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Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis and their dsRNA Leishmania virus 1. We show that parasite populations circulate in tropical rainforests and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites are geographically and ecologically more dispersed and associated with an increased prevalence, diversity and spread of viruses. Our results suggest that parasite gene flow and hybridization increased the frequency of parasite-virus symbioses, a process that may change the epidemiology of leishmaniasis in the region.
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Leishmania braziliensis , Leishmania , Leishmaniasis Cutánea , Humanos , Ecosistema , Leishmaniasis Cutánea/parasitología , Leishmania braziliensis/genética , Leishmania/genética , Perú/epidemiologíaRESUMEN
Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10-2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.
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OBJECTIVE: To evaluate the toxicity of three synthetic chalcones administered intraperitoneally to BALB/c mice. MATERIALS AND METHODS: The median lethal dose (LD50) was estimated by Dixon's Up-and-Down method. Subchronic toxicity of chalcones was evaluated at 20 and 40 mg/kg for 21 days. Behavioral, physiological, biochemical, and histological toxic effects were evaluated. RESULTS: Chalcone 43 produced mucus in feces, visceral damage (liver) and alterations in organ coefficient (kidney, p = 0.037 and brain, p = 0.008) when compared to the control group. In addition, histological analysis showed that this chalcone produced edema, inflammation and necrosis in the evaluated organs, although there was no significant difference with the control. None of the biochemical parameters differed significantly between the treatment groups at 40 mg/kg dose and the control. CONCLUSIONS: The LD50 for all three chalcones was greater than 550 mg/kg of body weight. Chalcones 40 and 42 were found to be relatively non-toxic. Both can be considered safe for intraperitoneal application in BALB/c mice and, consequently, are potential candidates for use in the treatment of leishmaniasis.
OBJETIVO: Evaluar la toxicidad de tres chalconas sintéticas administradas por vía intraperitoneal en ratones BALB/c. MATERIALES Y MÉTODOS: La dosis letal media (DL50) se estimó por el método Up-and-Down de Dixon. La toxicidad subcrónica de las chalconas se evaluó a 20 y 40 mg/kg por 21 días. Se evaluó el efecto tóxico a nivel de comportamiento, fisiológico, bioquímico e histológico. RESULTADOS: La chalcona 43 generó moco en las heces, daño visceral (hígado) y alteración en el coeficiente de órganos (riñón, p = 0,037 y cerebro, p = 0,008) en comparación con el grupo control. Además, en el análisis histológico se observó que esta chalcona produjo edema, inflamación y necrosis en los órganos evaluados, aunque no hubo diferencia significativa con el control. Todos los parámetros bioquímicos no difirieron significativamente entre los grupos de tratamiento a dosis de 40 mg/kg y el control. CONCLUSIONES: La DL50 para las tres chalconas fue superior a 550 mg/kg de peso corporal. Las chalconas 40 y 42 son relativamente no tóxicas. Ambas pueden considerarse seguras para la aplicación vía intraperitoneal en ratones BALB/c y, en consecuencia, son posibles candidatas para ser usadas en el tratamiento contra las leishmaniosis.
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Antiprotozoarios , Chalcona , Chalconas , Leishmaniasis , Animales , Antiprotozoarios/uso terapéutico , Chalconas/toxicidad , Ratones , Ratones Endogámicos BALB CRESUMEN
Here, we describe a detailed step-by-step protocol for the expression, purification, quantification, and activity determination of key enzymes for molecular detection of pathogens. Based on previous reports, we optimized the protocol for LbCas12a, Taq DNA polymerase, M-MLV reverse transcriptase, and TEV protease to make it compatible with minimal laboratory equipment, broadly available in low- and middle-income countries. The enzymes produced with this protocol have been successfully used for molecular detection applications. For complete details on the use and execution of this protocol, please refer to Alcántara et al. (2021a, 2021b).
