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.

Parasitological and molecular search for Leishmania natural infection in phlebotomine sand flies in peri-urban and rural sites of an Argentinean area endemic for tegumentary leishmaniasis.

Leishmaniases are neglected tropical diseases caused by Leishmania spp. parasites transmitted by the bite of phlebotomine sand flies. In Argentina, the most endemic area of American tegumentary leishmaniasis (ATL) has been Orán department, Province of Salta, where Leishmania (Viannia) braziliensis prevails and Nyssomyia neivai is considered as its vector, although there is no accurate and sufficient information in this regard. The aim of this work was to search for natural infection by Leishmania spp. in sand flies from peri-urban and rural sites with ATL background in Orán department. For this, sand flies were caught at five sites; female sand flies captured with Shannon trap were dissected to microscopically examine their gut contents, while females captured with CDC traps were molecularly analyzed by duplex PCR with two primer pairs to simultaneously amplify kinetoplast DNA (kDNA) and mammalian actin. A total of 1921 females were captured, with Ny. neivai being the most abundant species (89%), followed by Migonemyia migonei (6%) and cortelezzii complex (3%). No natural infection was found in any of them neither by dissection nor by PCR, although the detection limit of kDNA PCR was up to 25 promastigotes. The absence of infected females in peri-urban sites suggest that the transmission did not take place in those environments during the study period. Future searches for natural infection should focus on rural settings to deepen knowledge and elucidate the role of the circulating sand fly species as all have been linked to ATL transmission at other sites.

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.

Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity.

Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by Leishmania, and we applied this method to evaluate the activity in vitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of Leishmania (Leishmania) amazonensis. Parasites were selected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and the number of parasites was determined; then the growth in vitro and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess in vitro the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of L. amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however, in vivo the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC50 values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis.

Sand fly typing: a simple and morphologically-supported method based on polymorphism of 18S rRNA gene in a Leishmaniasis endemic area of Argentina.

Leishmaniases are vector-borne diseases that in the Americas are distributed from southern United States to northern Argentina. The vectors for this disease are small dipterans known as sand flies that are usually identified morphologically by observing structures with taxonomic value; but it is time-consuming, laborious, and requires entomological expertise. Then, this work was aimed at identifying sand flies with molecular techniques, using the morphological identification as a reference technique, in an endemic area of American Tegumentary Leishmaniasis (ATL) located in northern Argentina. For this, sand flies were caught at two patches of vegetation adjacent to rural areas in Orán department, Salta Province. Females were dissected with sterile needles; the head and last abdominal segments were analyzed for morphological identification. The remaining thorax and abdominal segments were used to extract DNA, which was amplified by PCR of the small subunit (SSU), 18S rRNA gene. PCR products were digested with CviQI and DdeI enzymes to identify sand fly species by Restriction Fragment Length Polymorphism (RFLP) analysis. Thus, the restriction pattern of each caught species was defined according to morphological identification. A total of 1501 females, belonging to four sand fly species, were captured. Nyssomyia neivai (1347/1501) was the most abundant species, followed by Migonemyia migonei (90/1501). From the total, 801 females were morphologically and molecularly identified, while 700 females were characterized only molecularly. For those females analyzed by both methods, there was total coincidence in the achieved result. Besides, the 5% (38/801) of females that could not be determined morphologically due to inadequate mounting were molecularly identified. All the females characterized just by PCR-RFLP, were successfully identified. Our results indicate that the explored method is capable of identifying the sand fly species that circulate in an ATL endemic area. Since this method is based on the analysis of markedly different patterns, the identification process might be more easily reproduced, as the bias introduced by the technician's lack of experience is removed.

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.

Development of a Multilocus sequence typing (MLST) scheme for Pan-Leishmania.

Since the description of the Leishmania genus, its identification and organization have been a challenge. A high number of molecular markers have been developed to resolve phylogenetic differences at the species level and for addressing key epidemiological and population genetics questions. Based on Multilocus enzyme electrophoresis (MLEE), Multilocus sequence typing (MLST) schemes have been developed using different gene candidates. From 38 original gene targets proposed by other authors, 27 of them were chosen. In silico selection was made by analyzing free access genomic sequence data of 33 Leishmania species, one Paraleishmania representative, and one outgroup, in order to select the best 15 loci. De novo amplifications and primers redesign of these 15 genes were analyzed over a panel of 20 reference strains and isolates. Phylogenetic analysis was made at every step. Two MLST schemes were selected. The first one was based on the analysis of three-gene fragments, and it is suitable for species assignment as well as basic phylogenetic studies. By the addition of seven-genes, an approach based on the analysis of ten-gene fragments was also proposed. This is the first work that two optimized MLST schemes have been suggested, validated against a phylogenetically diverse panel of Leishmania isolates. MLST is potentially a powerful phylogenetic approach, and most probably the new gold standard for Leishmania spp. characterization.

