Monitoring mosquito richness in an understudied area: can environmental DNA metabarcoding be a complementary approach to adult trapping?

Mosquito surveillance programmes are essential to assess the risks of local vector-borne disease outbreaks as well as for early detection of mosquito invasion events. Surveys are usually performed with traditional sampling tools (i.e., ovitraps and dipping method for immature stages or light or decoy traps for adults). Over the past decade, numerous studies have highlighted that environmental DNA (eDNA) sampling can enhance invertebrate species detection and provide community composition metrics. However, the usefulness of eDNA for detection of mosquito species has, to date, been largely neglected. Here, we sampled water from potential larval breeding sites along a gradient of anthropogenic perturbations, from the core of an oil palm plantation to the rainforest on São Tomé Island (Gulf of Guinea, Africa). We showed that (i) species of mosquitoes could be detected via metabarcoding mostly when larvae were visible, (ii) larvae species richness was greater using eDNA than visual identification and (iii) new mosquito species were also detected by the eDNA approach. We provide a critical discussion of the pros and cons of eDNA metabarcoding for monitoring mosquito species diversity and recommendations for future research directions that could facilitate the adoption of eDNA as a tool for assessing insect vector communities.

Intelligibility of pseudo-whispered speech after total laryngectomy.

This study aimed to assess the intelligibility of so-called 'pseudo-whispered speech' (pseudo-WS), as produced without voice nor pulmonic airstream by some alaryngeal patients prior to rehabilitation. Several perception tests were submitted to three experienced clinicians and three naive listeners, relying on the speech samples of 20 French native speakers: 10 alaryngeal speakers, solely using pseudo-WS when starting speech therapy up to six months after total laryngectomy, and 10 control speakers, recorded in the closest speech mode available, whispered speech (WS). Experts were asked to identify consonants (C) in the /a/+C+/a/ context and to rate intelligibility, unintended additive noise, and fluency on a likert-scale, while naive listeners completed a quantitative test of intelligibility. Intelligibility of WS was found to be high, with scores ranging from 46.33/54 to 53.67/54 (median 52.5, interquartile range 2.33) for the quantitative test, and segmental intelligibility ranging from 68.75% to 94.79% (median 87.5, interquartile range 17.71). Segmental confusion affected voicing in favour of unvoiced consonants, as previously reported in the literature. By contrast, intelligibility of pseudo-WS was found to be poor, with scores ranging from 1/54 (unintelligibility) to 28.33/54 (median 8.66, interquartile range 14.67) for the quantitative test, and segmental intelligibility ranging from 3.13% to 28.13% (median 9.24, interquartile range 14.58). Segmental intelligibility was not uniformly affected: stops, labials and unvoiced consonants were better identified than other categories. Finally, a significant correlation was found between global intelligibility and articulatory precision, while unintended additive noise and fluency seemed to play no role.

Can microplastics facilitate the emergence of infectious diseases?

Plastic pollution is a major environmental problem. Small plastic particles (called microplastics) have been reported to have pernicious effects on human and wildlife health, by altering physiological functions (e.g., immunity, metabolism) and interfering with commensal microorganisms. However, in addition to these direct toxic effects, we suggest that microplastic pollution might also exert deleterious effects, modifying (i) the exposure to pathogens (e.g., multi-drug resistant bacteria) and (ii) the dynamics of vector-borne diseases. Therefore, we argue that microplastics should be considered as a ubiquitous environmental hazard, potentially promoting the (re)emergence of infectious diseases. The implementation of multi- and interdisciplinary research projects are crucial to properly evaluate if microplastic pollution should be added to the current list of global health threats.

Clinical characteristics of singers attending a phoniatric outpatient clinic.

Purpose: Studies suggest that singers are over-represented in voice clinics and present a high risk of developing voice disorders. This retrospective study aims to describe the characteristics of 78 singers consulting a phoniatrician.Methods: In their medical files, data related to age, gender, occupational status, singing training, musical style, voice complaint, diagnosis, voice-quality grading (GRBAS) and treatment were gathered.Results: The patients were mostly female singers (87%). Non-professional singers (semi-professional included) represented 64%, professional singers 25% and students of singing 11%. The majority of singers were choristers (27%) and 22% were classical-style/oratorio-style singers. Two-thirds of the population had intensive vocal activity in speech or singing. Vocal endurance, somatosensory signs and difficulties with high pitches were the most frequent symptoms. Among the patients, 79% presented with singing-voice disorders with 85% of these having vocal fold lesions. Generally, their speaking voices were preserved. Vocal-folds nodules were the most prevalent pathology (37%) followed by sulcus (26%) and voice therapy was the main treatment.Conclusions: This study emphasizes the fact that singers have specific voice complaints related to their voice usage. The high occurrence of sulcus and other congenital-lesion suspicions, unusual in the general population consulting an ENT phoniatrician, seems to be rather specific for singers in agreement with the literature.

