A byproduct from the Valles Calchaquíes vineyards (Argentina) rich in phenolic compounds: a tool against endemic Leishmania dissemination.

Vineyard-derived pomace is a byproduct of the wine industry that can have a negative impact on the environment if it is only disposed of or used as a fertilizer. Owing to its polyphenol content, grape pomace is an alternative to biocontrol undesirable microorganisms. In the present study, we characterized the phenolic composition of red and white grape pomace from Valles Calchaquíes, Argentina, and explored its activity against Leishmania (Leishmania) amazonensis, an etiological agent of American tegumentary leishmaniasis, a neglected endemic disease in northern Argentina. Red and white pomace extracts similarly reduced Leishmania viability after a 48-h treatment, with the fractions containing a higher proportion of phenolic compounds being more active. Both extracts stimulated ATPase activity on the parasite plasma membranes, with white grape pomace having a stronger effect than red grape pomace. In addition, the extracts displayed fairly good anticholinesterase activity, which may have contributed to their anti-Leishmania activity. These results reinforce the potential applicability of grape pomace as an antimicrobial agent for the development of biopesticides.

Efficacy of topical Miltefosine formulations in an experimental model of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in ~ 90 countries, with an increasing incidence. Presently available pharmacotherapy implies the systemic administration of moderately/very toxic drugs. Miltefosine (Milt) is the only FDA-approved drug to treat CL via the oral route (Impavido®). It produces side effects; in particular, teratogenic effects are of concern. A topical treatment would have the great advantage of minimising the systemic circulation of the drug, preventing side effects. We prepared dispersions containing Milt and liposomes of different compositions to enhance/modulate trans-epidermal penetration and evaluated in vitro and in vivo efficacy and toxicity, in vitro release rate of the drug and particles size stability with time. Treatments were topically administered to BALB/c mice infected with Leishmania (Leishmania) amazonensis. The dispersions containing 0.5% Milt eliminated 99% of the parasites and cured the lesions with a complete re-epithelisation, no visible scar and re-growth of hair. Fluid liposomes decreased the time to heal the lesion and the time needed to eliminate viable amastigotes from the lesion site. Relapse of the infection was not found 1 month after treatment in any case. Ultraflexible liposomes on the other hand had no significant in vitro effect but decreased in vivo efficacy. A topical Milt formulation including fluid liposomes seems a promising treatment against CL.

Efficacy of topical risedronate and risedronate - Eudragit E complex in a model of cutaneous leishmaniasis induced by Leishmania (Leishmania) amazonensis.

An efficacious topical treatment for cutaneous leishmaniasis (CL) is highly desirable but still an ongoing challenge. Systemic risedronate (Ris) has been reported to have anti-leishmanial properties and Eudragit EPO (EuE) has shown in vitro activity against L. (L.) amazonensis. The aim of this work was to investigate the in vivo efficacy of topical Ris and EuE-Ris complexes on CL. Surface charge and Ris release kinetics from the different dispersions were analyzed. BALB/c mice were infected intradermally with promastigotes of L. (L.) amazonensis. Ulcers were treated with Ris or EuE-Ris hydrogels. All the lesions that received topical Ris or EuE-Ris showed an improvement with respect to control: reduction of ulcer average size, cicatrization, flattened edges and no signs of necrosis. In addition, a marked parasitic inhibition of 69.5 and 73.7% was observed in the groups treated with Ris and EuE-Ris, respectively, with the IgG2a levels indicating a tendency towards cure. The results are promising and the system should now be enhanced to achieve total parasite elimination.

High performance of an enzyme linked immunosorbent assay for American tegumentary leishmaniasis diagnosis with Leishmania (Viannia) braziliensis amastigotes membrane crude antigens.

