Noninvasive detection of Zika virus in mosquito excreta sampled from wild mosquito populations in French Guiana.

Arboviruses can be difficult to detect in the field due to relatively low prevalence in mosquito populations. The discovery that infected mosquitoes can release viruses in both their saliva and excreta gave rise to low-cost methods for the detection of arboviruses during entomological surveillance. We implemented both saliva and excreta-based entomological surveillance during the emergence of Zika virus (ZIKV) in French Guiana in 2016 by trapping mosquitoes around households of symptomatic cases with confirmed ZIKV infection. ZIKV was detected in mosquito excreta and not in mosquito saliva in 1 trap collection out of 85 (1.2%). One female Ae. aegypti L. (Diptera: Culicidae) was found with a ZIKV systemic infection in the corresponding trap. The lag time between symptom onset in a ZIKV-infected individual living near the trap site and ZIKV detection in this mosquito was 1 wk. These results highlight the potential of detection in excreta from trapped mosquitoes as a sensitive and cost-effective method to non invasively detect arbovirus circulation.

An annotated wing interferential pattern dataset of dipteran insects of medical interest for deep learning.

Several Diptera species are known to transmit pathogens of medical and veterinary interest. However, identifying these species using conventional methods can be time-consuming, labor-intensive, or expensive. A computer vision-based system that uses Wing interferential patterns (WIPs) to identify these insects could solve this problem. This study introduces a dataset for training and evaluating a recognition system for dipteran insects of medical and veterinary importance using WIPs. The dataset includes pictures of Culicidae, Calliphoridae, Muscidae, Tabanidae, Ceratopogonidae, and Psychodidae. The dataset is complemented by previously published datasets of Glossinidae and some Culicidae members. The new dataset contains 2,399 pictures of 18 genera, with each genus documented by a variable number of species and annotated as a class. The dataset covers species variation, with some genera having up to 300 samples.

Species identification of phlebotomine sandflies using deep learning and wing interferential pattern (WIP).

Sandflies (Diptera; Psychodidae) are medical and veterinary vectors that transmit diverse parasitic, viral, and bacterial pathogens. Their identification has always been challenging, particularly at the specific and sub-specific levels, because it relies on examining minute and mostly internal structures. Here, to circumvent such limitations, we have evaluated the accuracy and reliability of Wing Interferential Patterns (WIPs) generated on the surface of sandfly wings in conjunction with deep learning (DL) procedures to assign specimens at various taxonomic levels. Our dataset proves that the method can accurately identify sandflies over other dipteran insects at the family, genus, subgenus, and species level with an accuracy higher than 77.0%, regardless of the taxonomic level challenged. This approach does not require inspection of internal organs to address identification, does not rely on identification keys, and can be implemented under field or near-field conditions, showing promise for sandfly pro-active and passive entomological surveys in an era of scarcity in medical entomologists.

Wing Interferential Patterns (WIPs) and machine learning for the classification of some Aedes species of medical interest.

Hematophagous insects belonging to the Aedes genus are proven vectors of viral and filarial pathogens of medical interest. Aedes albopictus is an increasingly important vector because of its rapid worldwide expansion. In the context of global climate change and the emergence of zoonotic infectious diseases, identification tools with field application are required to strengthen efforts in the entomological survey of arthropods with medical interest. Large scales and proactive entomological surveys of Aedes mosquitoes need skilled technicians and/or costly technical equipment, further puzzled by the vast amount of named species. In this study, we developed an automatic classification system of Aedes species by taking advantage of the species-specific marker displayed by Wing Interferential Patterns. A database holding 494 photomicrographs of 24 Aedes spp. from which those documented with more than ten pictures have undergone a deep learning methodology to train a convolutional neural network and test its accuracy to classify samples at the genus, subgenus, and species taxonomic levels. We recorded an accuracy of 95% at the genus level and > 85% for two (Ochlerotatus and Stegomyia) out of three subgenera tested. Lastly, eight were accurately classified among the 10 Aedes sp. that have undergone a training process with an overall accuracy of > 70%. Altogether, these results demonstrate the potential of this methodology for Aedes species identification and will represent a tool for the future implementation of large-scale entomological surveys.

Deep learning and wing interferential patterns identify Anopheles species and discriminate amongst Gambiae complex species.

