Journal of Antimicrobial Chemotherapy (2003) 52, 464468 DOI: 10.

1093/jac/dkg356 Advance Access publication 29 July 2003

Amphotericin B lipid complex versus meglumine antimoniate in the treatment of visceral leishmaniasis in patients infected with HIV: a randomized pilot study
Fernando Laguna1*, Sebastin Videla2, Manuel E. Jimnez-Mejas3, Guillem Sirera4, Julin Torre-Cisneros5, Esteban Ribera6, Dolores Prados3, Bonaventura Clotet4, Mariano Sust2, Rogelio Lpez-Vlez7 and Jorge Alvar8 on behalf of the Spanish HIVLeishmania Study Group
de Enfermedades Infecciosas, Hospital Carlos III, Instituto de Salud Carlos III, C/Sinesio Delgado 12, 28029-Madrid; 2Laboratorios Dr Esteve, Barcelona; 3Hospital Virgen del Roco, Sevilla; 4Hospital Germans Trias i Pujol, Badalona; 5Hospital Reina Sofa, Crdoba; 6Hospital Vall dHebron, Barcelona; 7Hospital Ramn y Cajal, Madrid; 8Centro Nacional de Microbiologa, Majadahonda, Spain
Received 12 September 2002; returned 7 March 2003; revised 9 April 2003; accepted 4 June 2003
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Optimal treatment for HIV-related visceral leishmaniasis (VL) has still to be established. A pilot clinical trial was carried out in 57 HIVVL coinfected patients to compare the efficacy and safety of amphotericin B lipid complex (ABLC) versus meglumine antimoniate. The patients were randomized to receive either ABLC 3 mg/kg/day for 5 days (ABLC-5, 18 patients), ABLC 3 mg/kg/day for 10 days (ABLC-10, 20 patients) or meglumine antimoniate 20 mg Sbv /kg/day for 28 days (19 patients). Treatment was considered successful if parasites were not detected in a bone marrow aspirate after treatment. Parasitological cure was attained in 33% (95% CI: 13%59%) of the ABLC-5 group, in 42% (95% CI: 16%62%) of the ABLC-10 group and in 37% (95% CI: 16%62%) of the meglumine antimoniate group (P = 0.94). Eight out of 19 patients administered antimoniate discontinued treatment prematurely following serious adverse events, compared with one in the ABLC groups (P = 0.0006). The efficacy of ABLC is similar to meglumine antimoniate, but the severity of toxicity in the treatment of HIVVL is lower with ABLC. Keywords: clinical trials, Leishmania, anti-leishmanial drugs

Introduction
Visceral leishmaniasis (VL) is a severe disease caused by intracellular protozoa of the genus Leishmania. VL is common in patients with HIV infection living in endemic areas, and it is estimated that 2%9% of patients with AIDS in southern Europe will suffer new or reactivated VL.1 The pentavalent antimony (Sbv) salts, sodium stibogluconate and meglumine antimoniate, have been used for VL treatment both in immunocompetent and immunodepressed patients in most of the world.13 However, as evidence of efficacy from clinical trials in patients with HIV is scarce, first-line treatment of VL in this population remains controversial. In a previous clinical trial, the antileishmanial efficacy of amphotericin B deoxycholate was shown to be similar to meglumine antimoniate in HIV patients.4 However, both treatments are highly toxic, thus limiting their use. New lipid formulations of amphotericin B are less toxic than the conventional version5,6 and have shown good anti-leishmanial effic-

acy in experimental models.7 Some lipid formulations of amphotericin B have also been tested in immunocompetent patients with VL, and low toxicity and high efficacy were usually obtained.811 Also, previous results suggest that liposomal amphotericin B provides initially successful therapy of VL in HIV patients.9,1214 However, no data are available on the efficacy and toxicity of other amphotericin B lipid formulations in HIV patients with VL. The present pilot study was designed to investigate the efficacy and tolerability of two dosing schedules of amphotericin B lipid complex, as compared to a reference treatment, meglumine antimoniate, in patients with HIV during a first episode of VL.

