Detection of Wuchereria bancrofti DNA in wild caught vector and non-vector mosquitoes: implications for elimination of lymphatic filariasis.

BACKGROUND: Transmission Assessment Survey (TAS) is the WHO recommended method used for decision-making to stop or continue the MDA in lymphatic filariasis (LF) elimination programme. The WHO has also recommended Molecular Xenomonitoring (MX) of LF infection in vectors as an adjunct tool in settings under post-MDA or validation period. Screening of non-vectors by MX in post-MDA / validation settings could be useful to prevent a resurgence of LF infection, as there might be low abundance of vectors, especially in some seasons. In this study, we investigated the presence of LF infection in non-vectors in an area endemic for LF and has undergone many rounds of annual MDA with two drugs (Diethylcarbamazine and Albendazole, DA) and two rounds of triple drug regimens (Ivermectin + DA). METHODS AND RESULTS: Mosquitoes were collected from selected villages of Yadgir district in Karnataka state, India, during 2019. A total of 680 female mosquitoes were collected, identified morphologically by species and separated as pools. The female mosquitoes belonging to 3 species viz., Anopheles subpictus, Culex gelidus and Culex quinquefaciatus were separated, pooled, and the DNA extracted using less expensive method and followed by LDR based real-time PCR assay for detecting Wuchereria bancrofti infection in vector as well as non-vector mosquitoes. One pool out of 6 pools of An. subpictus, 2 pools out of 6 pools of Cx. gelidus, and 4 pools out of 8 pools of Cx. quinquefaciatus were found to be positive for W. bancrofti infection by RT-PCR. The infection rate in vectors and non-vectors was found to be 1.8% (95% CI: 0.5-4.2%) and 0.9% (95% CI: 0.2-2.3%), respectively. CONCLUSIONS: Our study showed that non-vectors also harbour W. bancrofti, thus opening an opportunity of using these mosquitoes as surrogate vectors for assessing risk of transmission to humans in LF endemic and post MDA areas.

Invasive snails, parasite spillback, and potential parasite spillover drive parasitic diseases of Hippopotamus amphibius in artificial lakes of Zimbabwe.

BACKGROUND: Humans impose a significant pressure on large herbivore populations, such as hippopotami, through hunting, poaching, and habitat destruction. Anthropogenic pressures can also occur indirectly, such as artificial lake creation and the subsequent introduction of invasive species that alter the ecosystem. These events can lead to drastic changes in parasite diversity and transmission, but generally receive little scientific attention. RESULTS: In order to document and identify trematode parasites of the common hippopotamus (Hippopotamus amphibius) in artificial water systems of Zimbabwe, we applied an integrative taxonomic approach, combining molecular diagnostics and morphometrics on archived and new samples. In doing so, we provide DNA reference sequences of the hippopotamus liver fluke Fasciola nyanzae, enabling us to construct the first complete Fasciola phylogeny. We describe parasite spillback of F. nyanzae by the invasive freshwater snail Pseudosuccinea columella, as a consequence of a cascade of biological invasions in Lake Kariba, one of the biggest artificial lakes in the world. Additionally, we report an unknown stomach fluke of the hippopotamus transmitted by the non-endemic snail Radix aff. plicatula, an Asian snail species that has not been found in Africa before, and the stomach fluke Carmyerius cruciformis transmitted by the native snail Bulinus truncatus. Finally, Biomphalaria pfeifferi and two Bulinus species were found as new snail hosts for the poorly documented hippopotamus blood fluke Schistosoma edwardiense. CONCLUSIONS: Our findings indicate that artificial lakes are breeding grounds for endemic and non-endemic snails that transmit trematode parasites of the common hippopotamus. This has important implications, as existing research links trematode parasite infections combined with other stressors to declining wild herbivore populations. Therefore, we argue that monitoring the anthropogenic impact on parasite transmission should become an integral part of wildlife conservation efforts.

Evaluating the Diagnostic Test Accuracy of Molecular Xenomonitoring Methods for Characterizing Community Burden of Lymphatic Filariasis.