We present a new and innovative identification method based on deep learning of the wing interferential patterns carried by mosquitoes of the Anopheles genus to classify and assign 20 Anopheles species, including 13 malaria vectors. We provide additional evidence that this approach can identify Anopheles spp. with an accuracy of up to 100% for ten out of 20 species. Although, this accuracy was moderate (> 65%) or weak (50%) for three and seven species. The accuracy of the process to discriminate cryptic or sibling species is also assessed on three species belonging to the Gambiae complex. Strikingly, An. gambiae, An. arabiensis and An. coluzzii, morphologically indistinguishable species belonging to the Gambiae complex, were distinguished with 100%, 100%, and 88% accuracy respectively. Therefore, this tool would help entomological surveys of malaria vectors and vector control implementation. In the future, we anticipate our method can be applied to other arthropod vector-borne diseases.

Wing Interferential Patterns (WIPs) and machine learning, a step toward automatized tsetse (Glossina spp.) identification.

A simple method for accurately identifying Glossina spp in the field is a challenge to sustain the future elimination of Human African Trypanosomiasis (HAT) as a public health scourge, as well as for the sustainable management of African Animal Trypanosomiasis (AAT). Current methods for Glossina species identification heavily rely on a few well-trained experts. Methodologies that rely on molecular methodologies like DNA barcoding or mass spectrometry protein profiling (MALDI TOFF) haven't been thoroughly investigated for Glossina sp. Nevertheless, because they are destructive, costly, time-consuming, and expensive in infrastructure and materials, they might not be well adapted for the survey of arthropod vectors involved in the transmission of pathogens responsible for Neglected Tropical Diseases, like HAT. This study demonstrates a new type of methodology to classify Glossina species. In conjunction with a deep learning architecture, a database of Wing Interference Patterns (WIPs) representative of the Glossina species involved in the transmission of HAT and AAT was used. This database has 1766 pictures representing 23 Glossina species. This cost-effective methodology, which requires mounting wings on slides and using a commercially available microscope, demonstrates that WIPs are an excellent medium to automatically recognize Glossina species with very high accuracy.

A New High-Throughput Tool to Screen Mosquito-Borne Viruses in Zika Virus Endemic/Epidemic Areas.

Mosquitoes are vectors of arboviruses affecting animal and human health. Arboviruses circulate primarily within an enzootic cycle and recurrent spillovers contribute to the emergence of human-adapted viruses able to initiate an urban cycle involving anthropophilic mosquitoes. The increasing volume of travel and trade offers multiple opportunities for arbovirus introduction in new regions. This scenario has been exemplified recently with the Zika pandemic. To incriminate a mosquito as vector of a pathogen, several criteria are required such as the detection of natural infections in mosquitoes. In this study, we used a high-throughput chip based on the BioMark™ Dynamic arrays system capable of detecting 64 arboviruses in a single experiment. A total of 17,958 mosquitoes collected in Zika-endemic/epidemic countries (Brazil, French Guiana, Guadeloupe, Suriname, Senegal, and Cambodia) were analyzed. Here we show that this new tool can detect endemic and epidemic viruses in different mosquito species in an epidemic context. Thus, this fast and low-cost method can be suggested as a novel epidemiological surveillance tool to identify circulating arboviruses.

Immunodetection and molecular determination of visceral and cutaneous Leishmania infection using patients' urine.

The diagnosis of leishmaniasis relies mainly on the use of invasive processes, to collect the biological material for detecting Leishmania parasites. Body fluids, which can be collected by non-invasive process, would greatly facilitate the leishmaniasis diagnosis. In the present study, we investigated the potency of urine immunoblotting to diagnose cutaneous and visceral leishmaniasis and we compared with routine molecular methods. A total of 80 samples, including 40 sera and their 40 corresponding urine samples were collected from 37 suspected patients with cutaneous and visceral leishmaniasis, and 3 healthy individuals (as control), in Ilam and Ardabil provinces of Iran. All sera and urine samples were analyzed, using immunoblotting. The confirmation of leishmaniasis infection was performed, using conventional and quantitative PCRs as well as by sequencing the amplicons. Among 37 suspected patients, 23 patients presented cutaneous lesions (CL) and 14 exhibited clinical symptoms reminiscent of visceral leishmaniasis (L. infantum). Among cutaneous patients, 15 were positive for zoonotic cutaneous leishmaniasis (L. major), and eight for anthroponotic cutaneous leishmaniasis (L. tropica). Molecular quantification of Leishmania parasites was performed on sera, urines and cutaneous biopsies of CL and VL patients, demonstrating that parasite load is lower in urines, compared to sera or biopsy. DNA can be detected in 20 out of 23 (86.9%) CL urine samples and in 13 out of 14 (92.8%) VL urine samples. Immunodetection analysis demonstrates that 22 out of 23 (95.6%) sera from CL patients and all patients suspected with VL are positive. For urine samples, 18 out of 23 (78.2%) urine of CL patients and 13 out of 14 (92.8%) urine of VL patients were positive, using Western blot. Therefore, immunodetection and molecular analysis using urine samples can be used as a diagnostic tool for surveying cutaneous and visceral leishmaniasis.