Subjects and methods

Fifteen major Spanish teaching hospitals took part. The study was approved by the Ethics Committee of each centre and authorized by the Spanish

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*Corresponding author. Tel: +34-91-453-25-05; Fax: +34-91-733-66-14; E-mail: flaguna@hotmail.com Members are listed in the Acknowledgements
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Visceral leishmaniasis in HIV

Health Authorities; it was conducted in accordance with Good Clinical Practice Guidelines. Written informed consent was obtained from all patients enrolled in the study. a 5 month follow-up period. Bone marrow aspirates or tissue biopsy samples were taken whenever there was a clinical suspicion of VL relapse. It was diagnosed if parasites were observed in tissue samples after the initial parasitological cure. If no relapse symptoms were observed 5 months after the end of therapy, a bone marrow aspirate was performed to confirm the absence of relapse.

Patient population and exclusion criteria

From August 1997September 1999, HIV patients who had undergone their first episode of VL were enrolled in the study. Male and female patients aged 18 years or older were eligible for inclusion if they had a diagnosis of HIV confirmed by Western blot, and a first episode of parasitologically confirmed VL. VL was diagnosed in patients showing compatible clinical symptoms and a positive Giemsa-stained smear or culture for Leishmania parasites in samples taken from bone marrow, spleen or liver. Exclusion criteria included patients with pancreatitis, prothrombin activity <40%, aminotransferase levels 10 the upper normal limit, myocardiopathy, heart failure, a Qt corrected interval >500 ms, creatinine levels >twice the upper normal limit, allergy to either ABLC or meglumine antimoniate, concomitant treatment with dideoxycytidine or dideoxyinosine and a life expectancy of <6 months. Women of childbearing potential were excluded if they were pregnant, might become pregnant, or were lactating. Active opportunistic infections were not an exclusion criterion.

Parasitological tests
Tissue samples were analysed at the microbiology, haematology or pathology departments of each hospital. In addition, all bone marrow samples were analysed in a central laboratory: the WHO Collaborating Centre of Leishmaniasis, at the Centro Nacional de Microbiologa, Instituto de Salud Carlos III, Majadahonda, Madrid. Demonstration of Leishmania amastigotes in tissue samples was performed using smears of May Grnwald-Giemsa stains examined at 1000 magnification. Bone marrow aspirates were cultured in NovyMcNeal Nicolle medium. Cultures were incubated at 25C and considered negative if no flagellates were observed after 4 weeks. The microbiologists, pathologists or haematologists who evaluated the tissue samples at the initial cure and relapse were blinded to the therapy received by each patient. In order to ascertain whether relapses were true relapses or reinfections, a genotyping test, using polymerase chain reaction plus restriction fragment length polymorphism was performed in all samples obtained before and after treatment, and is described elsewhere.15

Study design and protocol description

A multicentre, open-label, blinded, centrally randomized, parallel trial was carried out to compare meglumine antimoniate with two dosages of amphotericin B lipid complex in the treatment of first episodes of VL in HIV patients. A randomization list was prepared using the SAS program, which stratified patients into two groups, depending on the CD4 cell count at inclusion: above or below 200 cells/mm3. If this information was missing at the time of randomization, it was considered equivalent to the lymphocyte count: above or below 1000 cells/mm3. The randomization process was blinded and centralized. Each patient was treated on an open-label basis with one of the following three treatments: ABLC (Abelcet, The Liposome Company, Princeton, NJ, USA) 3 mg/kg body weight daily intravenously (iv) for 5 days, ABLC 3 mg/kg body weight daily iv for 10 days or meglumine antimoniate (Glucantime, Rhne-Poulenc, Madrid, Spain) 20 mg Sbv/kg body weight daily by parenteral route for 28 days as control. Treatment compliance was assessed by counting the number of vials in the returned boxes and by monitoring the clinical history. Treatment with other potentially anti-leishmanial drugs was forbidden during the study. Symptomatic treatment to prevent infusionrelated toxicity was carried out under the investigator criteria. Baseline evaluation included confirmation of HIV infection, VLrelated symptoms and physical examination. In addition, a CD4+ T lymphocyte count, a complete blood cell count, a coagulation test and a blood-chemistry panel test were carried out. During the treatment period, clinical evaluation was performed daily, and full blood counts and blood-chemistry tests were performed twice a week. These evaluations were also performed 1 and 5 months after therapy. Electrocardiograms were carried out at baseline, weekly, at the end of therapy and 5 months later. Adverse events were assessed each day during the treatment period, and at 1 and 5 months thereafter. The World Health Organisation (WHO) scale for toxicity was used. A toxicity level on the WHO scale of grade 2 or higher was considered to be an adverse event. The primary endpoint of the study was parasitological cure. This was defined as the presence of no Leishmania parasites in the smear or culture of the tissue biopsy sample, taken from the organ used at inclusion (bone marrow, spleen or liver) between 1 and 7 weeks after the completion of therapy. Therapy failure was defined as parasite persistence in any sample after a complete course of therapy, or the availability of no valid parasitological data after the treatment. VL relapses were studied during