BACKGROUND: Molecular xenomonitoring (MX), the detection of pathogen DNA in mosquitoes, is a recommended approach to support lymphatic filariasis (LF) elimination efforts. Potential roles of MX include detecting presence of LF in communities and quantifying progress towards elimination of the disease. However, the relationship between MX results and human prevalence is poorly understood. METHODS: We conducted a systematic review and meta-analysis from all previously conducted studies that reported the prevalence of filarial DNA in wild-caught mosquitoes (MX rate) and the corresponding prevalence of microfilaria (mf) in humans. We calculated a pooled estimate of MX sensitivity for detecting positive communities at a range of mf prevalence values and mosquito sample sizes. We conducted a linear regression to evaluate the relationship between mf prevalence and MX rate. RESULTS: We identified 24 studies comprising 144 study communities. MX had an overall sensitivity of 98.3% (95% confidence interval, 41.5-99.9%) and identified 28 positive communities that were negative in the mf survey. Low sensitivity in some studies was attributed to small mosquito sample sizes (<1000) and very low mf prevalence (<0.25%). Human mf prevalence and mass drug administration status accounted for approximately half of the variation in MX rate (R2 = 0.49, P < .001). Data from longitudinal studies showed that, within a given study area, there is a strong linear relationship between MX rate and mf prevalence (R2 = 0.78, P < .001). CONCLUSIONS: MX shows clear potential as tool for detecting communities where LF is present and as a predictor of human mf prevalence.

Development of a molecular xenomonitoring protocol to assess filariasis transmission.

According to the World Health Organization, lymphatic filariasis (LF), a mosquito-borne neglected tropical disease (NTD), should be eliminated as a public health concern by the end of 2020. To this end, the goals of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) include interrupting transmission through mass drug administration (MDA). After two decades, several countries have implemented MDA and are now ready to confirm whether transmission has been interrupted. The method for detecting the parasites in mosquito vectors known as xenomonitoring is a non-invasive tool for assessing the current transmission status of the filarial nematode Wuchereria bancrofti (which is responsible for 90% of cases) by their vectors. There are several methods available for detection of the worm in mosquito samples, such as dissection or polymerase chain reaction (PCR). However, most of these techniques still produce a considerable number of false-negative results. The present study describes a new duplex PCR protocol, which is an improvement on the traditional PCR methodology, enhanced by introducing the actin gene as an endogenous control gene. After adjusting the mosquito pool size, DNA extraction, and WbCx PCR duplex design, we achieved a reliable and sensitive molecular xenomonitoring protocol. This assay was able to eliminate 5% of false negative samples and detected less than one Wb larvae. This high sensitivity is particularly valuable after MDA, when prevalence declines. This new method could reduce the number of false-negative samples, which will enable us to improve our ability to generate accurate results and aid the monitoring strategies used by LF elimination programmes.

Screening of mosquitoes for filarioid helminths in urban areas in south western Poland-common patterns in European Setaria tundra xenomonitoring studies.

In recent years, numerous studies screening mosquitoes for filarioid helminths (xenomonitoring) have been performed in Europe. The entomological monitoring of filarial nematode infections in mosquitoes by molecular xenomonitoring might serve as the measure of the rate at which humans and animals expose mosquitoes to microfilariae and the rate at which animals and humans are exposed to the bites of the infected mosquitoes. We hypothesized that combining the data obtained from molecular xenomonitoring and phenological studies of mosquitoes in the urban environment would provide insights into the transmission risk of filarial diseases. In our search for Dirofilaria spp.-infected mosquitoes, we have found Setaria tundra-infected ones instead, as in many other European studies. We have observed that cross-reactivity in PCR assays for Dirofilaria repens, Dirofilaria immitis, and S. tundra COI gene detection was the rule rather than the exception. S. tundra infections were mainly found in Aedes mosquitoes. The differences in the diurnal rhythm of Aedes and Culex mosquitoes did not seem a likely explanation for the lack of S. tundra infections in Culex mosquitoes. The similarity of S. tundra COI gene sequences found in Aedes vexans and Aedes caspius mosquitoes and in roe deer in many European studies, supported by data on Ae. vexans biology, suggested host preference as the most likely cause of the mosquito genus-biased infections. High diversity of the COI gene sequences isolated in the city of Wroclaw in south western Poland and the presence of identical or almost identical sequences in mosquitoes and roe deer across Europe suggests that S. tundra has been established in most of Europe for a very long time.