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Enzimas , Escherichia coli , Proteínas Recombinantes , Cromatografía de Afinidad , Pruebas de Enzimas , Enzimas/genética , Enzimas/aislamiento & purificación , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Tipificación Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transformación BacterianaRESUMEN
Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 102 ge/µL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%-88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%-100%. The specificity was 96%-100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile laboratory Bento Lab, potentially enabling LMICs to implement detection services in unattended remote regions.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Países en Desarrollo , ARN Viral/genética , Sensibilidad y Especificidad , Técnicas de Amplificación de Ácido NucleicoRESUMEN
Here, we describe a detailed step-by-step protocol to detect SARS-CoV-2 RNA using RT-PCR-mediated amplification and CRISPR/Cas-based visualization. The optimized assay uses basic molecular biology equipment such as conventional thermocyclers and transilluminators for qualitative detection. Alternatively, a fluorescence plate reader can be used for quantitative measurements. The protocol detects two regions of the SARS-CoV-2 genome in addition to the human RNaseP sample control. Aiming to reach remote regions, this work was developed to use the portable molecular workstation from BentoLab. For complete details on the use and execution of this protocol, please refer to Alcántara et al., 2021.
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COVID-19/diagnóstico , Sistemas CRISPR-Cas , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/análisis , ARN Viral/genética , SARS-CoV-2/genética , COVID-19/genética , COVID-19/virología , Humanos , SARS-CoV-2/aislamiento & purificaciónRESUMEN
The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR-Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.
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Sistemas CRISPR-Cas , Endopeptidasa Clp/genética , Proteínas de Choque Térmico/genética , Leishmania braziliensis/genética , Proteínas Protozoarias/genética , Genética Inversa , Endopeptidasa Clp/metabolismo , Edición Génica , Marcación de Gen , Genes Protozoarios , Proteínas de Choque Térmico/metabolismo , Leishmania braziliensis/fisiología , Leishmania major/genética , Leishmania major/fisiología , Mutación , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/metabolismo , TermotoleranciaRESUMEN
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
BACKGROUND: Endemic regions of cutaneous leishmaniasis (CL) and intestinal helminthiasis overlap. CL treatment with systemic pentavalent antimonial drugs (Sb5+) fails in 10%-30% of patients. The study objective was to assess the etiological role of intestinal helminthiasis in CL treatment failure. METHODS: An unmatched case-control study was done in 4 CL treatment sites in Peru in 2012-2015. Cases were CL patients with Sb5+ treatment failure; controls were CL patients with Sb5+ treatment success. Patients with a parasitologically confirmed CL diagnosis who had received supervised Sb5+ treatment and could be classified as cases or controls were eligible. The main exposure variables were intestinal helminthiasis and strongyloidiasis, diagnosed through direct examination, rapid sedimentation, Baermann, Kato-Katz, or agar culture of stool samples. Additional exposure variables were other infections (HIV, human T-lymphotropic virus 1, tuberculosis, hepatitis B, intestinal protozoa) and noninfectious conditions (diabetes, renal insufficiency, and immunosuppressive medication). Age, gender, CL history, probable exposure place, and Leishmania species were treated as potential confounders in multiple logistic regression. RESULTS: There were 94 case and 122 control subjects. Overall, infectious and noninfectious comorbidities were frequent both among cases (64%) and controls (71%). The adjusted odds ratio (OR) for the association between any intestinal helminth infection and CL treatment failure was 0.65 (95% confidence interval [CI], 0.30-1.38), and the adjusted OR for the association between strongyloidiasis and CL treatment failure was 0.34 (95% CI, 0.11-0.92). CONCLUSIONS: In the Peruvian setting, high Sb5+ treatment failure rates are not explained by intestinal helminthiasis. On the contrary, strongyloidiasis had a protective effect against treatment failure.