Statistical analysis
Since this was a pilot study, no formal calculation was made of the sample size required. An expected sample of 20 patients per group was considered sufficient to provide a reasonable estimate of treatment effect. The proportions of patients with treatment success were compared among treatment groups by means of the Fishers Exact Test, and their 95% CIs estimated using binomial distribution. Safety analysis included all patients who had received at least one dose of drug. Proportions of patients showing adverse events were compared by the Fishers exact test.

Results
Fifty-seven HIV patients with a first episode of VL were included in the study. Of these 57, 18 were randomly assigned to receive ABLC for 5 days, 20 ABLC for 10 days and 19 meglumine antimoniate for 28 days. Baseline demographics and clinical characteristics are summarized in Table 1. No differences were found in demographic data, history of HIV disease, parasitological data for the diagnosis of LV, concurrent HIV medication, clinical symptoms or analytical data. Only 15 patients were on HAART therapy at VL diagnosis. There were no differences in CD4+ cell counts between patients with or without HAART therapy. Tissue samples used for the diagnosis of VL originated from the bone marrow in 54 cases (52 with a positive diagnosis by smears, two by culture and 33 with both techniques) and from the liver in two cases. One patient was incorrectly included with a positive palate biopsy, but Leishmania parasites were not detected in others samples. Patient outcome is shown in Table 2. The intention-to-treat population included 56 patients. Only one patient treated with ABLC-10 was excluded from this analysis for failing to meet a major entry criterion: the patient was randomized without a confirmed VL diagnosis. Thirteen patients were excluded from the on-treatment analysis for the following reasons: (1) Early withdrawal with lack of efficacy data: one patient in each group was lost to follow-up, and eight patients in the antimonial group were excluded prematurely follow-

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Table 1. Baseline characteristics of patients with VL ABLC-5 group (n = 18) Age in yearsa Gender (male/female) CD4 count (cells/mm3)b CD4 count (cells/mm3) <200 Patients on HAART therapy HIV stage A3 B3 C3
S.D. (range). NS, not significant.
aMean bMedian

ABLC-10 group (n = 20) 33.7 5.8 17/3 56 (0731) 16 3 2 5 13

Antimonial group (n = 19) 34.0 5.2 16/3 26 (1600) 17 7 1 2 16

Statistical analysis NS NS NS NS NS NS

34.1 6.0 15/3 41 (5366) 16 5 3 5 10

Table 2. Treatment outcome of patients with VL ABLC-5 group (n = 18) Early drop-out Death Adverse events Lost to follow-up Finished treatment Cured Not cured Protocol violation
aNot

ABLC-10 group (n = 20) 2 1a 1 18 8 (42%) 10

Antimonial group (n = 19) 9 3 5 1 10 7 (37%) 2 1

1 17 6 (33%) 10 1b

bExcluded

incorporated in intention-to-treat analysis because entry criteria not met. from on-treatment analysis.

