Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony.

INTRODUCTION: Evaluation of Leishmania drug susceptibility depends on in vitro Sb(V) susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of Sb(V)-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as Sb(V)-resistant and -sensitive, in order to identify potential resistance markers. METHODS: The differential expression of 13 genes involved in Sb(V) metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. RESULTS: Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of Sb(V)-resistant compared to the group of Sb(V)-sensitive parasites (P<0.01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. DISCUSSION: Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro Sb(V) resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro Sb(V) susceptibility assays, but interfering with the gene expression patterns.

A battery of 12 microsatellite markers for genetic analysis of the Leishmania (Viannia) guyanensis complex.

We used 12 microsatellite markers developed for Leishmania braziliensis to genotype 28 strains of the main species of the Leishmania guyanensis complex (i.e. L. guyanensis and L. panamensis) collected in Ecuador and Peru. The important heterozygote deficits observed in these populations are similar with the previous data obtained in L. braziliensis and raise again the debate on the reproductive mode of these protozoan parasites. The data showed genetic polymorphism and geographical differentiation giving information on population structure of the L. guyanensis complex. Regarding the two species, this study enhances again the debate on the taxonomic status of the different isolates belonging to L. guyanensis s.l. since the results showed substantial heterogeneity within this species complex. In conclusion, this study increases the number of available microsatellite loci for L. guyanensis species complex and raises fundamental biological questions. It confirms that microsatellite markers constitute good tools for population genetic studies on parasites of this complex.

Effects of point mutations in Plasmodium falciparum dihydrofolate reductase and dihydropterate synthase genes on clinical outcomes and in vitro susceptibility to sulfadoxine and pyrimethamine.

BACKGROUND: Sulfadoxine-pyrimethamine was a common first line drug therapy to treat uncomplicated falciparum malaria, but increasing therapeutic failures associated with the development of significant levels of resistance worldwide has prompted change to alternative treatment regimes in many national malaria control programs. METHODOLOGY AND FINDING: We conducted an in vivo therapeutic efficacy trial of sulfadoxine-pyrimethamine at two locations in the Peruvian Amazon enrolling 99 patients of which, 86 patients completed the protocol specified 28 day follow up. Our objective was to correlate the presence of polymorphisms in P. falciparum dihydrofolate reductase and dihydropteroate synthase to in vitro parasite susceptibility to sulfadoxine and pyrimethamine and to in vivo treatment outcomes. Inhibitory concentration 50 values of isolates increased with numbers of mutations (single [108N], sextuplet [BR/51I/108N/164L and 437G/581G]) and septuplet (BR/51I/108N/164L and 437G/540E/581G) with geometric means of 76 nM (35-166 nM), 582 nM (49-6890- nM) and 4909 (3575-6741 nM) nM for sulfadoxine and 33 nM (22-51 nM), 81 nM (19-345 nM), and 215 nM (176-262 nM) for pyrimethamine. A single mutation present in the isolate obtained at the time of enrollment from either dihydrofolate reductase (164L) or dihydropteroate synthase (540E) predicted treatment failure as well as any other single gene alone or in combination. Patients with the dihydrofolate reductase 164L mutation were 3.6 times as likely to be treatment failures [failures 85.4% (164L) vs 23.7% (I164); relative risk = 3.61; 95% CI: 2.14 - 6.64] while patients with the dihydropteroate synthase 540E were 2.6 times as likely to fail treatment (96.7% (540E) vs 37.5% (K540); relative risk = 2.58; 95% CI: 1.88 - 3.73). Patients with both dihydrofolate reductase 164L and dihydropteroate synthase 540E mutations were 4.1 times as likely to be treatment failures [96.7% vs 23.7%; RR = 4.08; 95% CI: 2.45 - 7.46] compared to patients having both wild forms (I164 and K540). CONCLUSIONS: In this part of the Amazon basin, it may be possible to predict treatment failure with sulfadoxine-pyrimethamine equally well by determination of either of the single mutations dihydrofolate reductase 164L or dihydropteroate synthase 540E. TRIAL REGISTRATION: ClinicalTrials.gov NCT00951106.

Treatment of cryptococcal meningitis in Peruvian AIDS patients using amphotericin B and fluconazole.