Natural-Product-Based Solutions for Tropical Infectious Diseases.

About half of the world's population and 80% of the world's biodiversity can be found in the tropics. Many diseases are specific to the tropics, with at least 41 diseases caused by endemic bacteria, viruses, parasites, and fungi. Such diseases are of increasing concern, as the geographic range of tropical diseases is expanding due to climate change, urbanization, change in agricultural practices, deforestation, and loss of biodiversity. While traditional medicines have been used for centuries in the treatment of tropical diseases, the active natural compounds within these medicines remain largely unknown. In this review, we describe infectious diseases specific to the tropics, including their causative pathogens, modes of transmission, recent major outbreaks, and geographic locations. We further review current treatments for these tropical diseases, carefully consider the biodiscovery potential of the tropical biome, and discuss a range of technologies being used for drug development from natural resources. We provide a list of natural products with antimicrobial activity, detailing the source organisms and their effectiveness as treatment. We discuss how technological advancements, such as next-generation sequencing, are driving high-throughput natural product screening pipelines to identify compounds with therapeutic properties. This review demonstrates the impact natural products from the vast tropical biome have in the treatment of tropical infectious diseases and how high-throughput technical capacity will accelerate this discovery process.

Metabarcoding options to study eukaryotic endoparasites of birds.

There is growing interest in the study of avian endoparasite communities, and metabarcoding is a promising approach to complement more conventional or targeted methods. In the case of eukaryotic endoparasites, phylogenetic diversity is extreme, with parasites from 4 kingdoms and 11 phyla documented in birds. We addressed this challenge by comparing different primer sets across 16 samples from 5 bird species. Samples consisted of blood, feces, and controlled mixes with known proportions of bird and nematode DNA. Illumina sequencing revealed that a 28S primer set used in combination with a custom blocking primer allowed detection of various plasmodiid parasites and filarioid nematodes in the blood, coccidia in the feces, as well as two potentially pathogenic fungal groups. When tested on the controlled DNA mixes, these primers also increased the proportion of nematode DNA by over an order of magnitude. An 18S primer set, originally designed to exclude metazoan sequences, was the most effective at reducing the relative number of avian DNA sequences and was the only one to detect Trypanosoma in the blood. Expectedly, however, it did not allow nematode detection and also failed to detect avian malaria parasites. This study shows that a 28S set including a blocking primer allows detection of several major and very diverse bird parasite clades, while reliable amplification of all major parasite groups may require a combination of markers. It helps clarify options for bird parasite metabarcoding, according to priorities in terms of the endoparasite clades and the ecological questions researchers wish to focus on.

Avian blood parasite richness decreases with major histocompatibility complex class I loci number.

Major histocompatibility complex (MHC) genes are among the most polymorphic in the vertebrate genome. The high allele diversity is believed to be maintained primarily by sexual and pathogen-mediated balancing selection. The number of MHC loci also varies greatly across vertebrates, most notably across birds. MHC proteins play key roles in presenting antigens on the cell surface for recognition by T cells, with class I proteins specifically targeting intracellular pathogens. Here, we explore the hypothesis that MHC class I diversity (measured as loci number) coevolves with haemosporidian parasite burden of the host. Using data on 54 bird species, we demonstrate that high-MHC class I diversity is associated with significantly lower richness of Plasmodium, Haemoproteus as well as overall haemosporidian parasite lineages, the former thus indicating more efficient protection against intracellular pathogens. Nonetheless, the latter associations were only detected when MHC diversity was assessed using cloning and not 454 pyrosequencing-based studies, nor across all genotyping methods combined. Our results indicate that high-MHC class I diversity might play a key role in providing qualitative resistance against diverse haemosporidian parasites in birds, but further clarification is needed for the origin of contrasting results when using different genotyping methods for MHC loci quantification.

Memory CD8+ T cell compartment associated with delayed onset of Plasmodium falciparum infection and better parasite control in sickle-cell trait children.