ing serious adverse events. (2) Major protocol violation: one patient in the antimonial group and one in the ABLC-5 group had their efficacy control performed outside the planned time interval (efficacy control performed the day of treatment discontinuation, efficacy control performed 113 days after treatment discontinuation). The on-treatment population consisted of 43 patients: 16 patients in the ABLC-5 group, 18 in the ABLC-10 group and nine in the meglumine antimoniate group. Intention-to-treat analysis of data showed similar efficacy for all treatments. Six (33%; 95% CI: 13%59%) of 18 patients in the ABLC-5 group, eight (42%; 95% CI: 20%67%) of 19 in the ABLC-10 group and seven (37%; 95% CI: 16%62%) of 19 in the antimonial group were parasitologically cured (P = 0.96). In an on-treatment analysis, six (38%; 95% CI: 15%65%) of 16 patients in the ABLC-5 group, eight (44%; 95% CI: 22%69%) of 18 in the ABLC-10 group and seven (78%; 95% CI: 40%97%) of nine in the antimonial group were cured. The incidence of at least one treatment-related adverse event was similar between the groups (Table 3). Eight out of 19 patients discontinued meglumine antimoniate treatment following serious adverse events: three patients died (one by sudden death, onea patient with chronic hepatitisfrom upper digestive bleeding and another from severe pneumonia), four patients had clinical pancreatitis and two others renal failure. Conversely, no deaths occurred in the ABLC

groups during the treatment period, and only one patient stopped the treatment, as a result of severe septic pneumonia (P = 0.0006). Infusion-related adverse events occurred only with ABLC and were mild. A total of 15 (40%) of 38 patients who received ABLC reported shivers and/or fever and/or vomiting with the infusion. However, the total number of patients showing adverse events not related to treatment infusion was higher in the antimonial treatment group (14 of 28, 50%) than in the ABLC groups (ABLC-5: four of 18, 22%; ABLC-10: seven of 20, 35%). Twenty-two patients initially considered as cured were followedup for 5 months. Only 19 of them had valid relapse data: seven in the antimonial group, four in the ABLC-5 group and eight in the ABLC10 group. The percentage of VL-free patients at 5 months of followup was 50% (95% CI: 7%93%) in the ABLC-5 group, 38% (95% CI: 9%76%) in the ABLC-10 group, and 57% (95% CI: 17%90%) in the antimonial group.

Discussion
This is the first comparative clinical trial testing amphotericin B entrapped in lipids versus a standard treatment in HIV patients with a first episode of VL. The results suggest that amphotericin B lipid complex presents a similar rate of initial cure, no treatment-related

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Table 3. Treatment-related adverse events reported during the clinical trial. Number of patients (%) ABLC-5 group (n = 18) Death Shivers and/or fever and/or vomiting with administration Phlebitis Vomiting Epigastric pain Pancreatitis Hyperamylasaemia Renal failure Itching Hypokalaemia Worsening anaemia Arthralgias ABLC-10 group (n = 20) Antimonial group (n = 19) 1 (5%) 7 (39%) 1 (6%) 1 (6%) 8 (40%) 1 (5%) 2 (10%)

1 (6%) 1 (6%) 1 (5%) 3 (15%)

4 (21%) 1 (5%) 4 (21%) 2 (11) 1 (5%)

1 (5%)

deaths and a lower rate of serious adverse events compared with meglumine antimoniate. The trial was designed before HAART had become a standard HIV treatment in Spain, but the inclusion of patients started at the beginning of the HAART era. This therapy increases the CD4 cell count and decreases the incidence of opportunistic infections in most patients as a result of the partial restoration of immunity. In fact, HAART, as with other opportunistic infections, reduced the incidence of primary VL episodes.16,17 This may have been why recruiting the number of patients scheduled in the protocol was drawn out. In our trial, however, most patients developed VL when they were not being treated with HAART, which could protect them if the induced immunological response is sufficient. The pentavalent antimonial was selected as the reference compound because it was first-line therapy for VL in HIV patients at the beginning of the trial1822 and is the treatment recommended by the WHO.23 In a comparative clinical trial with a similar design and dose schedules, we found an initial cure of 66% (95% CI, 50%79%) using parasitological criteria.4 We believe that the low response rate attained in the present trial, using pentavalent antimonials, is partially due to a high rate of treatment drop-out due to severe adverse events or early death. In fact, as with our previous trial and using strict parasitological criteria, more than 75% of patients who completed treatment attained a parasitological initial cure. Tolerability, incidence and type of adverse events reported for antimonial therapy correlated well with those previously described, frequently being pancreatic damage.4,24 In HIV patients, both slight and serious antimonial-related electrocardiographic alterations have been reported.4,18 In our study, no serious electrocardiographic changes were detected, although one unexpected sudden death was reported after 2 weeks of treatment, possibly due to antimonial toxicity. On the other hand, in the present trial, ABLC treatment was generally well tolerated with few infusion-related adverse events, and only one of 38 patients treated with ABLC showed renal failure. ABLC has been shown to be a successful treatment in immunocompetent Indian patients with VL.8,10 In the present trial, ABLC has been tested in a new setting, in severely immunosuppressed patients with VL caused by Leishmania infantum species, resulting in a low response with the doses used. We cannot explain these results. ABLC has not been tested as a treatment for VL in other countries, and we do

