Phase 2 trial of WR6026, an orally administered 8-aminoquinoline, in the treatment of visceral leishmaniasis caused by Leishmania chagasi.

There are no recognized orally administered treatments for any of the leishmaniases. The 8-aminoquinoline WR6026 is an orally administered analog of primaquine that cured 50% of patients with kala-azar in Kenya at a dose of 1 mg/kg/day for 28 days. A further phase 2, open-label, dose-escalating safety and efficacy study was performed for kala-azar in Brazil. Cure rates for Brazilian patients treated for 28 days were as follows: 1 mg/kg/day: 0 of 4 (0%); 1.5 mg/kg/day: 1 of 6 (17%); 2.0 mg/kg/day: 4 of 6 (67%); 2.5 mg/kg/day: 1 of 5 (20%); and 3.25 mg/kg/day: 0 of 1 (0%). Nephrotoxicity that was not anticipated from preclinical animal studies or from phase 1 studies was seen at 2.5 mg/kg/day in 2 patients and in the single patient administered 3.25 mg/kg/day. WR6026 demonstrated the unusual clinical features of lack of increased efficacy against Brazilian kala-azar with increased dosing above 2 mg/kg/day and toxicity that was not present in previous investigations.

Malaria in Brazilian military personnel deployed to Angola.

BACKGROUND: Malaria represents one of the most important infectious disease threats to deployed military forces; most personnel from developed countries are nonimmune personnel and are at high risk of infection and clinical malaria. This is especially true for forces deployed to highly-endemic areas in Africa and Southeast Asia where drug-resistant malaria is common. METHODS: We conducted an outbreak investigation of malaria cases in Angola where a total of 439 nonimmune Brazilian troops were deployed for a 6-month period in 1995-1996. A post-travel medical evaluation was also performed on 338 (77%) of the 439 soldiers upon return to Brazil. Questionnaire, medical record, thick/thin smear, and serum anti-Plasmodium falciparum antibody titer (by IFA) data were obtained. Peak serum mefloquine (M) and methylmefloquine (MM) metabolite levels were measured in a subsample of 66 soldiers (42 cases, 24 nonmalaria controls) who were taking weekly mefloquine prophylaxis (250 mg). RESULTS: Seventy-eight cases of malaria occurred among the 439 personnel initially interviewed in Angola (attack rate = 18%). Four soldiers were hospitalized, and 3 subsequently died of cerebral malaria. Upon return to Brazil, 63 (19%) of 338 soldiers evaluated were documented to have had clinical symptoms and a diagnosis of malaria while in Angola. In addition, 37 (11%) asymptomatically infected individuals were detected upon return (< 1% parasitemia). Elevated, post-travel anti-P. falciparum IFA titers (> or = 1:64) were seen in 101 (35%) of 292 soldiers tested, and was associated with a prior history of malaria in-country (OR = 3.67, 95% CI 1.98-6.82, p <.001). Noncompliance with weekly mefloquine prophylaxis (250 mg) was associated with a malaria diagnosis in Angola (OR = 3.75, 95% CI 0.97-17.41, p =.03) but not with recent P. falciparum infection (by IFA titer). Mean peak levels (and ratios) of serum M and MM were also found to be lower in those who gave a history of malaria while in Angola. CONCLUSIONS: Malaria was a significant cause of morbidity among Brazilian Army military personnel deployed to Angola. Mefloquine prophylaxis appeared to protect soldiers from clinical, but not subclinical, P. falciparum infections. Mefloquine noncompliance and an erratic chemoprophylaxis prevention policy contributed to this large outbreak in nonimmune personnel. This report highlights the pressing need for development of newer, more efficacious and practical, prophylactic drug regimens that will reduce the malaria threat to military forces and travelers.

Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis.

Mucosal leishmaniasis is arguably the most morbid sequelae of cutaneous leishmaniasis. The importance of early diagnosis for effective therapy, coupled with the difficulty of diagnosing the disease parasitologically, prompted this investigation of humoral immune markers of mucosal disease. Promastigote soluble antigens of Leishmania braziliensis, isolated from cutaneous and mucosal lesions, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis; antigens were identified by immunoblotting with parasite-specific IgG antibody-positive sera of patients with mucosal disease (n = 18) and cutaneous disease (n = 23). For antigens of the cutaneous parasite WR 2095, mucosal sera generally reacted intensely to antigens of 75, 66, and 45 kDa and weakly to 48-50-kDa antigens, whereas cutaneous sera generally detected weakly the first 3 antigens and intensely the latter doublet. The data suggest that the transition from the cutaneous antigenic profile to a mucosal antigenic profile could be used to predict mucosal disease in approximately half of mucosal patients. An additional finding was that antibodies present in the sera of patients with mucosal disease labeled a 66-kDa peptide of normal human lip mucosa more intensely than did cutaneous sera. Autoimmune processes stimulated by the reaction of IgG, originally directed against the 66-kDa of L. braziliensis, to the 66-kDa antigen of mucosal tissue may contribute to the clinical presentation of mucosal leishmaniasis.

