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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. Graphical Abstract

Background Intestinal schistosomiasis was confirmed endemic in Mangochi District, Malawi, in May of 2018 following an unexpected encounter with discreet populations of Biomphalaria spp. freshwater snails during routine malacological surveillance activities. Since then, only limited malacological surveillance of Biomphalaria has been carried out, and so the distribution of Biomphalaria populations in this area is currently unclear. Additionally, sites of active Schistosoma mansoni transmission in this area are also unknown. In the present study, through extensive malacological surveillance, we aimed to formally document the distribution of Biomphalaria in Mangochi District. We also aimed to identify active intestinal schistosomiasis transmission sites in this area through subjecting all collected Biomphalaria to a recently developed S. mansoni- specific molecular xenomonitoring PCR. Methods Three malacological surveys were carried out along the southern shoreline of Lake Malawi, Mangochi District, Malawi, in November 2021, July 2022 and October/November 2022. All collected Biomphalaria were subjected to cercarial shedding analysis to identify active Schistosoma infections. Shed cercariae were then genotyped to species level using a standard multi-locus PCR and Sanger sequencing protocol. Following this, a subset of Biomphalaria from each collection site were also genotyped to species level using a standard PCR and Sanger sequencing protocol. All collected Biomphalaria were then subjected to a recently developed S. mansoni- specific molecular xenomonitoring PCR to identify infected, but non-shedding, Biomphalaria. Results A total of 589 Biomphalaria were collected across all three surveys. One single Biomphalaria (0.17%) specimen was found to be actively shedding Schistosoma cercariae, which were molecularly confirmed as S. mansoni . All genotyped Biomphalaria ( n  = 42) were molecularly identified as B. pfeifferi . A further 19 Biomphalaria specimens, collected from four different surveillance sites, were found to be infected with S. mansoni through molecular xenomonitoring. Intestinal schistosomiasis transmission was therefore identified at four different foci in Mangochi District. Conclusions Our study highlights the importance of molecular approaches to investigate Biomphalaria populations and monitor Biomphalaria -associated intestinal schistosomiasis transmission in endemic areas. As such, the continued development and use of such approaches, in particular the development and use of molecular xenomonitoring assays that can be carried out in resource-poor schistosomiasis-endemic settings, is encouraged. The revision of ongoing schistosomiasis control programmes in Mangochi District, in line with WHO recommendations, is also encouraged. Graphical abstract

Our pilot parasitological investigation of cattle, supplemented with molecular DNA characterisation of encountered schistosomes, sheds first light upon bovine schistosomiasis on Unguja Island, Zanzibar. During February 2024, a total of 99 cattle were examined. Of these, 47 were exported animals from the Tanzanian mainland, designated for slaughter at two governmental abattoirs (Kisakasaka and Muwanda), and 52 were free-grazing animals sampled from four grazing locations within the island’s North and West-B regions. Upon visual inspection of 31 cattle carcasses at Kisakasaka for adult worms, the prevalence of bovine schistosomiasis was 51.6%; however, upon faecal miracidia hatching test (MHT) it was 80.6%. At Muwanda, only faecal MHT was used, finding a much lower prevalence of 12.5%. In free-grazing animals, the prevalence of bovine schistosomiasis by MHT was 0.0%. At Muwanda, the animal quarantine paddock was in disrepair, inclusive of a large pond now acting as a watering point. Here, numerous Bulinus forskalii sp. were found. Whilst no snails were observed to shed schistosome cercariae, molecular xenomonitoring did detect a pre-patent infection prevalence of 10.8%, with Schistosoma bovis firmly incriminated. Molecular DNA characterisation of adult schistosomes (n = 19) by real-time polymerase chain reaction (PCR) and high-resolution melt profiling, alongside DNA sequencing, also identified S. bovis, although two worms were putative S. bovis-S. mattheei hybrids. Atypical intrauterine eggs of S. bovis were noted upon microscopy of a worm pair. A broader screen of 92 miracidia confirmed S. bovis and three miracidia as S. bovis-S. mattheei hybrids. Contrasting with Pemba Island, Zanzibar, where autochthonous transmission of S. bovis can occur, bovine schistosomiasis on Unguja Island currently appears restricted to imported animals alone. However, the seminal detection of putative S. bovis-mattheei hybrids, alongside the current inadequate quarantine facilities at Muwanda, raises a new concern that such hybrid schistosomes may escape and enter the island’s hinterland. Should this happen, surveillance and control of urogenital schistosomiasis on Unguja would be compromised and further complicated. We therefore strongly recommend immediate repair and improved maintenance of governmental animal quarantine facilities. Future epidemiological surveys of imported cattle are now well justified, not only to better understand the full repertoire of hybrid schistosomes present but also to develop appropriate mitigating interventions.

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'.