Spatio-temporal distribution of vertically transmitted dengue viruses by Aedes aegypti (Diptera: Culicidae) from Arroyo Naranjo, Havana, Cuba.

OBJECTIVE: To study the distribution of vertical transmission of dengue viruses in field-collected Aedes aegypti larvae in the municipality of Arroyo Naranjo in Havana, Cuba. METHODS: Aedes aegypti larvae and pupae were collected monthly between September 2013 and July 2014 in the seven Municipal Health Areas of Arroyo Naranjo. Pools formed of 30-55 larvae were examined through PCR and sequencing to detect the presence of each serotype. RESULTS: We analysed 111 pools of larvae and pupae (4102 individuals) of which 37 tested positive for at least one DENV. More than one DENV type was observed in 10 of the 37 positive pools. Infected pools were detected every month, except in January, suggesting a sustained circulation of DENV in the vector populations. DENV-1 and DENV-3 were the most frequent and dispersed, though all four DENV types were detected. Nucleotide sequencing from positive pools confirmed RT-PCR results for DENV-1 (genotype V), DENV-3 (genotype III) and DENV-4 (genotype II). DENV-2 was detected by RT-PCR but could not be confirmed by nucleotide sequencing. CONCLUSION: Our study of the distribution of natural vertical transmission of dengue virus types highlights extrinsic virus activity patterns in the area and could be used as a new surveillance tool.

Xenomonitoring of different filarial nematodes using single and multiplex PCR in mosquitoes from Assiut Governorate, Egypt.

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.

Molecular xenomonitoring reveals Anopheles funestus and An. rivulorum as the primary vectors of lymphatic filariasis in coastal Kenya.

BACKGROUND: Lymphatic filariasis (LF) is an infectious neglected tropical disease caused by mosquito-borne nematodes such as Wuchereria bancrofti, Brugia malayi, and Brugia timori. Globally, LF affects 51 million people, with approximately 863 million at risk in 47 countries. In Kenya, filariasis is endemic along the entire coastal strip, and more recently, at the Kenya-Ugandan border. The World Health Organization (WHO) recommends mass drug administration to reduce disease transmission and morbidity. Monitoring the effectiveness of such interventions relies on robust surveillance, achieved through microscopic examination of microfilariae in nighttime blood, detection of circulating filarial antigens (CFA), and molecular xenomonitoring. We focused on molecular xenomonitoring along the Kenyan coast due to its noninvasive nature and the opportunity to identify new vectors. METHODS: In 2022, mosquitoes were collected from Kilifi, Kwale, and Taita-Taveta counties located within the LF endemic region in Kenya. Subsequently, genomic deoxyribonucleic acid (gDNA) was extracted from these mosquitoes for speciation and analysis of Wuchereria bancrofti infection rates. The impact of sociodemographic and household attributes on infection rates was assessed using generalized estimating equations. RESULTS: A total of 18,121 mosquitoes belonging to Culicinae (63.0%, n = 11,414) and Anophelinae (37.0%, n = 6707) subfamilies were collected. Morphological identification revealed that Anopheline mosquitoes were dominated by An. funestus (45.4%, n = 3045) and An. gambiae (42.8%, n = 2873). Wuchereria bancrofti infection rates were highest in Kilifi (35.4%; 95% CI 28.0-43.3%, n = 57/161) and lowest in Taita Taveta (5.3%; 95% CI 3.3-8.0%, n = 22/412). The major vectors incriminated are An. rivulorum, An. funestus sensu stricto, and An. arabiensis. Mosquitoes of the An. funestus complex were significantly associated with LF transmission (OR 18.0; 95% CI 1.80-180; p = 0.014). Additionally, a higher risk of transmission was observed outdoors (OR 1.74; 95% CI 1.08-2.82; p = 0.024) and in homesteads that owned livestock (OR 2.00; 95% CI 1.09-3.66; p = 0.025). CONCLUSIONS: In this study, we identified An. funestus s.l. sibling species, An. rivulorum and An. funestus s.s., as the primary vectors of lymphatic filariasis along the Kenyan coast. These findings also highlight that a significant portion of disease transmission potentially occurs outdoors where indoor-based vector control tools, including long-lasting insecticidal nets and indoor residual spray, may not be effective. Therefore, control measures targeting outdoor resting mosquitoes such as zooprophylaxis, larval source management, and attractive sugar baits may have potential for LF transmission reduction.