Inefficacy of allopurinol as monotherapy for Colombian cutaneous leishmaniasis. A randomized, controlled trial.

BACKGROUND: Hundreds of thousands of cases of cutaneous leishmaniasis occur each year worldwide. Available therapies are parenteral, moderately toxic, and costly. OBJECTIVE: To determine the efficacy of and tolerance for oral allopurinol as monotherapy for cutaneous leishmaniasis. DESIGN: Randomized, controlled trial. SETTING: Outpatient clinics in 11 regions of Colombia in which cutaneous leishmaniasis is endemic. PATIENTS: 187 otherwise healthy adults with cutaneous leishmaniasis. Eighty-four percent of patients were infected with or were from regions with Leishmania panamensis; 16% were infected or were from regions with L. braziliensis. INTERVENTION: Patients were randomly assigned to one of three treatment groups. The first group received allopurinol, three 100-mg tablets four times daily (20 mg/kg of body weight per day) for 28 days. The second group received three placebo tablets four times daily for 28 days. The third group received Glucantime, 20 mg of intramuscular antimony/kg per day for 20 days. MEASUREMENT: Complete cure was defined as complete clinical reepithelialization of all lesions at 3 months and no relapse during 12 months of follow-up. RESULTS: Of 182 patients whose data could be analyzed, 157 (86%) were evaluated. In the allopurinol group, 18 of 55 (33% [95% CI, 21% to 47%]) patients were cured; in the placebo group, 17 of 46 patients (37% [CI, 23% to 52%]) were cured (difference, 4% [CI, -14% to 22%]; P = 0.68); and in the Glucantime group, 52 of 56 patients (93% [CI, 83% to 98%]) were cured (P < 0.001 compared with the allopurinol and placebo groups combined). In most cases, therapy was considered to have failed because the lesion did not reepithelialize by 1.5 months after the end of therapy. Three cases of relapse (two in the allopurinol group and one in the placebo group) at the nasal mucosa (mucosal leishmaniasis) had occurred by the end of 12 months of follow-up. CONCLUSIONS: Allopurinol monotherapy has no effect on Colombian cutaneous disease primarily caused by L. panamensis and therefore is unlikely to be effective against cutaneous leishmaniasis in other endemic regions.

Evaluation of a direct immunofluorescent antibody (DIFMA) test using Leishmania genus-specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis.

A direct immunofluorescent antibody (DIFMA) test using a Leishmania genus-specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL) in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

Successful treatment of New World cutaneous leishmaniasis with a combination of topical paromomycin/methylbenzethonium chloride and injectable meglumine antimonate.

Colombian patients with New World cutaneous leishmaniasis were treated with a combination of a topical formulation (15% paromomycin sulfate/5% methylbenzethonium chloride, twice a day) and parenteral meglumine antimonate (20 mg of antimony [Sb]/kg.d]). Cohort 1 received topical therapy for 10 days and Sb for 7 days; 18 (90%) of the 20 patients were cured (follow-up, 12 months). Other clinical data suggested that neither the topical formulation alone nor the 7-day regimen of Sb alone would have cured many patients. In a subsequent cohort, which received topical therapy for 10 days and Sb for 3 days, the cure rate was 42% (eight of 19 patients). In Colombian cohorts (historical controls) treated with Sb alone for 10-15 days, the cure rate was 31%-36%. Side effects in cohort 1 patients consisted of local reactions to the topical formulation: burning and pruritus in 25% of patients and vesicle formation in 15% of patients. This is the first report that a regimen partially composed of topical antimicrobial agents can be highly effective for treatment of New World cutaneous leishmaniasis.

Limited efficacy of injectable aminosidine as single-agent therapy for Colombian cutaneous leishmaniasis.