OBJECTIVES: To describe the mycologic and clinical outcomes and factors associated with failure in Peruvian patients with AIDS-associated cryptococcal meningitis (CM) treated with amphotericin B deoxycholate (Amph B) followed by fluconazole. METHODS: Patients were treated with intravenous Amph B 0.7 mg/kg/day for 2 or 3 weeks followed by oral fluconazole 400mg/day for 7 or 8 weeks. Clinical and laboratory evaluations including cerebrospinal fluid (CSF) studies were performed at baseline and at weeks 2 and 10. RESULTS: The CSF cultures were negative in 25% and 68% of 47 patients at weeks 2 and 10, respectively. In the univariate analysis, baseline low body mass index (BMI), hyponatremia, low serum albumin, positive blood culture and CSF antigen titers >or=1024 were associated with a positive CSF culture at week 2. Baseline positive urine culture, positive blood culture, any positive extraneural culture and CSF opening pressure at week 2 >or=300 mm H2O were associated with a positive CSF culture at week 10. In the multivariate analysis no association was found. CONCLUSIONS: Therapy with Amph B and fluconazole, combined with aggressive management of elevated intracranial pressure (ICP), results in low CSF sterilization rates at week 2 and acceptable CSF sterilization rates at week 10 when compared with other series.

PERMANENT GENETIC RESOURCES: A set of 12 microsatellite loci for genetic studies of Leishmania braziliensis.

Twelve microsatellite loci of Leishmania braziliensis were examined, nine of which were developed in this work. Fifty-six Leishmania braziliensis were genotyped with these microsatellite loci. The 12 loci studied were polymorphic with the number of alleles ranging from five to 19, with a mean of 9.7 ± 4.1 and the observed heterozygosity averaging 0.425 ± 0.202. The important heterozygote deficits we observed (F(IS)  = 0.41, P value = 0.004) appear incompatible with the heterozygote excess expected in clonal diploids. This last result could revive the clonality/sexuality debate regarding Leishmania. This work validates the potential use of these microsatellites for population genetics analysis.

Differential polyadenylation of ribosomal RNA during post-transcriptional processing in Leishmania.

The protozoan parasite Leishmania belongs to the most ancient eukaryotic lineages and this is reflected in several distinctive biological features, such as eukaryotic polycistronic transcription and RNA trans-splicing. The disclosure of this organism's unusual characteristics leads to a better understanding of the origin and nature of fundamental biological processes in eukaryotes. Here we report another unusual phenomenon as we demonstrate that precursor ribosomal RNA can be extensively polyadenylated during post-transcriptional processingt. Furthermore, we demonstrate that the degree of precursor rRNA polyadenylation is variable in different strains and in the different life-stages of a strain.

Randomized, double-blind clinical trial of topical imiquimod 5% with parenteral meglumine antimoniate in the treatment of cutaneous leishmaniasis in Peru.

BACKGROUND: Current treatments for cutaneous leishmaniasis are limited by their toxicity, high cost, and discomfort and the emergence of drug resistance. New approaches, including combination therapies, are urgently needed. We performed a double-blind, randomized trial of therapy with parenteral antimony plus topical imiquimod, an innate immune-response modulator, versus therapy with antimony alone, in subjects with cutaneous leishmaniasis for whom an initial course of antimony therapy had failed. METHODS: Forty subjects with clinical resistance to antimony were recruited in Lima, Peru, between February 2001 and December 2002. All subjects received meglumine antimoniate (20 mg/kg/day im or iv) and were randomized to receive either topical imiquimod 5% cream (Aldara; 3M Pharmaceuticals) or vehicle control every other day for 20 days. Lesions and adverse events were evaluated during treatment and at 1, 2, 3, 6, and 12 months after the treatment period. RESULTS: The mean number of lesions was 1.2 per person; 71% of the lesions were facial and 76% were ulcerative. There were no major differences between the groups, and all but 2 subjects completed therapy. Mild adverse events were reported by 73% of the subjects, but only erythema occurred more commonly in the imiquimod group (P < or = .02). Lesions resolved more rapidly in the imiquimod group: 50% of the imiquimod group achieved cure at 1 month after the treatment period versus 15% of the vehicle cream group (P < or = .02); 61% of the imiquimod group at 2 months versus 25% of the vehicle cream group (P < or = .03); and 72% of the imiquimod group at 3 months versus 35% of the vehicle cream group (P < or = .02). Residual scarring in the imiquimod group was less prominent than in the vehicle cream group. CONCLUSIONS: Combined antimony plus imiquimod treatment was well tolerated, accelerated healing of lesions, and improved scar quality. This therapy may have particular advantages for subjects with facial lesions.