OBJECTIVES: Study of individuals with protection from Plasmodium falciparum (Pf) infection and clinical malaria, including individuals affected by the sickle-cell trait (HbAS), offers the potential to identify cellular targets that could be translated for therapeutic development. We previously reported the first involvement of cellular immunity in HbAS-associated relative protection and identified a novel subset of memory-activated NK cells that was enriched in HbAS children and associated with parasite control. We hypothesised that other memory cell subsets might distinguish the baseline profile of HbAS children and children with normal haemoglobin (HbAA). METHODS: Subsets of memory T cells and NK cells were analysed by flow cytometry in paired samples collected from HbAS and HbAA children, at baseline and during the first malaria episode of the ensuing transmission season. Correlations between cell frequencies and features of HbAS-mediated protection from malaria were determined. RESULTS: HbAS children displayed significantly higher frequency of memory CD8+ T cells at baseline than HbAA children. Baseline frequency of memory CD8+ T cells correlated with features of HbAS-mediated protection from malaria. Exploration of memory CD8+ T cell subsets revealed that central memory CD8+ T cell frequency was higher in HbAS children than in HbAA children. CONCLUSION: This study shows that HbAS children develop a larger memory CD8+ T cell compartment than HbAA children, and associates this compartment with better control of subsequent onset of infection and parasite density. Our data suggest that central memory CD8+ T cells may play an important role in the relative protection against malaria experienced by HbAS individuals, and further work to investigate this is warranted.

Th22 cells are efficiently recruited in the gut by CCL28 as an alternative to CCL20 but do not compensate for the loss of Th17 cells in treated HIV-1-infected individuals.

Gut CD4+ T cells are incompletely restored in most HIV-1-infected individuals on antiretroviral therapy, notably Th17 cells, a key subset in mucosal homeostasis. By contrast, gut Th22 cells are usually restored at normal frequencies. Th22 cells display a CCR6+CCR10+ phenotype and could thus respond to CCL20- and CCL28-mediated chemotaxis, while Th17 cells, which express CCR6 but not CCR10, depend on CCL20. Herein, we found that CCL28 is normally expressed by duodenal enterocytes of treated HIV-1-infected individuals, while CCL20 expression is blunted. Ex vivo, we showed that Th22 cells contribute to the reduction of CCL20 production by enterocytes through an IL-22- and IL-18-dependent mechanism. Th22 cells preferentially migrate via CCL20- rather than CCL28-mediated chemotaxis when both chemokines are available in the microenvironment. However, when the CCL20/CCL28 ratio drops, as in treated HIV-1-infected individuals, Th22 cells can migrate via the CCR10-CCL28 axis, as an alternative to CCR6-CCL20. This could explain the better reconstitution of gut Th22 compared with Th17 cells on antiretroviral therapy. Lastly, we assessed the relationships between the frequencies of gut Th17 and Th22 cells and inflammatory markers related to microbial translocation, and showed that Th22 cells do not compensate for the loss of Th17 cells in treated HIV-1-infected individuals.

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells.

Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8+ T cell peptide epitope hierarchy with as few as 1 × 104 cells. Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.

A novel population of memory-activated natural killer cells associated with low parasitaemia in Plasmodium falciparum-exposed sickle-cell trait children.

OBJECTIVES: The sickle-cell trait phenotype is associated with protection from malaria. Multiple molecular mechanisms have been proposed to explain this protection, but the role of the host immune system has been poorly investigated. We hypothesised that cellular immunity to malaria may develop differently in sickle-cell trait children (HbAS) and children with normal haemoglobin (HbAA) repeatedly exposed to Plasmodium falciparum (Pf). METHODS: Paired samples collected prior to the Pf transmission season and during the first malaria episode of the ensuing transmission season from HbAS and HbAA children were analysed by multiplex bead-based assay and comprehensive multi-dimensional flow cytometry profiling. RESULTS: Cellular immune profiles were enriched in HbAS relative to HbAA children before the start of the Pf transmission season, with a distinct NK subset. These cells were identified as a novel subset of memory-activated NK cells characterised by reduced expression of the ecto-enzyme CD38 as well as co-expression of high levels of HLA-DR and CD45RO. The frequency of this NK subset before the transmission season was negatively correlated with parasite density quantified during the first malaria episode of the ensuing transmission season. Functional assessment revealed that these CD38dim CD45RO+ HLA-DR+ NK cells represent a important source of IFN-γ. CONCLUSION: Our data suggest that this novel memory-activated NK cell subset may contribute to an accelerated and enhanced IFN-γ-mediated immune response and to control of parasite density in individuals with the sickle-cell trait. This distinct cellular immune profile may contribute to predispose HbAS children to a relative protection from malaria.

Perceptual Judgment of Voice Quality in Nondysphonic French Speakers: Effect of Task-, Speaker- and Listener-Related Variables.