Ninety military patients with cutaneous leishmaniasis in Colombia were randomly allocated to 3 treatment regimens of parenteral aminosidine sulphate: (i) 12 mg aminosidine base/kg/d for 7 d, (ii) 12 mg/kg/d for 14 d, and (iii) 18 mg/kg/d for 14 d. With the 89 evaluable patients, the cure rates 12 months after the end of treatment were 10%, 45%, and 50%, respectively. Fifty-eight of the 66 patients who were not cured had lesions that enlarged or were unchanged by 1.5 months after treatment follow up. The other 8 patients had lesions that relapsed between 3 and 12 months after therapy. Even in group (iii) the cure rate was inferior to that (> 90%) with antimony or pentamidine previously reported in this patient population. This study indicates that parenteral aminosidine alone is less likely to be successful in the treatment of cutaneous lesihmaniasis than visceral leishmaniasis, for which a 74% cure rate has been reported. Further trials might consider the combination of aminosidine with other antileishmanial drugs.

Successful treatment of Colombian cutaneous leishmaniasis with four injections of pentamidine.

We previously found that 2 mg of pentamidine isethionate/kg, administered every other day in seven injections, was 95% curative for Colombian cutaneous leishmaniasis. However, 17% of the patients had to prematurely terminate therapy due to drug toxicity and another 30% had mild-to-moderate toxic clinical reactions. In this report, we show that the same daily dose of drug, 2 mg/kg, but administered in only four every-other-day injections, resulted in an 84% cure rate in 38 patients. Twenty-one patients (55%) experienced side effects, three of which were moderate to severe. A higher daily dose of drug, 3 mg/kg, administered in four every-other-day injections, resulted in a 96% cure rate in 51 evaluable patients. Thirty-six of the treated patients (64%) experienced side effects, five of which were moderate to severe. Although hypotension and hypoglycemia were looked for in all patients, only one patient experienced hypoglycemia and it had normalized by follow-up. We propose that the regimen of 3 mg of pentamidine/kg every other day in four injections is optimal for Colombian cutaneous leishmaniasis and competitive with standard Glucantime therapy, in terms of cure rate, toxicity, length of time the patient has to be under medical supervision, and cost of drug plus medical attention. We suggest that such a short course of injectable agent be studied for the treatment of patients with cutaneous leishmaniasis from other endemic areas.

Pancreatitis induced by pentavalent antimonial agents during treatment of leishmaniasis.

Pentavalent antimony (Sbv), formulated as sodium stibogluconate or meglumine antimoniate, is the standard treatment for the leishmaniases. In 16 of 17 consecutive, prospectively observed patients in Washington D.C., serum levels of amylase and lipase rose to abnormal values after therapy with sodium stibogluconate was started; 12 of 17 had symptoms of pancreatitis. Sbv therapy was continued to completion in 7 of 17 patients and interrupted in 10 of 17. Pancreatitis improved in every patient after Sbv therapy was stopped. Sbv treatment was resumed after brief interruptions in 6 of 10 patients. All six of these patients had flares of pancreatitis, but each completed therapy. Subsequently, we measured amylase and lipase levels in stored sera from 32 patients treated in Peru with either sodium stibogluconate or meglumine antimoniate for mucosal leishmaniasis. In all 32 Peruvian patients, serum amylase and lipase rose to abnormal levels during Sbv therapy; 11 of 32 had symptoms of pancreatitis. Standard Sbv regimens induce pancreatitis in almost all patients, but continued therapy is often tolerated; pancreatitis subsides when therapy is stopped, and rechallenge may be tolerated after a brief halt in treatment.

Treatment of Brazilian kala-azar with a short course of amphocil (amphotericin B cholesterol dispersion).

Amphotericin B is an effective but toxic antileishmanial agent. Lipid-encapsulated amphotericin B should have a high therapeutic index for visceral leishmaniasis because reticuloendothelial cells, the sole site in which Leishmania is found, will phagocytize and concentrate the complex. Amphotericin B cholesterol dispersion (Amphocil; 2 mg/[kg.d] intravenously) was administered to 10 Brazilians with kala-azar for 10 days (cohort 1) and to 10 Brazilians with kala-azar for 7 days (cohort 2). All patients were successfully treated: 19 of the 20 patients were without visible parasites in the bone marrow; the mean time to afebrility was 4.2 days; spleen size regressed by a mean of 79% 2 months after therapy; and no patient had clinical or laboratory abnormalities by the end of 6-12 months of follow-up. Side effects were fever and chills accompanied by respiratory distress, but not nephrotoxicity, in children < 3 years of age.