A randomized, double-blind, parallel-group, dose-response study of micafungin compared with fluconazole for the treatment of esophageal candidiasis in HIV-positive patients.

BACKGROUND: Severely immunocompromised individuals are highly susceptible to Candida infection of the esophagus. This randomized, double-blind study assessed the dose-response relationship of the new echinocandin antifungal, micafungin, compared with that of standard fluconazole treatment. METHODS: A total of 245 patients (age, > or =18 years) with a prior diagnosis of acquired immunodeficiency syndrome/human immunodeficiency virus (HIV) infection and esophageal candidiasis, confirmed by endoscopy and culture, were randomized to receive micafungin (50, 100, or 150 mg per day) or fluconazole (200 mg per day). Both agents were administered once per day by a 1-h intravenous infusion for 14-21 days. The primary efficacy end point was endoscopic cure rate, defined as endoscopy grade of 0 at the end of therapy. RESULTS: The endoscopic cure rate (grade 0) was dose-dependent with 50, 100, and 150 mg of micafungin per day at 68.8%, 77.4%, and 89.8%, respectively. Symptoms improved or resolved rapidly (3-7 days of treatment in the majority of patients). The endoscopic cure rate for 100 and 150 mg of micafungin per day (83.5%) was comparable to that for 200 mg of fluconazole per day (86.7%; 95% confidence interval for the difference in endoscopic cure rate, -14.0% to 7.7%). The overall safety and tolerability was acceptable, with no important differences between micafungin (all doses) and fluconazole. CONCLUSIONS: The dose-response findings demonstrate a greater efficacy with micafungin at 100 and 150 mg per day than at 50 mg per day. This study also indicates that the efficacy of micafungin (at dosages of 100 and 150 mg per day) was comparable to that of fluconazole, suggesting that micafungin represents a valuable new treatment option for esophageal candidiasis in HIV-positive patients.

The influence of climate on the epidemiology of bartonellosis in Ancash, Peru.

The aim of this study was to evaluate the association between bartonellosis and selected climatic factors during the time periods 1983-1988 and 1995-99, which included two events of the El Niño phenomenon (1986-88, 1997-98), and to identify a reliable climate parameter to be used as an alert indicator for bartonellosis outbreaks in Ancash. The study site was Ancash and its province Carhuaz, Peru. Time-series cross-correlation analysis was used to assess the association between bartonellosis and climate parameters. A higher, almost 4-fold, monthly bartonellosis incidence risk in Ancash department was observed during the El Niño events of 1986-88 and 1997-98. At a regional (Ancash department) and local level (Carhuaz, Ancash), the best correlation was observed between bartonellosis and sea-surface temperature (SST). The results indicate that SST would be the best climate parameter to be used as an alert indicator for bartonellosis outbreaks in Ancash.

Environmental factors as determinants of malaria risk. A descriptive study on the northern coast of Peru.

We conducted a series of studies on the northern Pacific coast of Peru to determine environmental risk factors for malaria. We report in this paper the results of both a descriptive study of incidence and a prevalence survey of malaria. Both studies showed that the area was at low risk for malaria. The malaria incidence rate was 40/1000 p.a. during the study period, and the prevalence of infection was 0.9% (95% CI: 0.4-1.7) before and 1.4% (95% CI: 0.8-2.2) after the high incidence period. However, the risk of malaria varied according to season, village and even house within a single village. Incidence rates increased from February (2.6/1000 p.a.) to May (12.9/1000 p.a.) and decreased during the second part of the year. Most of the cases were clustered in four villages that constituted only 21% of the total population of the area. Houses where multiple cases were recorded were often located near a source of water. Our observations suggested that environmental factors, and particularly the presence of water for irrigation around villages and houses, played a major role in determining the risk of malaria. These observations were extended through an entomological study and a case-control study, to be published elsewhere.

Successful treatment of drug-resistant cutaneous leishmaniasis in humans by use of imiquimod, an immunomodulator.