Laboratory and field validation of the recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) for snail xenomonitoring for schistosomiasis.

Improvements in diagnostics for schistosomiasis in both humans and snail hosts are priorities to be able to reach the World Health Organization (WHO) goal of eliminating the disease as a public health problem by 2030. In this context, molecular isothermal amplification tests, such as Recombinase Polymerase Amplification (RPA), are promising for use in endemic areas at the point-of-need for their accuracy, robustness, simplicity, and time-effectiveness. The developed recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) was used to detect S. mansoni DNA from both laboratory and field Biomphalaria snails. Laboratory snails were experimentally infected and used at one, seven, and 28 days post-exposure (dpe) to 10 S. mansoni miracidia to provide samples in the early pre-patent infection stage. Field samples of Biomphalaria spp. were collected from the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil, which are endemic for S. mansoni. The sensitivity and specificity of the SmMIT-RPA assay were analysed and compared with existing loop-mediated isothermal amplification (LAMP), PCR-based methods, parasitological examination of the snails, and nucleotide sequencing. The SmMIT-RPA assay was able to detect S. mansoni DNA in the experimentally infected Biomphalaria glabrata as early as one dpe to 10 miracidia. It also detected S. mansoni infections (55.5% prevalence) in the field samples with the highest accuracy (100% sensitivity and specificity) compared with the other molecular tests used as the reference. Results from this study indicate that the SmMIT-RPA assay is a good alternative test to be used for snail xenomonitoring of S. mansoni due to its high sensitivity, accuracy, and the possibility of detecting early pre-patent infection. Its simplicity and portability also make it a suitable methodology in low-resource settings.

Surveillance of Culex spp. vectors and zoonotic arboviruses at a zoo in the United Kingdom.

The emergence of several zoonotic mosquito-borne pathogens in Europe, including West Nile virus, Sindbis virus and Usutu virus, has emphasised the importance of consistent surveillance. Considerable fieldwork effort is usually needed to detect low-prevalence pathogens in mosquitoes and screening vertebrate hosts and reservoirs is rarely done simultaneously with mosquito sampling. Zoological gardens offer an opportunity for the surveillance of pathogens, mosquitoes, hosts, and reservoirs concurrently; thus, the aim of this study was undertaking integrated surveillance for mosquito-borne pathogens of wild birds and mosquitoes in Chester Zoo (Cheshire) in the United Kingdom. Mosquitoes were collected in September 2020 and tested for zoonotic bird-hosted arboviruses (i.e., West Nile virus, Usutu virus and Sindbis virus) using RT-qPCRs. Of the 3316 mosquitoes trapped, 98% were identified as Culex spp. The average minimum prevalence of the viruses found in the literature was used to calculate the sample size needed for detecting these viruses with 99% confidence. The testing of 2878 Culex females found no evidence of presence of the three viruses. Significant differences were found in mosquito abundance per sampling site and collection date; furthermore, important sources of immature and resting mosquitoes were found near aviaries. Eighteen wild birds belonging to 11 species were found dead in the zoo from May to December 2020 and were RT-qPCR tested for West Nile virus and Usutu virus; all samples resulted negative for viral infection. It is unlikely that these viruses were present in the zoo during the sampling period; however, since they circulate in Europe and Usutu virus has been isolated in the United Kingdom and may overwinter here, continued monitoring of mosquitoes and wild birds is recommended as virus introduction and dissemination are possible. This study highlights the importance of regular and integrated arboviral surveillance of zoonotic pathogens in zoos providing baseline information to that end.

