A Point Mutation V419L in the Sodium Channel Gene from Natural Populations of Aedes aegypti Is Involved in Resistance to λ-Cyhalothrin in Colombia

Resistance to pyrethroids in mosquitoes is mainly caused by target site insensitivity known as knockdown resistance (kdr). In this work, we examined the point mutations present in portions of domains I, II, III, and IV of the sodium channel gene in Aedes aegypti mosquitoes from three Colombian municipalities. A partial region coding for the sodium channel gene from resistant mosquitoes was sequenced, and a simple allele-specific PCR-based assay (AS-PCR) was used to analyze mutations at the population level. The previously reported mutations, V1016I and F1534C, were found with frequencies ranging from 0.04 to 0.41, and 0.56 to 0.71, respectively, in the three cities. Moreover, a novel mutation, at 419 codon (V419L), was found in Ae. aegypti populations from Bello, Riohacha and Villavicencio cities with allelic frequencies of 0.06, 0.36, and 0.46, respectively. Interestingly, the insecticide susceptibility assays showed that mosquitoes from Bello were susceptible to λ-cyhalothrin pyrethroid whilst those from Riohacha and Villavicencio were resistant. A positive association between V419L and V1016I mutations with λ-cyhalothrin resistance was established in Riohacha and Villavicencio. The frequency of the F1534C was high in the three populations, suggesting that this mutation could be conferring resistance to insecticides other than λ-cyhalothrin, particularly type I pyrethroids. Further studies are required to confirm this hypothesis

Expansive and Diverse Phenotypic Landscape of Field Aedes aegypti (Diptera: Culicidae) Larvae with Differential Susceptibility to Temephos: Beyond Metabolic Detoxification

Arboviruses including dengue, Zika, and chikungunya are amongst the most significant public health concerns worldwide. Arbovirus control relies on the use of insecticides to control the vector mosquito Aedes aegypti (Linnaeus), the success of which is threatened by widespread insecticide resistance. The work presented here profiled the gene expression of Ae. aegypti larvae from field populations of Ae. aegypti with differential susceptibility to temephos originating from two Colombian urban locations, Bello and Cúcuta, previously reported to have distinctive disease incidence, socioeconomics, and climate. We demonstrated that an exclusive field-to-lab (Ae. aegypti strain New Orleans) comparison generates an over estimation of differential gene expression (DGE) and that the inclusion of a geographically relevant field control yields a more discrete, and likely, more specific set of genes. The composition of the obtained DGE profiles is varied, with commonly reported resistance associated genes including detoxifying enzymes having only a small representation. We identify cuticle biosynthesis, ion exchange homeostasis, an extensive number of long noncoding RNAs, and chromatin modelling among the differentially expressed genes in field resistant Ae. aegypti larvae. It was also shown that temephos resistant larvae undertake further gene expression responses when temporarily exposed to temephos. The results from the sampling triangulation approach here contribute a discrete DGE profiling with reduced noise that permitted the observation of a greater gene diversity, increasing the number of potential targets for the control of insecticide resistant mosquitoes and widening our knowledge base on the complex phenotypic network of the Ae. aegypti response to insecticides

Rapid Evaporative Ionization Mass Spectrometry (REIMS): a Potential and Rapid Tool for the Identification of Insecticide Resistance in Mosquito Larvae

Insecticide resistance is a significant challenge facing the successful control of mosquito vectors globally. Bioassays are currently the only method for phenotyping resistance. They require large numbers of mosquitoes for testing, the availability of a susceptible comparator strain, and often insectary facilities. This study aimed to trial the novel use of rapid evaporative ionization mass spectrometry (REIMS) for the identification of insecticide resistance in mosquitoes. No sample preparation is required for REIMS and analysis can be rapidly conducted within hours. Temephos resistant Aedes aegypti (Linnaeus) larvae from Cúcuta, Colombia and temephos susceptible larvae from two origins (Bello, Colombia, and the lab reference strain New Orleans) were analyzed using REIMS. We tested the ability of REIMS to differentiate three relevant variants: population source, lab versus field origin, and response to insecticide. The classification of these data was undertaken using linear discriminant analysis (LDA) and random forest. Classification models built using REIMS data were able to differentiate between Ae. aegypti larvae from different populations with 82% (±0.01) accuracy, between mosquitoes of field and lab origin with 89% (±0.01) accuracy and between susceptible and resistant larvae with 85% (±0.01) accuracy. LDA classifiers had higher efficiency than random forest with this data set. The high accuracy observed here identifies REIMS as a potential new tool for rapid identification of resistance in mosquitoes. We argue that REIMS and similar modern phenotyping alternatives should complement existing insecticide resistance management tools