Treatment failures for leishmaniasis with pentavalent antimonials, including meglumine antimonate, are increasingly common in many endemic areas. Imiquimod (Aldara; 3M Pharmaceuticals) is a novel immune response-activating compound, approved by the United States Food and Drug Administration, that is currently used to treat cervical warts and has been shown to activate macrophage killing of Leishmania species. Therefore, an open-label, prospective study was conducted of combined imiquimod plus meglumine antimonate therapy in 12 patients with cutaneous leishmaniasis who had previously not responded to meglumine antimonate therapy. All of the patients responded well to this combination therapy, and 90% were found to be cured at the 6-month follow-up period.

Atovaquone and proguani hydrochloride compared with chloroquine or pyrimethamine/sulfodaxine for treatment of acute Plasmodium falciparum malaria in Peru.

The efficacy and safety of a fixed-dose combination of atovaquone and proguanil hydrochloride (Malarone) were compared with chloroquine or pyrimethamine/sulfadoxine in patients with acute falciparum malaria in northern Peru. Patients were initially randomized to receive 1,000 mg atovaquone and 400 mg proguanil hydrochloride daily for 3 days (n=15) or 1,500 mg chloroquine (base) over a 3 day period (n=14) (phase 1). The cure rate with chloroquine was lower than expected and patients were subsequently randomized to receive a single dose of 75 mg pyrimethamine and 1,500 mg sulfadoxine (n=9) or atovaquone/proguanil as before (n=5) (phase 2). In phase 1, atovaquone/proguanil was significantly more effective than chloroquine (cure rate 100% [14/14] vs. 8% [1/13], P<0.0001). In phase 2, atovaquone/proguanil and pyrimethamine/sulfadoxine were both highly effective (cure rates 100% [5/5] and 100% [7/7]). There were no significant differences between treatment groups in parasite or fever clearance times. Adverse events were typical of malarial symptoms and did not differ significantly between groups. Overall efficacy of atovaquone/proguanil was 100% for treatment of acute falciparum malaria in a region with a high prevalence of chloroquine resistance.

Atovaquone and proguanil hydrochloride compared with choroquine or pyrimethamine/sulfadoxine for treatment of acute Plasmodium falciparum malaria in Peru

The efficacy and safety of a fixed-dose combination of atovaquone and proguanil hydrochloride (Malarone TM) were compared with chloroquine or pyrimethamine/sulfadoxine in patients with acute falciparum malaria in northern Peru. Patients were initially randomized to receive, 1,000 mg atovaquone and 400 mg mg proguanil hydrochloride daily for 3 days (n=15) or 1,500 mg chloroquine (base) over a 3 day period (n=14) (phase 1). The cure rate with chloroquine was lower than expected and patients were subsequently randomized to receive a single dose of 75 mg pyrimethamine and 1,500 mg sulfadoxine (n=9) or atovaquone/proguanil as before (n=5) (phase 2). In phase 1, atovaquone/proguanil was significantly more effective than chloriquine (cure rate 100 percent [14/14] versus 8 percent [1/13], P<0.0001). In phase 2, atovaquone/proguanil and pyrimethamine/sulfadoxine were both highly effective (cure rates 100 percent [5/5] and 100 percent [7/7]). There were no significant differences between treatment groups in parasite or fever clearance times. Adverse events were typical of malarium symptoms and did not differ significantly between groups. Overall efficacy of atovaquone/proguanil was 100 percent for treatment of acute falciparum malaria in a region with a high prevalence of chloroquine resistance.

Environmental risk factors for clinical malaria: a case-control study in the Grau region of Peru.

The role of environmental risk factors in clinical malaria has been studied mainly in Africa and Asia, few investigations have been carried out in Latin America. Field observations in northern coastal Peru, where the prevalence of malaria is high during the agricultural season, suggested that the risk of disease varied according to the characteristics of the house and the house environment. Environmental determinants of the risk of clinical malaria were therefore investigated through a case-control study: 323 clinical cases of malaria, recruited through community-based active case-finding, and 969 age-, sex- and village-matched controls were recruited into the study over a period of 12 months ending June 1997. Residual spraying of houses in the previous 6 months, living more than 100 m from a canal, a level of education equal to primary school or above and working in agriculture conferred significant protection from the risk of developing clinical malaria. The presence of spaces between the wall and roof in the subject's bedroom (eaves) and a house aged > 4 years statistically significantly increased the risk of disease. Based on these results we discuss possible control measures for malaria in this area of the country.

Survey of Bartonella species infecting intradomicillary animals in the Huayllacallán Valley, Ancash, Peru, a region endemic for human bartonellosis.