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BACKGROUND: Tegumentary leishmaniasis (TL) is a disease of skin and/or mucosal tissues caused by Leishmania parasites. TL patients may concurrently carry other pathogens, which may influence the clinical outcome of TL. METHODOLOGY AND PRINCIPAL FINDINGS: This review focuses on the frequency of TL coinfections in human populations, interactions between Leishmania and other pathogens in animal models and human subjects, and implications of TL coinfections for clinical practice. For the purpose of this review, TL is defined as all forms of cutaneous (localised, disseminated, or diffuse) and mucocutaneous leishmaniasis. Human immunodeficiency virus (HIV) coinfection, superinfection with skin bacteria, and skin manifestations of visceral leishmaniasis are not included. We searched MEDLINE and other databases and included 73 records: 21 experimental studies in animals and 52 studies about human subjects (mainly cross-sectional and case studies). Several reports describe the frequency of Trypanosoma cruzi coinfection in TL patients in Argentina (about 41%) and the frequency of helminthiasis in TL patients in Brazil (15% to 88%). Different hypotheses have been explored about mechanisms of interaction between different microorganisms, but no clear answers emerge. Such interactions may involve innate immunity coupled with regulatory networks that affect quality and quantity of acquired immune responses. Diagnostic problems may occur when concurrent infections cause similar lesions (e.g., TL and leprosy), when different pathogens are present in the same lesions (e.g., Leishmania and Sporothrix schenckii), or when similarities between phylogenetically close pathogens affect accuracy of diagnostic tests (e.g., serology for leishmaniasis and Chagas disease). Some coinfections (e.g., helminthiasis) appear to reduce the effectiveness of antileishmanial treatment, and drug combinations may cause cumulative adverse effects. CONCLUSIONS AND SIGNIFICANCE: In patients with TL, coinfection is frequent, it can lead to diagnostic errors and delays, and it can influence the effectiveness and safety of treatment. More research is needed to unravel how coinfections interfere with the pathogenesis of TL.
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Coinfección/epidemiología , Coinfección/parasitología , Leishmaniasis Cutánea/epidemiología , Animales , Argentina/epidemiología , Brasil/epidemiología , Modelos Animales de Enfermedad , Humanos , Piel/patologíaRESUMEN
Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.
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Leishmania braziliensis/virología , Leishmaniasis Mucocutánea/tratamiento farmacológico , Leishmaniasis Mucocutánea/virología , Leishmaniavirus , Antimonio/uso terapéutico , Antiprotozoarios/uso terapéutico , Bolivia/epidemiología , Estudios de Cohortes , Resistencia a Medicamentos , Humanos , Leishmaniasis Mucocutánea/epidemiología , Leishmaniasis Mucocutánea/parasitología , Leishmaniavirus/clasificación , Leishmaniavirus/genética , Perú/epidemiología , Insuficiencia del TratamientoRESUMEN
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
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
Conventional understanding suggests that simultaneous infection with more than one species of Leishmania is unlikely. In Peru, co-infections are clinically relevant because causative species dictates prognosis, treatment response, and follow-up. We describe a case of Leishmania (Viannia) braziliensis and L. (V.) lainsoni co-infection in a Peruvian patient with cutaneous leishmaniasis.
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Coinfección/parasitología , Leishmania braziliensis/patogenicidad , Leishmaniasis Cutánea/diagnóstico , Adulto , Gluconato de Sodio Antimonio/uso terapéutico , Coinfección/diagnóstico , Coinfección/tratamiento farmacológico , ADN Protozoario/análisis , Femenino , Humanos , Leishmania braziliensis/aislamiento & purificación , Leishmaniasis Cutánea/tratamiento farmacológico , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Protozoarias/análisis , Úlcera Cutánea/tratamiento farmacológico , Úlcera Cutánea/parasitología , Úlcera Cutánea/patologíaRESUMEN
BACKGROUND: Traditional methods of detecting Leishmania from cutaneous lesions involve invasive diagnostic procedures, such as scrapings, which cause discomfort, require technical expertise, and carry risks of invasive procedures. We compared the performance of 2 novel, molecular-based non-invasive methods for the diagnosis of cutaneous leishmaniasis (CL). METHODS: Consecutive patients presenting to the Leishmania Clinic at the Hospital Nacional Cayetano Heredia were enrolled. PCR was performed on filter paper lesion impressions (FPLIs), cytology brushes, and lancets for detection of Leishmania DNA. Smears from lesion scrapings and leishmanin skin test were also performed. Outcome measures were sensitivity and specificity. Composite reference standard was any 2 of 5 tests positive. Species identification was performed by PCR assays of positive specimens. RESULTS: Ninety patients with 129 lesions were enrolled, 117 of which fulfilled reference criteria for a diagnosis of CL. Of these 117 lesions, 113 were positive by PCR of lancets used for lesion scrapings versus 116 by PCR of FPLIs (p=0.930) or 116 by PCR of cytology brushes (p=0.930). Sensitivity and specificity of PCR on lancets were 96.6% [95% CI 93.3-99.9%] and 100%, respectively. Sensitivity and specificity of FPLI PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Sensitivity and specificity of cytology brush PCR were 99.1% [95% CI 97.4-100%] and 100%, respectively. Giemsa-stained lesion smear and leishmanin skin test had inferior sensitivities at 47.9% [95% CI 38.9-57.0%] and 82.3% [95% CI 73.9-90.7%], respectively, compared to PCR of invasive or non-invasive specimens (p<0.001). CONCLUSIONS: Cytology brush PCR constitutes a sensitive and specific alternative to traditional diagnostic assays performed on invasive specimens such as lesion scrapings. It performs comparatively to non-invasive FPLI PCR. This novel, rapid, and well-tolerated method has the potential for widespread use in the field and in pediatric populations where traditional specimen collection is difficult.