A first report of Pseudosuccinea columella (Say, 1817), an alien intermediate host for liver fluke, in Malawi.

Starting in October 2021, quarterly malacological surveys have been undertaken in Malawi, with the sampling of 12 specified freshwater habitats throughout a calendar year. Each survey monitors the presence of aquatic intermediate snail hosts of medical and veterinary importance. In March 2023, the alien lymnaeid species Pseudosuccinea columella was encountered for the first time in the surveys, in Nsanje District. This species identity was later confirmed upon DNA analysis of mitochondrial ribosomal 16S sequences. In July 2023, P. columella was also noted at single sites within Mangochi and Chikwawa Districts, and again in Nsanje District, with an additional location observed. Of particular importance, our sampled location in Mangochi District was directly connected to Lake Malawi, which expands the species list of invasive molluscs in this lake. While P. columella is a well-known intermediate snail host for human and animal fascioliasis, screening collected snails for trematode cercariae, alongside molecular xenomonitoring, did not yield equivocal evidence of active fluke infection. However, the newly recognized presence of this alien intermediate snail host within Lake Malawi, and along the Shire River Valley, flags a new concern in altered local transmission potential for human and animal fascioliasis.

Development, validation, and pilot application of a high throughput molecular xenomonitoring assay to detect Schistosoma mansoni and other trematode species within Biomphalaria freshwater snail hosts.

Schistosomiasis is a neglected tropical disease (NTD) caused by infection with parasitic trematodes of the genus Schistosoma that can lead to debilitating morbidity and mortality. The World Health Organization recommend molecular xenomonitoring of Biomphalaria spp. freshwater snail intermediate hosts of Schistosoma mansoni to identify highly focal intestinal schistosomiasis transmission sites and monitor disease transmission, particularly in low-endemicity areas. A standardised protocol to do this, however, is needed. Here, two previously published primer sets were selected to develop and validate a multiplex molecular xenomonitoring end-point PCR assay capable of detecting S. mansoni infections within individual Biomphalaria spp. missed by cercarial shedding. The assay proved highly sensitive and highly specific in detecting and amplifying S. mansoni DNA and also proved highly sensitive in detecting and amplifying non-S. mansoni trematode DNA. The optimised assay was then used to screen Biomphalaria spp. collected from a S. mansoni-endemic area for infection and successfully detected S. mansoni infections missed by cercarial shedding as well as infections with non-S. mansoni trematodes. The continued development and use of molecular xenomonitoring assays such as this will aid in improving disease control efforts, significantly reducing disease-related morbidities experienced by those in schistosomiasis-endemic areas.

One Health monitoring reveals invasive freshwater snail species, new records, and undescribed parasite diversity in Zimbabwe.