PURPOSE: Several perceptual scales have been developed to assess voice quality in dysphonic voices, among which the Grade Roughness Breathiness Asthenia Strain and a Rate of Dysphonia scale is probably the most frequently used. However, this clinical tool has not been properly validated with a normophonic population yet. The aim of the present study was to provide a first set of reference data gathered from a normal population, to serve as a basis of comparison for vocologists and laryngologists working with French-speaking patients. A second goal was to investigate the influence on this normal voice dataset, of variables known to affect perceptual judgments of pathological voice. MATERIAL AND METHODS: Sustained vowels and sentences produced by 80 healthy, normophonic French native speakers were perceptually assessed by a panel of 18 raters (nine students, nine experts) using the Grade Roughness Breathiness Asthenia Strain and a Rate of Dysphonia scale. RESULTS: The average overall grade was close to 1 on the (0 to 3) scale, questioning the notion of "normal" voice as opposed to dysphonic voice. Rating reliability as well as perceptual scores were affected by task-, speaker-, and listener-related factors: speech stimuli led to better rating reliability and were judged less severely than voice stimuli; experts were slightly more reliable and less severe than students; older speakers were unanimously considered as more dysphonic. Multiple interactions between these factors were observed, confirming the multidimensional nature of voice quality.

Deciphering host immunity to malaria using systems immunology.

A century of conceptual and technological advances in infectious disease research has changed the face of medicine. However, there remains a lack of effective interventions and a poor understanding of host immunity to the most significant and complex pathogens, including malaria. The development of successful interventions against such intractable diseases requires a comprehensive understanding of host-pathogen immune responses. A major advance of the past decade has been a paradigm switch in thinking from the contemporary reductionist (gene-by-gene or protein-by-protein) view to a more holistic (whole organism) view. Also, a recognition that host-pathogen immunity is composed of complex, dynamic interactions of cellular and molecular components and networks that cannot be represented by any individual component in isolation. Systems immunology integrates the field of immunology with omics technologies and computational sciences to comprehensively interrogate the immune response at a systems level. Herein, we describe the system immunology toolkit and report recent studies deploying systems-level approaches in the context of natural exposure to malaria or controlled human malaria infection. We contribute our perspective on the potential of systems immunity for the rational design and development of effective interventions to improve global public health.

Casting a Wide Net around Immunity to Malaria Catches p53.

The mechanisms underlying acquisition of naturally acquired immunity to malaria are poorly understood. In this issue of Immunity, Tran and colleagues (2019) demonstrate that systems immunology is a powerful tool to decipher molecular and cellular components contributing to this immunity.

Human challenge models: tools to accelerate the development of malaria vaccines.

INTRODUCTION: Malaria challenge models, where healthy human volunteers are intentionally infected with Plasmodium species parasites under controlled conditions, can be undertaken in several well-defined ways. These challenge models enable evaluation of the kinetics of parasite growth and clearance, host-pathogen interactions and the host immune response. They can facilitate discovery of candidate diagnostic biomarkers and novel vaccine targets. As translational tools they can facilitate testing of candidate vaccines and drugs and evaluation of diagnostic tests. AREAS COVERED: Until recently, malaria human challenge models have been limited to only a few Plasmodium falciparum strains and used exclusively in malaria-naïve volunteers in non-endemic regions. Several recent advances include the use of alternate P. falciparum strains and other species of Plasmodia, as well as strains attenuated by chemical, radiation or genetic modification, and the conduct of studies in pre-exposed individuals. Herein, we discuss how this diversification is enabling more thorough vaccine efficacy testing and informing rational vaccine development. EXPERT OPINION: The ability to comprehensively evaluate vaccine efficacy in controlled settings will continue to accelerate the translation of candidate malaria vaccines to the clinic, and inform the development and optimisation of potential vaccines that would be effective against multiple strains in geographically and demographically diverse settings.

Diversity, prevalence and host specificity of avian parasites in southern Tunisian oases.

Oases are semi-natural woodlots surrounded by an inhospitable desert environment. This insular-like habitat system is known to support a mixture of sedentary and migratory bird species originating from different areas. However, little is known about the interactions between these birds and parasites. In this study, we investigated the diversity, prevalence and host specificity of avian haemosporidian parasites in southern Tunisian oases in two sedentary and common bird species, the laughing dove Spilopelia senegalensis and hybrid sparrow Passer domesticus × hispaniolensis, in six sites that differ regarding vegetation structure and distance to the coast. Two new Haemoproteus lineages, related to other Haemoproteus transmitted by biting midges, were detected in doves. With regard to sparrows, all detected parasites have previously been reported in other sparrow populations, except for one new Haemoproteus lineage. Our results also showed that densely vegetated sites were characterized by the higher prevalence of Plasmodium but a lower prevalence of Haemoproteus compared with less-vegetated sites. This is the first study aiming to explore avian parasites in the oasis habitat. Gathering data on a larger sample of oases with different sizes and isolation levels will be the next step to better understand factors shaping the transmission dynamics of avian parasites in such ecosystems.