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Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/genética , Reacción en Cadena de la Polimerasa/métodos , ADN/metabolismo , ADN de Cinetoplasto/metabolismo , Humanos , Papel , Perú , Polimorfismo de Longitud del Fragmento de Restricción , Sensibilidad y Especificidad , Piel/química , Piel/microbiología , Pruebas Cutáneas/métodos , Especificidad de la Especie , Manejo de Especímenes/métodosRESUMEN
In order to understand the epidemiological dynamics of antimonial (Sb(V)) resistance in zoonotic tegumentary leishmaniasis and its link with treatment outcome, we analyzed the population structure of 24 Peruvian Leishmania braziliensis clinical isolates with known in vitro antimony susceptibility and clinical phenotype by multilocus microsatellite typing (14 microsatellite loci). The genetic variability in the Peruvian isolates was high and the multilocus genotypes were strongly differentiated from each other. No correlation was found between the genotypes and in vitro drug susceptibility or clinical treatment outcome. The finding of a polyphyletic pattern among the Sb(V)-resistant L. braziliensis might be explained by (i) independent events of drug resistance emergence, (ii) sexual recombination and/or (iii) other phenomena mimicking recombination signals. Interestingly, the polyphyletic pattern observed here is very similar to the one we observed in the anthroponotic Leishmania donovani (Laurent et al., 2007), hereby questioning the role of transmission and/or chemotherapeutic drug pressure in the observed population structure.
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Antimonio/farmacología , Antiprotozoarios/farmacología , Resistencia a Medicamentos/genética , Leishmania braziliensis/clasificación , Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/genética , Animales , Antimonio/uso terapéutico , Antiprotozoarios/uso terapéutico , Variación Genética , Genotipo , Humanos , Leishmania braziliensis/patogenicidad , Leishmaniasis Cutánea/tratamiento farmacológico , Repeticiones de Microsatélite , Pruebas de Sensibilidad Parasitaria , Perú , Resultado del TratamientoRESUMEN
BACKGROUND: Most of the Leishmania genome is reported to be constitutively expressed during the life cycle of the parasite, with a few regulated genes. Inter-species comparative transcriptomics evidenced a low number of species-specific differences related to differentially distributed genes or the differential regulation of conserved genes. It is of uppermost importance to ensure that the observed differences are indeed species-specific and not simply specific of the strains selected for representing the species. The relevance of this concern is illustrated by current study. METHODOLOGY/PRINCIPAL FINDINGS: We selected 5 clinical isolates of L. braziliensis characterized by their diversity of clinical and in vitro phenotypes. Real-time quantitative PCR was performed on promastigote and amastigote life stages to assess gene expression profiles at seven time points covering the whole life cycle. We tested 12 genes encoding proteins with roles in transport, thiol-based redox metabolism, cellular reduction, RNA poly(A)-tail metabolism, cytoskeleton function and ribosomal function. The general trend of expression profiles showed that regulation of gene expression essentially occurs around the stationary phase of promastigotes. However, the genes involved in this phenomenon appeared to vary significantly among the isolates considered. CONCLUSION/SIGNIFICANCE: Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics. Results obtained on an individual strain are not necessarily representative of a given species. Therefore, extreme care should be taken when comparing the profiles of different species and extrapolating functional differences between them.