not know the total doses of ABLC required to produce a high-level response in immunocompetent patients from other areas. Results from the different trials have shown that immunocompetent Indian and Kenyan patients with VL require lower doses of liposomal amphotericin B, as compared with European or Brazilian subjects, to attain parasitological cure.25 It has been suggested that these differences could be explained by different susceptibilities of the Leishmania species (Leishmania donovani in India and Kenya, L. infantum in Mediterranean VL, and Leishmania chagasi in Brazil) to amphotericin B, differences in the ages of patients and also in the splenic parasitic burden.25 Moreover, it seems that liposomal amphotericin B could be more efficacious than ABLC in immunocompetent Indian patients,11 although no formal comparative trial has been made between the drugs. If these results are true, it could be assumed that the total dose of ABLC required to attain a high parasitological initial cure in HIVVL coinfected Mediterranean patients must be higher than the lowest dose of liposomal amphotericin B with high efficacy in this population. At present, there is no general consensus about VL treatment in HIV patients in our country. However, pentavalent antimonials remain first-line therapy for new VL cases in most Spanish hospitals, because liposomal amphotericin B is expensive. If the patient develops severe toxicity, intolerance or treatment failure then liposomal amphotericin B is administered. Similarly, liposomal amphotericin B is the preferred drug in VL relapses. In summary, although both treatmentsmeglumine antimoniate for 28 days and amphotericin B lipid complex for 5 or 10 days showed a similar rate of initial cure, ABLC had a lower rate of serious adverse events, and no deaths occurred during the treatment period. Consequently, we believe that ABLC might to be an alternative to meglumine antimoniate therapy for VLHIV coinfection. However, more clinical trials with higher doses and pharmacoeconomic studies are necessary to support our proposal.

Acknowledgements
We are in debted to Csar Molinero, MD, PhD, for the initial study direction and to Maria Domingues for her secretarial work during the study. We also acknowledge the collaboration of the members of the Steering Committee: Juan Gonzlez-Lahoz, MD, Antonio Guerrero,