BACKGROUND: Snail-borne trematodes afflict humans, livestock, and wildlife. Recognizing their zoonotic potential and possible hybridization, a One Health approach is essential for effective control. Given the dearth of knowledge on African trematodes, this study aimed to map snail and trematode diversity, focusing on (i) characterizing gastropod snail species and their trematode parasites, (ii) determining infection rates of snail species as intermediate hosts for medically, veterinary, and ecologically significant trematodes, and (iii) comparing their diversity across endemic regions. METHODS: A cross-sectional study conducted in 2021 in Chiredzi and Wedza districts in Zimbabwe, known for high human schistosomiasis prevalence, involved malacological surveys at 56 sites. Trematode infections in snails were detected through shedding experiments and multiplex rapid diagnostic polymerase chain reactions (RD-PCRs). Morphological and molecular analyses were employed to identify snail and trematode species. RESULTS: Among 3209 collected snail specimens, 11 species were identified, including schistosome and fasciolid competent snail species. We report for the first time the invasive exotic snail Tarebia granifera in Zimbabwe, which was highly abundant, mainly in Chiredzi, occurring at 29 out of 35 sites. Shedding experiments on 1303 snails revealed a 2.24% infection rate, with 15 trematode species identified through molecular genotyping. Five species were exclusive to Chiredzi: Bolbophorus sp., Schistosoma mansoni, Schistosoma mattheei, Calicophoron sp., and Uvulifer sp. Eight were exclusive to Wedza, including Trichobilharzia sp., Stephanoprora amurensis, Spirorchid sp., and Echinostoma sp. as well as an unidentified species of the Plagiorchioidea superfamily. One species, Tylodelphys mashonensis, was common to both regions. The RD-PCR screening of 976 non-shedding snails indicated a 35.7% trematode infection rate, including the presence of schistosomes (1.1%) Fasciola nyanzae (0.6%). In Chiredzi, Radix natalensis had the highest trematode infection prevalence (33.3%), while in Wedza, R. natalensis (55.4%) and Bulinus tropicus (53.2%) had the highest infection prevalence. CONCLUSIONS: Our xenomonitoring approach unveiled 15 trematode species, including nine new records in Zimbabwe. Schistosoma mansoni persists in the study region despite six mass deworming rounds. The high snail and parasite diversity, including the presence of exotic snail species that can impact endemic species and biomedically important trematodes, underscores the need for increased monitoring.

Evaluating Molecular Xenomonitoring as a Tool for Lymphatic Filariasis Surveillance in Samoa, 2018-2019.

Molecular xenomonitoring (MX), the detection of filarial DNA in mosquitoes using molecular methods (PCR), is a potentially useful surveillance strategy for lymphatic filariasis (LF) elimination programs. Delay in filarial antigen (Ag) clearance post-treatment is a limitation of using human surveys to provide an early indicator of the impact of mass drug administration (MDA), and MX may be more useful in this setting. We compared prevalence of infected mosquitoes pre- and post-MDA (2018 and 2019) in 35 primary sampling units (PSUs) in Samoa, and investigated associations between the presence of PCR-positive mosquitoes and Ag-positive humans. We observed a statistically significant decline in estimated mosquito infection prevalence post-MDA at the national level (from 0.9% to 0.3%, OR 0.4) but no change in human Ag prevalence during this time. Ag prevalence in 2019 was higher in randomly selected PSUs where PCR-positive pools were detected (1.4% in ages 5-9; 4.8% in ages ≥10), compared to those where PCR-positive pools were not detected (0.2% in ages 5-9; 3.2% in ages ≥10). Our study provides promising evidence for MX as a complement to human surveys in post-MDA surveillance.

Presence of a cryptic Onchocerca species in black flies of northern California, USA.

BACKGROUND: Black flies (Diptera: Simuliidae) serve as arthropod vectors for various species of Onchocerca (Nematoda: Onchocercidae) that may be associated with disease in humans, domestic animals, and wildlife. The emergence of zoonotic Onchocerca lupi in North America and reports of cervid-associated zoonotic onchocerciasis by Onchocerca jakutensis highlight the need for increased entomological surveillance. In addition, there is mounting evidence that Onchocerca diversity in North America is far greater than previously thought, currently regarded as Onchocerca cervipedis species complex. This study reports new geographic records and black fly vector associations of an uncharacterized Onchocerca species. METHODS: To better understand the biodiversity and geographic distribution of Onchocerca, 485 female black flies (2015: 150, 2016: 335) were collected using CO2-baited traps from February to October 2015-2016 in Lake County, northern California, USA. Individual flies were morphologically identified and pooled (≤ 10 individuals) by species, collection date, and trap location. Black fly pools were processed for DNA extraction, and subsequent PCR and sequencing targeting of the NADH dehydrogenase subunit 5 gene of filarioids. RESULTS: Among the pools of black flies, there were 158 individuals of Simulium tescorum (2015: 57, 2016: 101), 302 individuals of Simulium vittatum (sensu lato [s.l.]) (2015: 82, 2016: 220), 16 individuals of Simulium clarum "black" phenotype (2015: 5, 2016: 11), and 13 individuals of S. clarum "orange" phenotype (2015: 6, 2016: 7). PCR analysis revealed the percentage of filarioid-positive pools were 7.50% (n = 3) for S. tescorum, 3.75% (n = 3) for S. vittatum (s.l., likely S. tribulatum), 7.69% (n = 1) for S. clarum "black" phenotype, and no positives for S. clarum "orange" phenotype. Genetic distance and phylogenetic analyses suggest that the northern California Onchocerca isolates belong to the same species reported in black flies from southern California (average pairwise comparison: 0.32%), and seem closely related to Onchocerca isolates of white-tailed deer from upstate New York (average pairwise comparison: 2.31%). CONCLUSION: A cryptic Onchocerca species was found in Lake County, California, and may be a part of a larger, continentally distributed species complex rather than a single described species of North America. In addition, there are at least three putative vectors of black flies (S. clarum, S. tescorum, S. vittatum) associated with this cryptic Onchocerca species. A comprehensive reassessment of North American Onchocerca biodiversity, host, and geographic range is necessary.

