SYBR Green-based Real-Time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases

<p>Abstract</p> <p>Background</p> <p>Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between <it>Leishmania </it>isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect <it>Leishmania </it>infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk.</p> <p>Results</p> <p>A SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between <it>Leishmania </it>subgenera. A panel of reference strains representing subgenera <it>Leishmania </it>and <it>Viannia </it>was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95°C ± 0.01 and 77.36°C ± 0.02 for <it>Leishmania </it>and <it>Viannia</it>, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together <it>L</it>. (<it>V</it>.) <it>braziliensis </it>and <it>L</it>. (<it>V</it>.) <it>shawii </it>with a bootstrap value of 100%; while for <it>L</it>. (<it>L</it>.) <it>infantum </it>and <it>L</it>. (<it>L</it>.) <it>amazonensis</it>, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus <it>Viannia </it>amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus <it>Leishmania </it>(average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected <it>Lutzomyia </it>spp. captured in two Brazilian localities.</p> <p>Conclusions</p> <p>For all tested samples, a characteristic amplicon melting profile was evidenced for each <it>Leishmania </it>subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.</p

Occurrence of triatomines (Hemiptera: Reduviidae) in domestic and natural environments in novo remanso, itacoatiara, amazonas, Brazil

Introduction: The present study reports the presence of triatomines in natural, peridomestic, and intradomicile environments in Itacoatiara municipality, state of Amazonas, a non-endemic region for Chagas disease. Methods: Active search was performed inside tree trunks, and palm trees, residences, and peridomiciles localized near the forest area. Results: Twenty adults and ten triatomines nymphs were collected, fifteen of which were from natural forests, thirteen from intradomiciles, and two from peridomicile areas. Conclusions: The new records of adults and nymphs of triatomines in the intra-and peridomiciles suggest the adoption of prophylactic measures for vector surveillance in the study area. © 2019, Sociedade Brasileira de Medicina Tropical. All rights reserved

Trypsin-like serine peptidase profiles in the egg, larval, and pupal stages of Aedes albopictus

BACKGROUND: Aedes albopictus, a ubiquitous mosquito, is one of the main vectors of dengue and yellow fever, representing an important threat to public health worldwide. Peptidases play key roles in processes such as digestion, oogenesis, and metamorphosis of insects. However, most of the information on the proteolytic enzymes of mosquitoes is derived from insects in the adult stages and is often directed towards the understanding of blood digestion. The aim of this study was to investigate the expression of active peptidases from the preimaginal stages of Ae. albopictus. METHODS: Ae. albopictus eggs, larvae, and pupae were analyzed using zymography with susbtrate-SDS-PAGE. The pH, temperature and peptidase inhibitor sensitivity was evaluated. In addition, the proteolytic activities of larval instars were assayed using the fluorogenic substrate Z-Phe-Arg-AMC. RESULTS: The proteolytic profile of the larval stage was composed of 8 bands ranging from 17 to 130 kDa. These enzymes displayed activity in a broad range of pH values, from 5.5 to 10.0. The enzymatic profile of the eggs was similar to that of the larvae, although the proteolytic bands of the eggs showed lower intensities. The pupal stage showed a complex proteolytic pattern, with at least 6 bands with apparent molecular masses ranging from 30 to 150 kDa and optimal activity at pH 7.5. Peptidases from larval instars were active from 10°C to 60°C, with optimal activity at temperatures between 37°C and 50°C. The proteolytic profile of both the larval and pupal stages was inhibited by phenyl-methyl sulfonyl-fluoride (PMSF) and Nα-Tosyl L-lysine chloromethyl ketone hydrochloride (TLCK), indicating that the main peptidases expressed during these developmental stages are trypsin-like serine peptidases. CONCLUSION: The preimaginal stages of Ae. albopictus exhibited a complex profile of trypsin-like serine peptidase activities. A comparative analysis of the active peptidase profiles revealed differential expression of trypsin-like isoforms among the preimaginal stages, suggesting that some of these enzymes are stage specific. Additionally, a comparison of the peptidase expression between larvae from eggs collected in the natural environment and larvae obtained from the eggs of female mosquitoes maintained in colonies for a long period of time demonstrated that the proteolytic profile is invariable under such conditions

Parasite persistence in treated chagasic patients revealed by xenodiagnosis and polymerase chain reaction