The diagnosis of American tegumentary leishmaniasis (ATL) still requires the design of more effective tools. Leishmania (Viannia) braziliensis is the causal agent of the 90% of Argentinean ATL cases. Considering the current knowledge, an ELISA based crude antigen (CA) for the diagnosis was designed. Ninety-nine subjects diagnosed as ATL, 27 as no-ATL, and 84 donors from non-ATL-endemic areas were included in this study. The current ATL diagnosis was based four techniques, dermal smear microscopic examination (parasitological test), PCR, Leishmanin skin test, and clinical records. We obtained CA extracts from promastigotes and amastigotes from macrophage cultures of different zymodemes of endemic Leishmania species circulating in the study area. Crude antigens from the 'local' main zymodeme of L. (V.) braziliensis showed the highest reactivity against anti-Leishmania antibodies compared to the other included species. The CA of amastigotes of this zymodeme was 3.4 fold more reactive than promastigotes one. Moreover, amastigote-membrane CA (MCA) were 3.6 fold more reactive than the soluble antigens. The MCA-ELISA reached a sensitivity and specificity of 98% (CI = 94.7%-100%) and 63.6% (53.9-73.1), respectively. When anti-Trypanosoma cruzi reactive sera were excluded, the specificity reached 98.4% (94.4-100), while the sensitivity was similar, with a positive predictive value (PV) of 98.6% (94.6-100) and negative PV of 96.3% (91.6-100). The performance of the MCA-ELISA results strongly contribute to the final diagnostic decision, since a non-reactive serological result almost discards the suspected ATL, because of its high negative PV. The developed MCA-ELISA showed a high diagnostic performance, which makes it a good candidate for ATL diagnosis, for seroprevalence studies, or for monitoring treatments efficacy.

Efficacy of Topical Treatment with (-)-Epigallocatechin Gallate, A Green Tea Catechin, in Mice with Cutaneous Leishmaniasis.

The treatment of leishmaniasis includes pentavalent antimony drugs but, because of the side effects, toxicity and cases of treatment failure or resistance, the search of new antileishmanial compounds are necessary. The aims of this study were to evaluate and compare the in vitro antileishmanial activity of four green tea catechins, and to assess the efficacy of topical (-)-epigallocatechin gallate in a cutaneous leishmaniasis model. The antileishmanial activity of green tea catechins was evaluated against intracellular amastigotes, and cytotoxicity was performed with human monocytic cell line. BALB/c mice were infected in the ear dermis with Leishmania (Leishmania) amazonensis and treated with topical 15% (-)-epigallocatechin gallate, intraperitoneal Glucantime, and control group. The efficacy of treatments was evaluated by quantifying the parasite burden and by measuring the lesions size. (-)-Epigallocatechin gallate and (-)-epigallocatechin were the most active compounds with IC50 values <59.6 µg/mL and with a selectivity index >1. Topical treatment with (-)-epigallocatechin gallate decreased significantly both lesion size and parasite burden (80.4% inhibition) compared to control group (p < 0.05), and moreover (-)-epigallocatechin gallate showed a similar efficacy to Glucantime (85.1% inhibition), the reference drug for leishmaniasis treatment.

Genetic and clinical characterization of canine leishmaniasis caused by Leishmania (Leishmania) infantum in northeastern Argentina.

Leishmaniases comprise zoonotic diseases caused by protozoan flagellates of the Leishmania genus. They are endemic to South America, and the visceral form has been recently reported in Argentina. Dogs can play different roles in the Leishmania transmission cycles, depending mainly on the species of parasite involved. Here we focused on the clinical characterization of canine leishmaniasis (CanL) in Northeast Argentina and on the molecular typing of its etiological agent. The nested polymerase chain reaction and sequence analysis of the Leishmania cytochrome b (cyt b) gene was performed on DNA templates purified from lymph nodes, bone marrow or spleen aspirates obtained from 48 dogs previously diagnosed by the observation of Leishmania amastigotes on smears from these aspirates. Their clinical and epidemiological data were also recorded. Systemic abnormalities were observed in 46 subjects (95.8%), most frequently lymphadenopathy, and emaciation (89.6 and 75%). Furthermore, 87% also presented tegumentary abnormalities, such as alopecia (54.2%) or secondary skin lesions (47.9%), among others. Twenty three dogs were positive for cyt b amplification. The sequence analysis showed the presence of two genotypes, LiA1 and LiA2, assigned to Leishmania (Leishmania) infantum, with 99.9 and 100% homology with the reference strain MHOM/TN/80/IPT1 respectively. LiA1 was identified in 18 cases (78.3%) and LiA2 in five (21.7%). Two cyt b variants of L. (L.) infantum were incriminated as the causative agents of CanL cases from three cities: Posadas, Garupá, and Ituzaingó. All three cities are located in the northeastern area of the country, where these parasites seem to be spreading in urban areas.