The use of molecular xenomonitoring for surveillance of mosquito-borne diseases.

The scientific community recognizes that molecular xenomonitoring (MX) can allow infected mosquitoes to serve as a proxy for human infection in vector-borne disease surveillance, but developing reliable MX systems for programmatic use has been challenging. The primary aim of this article is to examine the available evidence to recommend how MX can best be used for various purposes. Although much of the literature published within the last 20 years focuses on using MX for lymphatic filariasis elimination, a growing body of evidence supports its use in early warning systems for emerging infectious diseases (EIDs). An MX system design must consider the goal and target (e.g. diseases targeted for elimination versus EIDs), mosquito and pathogen characteristics, and context (e.g. setting and health system). MX is currently used as a 'supplement' to human surveillance and will not be considered as a 'replacement' until the correlation between pathogen-infection rates in human and mosquito populations is better understood. Establishing such relationships may not be feasible in elimination scenarios, due to increasingly dwindling human infection prevalence after successful control, but may still be possible for EIDs and in integrated disease surveillance systems. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

A Comparison of Adult Mosquito Trapping Methods to Assess Potential West Nile Virus Mosquito Vectors in Greece during the Onset of the 2018 Transmission Season.

West Nile virus (WNV) threatens the health of humans and equines worldwide. Culex (Cx.) pipiens complex mosquitoes are major vectors but numerous other species have been implicated. Due to variations in blood-feeding behaviour, Cx. pipiens biotypes and hybrids influence transmission, from enzootic cycles (between mosquitoes and birds), to spill-over transmission to humans and equines. In this study, mosquitoes were collected in May-June 2018 during the early period of the transmission season from two regional units of Greece, where WNV cases had been reported in the previous four years (Palaio Faliro and Argolida). A total of 1062 mosquitoes were collected with Biogents Sentinel 2 traps collecting both a greater number of all mosquito species and the Cx. pipiens complex than CDC miniature light traps or Heavy Duty EVS traps. Molecular identification confirmed additional species including Aedes albopictus. The proportion of Cx. pipiens biotypes in Palaio Faliro was 54.5% pipiens, 20.0% molestus and 25.5% hybrids. In Argolida, the collection comprised 68.1% pipiens biotype, 8.3% molestus biotype and 23.6% hybrids. Screening resulted in WNV detection in three females of the pipiens biotype and in one hybrid. As hybrids play a role in spill-over transmission, these findings highlight the importance of entomological surveillance programs incorporating molecular xenomonitoring as an early warning before human cases at the onset of the transmission season.

Monitoring the presence of trypanosomes' DNA - Including Trypanosoma brucei gambiense DNA - From the midguts of riverine Glossina trapped in the south east outskirts of Kinshasa City (Democratic Republic of Congo).