Lutzomyia longipalpis (Diptera: Psychodidae) Argentina-Bolivia border: new report and genetic diversity.

American visceral leishmaniasis (AVL) has two main scenarios of transmission as follows: scattered cases in rural areas and urban outbreaks. Urban AVL is in active dispersion from the northeastern border of Argentina-Paraguay-Brazil to the South. The presence of Lutzomyia longipalpis was initially reported in urban environments in the northwestern border of the country. The presence of Lu. longipalpis, environmental variables associated with its distribution, and its genetic diversity were assessed in Salvador Mazza, Argentina, on the border with Bolivia. The genetic analysis showed high haplotype diversity, low nucleotide diversity, and low nucleotide polymorphism index. We discuss the hypothesis of an expanding urban population with introgressive hybridisation of older haplogroups found in their path in natural forest or rural environments, acquiring a new adaptability to urban environments, and the possibility of changes in vector capacity.

Tegumentary leishmaniasis and sand flies in a border area between Argentina and Bolivia.

Background: Some sand flies are of medical importance because they are vectors of Leishmania parasites that are responsible for leishmaniasis. The aim of this study was to make a retrospective epidemiological analysis of tegumentary leishmaniasis (TL), to identify Leishmania spp. from patient isolates and to describe the diversity of sand flies from a border area between Bolivia and Argentina. Methods: TL cases included in the study were diagnosed in an endemic area of the north of Argentina from 1985 to 2017. The parasites isolated were characterized by the cytochrome B method. Sand flies were captured with Centers for Disease Control traps in Aguas Blancas and Media Luna-Algarrobito localities. Results: A total of 118 cases of TL were analysed. Eight isolates were characterized as Leishmania (Viannia) braziliensis. A total of 1291 sand flies were captured, including Nyssomyia neivai, Cortelezzii complex, Evandromyia sallesi, Migonemyia migonei and Micropygomyia quinquefer. Within the area, sand flies were found in the backyards of houses. Conclusions: In this region there exists the possibility of peridomestic transmission of TL in the neighbourhoods peripheral to the urban area and in rural environments as well as the risk of transmission to travellers that pass through the customs offices.

Lutzomyia longipalpis (Diptera: Psychodidae) Argentina-Bolivia border: new report and genetic diversity

American visceral leishmaniasis (AVL) has two main scenarios of transmission as follows: scattered cases in rural areas and urban outbreaks. Urban AVL is in active dispersion from the northeastern border of Argentina-Paraguay-Brazil to the South. The presence of Lutzomyia longipalpis was initially reported in urban environments in the northwestern border of the country. The presence of Lu. longipalpis, environmental variables associated with its distribution, and its genetic diversity were assessed in Salvador Mazza, Argentina, on the border with Bolivia. The genetic analysis showed high haplotype diversity, low nucleotide diversity, and low nucleotide polymorphism index. We discuss the hypothesis of an expanding urban population with introgressive hybridisation of older haplogroups found in their path in natural forest or rural environments, acquiring a new adaptability to urban environments, and the possibility of changes in vector capacity.

Molecular Identification of Leishmania spp. DNA from Archived Giemsa-Stained Slides of Patients from Salta, Argentina.

Cutaneous leishmaniasis is endemic in Salta province, which belongs to the northwest of Argentina. Leishmania spp. DNA from Giemsa-stained slides of up to 12 years in storage of patients from Salta was characterized through polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP). One hundred smears positive for microscopy, classified in a semiquantitative scale for amastigote density, were analyzed. Also, Leishmanin skin test (LST) results were included. DNA extraction was carried out applying lysis buffer with proteinase K, and then DNA was amplified with ribosomal internal transcribed spacer 1 primers. PCR products were digested with HaeIII enzyme. All PCR-positive smears (74/100) belonged to Viannia subgenus. A statistically significant, directly proportional relationship between semiquantitative microscopy and PCR results was detected. All patients had LST-positive results (induration ≥ 5 mm), and the smears of those with smaller induration (LST < 19 mm) gave a higher proportion of positive PCR results. This study determined that smear age did not affect PCR positivity, which allows retrospective analyzes and suggests smears might be useful for molecular complementary diagnosis. Because Leishmania (Viannia) braziliensis is the main circulating species in the study area, determining Viannia subgenus in all analyzed samples confirms previous findings. PCR positivity showed statistically significant differences according to semiquantitative microscopy, highlighting the importance of parasite burden in the diagnostic sensitivity of the method. Considering that smears of patients with smaller LST induration were more positive in PCR, a negative smear from patients with positive LST response, but < 19 mm, could actually represent a false-negative result.

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.

Desarrollo de una PCR en tiempo real para la cuantificación de leishmania SP en muestras de pacientes con leishmaniasis tegumentaria americana / Development of a real time PCR assay for the quantification of leishmania SP in samples from patients with american tegumentary leishmaniasis

INTRODUCCION: La leishmaniasis tegumentaria americana (LTA) es una enfermedad endémica reemergente en la provincia de Salta. Los recursos terapéuticos disponibles presentan serias limitaciones. Se han detectado numerosos casos de fallas terapéuticas debido a que el criterio de curación se basa en la evolución clínica de las lesiones.OBJETIVO: A fin de contar con un sistema adecuado para monitorear la enfermedad, se propuso optimizar una PCR en tiempo real (RT-PCR) basada en el uso de un agente intercalante y oligonucleótidos que amplifican secuencias del ADN del kinetoplasto (KADN), directamente de muestras de raspados de lesiones (en pacientes con LTA) contenidas en buffer TE (Tris-EDTA), sin extracción de ADN.METODOS: Para obtener una curva estándar (CE) se realizaron diluciones seriadas de parásitos (p) de cepas de referencia de especies que circulan en la zona. También se compararon 3 métodos de procesamiento de muestras (PM): Buffer de lisis, Insta-GeneTM Matrix (BIO-RAD) y TE. Luego se analizaron negativos para evaluar la especificidad y sensibilidad del sistema. Estas muestras provenían de raspados de lesiones cutáneas o mucocutáneas, tomados con palillos de madera en 300 μl TE.RESULTADOS: El límite de detección fue de 0,001 p/300 μl TE. La CE construida a partir de una cepa de Leishmania Viannia braziliensis mostró una pendiente de -3,40, eficiencia de amplificación de 96,66%, coeficiente de Pearson (R2) de 0,997 e intersección en la ordenada de 44,079. Al comparar los PM, el método de buffer TE fue el más eficiente. La RT-PCR desarrollada mostró un 100% de especificidad frente a muestras controles negativos y un 100% de sensibilidad frente a controles positivos.CONCLUSIONES: El sistema analizado es altamente sensible y permite detectar parásitos de Leishmania sp directamente de muestras clínicas provenientes de raspados de lesiones.

INTRODUCTION: The american tegumentary leishmaniasis (ATL) is an endemic, re-emergent disease in the Province of Salta. The therapeutic resources which are available have serious limitations. Numerous treatment failures have been detected due to the fact that the evaluation of chemotherapy is based on the clinical outcome of lesionsOBJECTIVE: In order to find a system for the correct follow-up of the disease, the study aimed at optimized the real time PCR (RT-PCR) based on intercalating agent and primers that amplify kinetoplastic DNA sequences (KDNA), directly on samples from skin lesion scrappings (from patients with ATL) contained in buffer TE, without DNA purification..METHODS: To obtain a stardard curve (SC) serial dilutions of parasites (p) were made (from reference strains of species that circulate in the region). 3 different methods of sample processing (SP) were compared: Lysis buffer, Insta-GeneTM Matrix (BIO-RAD) and TE (Tris-EDTA). The study evaluated the specificity and sensibility of the system by analyzing positive (by conventional PCR) and negative clinical samples. These samples were from cutaneous or mucocutaneous lesion scrapings, taken with toothpicks in 300 μl TE.RESULTS: The detection limit was 0.001 p/300 μl TE. The SC built with a Leishmania Viannia braziliensis strain showed a slope of -3.40, amplification efficiency of 96.66%, Pearson coefficient (R2) of 0.997 and 44.079 x-intersection. When comparing the SP, the buffer TE method was the most efficient one. With respect to positive and negative control samples, this RT-PCR showed a sensitivity and specificity of 100% respectively.CONCLUSIONS: This system is highly sensitive and allows to detect Leishmania sp parasites directly from clinical samples of lesion scrapings.