Polymerase chain reaction (PCR) was compared with xenodiagnosis performed 20 years after trypanocidal chemotherapy to investigate parasite clearance. Eighty-five seropositive individuals for Chagas disease presenting a positive xenodiagnosis were treated with specific drugs; 37 in the acute phase and 48 in the chronic phase. Fifteen chronic assymptomatic patients received a placebo. Treatment in the acute phase led to PCR negative results in 73% of the cases, while xenodiagnosis was negative in 86%. In the chronic phase, PCR was negative in 65% of the patients and 83% led to xenodiagnosis negative results. Regarding the untreated group (placebo), 73% gave negative results by xenodiagnosis, of which 36% were positive by PCR. Individuals that were considered seronegative (n=10), presented unequivocally negative results in the PCR demonstrating the elimination of parasite DNA. Seventeen individuals had their antibodies titers decreased to such a level that the final results were considered as doubtful and 16 of them presented negative PCR. The molecular method represents a clear advantage over conventional techniques to demonstrate persistent infections in Chagas disease patients that underwent chemotherapy

Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence

Accurate diagnostics tools and surrogate markers of parasitological response to treatment are priority needs for management of Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a Satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (Tc I) and CL-Brener (Tc VI) stocks similar LOD95 values (0.903 (0.745-1.497) and 0.667 (CI 0.113-3.927) copy numbers/μL, respectively), when synthetic DNA was the standard for quantification, thus allowing direct comparison of loads in samples infected with different DTUs. This standard curve was evaluated in 205 samples from 38 acute oral and 19 chronic CD patients from different geographical areas infected with different genotypes, including samples obtained during treatment follow-up, and high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase III clinical trials, to follow-up patients under treatment or at risk of reactivation and in experimental models using different parasite strains.Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Silva Gomess, Natalia Lins. Fundación Oswaldo Cruz; BrasilFil: Apodaca, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; VenezuelaFil: Díaz Bello, Zoraida. Universidad Central de Venezuela; VenezuelaFil: Quintino Souza, Leticia Rocha. Fundación Oswaldo Cruz; BrasilFil: Tavares Costa, Alexandre Dias. Fundación Oswaldo Cruz; BrasilFil: Britto, Constança. Fundación Oswaldo Cruz; BrasilFil: Moreira, Otacilio Cruz. Fundación Oswaldo Cruz; BrasilFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

In Vitro and In Vivo Investigation of the Efficacy of Arylimidamide DB1831 and Its Mesylated Salt Form - DB1965 - against Trypanosoma cruzi Infection

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA - DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T.cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC50 value/48 h of 5–40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T.cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T.cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole

Combined Treatment of Heterocyclic Analogues and Benznidazole upon Trypanosoma cruzi In Vivo

Chagas disease caused by Trypanosoma cruzi is an important cause of mortality and morbidity in Latin America but no vaccines or safe chemotherapeutic agents are available. Combined therapy is envisioned as an ideal approach since it may enhance efficacy by acting upon different cellular targets, may reduce toxicity and minimize the risk of drug resistance. Therefore, we investigated the activity of benznidazole (Bz) in combination with the diamidine prodrug DB289 and in combination with the arylimidamide DB766 upon T. cruzi infection in vivo. The oral treatment of T.cruzi-infected mice with DB289 and Benznidazole (Bz) alone reduced the number of circulating parasites compared with untreated mice by about 70% and 90%, respectively. However, the combination of these two compounds decreased the parasitemia by 99% and protected against animal mortality by 100%, but without providing a parasitological cure. When Bz (p.o) was combined with DB766 (via ip route), at least a 99.5% decrease in parasitemia levels was observed. DB766+Bz also provided 100% protection against mice mortality while Bz alone provided about 87% protection. This combined therapy also reduced the tissular lesions induced by T. cruzi infection: Bz alone reduced GPT and CK plasma levels by about 12% and 78% compared to untreated mice group, the combination of Bz with DB766 resulted in a reduction of GPT and CK plasma levels of 56% and 91%. Cure assessment through hemocultive and PCR approaches showed that Bz did not provide a parasitological cure, however, DB766 alone or associated with Bz cured ≥13% of surviving animals