Even if the number of Human African Trypanosomiasis (HAT) cases from Kinshasa province in DRC is going towards elimination for the last decade, cases still occur in the periphery of the city. The diagnosis of 21 cases in the south periphery of Kinshasa, between 2015 and 2017 gives evidence of the existence of an active focus in this area. Here, we present the results of a punctual entomological survey that was realized in july 2014 in the outskirts of the southeast of Kinshasa. Using pyramidal traps, we caught tsetse flies during 2 days, dissecting the fresh ones for further molecular analysis. The average Apparent Density of flies per Trap and per Day was three with a maximum of 5.6 flies in Nganda PIO. Polymerase chain reaction analysis of the midguts provided evidence of a high prevalence (57.2%) of infected flies. Ninety three percent of the trypanosomes that were identified belonged to the Nanomonas species, but Trypanozoon trypanosomes were also present in 24% of the infected flies, including mixed infections with Nanomonas, including 3 flies carrying Trypanosoma brucei gambiense, the human pathogen of trypanosomiasis. These results show that at the time of the field's study there was an active reservoir of trypanosomes, closed to pigsties, knowing that pig is a potential animal reservoir. It also demonstrates that xenomonitoring using the entomological approach can be an efficient tool for monitoring sleeping sickness. Finally, results are discussed in the frame of WHO's HAT elimination project. Regarding Kinshasa, it points out the need of regular epidemiologic surveys.

Exposing the Barcoding Void: An Integrative Approach to Study Snail-Borne Parasites in a One Health Context.

Trematodes are snail-borne parasites of major zoonotic importance that infect millions of people and animals worldwide and frequently hybridize with closely related species. Therefore, it is desirable to study trematodiases in a One Health framework, where human and animal trematodes are considered equally important. It is within this framework that we set out to study the snail and trematode communities in four artificial lakes and an abattoir in Zimbabwe. Trematode infections in snails were detected through multiplex PCR protocols. Subsequently, we identified snails by sequencing a partial mitochondrial cytochrome c oxidase subunit I (COI) fragment, and trematodes (adults from the abattoir and larval stages detected in snails) using COI and nuclear rDNA markers. Of the 1,674 collected snails, 699 were molecularly analyzed, in which we identified 12 snail and 19 trematode species. Additionally, three parasite species were sampled from the abattoir. Merely four trematode species were identified to species level through COI-based barcoding. Moreover, identification of members of the superfamilies Opisthorchioidea and Plagiorchioidea required a phylogenetic inference using the highly conserved 18S rDNA marker, as no related COI reference sequences were present in public databases. These barcoding challenges demonstrate a severe barcoding void in the available databases, which can be attributed to the neglected status of trematodiases. Adding to this, many available sequences cannot be used as different studies use different markers. To fill this gap, more studies on African trematodes, using a standardized COI barcoding region, are desperately needed.

Assessing the Presence of Wuchereria bancrofti Infections in Vectors Using Xenomonitoring in Lymphatic Filariasis Endemic Districts in Ghana.

Mass drug administration (MDA) is the current mainstay to interrupt the transmission of lymphatic filariasis. To monitor whether MDA is effective and transmission of lymphatic filariasis indeed has been interrupted, rigorous surveillance is required. Assessment of transmission by programme managers is usually done via serology. New research suggests that xenomonitoring holds promise for determining the success of lymphatic filariasis interventions. The objective of this study was to assess Wuchereria bancrofti infection in mosquitoes as a post-MDA surveillance tool using xenomonitoring. The study was carried out in four districts of Ghana; Ahanta West, Mpohor, Kassena Nankana West and Bongo. A suite of mosquito sampling methods was employed, including human landing collections, pyrethrum spray catches and window exit traps. Infection of W. bancrofti in mosquitoes was determined using dissection, conventional and real-time polymerase chain reaction and loop mediated isothermal amplification assays. Aedes, Anopheles coustani, An. gambiae, An. pharoensis, Culex and Mansonia mosquitoes were sampled in each of the four study districts. The dissected mosquitoes were positive for filarial infection using molecular assays. Dissected An. melas mosquitoes from Ahanta West district were the only species found positive for filarial parasites. We conclude that whilst samples extracted with Trizol reagent did not show any positives, molecular methods should still be considered for monitoring and surveillance of lymphatic filariasis transmission.