Insularity effects on the assemblage of the blood parasite community of the birds from the Gulf of Guinea.

AIM: Lower species diversity, increased population densities and ecological niche enlargement are common characteristics of island faunas. However it remains to be determined if they extend to the parasite community. We tested if Haemosporidia parasite pressure varies between islands and the mainland with two different levels of analysis: i) at the host community level, and ii) with paired-species comparisons between islands and the mainland. LOCATION: Gulf of Guinea, West Africa. METHODS: We used molecular-based methods to identify avian Haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) to describe their diversity, prevalence, host specificity and their phylogenetic relationships in five islands of the Gulf of Guinea and in nearby mainland areas. RESULTS: We found reduced Haemosporidia diversity on islands for Haemoproteus and Leucocytozoon, but not for Plasmodium. In addition, lower parasite prevalence on islands was found using a paired-species approach. Although the mean host specificity of the parasite community on islands did not differ from the mainland, we found a very distinct parasite species assemblage on the islands, which was composed of both the most generalist and the most specialist lineages. MAIN CONCLUSIONS: This study supports the hypothesis that parasite pressure is reduced on islands. Colonization is made by generalists with high host switching capacities, with some subsequently evolving into highly specialised parasites. This suggests that 'taxon cycle' dynamics may explain the assemblage of insular parasite communities.

First record of Aedes albopictus (Skuse 1894) on São tomé island.

Biological invasions have critical impacts on native biodiversity and human societies and especially on oceanic islands that are fragile and threatened ecosystems. The invasive tiger mosquito Aedes (Stegomyia) albopictus (Skuse, 1894) native to Southeast Asia has been introduced during the past 30 years almost everywhere in the world, including the Americas, the Pacific, Europe and Africa. It has been reported for the first time in the Gulf of Guinea in 2000, first in Cameroon, then in Bioko Island in 2003 and more recently in Gabon in 2007. Here we report the first record of Ae. albopictus on São Tomé Island. Although we cannot estimate precisely the year of introduction on São Tomé Island, it most likely arrived within the last 10 years. By sequencing the mitochondrial cytochrome c oxidase gene from individual adults, we detected three haplotypes already present in mainland Africa. More studies are needed to explore the dynamics of its expansion and competition with insular native mosquitoes.

Avian malaria in a boreal resident species: long-term temporal variability, and increased prevalence in birds with avian keratin disorder.

The prevalence of vector-borne parasitic diseases is widely influenced by biological and ecological factors. Environmental conditions such as temperature and precipitation can have a marked effect on haemosporidian parasites (Plasmodium spp.) that cause malaria and those that cause other malaria-like diseases in birds. However, there have been few long-term studies monitoring haemosporidian infections in birds in northern latitudes, where weather conditions can be highly variable and the effects of climate change are becoming more pronounced. We used molecular methods to screen more than 2,000 blood samples collected from black-capped chickadees (Poecile atricapillus), a resident passerine bird. Samples were collected over a 10 year period, mostly during the non-breeding season, at seven sites in Alaska, USA. We tested for associations between Plasmodium prevalence and local environmental conditions including temperature, precipitation, site, year and season. We also evaluated the relationship between parasite prevalence and individual host factors of age, sex and presence or absence of avian keratin disorder. This disease, which causes accelerated keratin growth in the beak, provided a natural study system in which to test the interaction between disease state and malaria prevalence. Prevalence of Plasmodium infection varied by year, site, age and individual disease status but there was no support for an effect of sex or seasonal period. Significantly, birds with avian keratin disorder were 2.6 times more likely to be infected by Plasmodium than birds without the disorder. Interannual variation in the prevalence of Plasmodium infection at different sites was positively correlated with summer temperatures at the local but not statewide scale. Sequence analysis of the parasite cytochrome b gene revealed a single Plasmodium spp. lineage, P43. Our results demonstrate associations between prevalence of avian malaria and a variety of biological and ecological factors. These results also provide important baseline data that will be informative for predicting future changes in Plasmodium prevalence in the subarctic.