Background & objectives: Annual mass drug administration (MDA) is the main strategy to interrupt the transmission of lymphatic filariasis (LF) in the community. The main aim of monitoring the MDA program, for its effectiveness and interruption of LF is the post-MDA surveillance using antigen survey in children born after MDA. The latest technique of new research suggests that xenomonitoring is an effective tool for monitoring LF intervention. The objective of this study was to assess the W. bancrofti infection/or infectivity in vector mosquitoes by xenomonitoring during post-MDA surveillance. Methods: A descriptive cross-sectional study was conducted in the hotspots of selected four districts of Central Nepal. A gravid trap technique was used for sampling mosquitoes. Infection/or infectivity was determined via the dissection of vector mosquitoes. Anopheles, Aedes, Armigerus and Culex species were collected from hotspots of four endemic districts, two from the hilly region (Lalitpur and Dhading) and two from Terai region (Bara and Mahottari) of Central Nepal. Results: A total of 4450 mosquitoes belonging to four genera, Anopheles, Culex, Armigeres, and Aedes were collected from four hotspots. The distribution of Culex quinquefasciatus was found to be the highest, 88.9% (n=3955/4450) followed by Cx. vishnui (4.5%), Armigeres sp (5.8%), An. culicifascies (0.2%), Aedes spp (0.8%). The proportion of female mosquitoes trapped is significantly higher. A total of 3344 parous Cx. quinquefasciatus mosquitoes were dissected for any larval stage of W. bancrofti. We could not find any filarial infection in dissected mosquito samples. Interpretation & conclusion: We conclude that the gravid trap is an efficient tool for the collection of gravid Cx. quinquefasciatus mosquitoes for xenomonitoring studies of filariasis endemic regions. Vector composition indicated a maximum number of vector mosquitoes of lymphatic filariasis were trapped compared with the other three species. Distribution and density of Cx. quinquefasciatus was found highest in four hotspots of endemic districts. None of the Cx. quinquefasciatus dissected were found to be infected by larval forms of filaria. Since the low levels of infection persistence in the human population in these hot spots, vector infection and infectivity can't be ignored. Microscopic xenomonitoring at a low level of infection persistent is less likely to be efficient so molecular xenomonitoring along with a large sample should be required in each of the hot spots of the districts. Additionally, area is receptive so further vector control intervention should be required to reduce the risk of resurgence of infection.

Background Lymphatic filariasis (LF) is a mosquito-borne filarial disease targeted for elimination by the year 2020. The Republic of Togo undertook mass treatment of entire endemic communities from 2000 to 2009 to eliminate the transmission of the disease and is currently the first sub-Saharan African country to be validated by WHO for the elimination of LF as a public health problem. However, post-validation surveillance activities are required to ensure the gains achieved are sustained. This survey assessed the mosquito vectors of the disease and determined the presence of infection in these vectors, testing the hypothesis that transmission has already been interrupted in Togo. Method Mosquitoes were collected from 37 villages located in three districts in one of four evaluation units in the country. In each district, 30 villages were selected based on probability proportionate to size; eight villages (including one of the 30 villages already selected) where microfilaremia-positive cases had been identified during post-treatment surveillance activities were intentionally sampled. Mosquitoes were collected using pyrethrum spray collections (PSC) in households randomly selected in all villages for five months. In the purposefully selected communities, mosquitoes were also collected using human landing collections (HLC) and exit traps (ET). Collected mosquitoes were identified morphologically, and the identification of Wuchereria bancrofti DNA in the mosquitoes was based on the pool screening method, using the LAMP assay. Results A total of 15,539 mosquitoes were collected during the study. Anopheles gambiae (72.6%) was the predominant LF vector collected using PSC. Pool screen analysis of 9191 An . gambiae in 629 pools revealed no mosquitoes infected with W. bancrofti (0%; CI: 0–0.021). Conclusions These results confirm the findings of epidemiological transmission assessment surveys conducted in 2012 and 2015, which demonstrated the absence of LF transmission in Togo. The challenges of implementing molecular xenomonitoring are further discussed.

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.

Biomphalaria pfeifferi snails serve as the major intermediate host for intestinal schistosomiasis in Sudan. The genetic structure and infection status of 163 B. pfeifferi collected from six localities in Gezira State, Sudan (East Gezira, Greater Wadmedani, Hasahisa, North Umelgura, South Gezira, and Managil) were characterized. Cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA) mitochondrial genes were used for B. pfeifferi molecular identification and genetic diversity investigation. Schistosoma mansoni infection was detected using the traditional cercarial shedding and molecular methods (SmF/R primers). Five COI haplotypes and ten 16S haplotypes were identified, with haplotype diversity of 0.50 for COI and 0.11 for 16S. High evolutionary divergence was observed between groups (Fst = 0.94) for the COI, and low genetic divergence (Fst = 0.04) for the 16S, indicating genetic divergence among Sudanese B. pfeifferi, with the 16S showing lower divergence than the COI, consistent with a post-bottleneck population expansion. Cercarial shedding detected an overall infection prevalence of 3.6% (8/219), with only two snails from Hasahisa shedding S. mansoni cercariae. The SmF/R primers revealed a higher infection prevalence of 7.4% (12/163), with all S. mansoni positive samples found at the Hasahisa site. Findings highlight the value of molecular diagnostic tools for accurate surveillance and emphasize the need for site-specific control strategies.

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.

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. Graphical abstract