associations occurred at IFNG. Global association with haplotypes comprising all four markers at IFNGR1 (chi(2)(10df)=21.97, P=0.015) was observed, associated with a significant (chi(2)(1df)=4.54, P=0.033) bias in transmission of the haplotype insTT T T T and less (chi(2)(1df)=5.59, P=0.018) than expected transmission of insTT C C C. When compared with data on malaria associations from Gambia, the results suggest a complex pattern of haplotypic variation at the IFNGR1 promoter locus associated with different infectious disease in African populations that reflect the complex roles of IFN-gamma in parasite killing versus inflammation and pathogenesis.

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.

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

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.

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.

Dichotomy of protective cellular immune responses to human visceral leishmaniasis.

Healing/protective responses in human visceral leishmaniasis (VL) are associated with stimulation/production of Th1 cytokines, such as interferon IFN-gamma, and conversion in the leishmanin skin test (LST). Such responses were studied for 90 days in 44 adult healthy volunteers from VL non-endemic areas, with no past history of VL/cutaneous leishmaniasis (CL) and LST non-reactivity following injection with one of four doses of Alum-precipitated autoclaved Leishmania major (Alum/ALM) +/- bacille Calmette-Guerin (BCG), a VL candidate vaccine. The vaccine was well tolerated with minimal localized side-effects and without an increase in antileishmanial antibodies or interleukin (IL)-5. Five volunteers (5/44; 11.4%) had significant IFN-gamma production by peripheral blood mononuclear cells (PBMCs) in response to Leishmania antigens in their prevaccination samples (P = 0.001) but were LST non-reactive. On day 45, more than half the volunteers (26/44; 59.0%) had significantly high LST indurations (mean 9.2 +/- 2.7 mm) and high IFN-gamma levels (mean 1008 +/- 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-gamma production (mean 873 +/- 290; median 902; P = 0.001), but were non-reactive in LST. An additional five volunteers (5/44; 11.4%) had low IFN-gamma levels (mean 110 +/- 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-gamma levels, but significant LST induration (mean 10 +/- 2.9 mm; median 11). By day 90 the majority of volunteers (27/44; 61.4%) had significant LST induration (mean 10.8 +/- 9.9 mm; P < 0.001), but low levels of L. donovani antigen-induced IFN-gamma (mean 66.0 +/- 62 pg/ml; P > 0.05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-gamma and LST induration, while five volunteers had low levels of IFN-gamma (<100 pg/ml) and no LST reactivity (00 mm). One volunteer was lost to follow-up. In conclusion, it is hypothesized that cellular immune responses to human VL are dichotomatous, and that IFN-gamma production and the LST response are not in a causal relationship. Following vaccination and probably cure of VL infection, the IFN-gamma response declines with time while the LST response persists. LST is a simple test that can be used to assess candidate vaccine efficacy.

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.

Allele frequency and genotype distribution of polymorphisms within disease-related genes is influenced by ethnic population sub-structuring in Sudan.

Four single nucleotide polymorphisms (SNPs) and a variable number of tandem repeats (VNTR) polymorphism located within disease associated/causing genes were typed in four populations of different tribal and ethnic affiliation from the Sudan. The genotype and allele frequencies were compared with those of other groups from published and unpublished data of world populations. The combined Sudanese sample conformed with Hardy-Weinberg equilibrium (HWE) expectation. However, population sub-structuring according to ethnic/linguistic group indicated at least two SNPs in departure from HWE. Differences in allele frequencies and genotype distribution between groups was also noted in three of the four SNPs. The other loci were distributed homogeneously within the populations studied with genotype frequencies in agreement with HWE expectation. These results highlight the importance of inter-population stratification for polymorphic markers, as well as the potential influence of evolutionary history and ethnic variation of loci, in the general distribution of SNPs and other polymorphisms.

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.

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.

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.

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.