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.

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.

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.

Extensive polymorphism at the Gp63 locus in field isolates of Leishmania peruviana.

Genetic diversity within and between tandemly arrayed copies of the Gp63 gene occurs in laboratory isolates of Leishmania spp., but the extent to which this represents natural genetic diversity has not been assessed. Here, the Gp63 locus is examined in 58 fresh isolates of L. peruviana, and clones derived from them, collected throughout the Peruvian Andes. Extensive polymorphism is observed, both in size of Gp63 containing chromosomes, and for restriction-fragment-length polymorphisms (RFLPs) at the Gp63 locus. All clones within an isolate are identical, including those with two distinct Gp63-hybridising chromosomal-sized pulsed-field gel electrophoresis (PFGE) bands, consistent with diploidy but with size differences in homologous chromosomes. For RFLP analysis, three enzymes were selected to cut within the coding region (PstI), in the intergenic region (SalI) and outside (EcoRI) the Gp63 gene cluster. PstI gave identical banding patterns across all isolates/clones. For EcoRI and SalI, all clones within an isolate were identical, but isolates were polymorphic for fragments at 13 (2-30 kb) and 8 (2.6-8.8 kb) different molecular mass locations generating 19 and 16 distinct RFLP patterns or genotypes for each enzyme, respectively. EcoRI restriction patterns, analysed by PFGE, were consistent with the presence of two clusters of Gp63 genes on each homologous chromosome, one contained within EcoRI fragments large enough to carry from 3 to 10 copies of the Gp63 gene, the second on fragments which could carry 1 or 2 copies of the gene. SalI patterns indicated variable restriction sites within clusters, but not within every intergenic region. A hierarchical analysis of variance of allele frequencies, expressed in terms of Wright's F-statistic, indicated significant barriers to gene flow at all levels, valleys within regions (north/south), villages within valleys, and individuals within villages. This extreme polymorphism at the Gp63 locus of L. peruviana demonstrates the great potential for generation of genetic diversity in parasite populations.

A comparative field study of the relative importance of Lutzomyia peruensis and Lutzomyia verrucarum as vectors of cutaneous leishmaniasis in the Peruvian Andes.

A two-year field study of Andean cutaneous leishmaniasis (uta) in the valley of Purisima, Ancash Department, Peru has provided quantitative epidemiologic and entomologic evidence for the predominant role of Lutzomyia peruensis in the transmission of Leishmania peruviana in this endemic area. The monthly incidence in the valley was greatest in the wet season (from December to May), when Lu. peruensis was particularly endophilic. A significant correlation was detected between intradomiciliary (but not extradomiciliary) Lu. peruensis abundance and the monthly incidence of uta in the valley following a one-month time lag. In contrast, no significant correlation was detected between any measure of Lu. verrucarum abundance and the incidence of uta. Lutzomyia peruensis and Lu. verrucarum comprise more than 98% of all the sand fly captures made in this valley. The increase in incidence of uta with altitude, which reached a peak rate between 2,250 and 2,750 meters above sea level, was associated with an increase in the relative abundance of Lu. peruensis as compared with Lu. verrucarum. Seasonal and altitudinal variation was also detected in the peak time of activity for both sand fly species, a phenomenon that could significantly influence the transmission rate: later host-seeking sand flies being more likely to find sleeping, nondefensive, human hosts.

Diagnosis of Leishmania using the polymerase chain reaction: a simplified procedure for field work.

Oligonucleotide primers directed to the minicircle kinetoplast DNA of Leishmania strains supported enzymatic amplification of this DNA by the polymerase chain reaction (PCR). A single product of 70 basepairs was obtained from parasites belonging exclusively to the L. braziliensis complex. Direct sequencing of the amplified product confirmed its minicircle origin. Skin biopsy specimens from human patients were used directly for the PCR. A pulse incubation of such specimens with deoxyribonuclease I prior to the PCR increased the reliability of the assay. Nuclease disruption of the kinetoplast network was expected to make more copies of the minicircle accessible to amplification. Comparative results between the PCR and conventional parasitologic detection procedures indicate that the DNA detection approach presented is by far more sensitive for diagnostic purposes. Innovations in the PCR protocol are presented that adapt the diagnosis of leishmaniasis to settings with minimal equipment and that are distant from central laboratories.

Genetic heterogeneity in Peruvian Leishmania isolates.

Leishmania were isolated from Peruvian patients with uta or espundia; genomic DNA was examined for restriction fragment length differences by Southern blot analysis using DNA probes for beta-tubulin and for the major surface antigen gp63. Using 5 different restriction endonucleases, Peruvian isolates show homogeneity when examined at the beta-tubulin locus. In contrast, the organisms demonstrated heterogeneity both within and between disease groups when examined for restriction site differences within the gp63 locus. The differences observed did not correlate with the 2 disease groups. Comparison of these Peruvian isolates to New World reference strains of the Leishmania braziliensis complex reveals no consistent pattern of identity with either L. b. guyanensis, L. b. panamensis, or L. b. braziliensis.