Isolation of Leishmania mexicana (Kinetoplastida: Trypanosomatidae) from Lutzomyia anthophora (Diptera: Psychodidae) collected in Texas.

Three of 27 female Lutzomyia anthophora (Addis) collected in Texas from the nest of a southern plains woodrat, Neotoma micropus Baird, during October 1991 were infected with flagellate protozoans. Isolates were grown in Schneider's Drosophila medium supplemented with 20% fetal bovine serum, and isozyme analysis of two of the isolates determined the parasites to be Leishmania mexicana (Biagi). These are the first isolations of Leishmania from field-collected sand flies in North America north of Mexico. Possible reasons for the lack of human cases near the focus are presented.

Evaluation of pentamidine for the treatment of cutaneous leishmaniasis in Colombia.

Ninety-two patients in Colombia with cutaneous leishmaniasis were randomly assigned to groups to be treated with meglumine antimonate (infected control subjects; 10 mg of antimony/kg intramuscularly, twice a day for 20 days), pentamidine (2 mg/kg every other day intramuscularly, for a total of seven injections), or itraconazole (200 mg orally, twice a day for 28 days) or to receive no treatment (negative control subjects). In the group treated with meglumine antimonate, 21 of 23 patients healed by the first follow-up visit, 1.5 months after the end of therapy, and did not relapse (91% cure rate). In the pentamidine-treated group, 23 of 24 patients healed and did not relapse (96%). Four of the 23 pentamidine-treated patients who ultimately were cured had terminated therapy prematurely (after receiving 4-6 injections) because of hypotension (2 patients), hypoglycemia (1 patient), or headache and myalgias (1 patient). In a subsequent group of 19 patients who were administered 2 mg of pentamidine/kg every other day for a total of only four injections, 14 healed without relapse (74% cure rate). The itraconazole-treated group was similar to the no-treatment control group in terms of the number of patients for whom therapy failed (75% and 64%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Placebo controlled treatment of Ecuadorian cutaneous leishmaniasis.

Pentavalent antimony has been considered to be the standard treatment for leishmaniasis, but more recently, the orally administrable agent allopurinol ribonucleoside has been the subject of several clinical trials. In this study, these two agents were evaluated in patients with Ecuadorian cutaneous leishmaniasis. Patients were randomly assigned to the two treatment groups. The mean reduction in lesion size for the 28 patients treated with Pentostam (20 mg Sb/kg/day intramuscularly for 20 days) was 61%, 23%, and 11% after one, two, and three weeks, respectively. There was a wide range in the individual values, and some lesions markedly enlarged in the first week of therapy. An initially healed lesion was defined as one that had greater than 80% re-epithelialized by the 1.5-month post-treatment followup. All Pentostam patients demonstrated this degree of lesion resolution (100% initial healing rate), but one patient showed evidence of relapse at the three month followup resulting in a 96% complete healing rate for the 12 month observation period. Patients in the untreated control group demonstrated a strikingly high rate of healing with 9 of 12 patients having re-epithelialized all lesions after 1.5 months observation (75% initial healing rate). The mean reduction in lesion size for the untreated patients was 56%, 29%, and 25% after one, two, and three weeks, respectively. Twenty-one patients received allopurinol ribonucleoside (1,500 mg QID) plus probenecid (500 mg QID) for 28 days. Lesions in nine of these patients were healed at the time of the 1.5 month followup (41% healing rate).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Leishmania colombiensis sp. n (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals, and phlebotomine sand flies in Colombia and Panama.

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Isolation of Leishmania mexicana from Neotoma micropus collected in Texas.

A female Neotoma micropus infected with Leishmania was collected in Zavala County, Texas, on 15 January 1990. The infection was limited to lesions at the bases of the ears, and the parasite grew readily in Schneider's Drosophila medium supplemented with 20% fetal bovine serum. Isozyme analysis determined the parasite to be Leishmania mexicana.

Structure-activity relationships of analogs of pentamidine against Plasmodium falciparum and Leishmania mexicana amazonensis.