Leishmania infections: Molecular targets and diagnosis.

Progress in the diagnosis of leishmaniases depends on the development of effective methods and the discovery of suitable biomarkers. We propose firstly an update classification of Leishmania species and their synonymies. We demonstrate a global map highlighting the geography of known endemic Leishmania species pathogenic to humans. We summarize a complete list of techniques currently in use and discuss their advantages and limitations. The available data highlights the benefits of molecular markers in terms of their sensitivity and specificity to quantify variation from the subgeneric level to species complexes, (sub) species within complexes, and individual populations and infection foci. Each DNA-based detection method is supplied with a comprehensive description of markers and primers and proposal for a classification based on the role of each target and primer in the detection, identification and quantification of leishmaniasis infection. We outline a genome-wide map of genes informative for diagnosis that have been used for Leishmania genotyping. Furthermore, we propose a classification method based on the suitability of well-studied molecular markers for typing the 21 known Leishmania species pathogenic to humans. This can be applied to newly discovered species and to hybrid strains originating from inter-species crosses. Developing more effective and sensitive diagnostic methods and biomarkers is vital for enhancing Leishmania infection control programs.

Molecular characterization of Wolbachia infection in bed bugs (Cimex lectularius) collected from several localities in France.

Wolbachia symbionts are maternally inherited intracellular bacteria that have been detected in numerous insects including bed bugs. The objective of this study, the first epidemiological study in Europe, was to screen Wolbachia infection among Cimex lectularius collected in the field, using PCR targeting the surface protein gene (wsp), and to compare obtained Wolbachia strains with those reported from laboratory colonies of C. lectularius as well as other Wolbachia groups. For this purpose, 284 bed bug specimens were caught and studied from eight different regions of France including the suburbs of Paris, Bouches-du-Rhône, Lot-et-Garonne, and five localities in Alpes-Maritimes. Among the samples, 166 were adults and the remaining 118 were considered nymphs. In all, 47 out of 118 nymphs (40%) and 61 out of 166 adults (37%) were found positive on wsp screening. Among the positive cases, 10 samples were selected randomly for sequencing. The sequences had 100% homology with wsp sequences belonging to the F-supergroup strains of Wolbachia. Therefore, we confirm the similarity of Wolbachia strains detected in this epidemiological study to Wolbachia spp. reported from laboratory colonies of C. lectularius.

Experimental infection of Phlebotomus perniciosus by bioluminescent Leishmania infantum using murine model and artificial feeder

Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. In the present study, we carried out a screening on the experimental infection of Phlebotomus pernioucus by bioluminescent Leishmania infantum using murine model and artificial feeder. We developed a real-time polymerase chain reaction (RT-PCR)-based method to determine individually the number of Leishmania promastigotes fed by infected flies. Among 1840 new emerged female sand flies, 428 were fed on the infected mice. After their death, they were analysed individually by RT-PCR. Our results demonstrated just a single Leishmania positive female at sixth day post meal. A total of 1070 female sand flies were exposed in contact with artificial feeder containing the human blood with two different quantities of Leishmania parasites: 2.106/mL and 1.107/mL. A blood meal including 1.107/mL LUC-promastigotes was proposed to 270 females and 75 (28%) flies were engorged. Among them, 44 (59%) were positive by RT-PCR analysis, with a relative average of 50551 Leishmania parasites. In case of blood feeding of females with 2.106/mL promastigotes, 57 out of 800 (7%) females succeed to feed from artificial feeder which 22 (39%) were positive with a relative average of 6487 parasites.

Experimental infection of Phlebotomus perniciosus by bioluminescent Leishmania infantum using murine model and artificial feeder.

Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. In the present study, we carried out a screening on the experimental infection of Phlebotomus pernioucus by bioluminescent Leishmania infantum using murine model and artificial feeder. We developed a real-time polymerase chain reaction (RT-PCR)-based method to determine individually the number of Leishmania promastigotes fed by infected flies. Among 1840 new emerged female sand flies, 428 were fed on the infected mice. After their death, they were analysed individually by RT-PCR. Our results demonstrated just a single Leishmania positive female at sixth day post meal. A total of 1070 female sand flies were exposed in contact with artificial feeder containing the human blood with two different quantities of Leishmania parasites: 2.106/mL and 1.107/mL. A blood meal including 1.107/mL LUC-promastigotes was proposed to 270 females and 75 (28%) flies were engorged. Among them, 44 (59%) were positive by RT-PCR analysis, with a relative average of 50551 Leishmania parasites. In case of blood feeding of females with 2.106/mL promastigotes, 57 out of 800 (7%) females succeed to feed from artificial feeder which 22 (39%) were positive with a relative average of 6487 parasites.

A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies.

BACKGROUND: The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. METHODOLOGY AND PRINCIPAL FINDINGS: Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? CONCLUSIONS AND SIGNIFICANCE: We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

Spatial genetic structure and restricted gene flow in bed bugs (Cimex lectularius) populations in France.

Bed bugs (Cimex lectularius) are resurgent blood-sucking ectoparasites that are currently increasing at a rapid rate, particularly in industrialized countries, such as France. Despite the rapid spread of bed bugs, there is a lack of knowledge concerning the population structure and gene flow among C. lectularius populations in France. To fill this gap, a genetic study was conducted using 183 C. lectularius from 14 populations of bed bugs collected in a hotel and in individual apartments in the French Riviera and in the Saint Ouen suburb of Paris. The samples were genotyped using an isolated set of six polymorphic microsatellite loci, including five new loci which were newly isolated and chosen based on prior successful amplification, and one previously described loci (bb15b). The low genetic diversity observed in the samples (of one to five alleles) suggested that most of prospected populations were established by only a few individuals, possibly from a single mated female. The overall genetic differentiation was high and statistically significant (FST=0.556, p<0.0001). Pairwise analysis of the populations indicated significant genetic differentiation for 24 out of the 45 (53%) population pairs associated with FST, ranging from 0.0042 to 0.862. No obvious relationship between the level of genetic differentiation and the geographic distance was observed when considering all samples. Analysis with Structure software identified nine distinct genetic clusters within the dataset. These preliminary results help to elucidate the genetic structure and gene flow of C. lectularius populations in France; however, the available information should be expanded in further studies.

A review of data on laboratory colonies of bed bugs (Cimicidae), an insect of emerging medical relevance.

Cimicidae are hematophagous Heteroptera, feeding on human blood, that have been the subject of significant medical investigation. In particular, they have been colonized under laboratory conditions to study their medical relevance. Laboratory colonization of these bugs is a multifactorial phenomenon. Our goal was to conduct a comparative literature review to classify the published data, demonstrating preferred bed bug colony conditions. We show that physical factors including temperature, relative humidity and photoperiod, and physiological factors such as type and frequency of blood meals play important roles in laboratory colonies. Any change in these factors produces changes in life-cycle duration. Temperature and blood meal are the most important factors, with a marked impact on the life-cycle of laboratory populations, depending on the species. A wide range of temperatures (15-34 °C) and relative humidity (46-75%) with an average of 25 °C and 59% were found for these colonies. Two widely used blood sources for the colonies were rabbits and humans.

New microsatellite markers for multi-scale genetic studies on Phlebotomus ariasi Tonnoir, vector of Leishmania infantum in the Mediterranean area.

The population structure of Phlebotomus ariasi, a proven vector of Leishmania infantum in the Mediterranean area, is still poorly understood. Previously, only two microsatellite loci had been developed to study the population genetics of this species. Herein we use these loci and determined fourteen novel microsatellite loci, useful for the characterization of P. ariasi populations. These loci were tested on three populations of P. ariasi, two from France and one from Portugal. In addition, the usefulness of these markers was also evaluated on seven other sandfly species. We show, that for P. ariasi, 15 of the 16 loci selected were polymorphic, with a mean of 4.25 alleles and an observed heterozygosity of 0.299. Within the P. ariasi population of France, 11 loci were polymorphic, with an average of 2.44 alleles and an observed heterozygosity of 0.2177. The fixation index was moderate among the French populations but high between French and Portuguese populations. In addition, eight loci were also found to be amplifiable in six other Phlebotomus species. These results demonstrate the usefulness of this new set of microsatellite loci for population structure and molecular ecology studies of P. ariasi at various spatial scales, but also of other sandfly species.