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

A century after its discovery, Chagas disease, caused by the parasite Trypanosoma cruzi, still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The polymerase chain reaction (PCR) has been proposed as a sensitive laboratory tool for detection of T. cruzi infection and monitoring of parasitological treatment outcome. However, high variation in accuracy and lack of international quality controls has precluded reliable applications in the clinical practice and comparisons of data among cohorts and geographical regions. In an effort towards harmonization of PCR strategies, 26 expert laboratories from 16 countries evaluated their current PCR procedures against sets of control samples, composed by serial dilutions of T.cruzi DNA from culture stocks belonging to different lineages, human blood spiked with parasite cells and blood samples from Chagas disease patients. A high variability in sensitivities and specificities was found among the 48 reported PCR tests. Out of them, four tests with best performance were selected and further evaluated. This study represents a crucial first step towards device of a standardized operative procedure for T. cruzi PCR.Fil: Schijman, Alejandro G. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh); Argentina.Fil: Bisio, Margarita. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh); Argentina.Fil: Orellana, Liliana. Universidad de Buenos Aires. Instituto de Cálculo; Argentina.Fil: Sued, Mariela. Universidad de Buenos Aires. Instituto de Cálculo; Argentina.Fil: Duffy, Tomás. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh); Argentina.Fil: Mejia Jaramillo, Ana M. Universidad de Antioquia. Grupo Chagas; Colombia.Fil: Cura, Carolina. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET). Laboratorio de Biología Molecular de la Enfermedad de Chagas (LabMECh); Argentina.Fil: Auter, Frederic. French Blood Services; Francia.Fil: Veron, Vincent. Universidad de Parasitología. Laboratorio Hospitalario; Guayana Francesa.Fil: Qvarnstrom, Yvonne. Centers for Disease Control. Department of Parasitic Diseases; Estados Unidos.Fil: Deborggraeve, Stijn. Institute of Tropical Medicine; Bélgica.Fil: Hijar, Gisely. Instituto Nacional de Salud; Perú.Fil: Zulantay, Inés. Facultad de Medicina; Chile.Fil: Lucero, Raúl Horacio. Universidad Nacional del Nordeste; Argentina.Fil: Velázquez, Elsa. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Fil: Tellez, Tatiana. Universidad Mayor de San Simon. Centro Universitario de Medicina Tropical; Bolivia.Fil: Sanchez Leon, Zunilda. Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud; Paraguay.Fil: Galvão, Lucia. Faculdade de Farmácia; Brasil.Fil: Nolder, Debbie. Hospital for Tropical Diseases. London School of Tropical Medicine and Hygiene Department of Clinical Parasitology; Reino Unido.Fil: Monje Rumi, María. Universidad Nacional de Salta. Laboratorio de Patología Experimental; Argentina.Fil: Levi, José E. Hospital Sirio Libanês. Blood Bank; Brasil.Fil: Ramirez, Juan D. Universidad de los Andes. Centro de Investigaciones en Microbiología y Parasitología Tropical; Colombia.Fil: Zorrilla, Pilar. Instituto Pasteur; Uruguay.Fil: Flores, María. Instituto de Salud Carlos III. Centro de Mahahonda; España.Fil: Jercic, Maria I. Instituto Nacional De Salud. Sección Parasitología; Chile.Fil: Crisante, Gladys. Universidad de los Andes. Centro de Investigaciones Parasitológicas J.F. Torrealba; Venezuela.Fil: Añez, Néstor. Universidad de los Andes. Centro de Investigaciones Parasitológicas J.F. Torrealba; Venezuela.Fil: De Castro, Ana M. Universidade Federal de Goiás. Instituto de Patologia Tropical e Saúde Pública (IPTSP); Brasil.Fil: Gonzalez, Clara I. Universidad Industrial de Santander. Grupo de Inmunología y Epidemiología Molecular (GIEM); Colombia.Fil: Acosta Viana, Karla. Universidad Autónoma de Yucatán. Departamento de Biomedicina de Enfermedades Infecciosas y Parasitarias Laboratorio de Biología Celular; México.Fil: Yachelini, Pedro. Universidad Católica de Santiago del Estero. Instituto de Biomedicina; Argentina.Fil: Torrico, Faustino. Universidad Mayor de San Simon. Centro Universitario de Medicina Tropical; Bolivia.Fil: Robello, Carlos. Instituto Pasteur; Uruguay.Fil: Diosque, Patricio. Universidad Nacional de Salta. Laboratorio de Patología Experimental; Argentina.Fil: Triana Chavez, Omar. Universidad de Antioquia. Grupo Chagas; Colombia.Fil: Aznar, Christine. Universidad de Parasitología. Laboratorio Hospitalario; Guayana Francesa.Fil: Russomando, Graciela. Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud; Paraguay.Fil: Büscher, Philippe. Institute of Tropical Medicine; Bélgica.Fil: Assal, Azzedine. French Blood Services; Francia.Fil: Guhl, Felipe. Universidad de los Andes. Centro de Investigaciones en Microbiología y Parasitología Tropical; Colombia.Fil: Sosa Estani, Sergio. ANLIS Dr.C.G.Malbrán. Centro Nacional de Diagnóstico e Investigación en Endemo-Epidemias; Argentina.Fil: DaSilva, Alexandre. Centers for Disease Control. Department of Parasitic Diseases; Estados Unidos.Fil: Britto, Constança. Instituto Oswaldo Cruz/FIOCRUZ. Laboratório de Biologia Molecular e Doenças Endêmicas; Brasil.Fil: Luquetti, Alejandro. Laboratório de Pesquisa de Doença de Chagas; Brasil.Fil: Ladzins, Janis. World Health Organization (WHO). Special Programme for Research and Training in Tropical Diseases (TDR); Suiza