The antiprotozoal compound 1,5-di(4-amidinophenoxy)pentane (pentamidine) and 36 of its analogs were screened for in vitro activity against Leishmania mexicana amazonensis clone 669 C4S (MHOM/BR/73/M2269) and Plasmodium falciparum clones W2 (Indochina III/CDC) and D6 (Sierra Leone I/CDC). Pentamidine and each of the analogs tested exhibited activity in vitro against L. m. amazonensis and P. falciparum. The pentamidine analogs were more effective against the P. falciparum clones than against L. m. amazonensis. P. falciparum was extremely susceptible to these compounds, with 50% inhibitory concentrations as low as 0.03 microM. While none of the analogs exhibited marked improvement in antileishmanial activity compared with pentamidine, 12 of the pentamidine analogs showed activity approximately equal to or greater than that of the parent compound. From the promising activity exhibited by the pentamidine analogs in this in vitro study and their potential for reduced toxicity relative to the parent drug, pentamidine-related compounds hold promise as new agents for the treatment of protozoal infections.

Human cutaneous leishmaniasis in Ecuador: identification of parasites by enzyme electrophoresis.

Twenty-six strains of Leishmania were isolated from cutaneous lesions in humans in 3 different geographical areas of Ecuador. The species were identified by enzyme electrophoresis as Leishmania braziliensis, L. panamensis, L. guyanensis, L. mexicana, and L. amazonensis.

Mucosal leishmaniasis in Brazil.

The clinical diagnosis and laboratory identification of Leishmania braziliensis braziliensis, a parasitic disease affecting the upper aerodigestive tract, is difficult. A retrospective computer-assisted analysis of patient records was done after examination of 58 patients with mucosal leishmaniasis in an endemic area of L. braziliensis braziliensis in Bahia, Brazil during January 1987. Biopsies of clinically active and clinically inactive mucosal patients were examined for parasites using routine hematoxylin and eosin histopathology and a new technique for rapid detection of Leishmania amastigotes using a genus-specific indirect immunofluorescent assay. No amastigotes were found in specimens from seven patients with clinically inactive mucosal disease using immunofluorescent monoclonal assay techniques, whereas specimens from seven out of 14 patients with clinically active mucosal disease were positive. These results suggest that the immunofluorescent antibody technique is markedly superior in identifying the intracellular amastigote in tissue sections of mucosal biopsies when compared to histopathology techniques or with other standard tests done in rural areas of Brazil. Various clinical and laboratory test data of the entire group of patients were examined and the efficacy of treatment evaluated. The median interval of time noted between cutaneous and mucosal disease was 4.5 years. Relapse was noted in 31% of patients treated with a low dose of meglumine antimoniate (10 mg per kg of body weight). Patients treated with a high dose of meglumine antimoniate (20 mg per kg of body weight) had a relapse rate of 27.3%. A chi-square statistical analysis revealed no significant difference (chi 2 = 0.049) between the two groups. Patients were considered cured if mucosal granulations were clinically absent after 4.6 years.

Leishmania mexicana: chemistry and biochemistry of sodium stibogluconate (Pentostam).

The chemical properties of the primary antileishmanial agent sodium stibogluconate (Pentostam), and the interaction of Pentostam with Leishmania mexicana amastigotes, have been investigated with the aid of [125Sb]Pentostam. The molecular weight by P2 chromatography showed [125Sb]Pentostam to be of multiple species of MW = 100-4000 Da, rather than the one species of 746 Da predicted by the commonly hypothesized structural formula. Nonradioactive Pentostam had a lower osmolarity (789 mOsm for a 100 mg Sb/ml solution) than predicted (1644 mOsm), which indicates that the multiple components of Pentostam (Sb and derivatives of gluconic acid) are more closely complexed with each other than previously thought. When incubated with L. mexicana amastigotes, labeled drug was bound to at least six polypeptides of molecular weights ranging from 14,000 to 68,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Interaction with the polypeptides is presumed to contribute to the antileishmanial action of Pentostam.

Leishmania braziliensis: protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro.

When Leishmania species are grown in vitro, parasites from the stationary phase differ from those in log phase growth in being more infective and more resistant to complement and macrophage mediated killing. In the present study, log phase and stationary phase promastigotes of Leishmania braziliensis panamensis were compared at the molecular level. Differences in polypeptide and glycoprotein composition and antigenicity between log and stationary phase promastigotes of L. b. panamensis were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the former showed that two polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. There were also differences in surface lectin binding characteristics of log and stationary phase promastigotes. Live stationary phase promastigotes bound more concanavalin and lentil lectin than log phase promastigotes, indicating a greater number of mannose residues on their surfaces.