The natural cycle of Bartonella bacilliformis remains uncertain, and the suspected existence of animal reservoirs for the bacterium has never been convincingly demonstrated. We conducted a survey of Bartonella species infecting intradomicillary animals in a bartonellosis-endemic region of Peru, obtaining blood from 50 animals living in the homes of 11 families whose children had recently had bartonellosis. Bartonella-like bacteria were recovered from four of nine small rodents included in the study, but from none of the 41 domesticated animals. Identification and comparison of these isolates, and two Bartonella-like isolates obtained from Phyllotis mice in a different endemic region of Peru using serologic and genotypic methods indicated that although none were strains of B. bacilliformis, five were probably representatives of three previously unrecognized Bartonella species and one was a likely strain of the pathogenic species B. elizabethae.

The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia.

In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63-RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR-RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni; reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63-RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63-RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63-RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR-RFLP). PCR-RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63-RFLP and PCR-RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.

Short report: detection of Leishmaniavirus in human biopsy samples of leishmaniasis from Peru.

Leishmaniavirus is a double-stranded RNA virus that persistently infects some strains of the protozoan parasite Leishmania. There is considerable interest in the possibility that the presence of this virus alters parasite phenotype and may affect disease pathogenesis. If so, the virus marker could provide a valuable prognostic indicator for human leishmaniasis, particularly in those cases caused by New World parasite strains. The virus has been detected in cultured L. braziliensis, L. b. guyanensis, and L. major. To date there has been no information as to the extent of infection in samples prior to culturing in the laboratory. This study demonstrates, through the reverse transcription-polymerase chain reaction, that Leishmaniavirus exists in human biopsy samples of leishmaniasis prior to manipulation in culture.

Efficacy of sodium stibogluconate alone and in combination with allopurinol for treatment of mucocutaneous leishmaniasis.

A randomized, open, controlled clinical trial was designed to evaluate the efficacy, tolerance, and safety of sodium stibogluconate plus allopurinol and sodium stibogluconate alone as treatment of patients with mucocutaneous leishmaniasis. In phase 1 of the study, all 22 patients with severe disease had improvement of their lesions, but only two had clinical cure (both of these patients received sodium stibogluconate alone). In phase 2, which included 59 patients with moderate disease, the cure rate among sodium stibogluconate recipients was 75% (21 of 28) compared with 63.6% (14 of 22) among the sodium stibogluconate plus allopurinol recipients. The rates of clinical adverse events were similar among both groups. Thrombocytopenia was more frequent in the sodium stibogluconate plus allopurinol recipients, but the difference was not statistically significant. Eight patients (two sodium stibogluconate recipients and six sodium stibogluconate plus allopurinol recipients) withdrew from the study because of severe thrombocytopenia. In this study, the addition of allopurinol to sodium stibogluconate provided no clinical benefit as treatment of mucocutaneous leishmaniasis.

Pharmacokinetic profile of ABELCET (amphotericin B lipid complex injection): combined experience from phase I and phase II studies.

Amphotericin B (AmB) has been the most effective systemic antifungal agent, but its use is limited by the dose-limiting toxicity of the conventional micellar dispersion formulation (Fungizone). New formulations with better and improved safety profiles are being developed and include ABELCET (formerly ABLC), but their dispositions have not been well characterized; hence, the reason for their improved profiles remains unclear. This report details the pharmacokinetics of ABELCET examined in various pharmacokinetic and efficacy studies by using whole-blood measurements of AmB concentration performed by high-pressure liquid chromatography. The data indicated that the disposition of AmB after administration of ABELCET is different from that after administration of Fungizone, with a faster clearance and a larger volume of distribution. It exhibits complex and nonlinear pharmacokinetics with wide interindividual variability, extensive distribution, and low clearance. The pharmacokinetics were unusual. Clearance and volume of distribution were increased with dose, peak and trough concentrations after multiple dosings increased less than proportionately with dose, steady state appeared to have been attained in 2 to 3 days, despite an estimated half-life of up to 5 days, and there was no evidence of significant accumulation in the blood. The data are internally consistent, even though they were gathered under different conditions and circumstances. The pharmacokinetics of ABELCET suggest that lower concentrations in blood due to higher clearance and greater distribution may be responsible for its improved toxicity profile compared to those of conventional formulations.