Efficacy of 28-day and 40-day regimens of sodium stibogluconate (Pentostam) in the treatment of mucosal leishmaniasis.

The efficacy and toxicity of two regimens of antimony, 28 and 40 days of 20 mg of antimony/kg/day, were compared in the treatment of culture-positive mucosal leishmaniasis involving more than one anatomic site. Forty consecutive eligible Peruvians with infiltrative or ulcerative mucosal disease of the lips, nose, palate-uvula-pharynx, or larynx-epiglottis were randomized to receive either 28 days (P28) or 40 days (P40) of sodium stibogluconate (Pentostam). Treatment was prematurely terminated due to thrombocytopenia in three patients and two patients did not complete six months of follow-up. At one month post-treatment, 13% (2 of 16) of the P28 patients and 16% (3 of 19) of the P40 patients no longer had infiltrates or ulcers and were initially considered cured. During a further 11 months of follow-up, infiltrated lesions healed in eight more P28 patients and in 10 more P40 patients. The cure rate after 12 months of follow-up was therefore 63% for both groups (10 of 16 in the P28 group and 12 of 19 in the P40 group). The total of 13 patients who had infiltrates or ulcers at the 9-12-month follow-up were considered failures. All seven patients (three in the P28 group and four in the P40 group) whose lesions were culture-positive for Leishmania at some point in the 12 months after treatment, and who were thereby parasitologic failures, were also clinical failures.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of nonradioactive DNA probes for the characterization of Leishmania isolates from Peru.

We describe conditions for dot blot DNA hybridization studies using biotinylated kDNA probes from Leishmania. The sensitivity and specificity attained with biotinylated or 32P-labeled probes were equivalent. The lower level of detection obtained was 100 parasites that were blotted on nitrocellulose paper and then treated with Proteinase K. Studies were performed with 112 Leishmania isolates from Andean (uta) and sylvatic mucocutaneous (espundia) patients and all were determined to belong to the Leishmania braziliensis complex.

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.

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.

Lutzomyia verrucarum can transmit Leishmania peruviana, the aetiological agent of Andean cutaneous leishmaniasis.

In much of the endemic area for cutaneous leishmaniasis (uta) in the Peruvian Andes, the only 2 anthropophilic sandfly species present are Lutzomyia peruensis and Lu. verrucarum. On the basis of a single confirmed isolation of Leishmania peruviana (the aetiological agent of uta) from a wild Lu. peruensis, and apparent associations between sandfly abundance and the incidence of uta, it is generally believed that Lu. peruensis is the most important vector. In this paper, a potential role for Lu. verrucarum in the transmission of uta is indicated by laboratory experiments which show that this species is vectorially competent for L. peruviana. Individual or pooled colonized sandflies were permitted to take a second blood meal on 22 susceptible golden hamsters at varying intervals after feeding on hamsters previously infected with L. peruviana. Transmission was achieved by a single infected sandfly (of a total of 59) following a 15 d incubation period. Transmission was recognized by the characteristic clinical response (footpad swelling) associated with hamsters which have been inoculated with L. peruviana, and by the presence of parasites in aspirates made from the swollen footpad, detected by the polymerase chain reaction (PCR) and by parasite isolations in biphasic blood-agar culture medium. The identity of the parasite isolates was also confirmed by PCR (specific for parasites in the L. braziliensis complex). This is the first reported experimental transmission of L. peruviana by any sandfly species.

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 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.

The fall and rise of Andean cutaneous leishmaniasis: transient impact of the DDT campaign in Peru.

A retrospective analysis was carried out on census data collected from house-to-house surveys during 1991-1992 in 4 areas endemic for Andean cutaneous leishmaniasis (uta) in the Department of Lima, Peru. Major changes in mean annual incidence in susceptible persons have taken place in these sites during the last 60 years. In particular, there is strong support for the hypothesis that, from the 1950s to the 1970s, the transmission rate was temporarily suppressed, largely as a by-product of the DDT house spraying campaign against malaria. These results are consistent with (i) anecdotal evidence, contemporary with the spraying campaign, and (ii) the official Ministry of Health records for the annual number of uta cases in the Departments of Lima and Ancash.

Putative Leishmania hybrids in the Eastern Andean valley of Huanuco, Peru.

During an outbreak of tegumentary leishmaniasis that developed in the 1990s in the Eastern Andean valley of Huanuco, Peru, the coexistence of Andean (uta) and sylvatic leishmaniases was suspected for ecological and geographical reasons, and sympatric sampling was carried out. Seven human isolates of Leishmania were characterized by multilocus enzyme electrophoresis, random amplification of polymorphic DNA and molecular karyotyping. The three methods identified 3 isolates as L. braziliensis, and 4 isolates as putative hybrids with characters of L. braziliensis and L. peruviana. Data from Huanuco are compared to previous results from other areas endemic for uta. Biological and epidemiological implications are discussed.

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.