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MD and Albert Pahissa, MD. We also thank Laboratorios Pen S.A (Barcelona, Spain) for financial support (Clinical Trial ESCLIN 005/96). The study was conducted under the joint authorship of: Dr Pompeyo Viciana, Hospital Virgen del Rocio (Sevilla); Dr Toms Navarro, Hospital German Trias i Pujol (Badalona); Dr Jos M. Kindeln, Hospital Reina Sofa (Crdoba); Dr Carlos Azuaje, Hospital Vall dHebron (Barcelona); Dr Jordi Alts, Hospital Comarcal de lAlt Peneds (Barcelona); Dr Jos R. Arribas, Hospital La Paz (Madrid); Dr Vicente Boix, Hospital General (Alicante); Dr Josep M. Gatell, Hospital Clnic (Barcelona); Dr Miguel de Grgolas, Fundacin Jimnez Daz (Madrid); Dr Manuel Mrquez, Hospital Virgen de la Victoria (Mlaga); Dr Federico Pulido, Hospital 12 de Octubre (Madrid); Dr Rafael Rubio, Hospital 12 de Octubre (Madrid); Dra Ana Salas, Hospital de Son Dureta (Mallorca); Dr Jess Sanz, Hospital de La Princesa (Madrid); Dra Carmen Caavate, Instituto de Salud Carlos III, Majadahonda (Madrid); Dra Carmen Chicharro, Instituto de Salud Carlos III, Majadahonda (Madrid); Dr Israel Cruz, Instituto de Salud Carlos III, Majadahonda (Madrid); Dr Miguel A. Morales, Instituto de Salud Carlos III, Majadahonda (Madrid); Ms Soledad Casals, Laboratorios Dr Esteve (Barcelona); Dra Carmen Garca, Laboratorios Dr Esteve (Barcelona); Mr Artur Sans, Laboratorios Dr Esteve (Barcelona); Mr Alberto Puyada, Laboratorios Dr Esteve (Barcelona); Mr Ignasi Tolr, Laboratorios Dr Esteve (Barcelona).
10. Sundar, S. & Murray, H. W. (1996). Cure of antimony-unresponsive Indian visceral leishmaniasis with amphotericin B lipid complex. Journal of Infectious Diseases 173, 7625. 11. Sundar, S., Agrawal, G., Rai, M. et al. (2001). Treatment of Indian visceral leishmaniasis with single or daily infusions of low dose liposomal amphotericin B: randomised trial. British Medical Journal 323, 41922. 12. Russo, R., Nigro, L. C., Minniti, S. et al. (1996). Visceral leishmaniasis in HIV infected patients: treatment with high dose liposomal amphotericin B (AmBisome). Journal of Infection 32, 1337. 13. Torre-Cisneros, J., Villanueva, J. L. & Kindelan, J. M. (1993). Successful treatment of antimony-resistant visceral leishmaniasis with liposomal amphotericin B in patients infected with human immunodeficiency virus. Clinical Infectious Diseases 17, 6257. 14. Laguna, F., Torre-Cisneros, J., Moreno, V. et al. (1995). Efficacy of intermittent liposomal amphotericin B in the treatment of visceral leishmaniasis in patients infected with human immunodeficiency virus. Clinical Infectious Diseases 21, 7112. 15. Morales, M. A., Cruz, I., Rubio, J. M. et al. (2002). Relapses versus reinfections in patients coinfected with Leishmania infantum and human immunodeficiency virus type 1. Journal of Infectious Diseases 185, 15337. 16. Tumbarello, M., Tacconelli, E., Bertagnolio, S. et al. (2000). Highly active antiretroviral therapy decreases incidence of visceral leishmaniasis in HIV-infected individuals. AIDS 14, 29489. 17. Lpez Vlez, R., Casado, J. L. & Pintado, V. (2001). Decline of a visceral leishmaniasis epidemic in HIV-infected patients after the introduction of highly active antiretroviral therapy (HAART). Clinical Microbiology and Infection 7, 3945. 18. Lpez-Vlez, R., Prez Molina, J. A., Guerrero, A. et al.(1998). Clinicoepidemiologic characteristics, prognostic factors, and survival analysis of patients coinfected with human immunodeficiency virus and Leishmaniasis in an area of Madrid, Spain. American Journal of Tropical Medicine and Hygiene 58, 43643. 19. Medrano, F. J., Hernndez-Quero, J., Jimnez, E. et al. (1992). Visceral leishmaniasis in HIV-infected individuals: a common opportunistic infection in Spain? AIDS 6, 1499503. 20. Montalban, C., Calleja, J. L., Erice, A. et al. (1990). Visceral leishmaniasis in patients infected with human immunodeficiency virus. Journal of Infection 21, 26170. 21. Laguna, F., Adrados, M., Alvar, J. et al. (1997). Visceral leishmaniasis in patients infected with the human immunodeficiency virus. European Journal of Clinical Microbiology and Infectious Diseases 16, 898903. 22. Pintado, V., Martn-Rabadn, P., Rivera, M. L. et al. (2001). Visceral leishmaniasis in human-immunodeficiency virus (HIV)-infected and nonHIV-infected patients. Medicine 80, 5473. 23. Gradoni, L., Bryceson, A. & Desjeux, P. (1995). Treatment of mediterranean visceral leishmaniasis. Bulletin of the World Health Organisation 73, 1917. 24. Aronson, N. E., Wortmann, G. W., Johnson, S. C. et al. (1998). Safety and efficacy of intravenous sodium stibogluconate in the treatment of leishmaniasis: recent U. S. military experience. Clinical Infectious Diseases 27, 145764. 25. Berman, J. D., Badar, R., Thakur, C. P. et al. (1998). Efficacy and safety of liposomal amphotericin B (AmBisome) for visceral leishmaniasis in endemic developing countries. Bulletin of the World Health Organisation 76, 2532.

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