Glycosylation of Trypanosoma cruzi TcI antigen reveals recognition by chagasic sera

Chagas disease is considered the most important parasitic disease in Latin America. The protozoan agent, Trypanosoma cruzi, comprises six genetic lineages, TcI-TcVI. Genotyping to link lineage(s) to severity of cardiomyopathy and gastrointestinal pathology is impeded by the sequestration and replication of T. cruzi in host tissues. We describe serology specific for TcI, the predominant lineage north of the Amazon, based on expression of recombinant trypomastigote small surface antigen (gTSSA-I) in the eukaryote Leishmania tarentolae, to allow realistic glycosylation and structure of the antigen. Sera from TcI-endemic regions recognised gTSSA-I (74/146; 50.7%), with no cross reaction with common components of gTSSA-II/V/VI recombinant antigen. Antigenicity was abolished by chemical (periodate) oxidation of gTSSA-I glycosylation but retained after heat-denaturation of conformation. Conversely, non-specific recognition of gTSSA-I by non-endemic malaria sera was abolished by heat-denaturation. TcI-specific serology facilitates investigation between lineage and diverse clinical presentations. Glycosylation cannot be ignored in the search for immunogenic antigens

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

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

Association of Midgut Bacteria and Their Metabolic Pathways with Zika Infection and Insecticide Resistance in Colombian <i>Aedes aegypti</i> Populations

Introduction: Aedes aegypti is the vector of several arboviruses such as dengue, Zika, and chikungunya. In 2015–16, Zika virus (ZIKV) had an outbreak in South America associated with prenatal microcephaly and Guillain-Barré syndrome. This mosquito’s viral transmission is influenced by microbiota abundance and diversity and its interactions with the vector. The conditions of cocirculation of these three arboviruses, failure in vector control due to insecticide resistance, limitations in dengue management during the COVID-19 pandemic, and lack of effective treatment or vaccines make it necessary to identify changes in mosquito midgut bacterial composition and predict its functions through the infection. Its study is fundamental because it generates knowledge for surveillance of transmission and the risk of outbreaks of these diseases at the local level. Methods: Midgut bacterial compositions of females of Colombian Ae. aegypti populations were analyzed using DADA2 Pipeline, and their functions were predicted with PICRUSt2 analysis. These analyses were done under the condition of natural ZIKV infection and resistance to lambda–cyhalothrin, alone and in combination. One-step RT-PCR determined the percentage of ZIKV-infected females. We also measured the susceptibility to the pyrethroid lambda–cyhalothrin and evaluated the presence of the V1016I mutation in the sodium channel gene. Results: We found high ZIKV infection rates in Ae. aegypti females from Colombian rural municipalities with deficient water supply, such as Honda with 63.6%. In the face of natural infection with an arbovirus such as Zika, the diversity between an infective and non-infective form was significantly different. Bacteria associated with a state of infection with ZIKV and lambda–cyhalothrin resistance were detected, such as the genus Bacteroides, which was related to functions of pathogenicity, antimicrobial resistance, and bioremediation of insecticides. We hypothesize that it is a vehicle for virus entry, as it is in human intestinal infections. On the other hand, Bello, the only mosquito population classified as susceptible to lambda–cyhalothrin, was associated with bacteria related to mucin degradation functions in the intestine, belonging to the Lachnospiraceae family, with the genus Dorea being increased in ZIKV-infected females. The Serratia genus presented significantly decreased functions related to phenazine production, potentially associated with infection control, and control mechanism functions for host defense and quorum sensing. Additionally, Pseudomonas was the genus principally associated with functions of the degradation of insecticides related to tryptophan metabolism, ABC transporters with a two-component system, efflux pumps, and alginate synthesis. Conclusions: Microbiota composition may be modulated by ZIKV infection and insecticide resistance in Ae. aegypti Colombian populations. The condition of resistance to lambda–cyhalothrin could be inducing a phenome of dysbiosis in field Ae. aegypti affecting the transmission of arboviruses