Visceral Leishmaniasis Caused by Leishmania infantum in Salta, Argentina: Possible Reservoirs and Vectors.

Cases of human visceral leishmaniasis (HVL) were not recorded until recently in the Chaco region of northwestern Argentina. Dogs were surveyed at the sites of infection of two HVL index cases in the Chaco region of Salta province. Canine cases (CanL) were diagnosed by two parasitological methods, two molecular methods targeting mini- and maxicircle DNA, and immunochromatographic dipstick. Among 77 dogs studied, 10 (13%) were found infected with Leishmania spp. In seven dogs and two humans, the infecting species was typed as Leishmania (Leishmania) infantum. The same genotype was detected in the human and two of the CanL. Although several diagnostic methods displayed weak or moderate agreement, the concordance values for serology versus maxicircle PCR were very good (Kappa index = 0.84). Sandflies captured in the area were identified as Lutzomyia migonei and Lu. cortelezzii/Lu. sallesi (cortelezzii complex). The focal appearance of leishmaniasis in dogs and humans in a sylvatic region and its relatively low prevalence of infection suggests that L. (L.) infantum transmission to dogs and humans may, in this region, stem from sylvatic reservoirs.

Multilocus sequence typing approach for a broader range of species of Leishmania genus: describing parasite diversity in Argentina.

Leishmaniasis is a vector-borne protozoan infection affecting over 350 million people around the world. In Argentina cutaneous leishmaniasis is endemic in nine provinces and visceral leishmaniasis is spreading from autochthonous transmission foci in seven provinces. However, there is limited information about the diversity of the parasite in this country. Implementation of molecular strategies for parasite typing, particularly multilocus sequence typing (MLST), represents an improved approach for genetic variability and population dynamics analyses. We selected six loci as candidates implemented in reference strains and Argentinean isolates. Phylogenetic analysis showed high correlation with taxonomic classification of the parasite. Autochthonous Leishmania (Viannia) braziliensis showed higher genetic diversity than L. (Leishmania) infantum but low support was obtained for intra-L. braziliensis complex variants suggesting the need of new loci that contribute to phylogenetic resolution for an improved MLST or nested-MLST scheme. This study represents the first characterization of genetic variability of Leishmania spp. in Argentina.

The isolation and molecular characterization of Leishmania spp. from patients with American tegumentary leishmaniasis in northwest Argentina.

American tegumentary leishmaniasis (ATL) is a group of zoonotic diseases caused by kinetoplastid flagellates of the genus Leishmania. A total of 66 patients diagnosed as positive ATL cases from northwest Argentina were included in this study. Leishmania stocks were isolated in vitro and analyzed over promastigote cultures sown on FTA through nested PCR and sequence of cytochrome b (cyt b). The molecular analysis resulted in the incrimination of L. (Viannia) braziliensis as the predominant species in the studied area, identifying two genotypes of L. (V.) braziliensis, 24 cases of Ab-1 cyt b and 41 cases of Ab-2 cyt b. One L. (V.) guyanensis strain was obtained from a traveler from the Brazilian Amazon. The prevalence of different genotypes was in agreement with previous studies, suggesting the necessity for new systems to study the genetic diversity in more detail. Most of the cases typified in this study were registered in the area of Zenta Valley (Orán, Hipólito Yrigoyen, and Pichanal cities), pointing a link between genotype and geographical origin of the sample. Sex and age distribution of the patients indicate that the transmission was predominantly associated with rural areas or rural activities, although the results might not exclude the possibility of peri-urban transmission. This work represents, so far, the largest isolation and molecular characterization of ATL cases in Argentina.