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Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Leishmania braziliensis/crecimiento & desarrollo , Leishmania braziliensis/genética , ADN Protozoario/química , ADN Protozoario/genética , Datos de Secuencia Molecular , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
In Brazil, cutaneous leishmaniasis represents a serious public health problem, and chemotherapy is an important element of the clinical management of this disease. However, treatment efficacy is variable, a phenomenon that might be due to host and parasite (e.g., drug resistance) factors. To better understand the possible contribution of parasite factors to this phenomenon, we characterised 12 Leishmania braziliensis (LB) and 25 Leishmania guyanensis (LG) isolates collected from patients experiencing different antimonial treatment outcomes. For each isolate, promastigote cultures were grown in duplicate and were harvested at the late-log and stationary phases of growth. The RNA expression profiles of six genes encoding proteins with roles in antimony metabolism (AQP1, MRPA, GSH1, GSH2, TRYR and TDR1) were assessed by means of real-time quantitative PCR. Molecular data were compared to the clinical phenotypes. Within LB, we did not find statistically significant differences in the expression levels of the examined genes among isolates from patients with different treatment outcomes. In LG, GSH1 (encoding gamma-glutamylcysteine synthetase, gamma-GCS) was overexpressed in therapeutic failure isolates regardless of the growth curve phase. This finding reveals the predictive potential of promastigote expression curves for the prognosis of cutaneous leishmaniasis caused by LG in Brazil.
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Antimonio/uso terapéutico , Perfilación de la Expresión Génica , Leishmania braziliensis/genética , Leishmania guyanensis/genética , Leishmaniasis Cutánea/tratamiento farmacológico , Animales , Antiprotozoarios/uso terapéutico , Brasil , Resistencia a Medicamentos/genética , Humanos , Leishmania braziliensis/aislamiento & purificación , Leishmania guyanensis/aislamiento & purificación , Leishmaniasis Cutánea/parasitología , Meglumina/uso terapéutico , Antimoniato de Meglumina , Compuestos Organometálicos/uso terapéutico , Parásitos/genética , Parásitos/aislamiento & purificación , Resultado del TratamientoRESUMEN
Human T-lymphotropic virus 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a complication that affects up to 5% of HTLV-1-infected individuals. Several host genetic and viral factors have been associated with the risk of HAM/TSP. The aim of this study was to evaluate the performance of a prognostic model for HAM/TSP developed in Japan in a Peruvian population of 71 HAM/TSP patients and 94 asymptomatic carriers (ACs). This model included age, proviral load (PVL), the presence of HLA-A*02 and HLA-Cw*08 alleles, SDF-1 +801, and TNF-alpha -863 polymorphisms, and viral subgroup. We describe frequencies for the four host genetic markers and demonstrate the presence of the HTLV-1 tax B subgroup in Peru. Using cross-validation, we show that the predictive ability of the prognostic model, as characterized by the area under the receiver-operating characteristic curve (AUC), does not differ from a model containing PVL only (both AUC = 0.74). We found some suggestive evidence of a protective effect of the HLA-A*02 allele but failed to replicate the associations with the other three genetic markers and with viral subgroup. A logistic model containing PVL, age, gender, and HLA-A*02 provided the best predictive ability in the Peruvian cohort (AUC = 0.79). J. Med. Virol. 82:460-466, 2010. (c) 2010 Wiley-Liss, Inc.
Asunto(s)
Antígenos HLA/genética , Infecciones por HTLV-I/complicaciones , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Paraparesia Espástica Tropical/diagnóstico , Polimorfismo Genético , Factor de Necrosis Tumoral alfa/genética , Adolescente , Adulto , Factores de Edad , Anciano , Biomarcadores , Femenino , Infecciones por HTLV-I/genética , Infecciones por HTLV-I/virología , Humanos , Japón , Masculino , Persona de Mediana Edad , Perú , Valor Predictivo de las Pruebas , Pronóstico , Provirus/aislamiento & purificación , Factores de Riesgo , Carga Viral , Adulto JovenRESUMEN
Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-microl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, <0.001). The mean times to culture positivity were 4.2 days by microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture.