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Introduction: Resistance against anti-Leishmania drugs (DR) has been studied for years, giving important insights into long-term adaptations of these parasites to drugs, through genetic modifications. However, microorganisms can also survive lethal drug exposure by entering into temporary quiescence, a phenomenon called drug tolerance (DT), which is rather unexplored in Leishmania. Methods: We studied a panel of nine Leishmania braziliensis strains highly susceptible to potassium antimonyl tartrate (PAT), exposed promastigotes to lethal PAT pressure, and compared several cellular and molecular parameters distinguishing DT from DR. Results and discussion: We demonstrated in vitro that a variable proportion of cells remained viable, showing all the criteria of DT and not of DR: i) signatures of quiescence, under drug pressure: reduced proliferation and significant decrease of rDNA transcription; ii) reversibility of the phenotype: return to low IC50 after removal of drug pressure; and iii) absence of significant genetic differences between exposed and unexposed lineages of each strain and absence of reported markers of DR. We found different levels of quiescence and DT among the different L. braziliensis strains. We provide here a new in-vitro model of drug-induced quiescence and DT in Leishmania. Research should be extended in vivo, but the current model could be further exploited to support R&D, for instance, to guide the screening of compounds to overcome the quiescence resilience of the parasite, thereby improving the therapy of leishmaniasis.
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Leishmania braziliensis , Leishmania , Leishmaniasis Cutánea , Humanos , Leishmania braziliensis/genética , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/parasitologíaRESUMEN
The implementation of prospective drug resistance (DR) studies in the research-and-development (R&D) pipeline is a common practice for many infectious diseases but not for neglected tropical diseases (NTDs). Here, we explored and demonstrated the importance of this approach using as paradigms Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GlaxoSmithKline (GSK) "Leishbox" to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at the genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross-resistance to these drugs, suggesting a new and unique mechanism. By whole-genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at the highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/reduced susceptibility of L. donovani to TCMDC-143345. IMPORTANCE Humans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R&D pipeline, a common practice for many infectious diseases but not for NTDs. Here, using Leishmania donovani, the etiological agent of visceral leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK Leishbox to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1-like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.
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Antiprotozoarios , Resistencia a Medicamentos , Dinamina I , Leishmania donovani , Leishmaniasis Visceral , Humanos , Antiprotozoarios/inmunología , Dinamina I/genética , Dinamina I/inmunología , Genómica , Leishmania donovani/genética , Leishmania donovani/inmunología , Leishmania donovani/parasitología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Filogenia , Estudios Retrospectivos , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/inmunologíaRESUMEN
Microorganisms can adopt a quiescent physiological condition which acts as a survival strategy under unfavorable conditions. Quiescent cells are characterized by slow or non-proliferation and a deep downregulation of processes related to biosynthesis. Although quiescence has been described mostly in bacteria, this survival skill is widespread, including in eukaryotic microorganisms. In Leishmania, a digenetic parasitic protozoan that causes a major infectious disease, quiescence has been demonstrated, but the molecular and metabolic features enabling its maintenance are unknown. Here, we quantified the transcriptome and metabolome of Leishmania promastigotes and amastigotes where quiescence was induced in vitro either, through drug pressure or by stationary phase. Quiescent cells have a global and coordinated reduction in overall transcription, with levels dropping to as low as 0.4% of those in proliferating cells. However, a subset of transcripts did not follow this trend and were relatively upregulated in quiescent populations, including those encoding membrane components, such as amastins and GP63, or processes like autophagy. The metabolome followed a similar trend of overall downregulation albeit to a lesser magnitude than the transcriptome. It is noteworthy that among the commonly upregulated metabolites were those involved in carbon sources as an alternative to glucose. This first integrated two omics layers afford novel insight into cell regulation and show commonly modulated features across stimuli and stages.
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American Tegumentary Leishmaniasis (ATL) is an endemic and neglected disease of South America. Here, mucosal leishmaniasis (ML) disproportionately affects up to 20% of subjects with current or previous localised cutaneous leishmaniasis (LCL). Preclinical and clinical reports have implicated the Leishmania RNA virus-1 (LRV1) as a possible determinant of progression to ML and other severe manifestations such as extensive cutaneous and mucosal disease and treatment failure and relapse. However, these associations were not consistently found in other observational studies and are exclusively based on cross-sectional designs. In the present study, 56 subjects with confirmed ATL were assessed and followed out for 24-months post-treatment. Lesion biopsy specimens were processed for molecular detection and quantification of Leishmania parasites, species identification, and LRV1 detection. Among individuals presenting LRV1 positive lesions, 40% harboured metastatic phenotypes; comparatively 58.1% of patients with LRV1 negative lesions harboured metastatic phenotypes (p = 0.299). We found treatment failure (p = 0.575) and frequency of severe metastatic phenotypes (p = 0.667) to be similarly independent of the LRV1. Parasite loads did not differ according to the LRV1 status (p = 0.330), nor did Leishmanin skin induration size (p = 0.907) or histopathologic patterns (p = 0.780). This study did not find clinical, parasitological, or immunological evidence supporting the hypothesis that LRV1 is a significant determinant of the pathobiology of ATL.
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Leishmania/patogenicidad , Leishmania/virología , Leishmaniasis Cutánea/parasitología , Leishmaniavirus/aislamiento & purificación , Adulto , Estudios de Cohortes , Humanos , Leishmania/clasificación , Leishmaniasis Cutánea/patología , Leishmaniasis Mucocutánea/parasitología , Leishmaniasis Mucocutánea/patología , Leishmaniavirus/genética , Masculino , Persona de Mediana Edad , Fenotipo , Estudios Prospectivos , Insuficiencia del TratamientoRESUMEN
The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean Leishmania as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles-but not maxicircles-show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.
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Genoma Mitocondrial/genética , Interacciones Huésped-Parásitos/genética , Leishmania braziliensis/genética , Leishmaniasis Cutánea/genética , Ecosistema , Bosques , Especiación Genética , Humanos , Leishmania braziliensis/patogenicidad , Leishmaniasis Cutánea/epidemiología , Leishmaniasis Cutánea/parasitología , Perú/epidemiología , FilogeografíaRESUMEN
Here, we report a pilot study paving the way for further single cell genomics studies in Leishmania. First, the performances of two commercially available kits for Whole Genome Amplification (WGA), PicoPLEX and RepliG were compared on small amounts of Leishmania donovani DNA, testing their ability to preserve specific genetic variations, including aneuploidy levels and SNPs. We show here that the choice of WGA method should be determined by the planned downstream genetic analysis, PicoPLEX and RepliG performing better for aneuploidy and SNP calling, respectively. This comparison allowed us to evaluate and optimize corresponding bio-informatic methods. As PicoPLEX was shown to be the preferred method for studying single cell aneuploidy, this method was applied in a second step, on single cells of L. braziliensis, which were sorted by fluorescence activated cell sorting (FACS). Even sequencing depth was achieved in 28 single cells, allowing accurate somy estimation. A dominant karyotype with three aneuploid chromosomes was observed in 25 cells, while two different minor karyotypes were observed in the other cells. Our method thus allowed the detection of aneuploidy mosaicism, and provides a solid basis which can be further refined to concur with higher-throughput single cell genomic methods.
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Biología Computacional/métodos , Genoma de Protozoos , Cariotipificación/métodos , Leishmania/genética , Análisis de la Célula Individual/métodos , Aneuploidia , Citometría de Flujo/métodosRESUMEN
Under stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently discovered in Leishmania. We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.
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ADN Protozoario , ADN Ribosómico , Sitios Genéticos , Proteínas Fluorescentes Verdes , Leishmania braziliensis , Leishmania mexicana , Microorganismos Modificados Genéticamente , Animales , ADN Protozoario/genética , ADN Protozoario/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Leishmania braziliensis/citología , Leishmania braziliensis/crecimiento & desarrollo , Leishmania braziliensis/metabolismo , Leishmania mexicana/citología , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , RatonesRESUMEN
It was recently hypothesized that Leishmania amastigotes could constitute a semi-quiescent stage characterized by low replication and reduced metabolic activity. This concept developed with Leishmania (Leishmania) mexicana and Leishmania (Leishmania) major models might explain numerous clinical and sub-clinical features of Leishmania (Viannia) braziliensis infections, like reactivation of the disease, non-response to chemotherapy or asymptomatic infections. We compared here in vitro the proliferative capability of L. (V.) braziliensis amastigotes and promastigotes, assessed the expression of key molecular parameters and performed metabolomic analysis. We found that contrary to the highly proliferative promastigotes, amastigotes (axenic and intracellular) do not show evidence of extensive proliferation. In parallel, amastigotes showed a significant decrease of (i) the kDNA mini-circle abundance, (ii) the intracellular ATP level, (iii) the ribosomal components: rRNA subunits 18S and 28S α and ribosomal proteins RPS15 and RPL19, (iv) total RNA and protein levels. An untargeted metabolomic study identified clear differences between the different life stages: in comparison to logarithmic promastigotes, axenic amastigotes showed (a) a strong decrease of 14 essential and non-essential amino acids and eight metabolites involved in polyamine synthesis, (b) extensive changes in the phospholipids composition and (c) increased levels of several endogenous and exogenous sterols. Altogether, our results show that L. (V.) braziliensis amastigotes can show a phenotype with negligible rate of proliferation, a lower capacity of biosynthesis, a reduced bio-energetic level and a strongly altered metabolism. Our results pave the way for further exploration of quiescence among amastigotes of this species.
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Leishmania braziliensis/crecimiento & desarrollo , Leishmania braziliensis/metabolismo , Leishmaniasis Cutánea/parasitología , Metaboloma , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Vías Biosintéticas , Células Cultivadas , Femenino , Ratones Endogámicos BALB C , Proteínas Protozoarias/análisis , Proteínas Protozoarias/metabolismo , ARN Protozoario/análisis , ARN Protozoario/metabolismo , Proteínas Ribosómicas/análisis , Proteínas Ribosómicas/metabolismoRESUMEN
Mucosal leishmaniasis (ML) is a disfiguring manifestation of Leishmania (Viannia) infection. We evaluated parasite load (PL) over time as a potential biomarker of treatment outcome in ML. PL was assessed with kinetoplast DNA quantitative real-time polymerase chain reaction (kDNA-qPCR) at enrollment, days 14 and 21-28 of therapy and 3, 6, 12-18, and 18-24 months after treatment of ML and correlated to demographic, clinical, and parasitologic factors. Forty-four patients were enrolled: 30 men and 14 women. Enrollment PL differed significantly by causative species (P < 0.001), and was higher in patients with severe ML (nasal and laryngeal involvement) compared with those with only isolated nasal involvement (median = 1,285 versus 51.5 parasites/µg tissue DNA; P = 0.005). Two patterns of PL emerged: pattern 1 (N = 23) was characterized by a sequential decline in PL during and after therapy until kDNA was undetectable. Pattern 2 (N = 18) was characterized by clearance of detectable kDNA during treatment, followed by an increased PL thereafter. All patients who failed treatment (N = 4) demonstrated pattern 1. Leishmania (Viannia) braziliensis was overrepresented among those with pattern 2 (P = 0.019). PL can be quantified by cytology brush qPCR during and after treatment in ML. We demonstrate that treatment failure was associated with undetectable PL, and L. (V.) braziliensis infection was overrepresented in those with rebounding PL.
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Anfotericina B/uso terapéutico , Gluconato de Sodio Antimonio/uso terapéutico , ADN de Cinetoplasto/genética , Leishmaniasis Mucocutánea/parasitología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anfotericina B/administración & dosificación , Gluconato de Sodio Antimonio/administración & dosificación , Biomarcadores , Niño , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Reacción en Cadena en Tiempo Real de la Polimerasa , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion. METHODOLOGY/PRINCIPAL FINDINGS: We applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4). CONCLUSION/SIGNIFICANCE: Our results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.
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ADN de Cinetoplasto/genética , Leishmania/genética , Leishmaniasis Cutánea/parasitología , Carga de Parásitos , Úlcera Cutánea/parasitología , Adolescente , Adulto , Anciano , Femenino , Humanos , Leishmania/clasificación , Leishmaniasis Cutánea/patología , Masculino , Persona de Mediana Edad , Úlcera Cutánea/patología , Especificidad de la Especie , Adulto JovenRESUMEN
Background: Risk of malnutrition is elevated among oncologic patients, and this increases postoperative morbidity and mortality. Aim: To study the association between nutritional status and postoperative outcomes in a group of patients with gastrointestinal cancers. Patients and Methods: We studied 129 patients with diagnosis of digestive cancer, previous to potentially curative surgery. Nutritional status was evaluated through anthropometric measures, Subjective Global Assessment (SGA), dietary intake recalls and routine biochemical parameters. Functional performance was assessed by the Karnofsky index (KI). Cancer stage was classified according to TNM4. During the postoperative period, complications, length of stay at the critical care ward and duration of hospitalization were registered. Thirty days after discharge, patients were contacted, and the appearance of new complications was listed. Results: According to SGA 14.7% of patients were classified as well nourished (A), 57.3% as moderately undernourished or at risk of malnutrition (B) and 27.9% as severely malnourished (C). The incidence of total complications was 25.5%. Nutritional status was not associated with cancer stage. The frequency of complications among patients classified as A, B and C were 5.5, 25.3 and 37.1% respectively (p = 0.03). Conclusions: We detected a high frequency of malnutrition in this group of patients. Overall the frequency of postoperative complications was low, however malnourished patients exhibited a higher rate of surgical complications.
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Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Gastrointestinales/cirugía , Estado Nutricional , Complicaciones Posoperatorias/mortalidad , Antropometría , Proteína C-Reactiva/análisis , Métodos Epidemiológicos , Neoplasias Gastrointestinales/complicaciones , Neoplasias Gastrointestinales/mortalidad , Tiempo de Internación , Desnutrición/etiología , Desnutrición/mortalidad , Estadificación de Neoplasias , Evaluación Nutricional , Periodo Preoperatorio , Factores de TiempoRESUMEN
BACKGROUND: Risk of malnutrition is elevated among oncologic patients, and this increases postoperative morbidity and mortality. AIM: To study the association between nutritional status and postoperative outcomes in a group of patients with gastrointestinal cancers. PATIENTS AND METHODS: We studied 129 patients with diagnosis of digestive cancer, previous to potentially curative surgery. Nutritional status was evaluated through anthropometric measures, Subjective Global Assessment (SGA), dietary intake recalls and routine biochemical parameters. Functional performance was assessed by the Karnofsky index (KI). Cancer stage was classified according to TNM4. During the postoperative period, complications, length of stay at the critical care ward and duration of hospitalization were registered. Thirty days after discharge, patients were contacted, and the appearance of new complications was listed. RESULTS: According to SGA 14.7% of patients were classified as well nourished (A), 57.3% as moderately undernourished or at risk of malnutrition (B) and 27.9% as severely malnourished (C). The incidence of total complications was 25.5%. Nutritional status was not associated with cancer stage. The frequency of complications among patients classified as A, B and C were 5.5, 25.3 and 37.1% respectively (p = 0.03). CONCLUSIONS: We detected a high frequency of malnutrition in this group of patients. Overall the frequency of postoperative complications was low, however malnourished patients exhibited a higher rate of surgical complications.
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Neoplasias Gastrointestinales/cirugía , Estado Nutricional , Complicaciones Posoperatorias/mortalidad , Anciano , Antropometría , Proteína C-Reactiva/análisis , Métodos Epidemiológicos , Femenino , Neoplasias Gastrointestinales/complicaciones , Neoplasias Gastrointestinales/mortalidad , Humanos , Tiempo de Internación , Masculino , Desnutrición/etiología , Desnutrición/mortalidad , Persona de Mediana Edad , Estadificación de Neoplasias , Evaluación Nutricional , Periodo Preoperatorio , Factores de TiempoRESUMEN
Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r = 0.87; P < 0.0001), but the former showed higher sensitivity (P = 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P = 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P = 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.
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Leishmania/aislamiento & purificación , Leishmaniasis/parasitología , Membrana Mucosa/parasitología , Carga de Parásitos/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Piel/parasitología , Adulto , ADN de Cinetoplasto/análisis , ADN de Cinetoplasto/genética , ADN Protozoario/análisis , ADN Protozoario/genética , Femenino , Humanos , Leishmaniasis/patología , Masculino , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
Protozoa of the Leishmania genus are transmitted to humans by the bite of infected sandflies, and are the causative agents of leishmaniasis which ranges from cutaneous to visceral clinical forms. The definitive diagnosis of leishmaniasis has relied traditionally on parasite demonstration, either by microscopy or culture; in the last years, diagnosis based on PCR methods has overcome some drawbacks of traditional methods, increasing sensitivity and allowing using less invasive sampling for diagnosis. However, there are not defined protocols and almost each laboratory applies its own in-house method. Although there are several studies comparing the performance of different methods within the same laboratory, those addressing interlaboratory comparison are scarce, in spite of the growing number of collaborative projects between partners from different leishmaniasis endemic and non-endemic countries. In this work we propose a protocol for interlaboratory comparison of conventional and real-time PCR methods involving four participant laboratories from four different endemic regions in four continents; the protocol includes a quality control step and reduces the variability among the samples tested by each participant. A panel of 77 samples from human origin and 9 from different parasite strains was blindly tested by the participants, aiming to assess the sensitivity of the different methods as well as their usefulness for species identification. Real-time PCR methods targeting the kDNA minicircles returned the highest sensitivity, while both PCR targeting ITS-1 and further HaeIII digestion and a combined algorithm including hsp70 PCR and restriction fragment length polymorphism analysis were the most appropriate approaches for species identification.