The Midgut Microbiota of Colombian Aedes aegypti Populations with Different Levels of Resistance to the Insecticide Lambda-cyhalothrin

Insecticide resistance in Aedes aegypti populations is a problem that hinders vector control and dengue prevention programs. In this study, we determined the susceptibility of Ae. aegypti populations from six Colombian regions to the pyrethroid lambda-cyhalothrin and evaluated the presence of the V1016I mutation in the sodium channel gene, which has been broadly involved in the resistance to this insecticide. The diversity of the gut microbiota of these mosquito populations was also analyzed. Only mosquitoes from Bello were susceptible to lambda-cyhalothrin and presented a lower allelic frequency of the V1016I mutation. Remarkably, there was not an important change in allelic frequencies among populations with different resistance ratios, indicating that other factors or mechanisms contributed to the resistant phenotype. Treatment of mosquitoes with antibiotics led us to hypothesize that the intestinal microbiota could contribute to the resistance to lambda-cyhalothrin. Beta diversity analysis showed significant differences in the species of bacteria present between susceptible and resistant populations. We identified 14 OTUs of bacteria that were unique in resistant mosquitoes. We propose that kdr mutations are important in the development of resistance to lambda-cyhalothrin at low insecticide concentrations but insect symbionts could play an essential role in the metabolization of pyrethroid insecticides at higher concentrations, contributing to the resistant phenotype in Ae. aegypti

Acute Pediatric Chagas Disease in Antioquia, Colombia: A Geographic Location of Suspected Oral Transmission

Chagas disease, Trypanosoma cruzi infection, is an insidious cause of heart failure in Latin America. Early diagnosis and treatment are critical to prevent irreversible myocardial damage that progressively accumulates over decades. Several structural barriers account for the less than 1% of cases in Colombia being treated, including poor physician knowledge, especially considering that some regions are considered non-endemic. The two cases reported here represent an emerging epidemiologic scenario associated with pediatric Chagas disease. Both cases are suspected oral transmitted parasitic infection in a geographic region of Colombia (Andean region of Antioquia) where no previous oral transmission of Chagas disease had been reported. Their clinical histories and course of disease are presented here to increase physician awareness of the epidemiologic risk factors and clinical manifestations associated with pediatric oral Chagas disease in Antioquia department, Colombia

Genomic Analysis of Colombian Leishmania panamensis strains with different level of virulence

Abstract The establishment of Leishmania infection in mammalian hosts and the subsequent manifestation of clinical symptoms require internalization into macrophages, immune evasion and parasite survival and replication. Although many of the genes involved in these processes have been described, the genetic and genomic variability associated to differences in virulence is largely unknown. Here we present the genomic variation of four Leishmania (Viannia) panamensis strains exhibiting different levels of virulence in BALB/c mice and its application to predict novel genes related to virulence. De novo DNA sequencing and assembly of the most virulent strain allowed comparative genomics analysis with sequenced L. (Viannia) panamensis and L. (Viannia) braziliensis strains, and showed important variations at intra and interspecific levels. Moreover, the mutation detection and a CNV search revealed both base and structural genomic variation within the species. Interestingly, we found differences in the copy number and protein diversity of some genes previously related to virulence. Several machine-learning approaches were applied to combine previous knowledge with features derived from genomic variation and predict a curated set of 66 novel genes related to virulence. These genes can be prioritized for validation experiments and could potentially become promising drug and immune targets for the development of novel prophylactic and therapeutic interventions