Polymorphism-specific PCR enhances the diagnostic performance of American tegumentary leishmaniasis and allows the rapid identification of Leishmania species from Argentina.

BACKGROUND: The diagnosis of the leishmaniases poses enormous challenges in Argentina. The Polymorphism-Specific PCR (PS-PCR) designed and validated in our laboratories has been proven effective for typifying the Leishmania genus from cultured material. Here we evaluated the performance of this method in the diagnosis of American tegumentary leishmaniasis (ATL) and the rapid identification of Leishmania spp. directly from clinical specimens. METHODS: A total of 63 patients from northwestern Argentina, with cutaneous or mucocutaneous lesions, underwent an ATL diagnosis protocol which included clinical examination, Leishmanin skin test, and microscopic examination of dermal smears. In addition, we performed PS-PCR on DNA directly extracted from the specimens scraped from the lesions. RESULTS: Out of the 63 patients, 44 were classified as ATL cases and 19 as non-ATL cases. The diagnostic sensitivity of the microscopic analysis of dermal smears and PS-PCR individually were 70.5% and 81%, respectively. When performing both tests in parallel, this parameter increased significantly to 97.6% (p = 0.0018). The specificities, on the other hand, were 100%, 84.2%, and 83.3% for the combination, respectively (p > 0.05). Using the PS-PCR analysis we successfully identified the Leishmania spp. in 31 out of the 44 ATL cases. Twenty-eight (90.3%) cases were caused by L. (V.) braziliensis, two (6.5%) by L. (V.) guyanensis, and one (3.2%) by L. (V.) panamensis. CONCLUSIONS: The efficacy of the ATL diagnosis was significantly improved by combining the dermal smear examination with a PS-PCR analysis. Our strategy allowed us to reach the diagnosis of ATL with high accuracy regarding the species of the etiological agent in 70.5% of the cases. Moreover, we diagnosed two cases of the disseminated cutaneous form caused by L. (V.) braziliensis and a cutaneous case due to L. (V.) panamensis infection, both findings reported for the first time in Argentina.

Molecular mass screening to incriminate sand fly vectors of Andean-type cutaneous leishmaniasis in Ecuador and Peru.

Sand flies from the Andean areas of Ecuador and Peru were examined for Leishmania infections by using our recently established molecular mass screening method. Leishmanial minicircle DNA-positive sand flies were detected in 3 of 192 and 1 of 462 samples from Ecuador and Peru, respectively. Sand fly species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 18S ribosomal RNA (rRNA) gene, and the positive flies were Lutzomyia (Lu.) ayacuchensis and Lu. peruensis, respectively. Furthermore, cytochrome b and mannose-phosphate isomerase gene sequence analyses identified the parasites from Ecuador and Peru as Leishmania (Leishmania) mexicana and L. (Viannia) peruviana, respectively. Thus, the mass screening method was confirmed to be a powerful tool for sand fly research.

Natural infection of Lutzomyia tortura with Leishmania (Viannia) naiffi in an Amazonian area of Ecuador.

Natural infection of sand flies with Leishmania parasites was surveyed in an Amazonian area in Ecuador where leishmaniasis is endemic. Seventy-one female sand flies were dissected and one was positive for Leishmania protozoa. The species of this sand fly was identified as Lutzomyia (Lu.) tortura on the basis of morphologic characteristics. Analysis of the cytochrome b gene sequence identified the parasite as L. (Viannia) naiffi. We report the distribution of L. (V.) naiffi in Ecuador and detection of a naturally infected sand fly in the Ecuadorian Amazon and natural infection of Lu. tortura with Leishmania parasites in the New World.

Leishmania isoenzyme polymorphisms in Ecuador: relationships with geographic distribution and clinical presentation.

BACKGROUND: Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL) from Ecuador were analyzed. METHODS: Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. RESULTS: Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia) panamensis (21 isolates, 7 zymodemes), L. (V.) guyanensis (7 isolates, 4 zymodemes), L. (V.) braziliensis (5 isolates, 3 zymodemes), L. (Leishmania) mexicana (11 isolates, 4 zymodemes), L. (L.) amazonensis (10 isolates, 2 zymodemes) and L. (L.) major (2 isolates, 1 zymodeme). L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL); eight cases of leishmaniasis recidiva cutis (LRC) found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. CONCLUSION: Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC) and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador.

Are cytochrome B gene sequencing and polymorphism-specific polymerase chain reaction as reliable as multilocus enzyme electrophoresis for identifying Leishmania spp. from Argentina?

Recently, two techniques, polymerase chain reaction (PCR) amplification and sequencing of cytochrome b gene (cyt b gene sequencing) and polymorphism-specific PCR (PS-PCR) were recommended for Leishmania species identification. Before this study, however, the accuracy of these methods had not been tested against the multilocus enzyme electrophoresis, the current gold standard technique on this task. Therefore, a trial was done for the first time to compare the results obtained by these techniques, using 17 Argentinean Leishmania stocks in independent assays. For all the stocks examined, the same results at species level were obtained by the three techniques. Among them, 14 were assigned to L. (Viannia) braziliensis, and three to L. (V.) guyanensis. The two techniques, cyt b gene sequencing and PS-PCR, were able to distinguish between all the proven species responsible for leishmaniases in Argentina. Thus, both techniques were validated and could be used independently for the species designation of Leishmania parasites in the country.

Multilocus enzyme electrophoresis and cytochrome B gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan.

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

Efficacy of vaccination with a combination of Leishmania amastigote antigens and the lipid A-analogue ONO-4007 for immunoprophylaxis and immunotherapy against Leishmania amazonensis infection in a murine model of New World cutaneous leishmaniasis.

Activation of innate immunity using adjuvants that activate Toll-like receptor 4 pathways have great potential for improving the protection induced by parasite vaccines. We investigated protective and therapeutic effects of a vaccine against leishmaniasis containing a combination of an adjuvant synthetic lipid A-analogue, ONO-4007 and Leishmania amazonensis antigens. ONO-4007 was co-injected with soluble and membrane-enriched L. amazonensis-amastigote antigens into BALB/c mice that had either already been infected with 1 x 10(6) L. amazonensis promastigotes (immunotherapy study) or before challenge with the same infectious dose (immunoprophylaxis study). Sixty percent of mice vaccinated before infectious challenge controlled their Leishmania infections - defined by the absence of footpad-swelling and negative Leishmania cultures - compared to 0% of controls, and 40% of mice vaccinated after infection resolved their infections compared to 0% of controls. Protective immunity in both immunoprophylaxis and immunotherapy models was associated with increased protein production of IL-12 and IFN-gamma. These data suggest that vaccination with a combination of ONO-4007 and amastigote antigens of L. amazonensis may be useful for the prevention and treatment of leishmaniasis, and that the protective immunity induced is associated with the production of type-1 cytokines.

Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina.

Sixteen Leishmania stocks, 15 isolated from patients with cutaneous (CL), mucocutaneous (MCL), or recurrent cutaneous leishmaniasis, plus one from a dog with CL in Salta and Corrientes Provinces, Argentina, were studied by multilocus enzyme electrophoresis. Thirteen of the stocks from humans were grouped in two zymodemes; nine termed as KMS 1, four as KMS 2, and assigned to Leishmania (Viannia) braziliensis. Two additional stocks from CL cases expressed a KMS 4 enzyme profile, corresponding to L. (V.) guyanensis. Although the parasites from the dog were also assigned to L. (V.) braziliensis, its zymodeme, KMS 3, was not expressed in any of the current human isolates. The characterization of Leishmania from a dog was done for the first time in Argentina. The importance of the intraspecific polymorphism in the induction of clinical forms and in the host-reservoir concept is briefly discussed, based on the zymodeme data of isolates from humans and dogs. The presence of L. (V.) guyanensis was confirmed in the country.

Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction.

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.