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In southern Zambia, malaria transmission is low, with outdoor biting Anopheles mosquitoes playing a significant role in malaria transmission. Locals cook in outdoor, open-walled kitchen shelters, exposing them to these outdoor biting vectors and malaria. Volatile pyrethroid spatial emanators (SE) operate through a mode of action which could provide local protection around these kitchens. In this study, SE devices containing the pyrethroid transfluthrin were deployed to local kitchens, where human landing collection (HLC) was utilized to determine differences in host-landing rates associated with protection. Forty-one households from two villages were enrolled in the study in clusters of five (or six) and randomly assigned a treatment by cluster. Local mosquito collectors were recruited and trained to conduct HLCs from 18:00-06:00 once per week for 15 weeks. SE and placebo devices were replaced monthly. Following collection, mosquitoes were returned to the lab for morphological identification. Results were analyzed in R with negative binomial generalized linear mixed models (GLMM) considering all-night and per-hour capture. A total of 3021 mosquitoes were collected during the follow-up period, with Anopheles species composing roughly half of all specimens. Anopheles activity was lower in SE protected kitchens based on all night and hourly comparisons. Mosquito activity was highest in the middle of the night, and SE was not significantly associated with protection between 18:00-21:00. SE usage appeared to reduce mosquito host-seeking activity by approximately 65-70%, with this efficacy appearing to diminish gradually over time to approximately 20-25% four weeks after opening, at which point they were replaced. Culicine mosquito behavior was not significantly impacted by the SE. The SE device provided protection to individuals within protected kitchen structures overnight and during most hours of the night. The number of mosquitoes was lowest during the early collection hours between 18:00-21:00, a period in which the SE devices did not significantly impact mosquito host-seeking behavior. This result has implications for this use-case and should be further explored.

Background Attractive targeted sugar bait (ATSB) stations are a promising new approach to malaria vector control that could compliment current tools by exploiting the natural sugar feeding behaviors of mosquitoes. Recent proof of concept work with a prototype ATSB ® Sarabi Bait Station (Westham Co., Hod-Hasharon, Israel) has demonstrated high feeding rates and significant reductions in vector density, human biting rate, and overall entomological inoculation rate for Anopheles gambiae sensu lato ( s.l.) in the tropical savannah of western Mali. The study reported here was conducted in the more temperate, rainier region of Western Province, Zambia and was designed to confirm the primary vector species in region and to estimate corresponding rates of feeding from prototype attractive sugar bait (ASB) Sarabi Bait Stations. Methods The product evaluated was the Sarabi v1.1.1 ASB station, which did not include insecticide but did include 0.8% uranine as a dye allowing for the detection, using UV fluorescence light microscopy, of mosquitoes that have acquired a sugar meal from the ASB. A two-phase, crossover study design was conducted in 10 village-based clusters in Western Province, Zambia. One study arm initially received 2 ASB stations per eligible structure while the other initially received 3. Primary mosquito sampling occurred via indoor and outdoor CDC Miniature UV Light Trap collection from March 01 through April 09, 2021 (Phase 1) and from April 19 to May 28, 2021 (Phase 2). Results The dominant vector in the study area is Anopheles funestus s.l., which was the most abundant species group collected (31% of all Anophelines; 45,038/144,5550), had the highest sporozoite rate (3.16%; 66 positives out of 2,090 tested), and accounted for 94.3% (66/70) of all sporozoite positive specimens. Of those An. funestus specimens further identified to species, 97.2% (2,090/2,150) were An. funestus sensu stricto ( s.s .). Anopheles gambiae s.l. (96.8% of which were Anopheles arabiensis ) is a likely secondary vector and Anopheles squamosus may play a minor role in transmission. Overall, 21.6% (9,218/42,587) of An. funestus specimens and 10.4% (201/1,940) of An. gambiae specimens collected were positive for uranine, translating into an estimated daily feeding rate of 8.9% [7.7–9.9%] for An. funestus (inter-cluster range of 5.5% to 12.7%) and 3.9% [3.3–4.7%] for An. gambiae (inter-cluster range of 1.0–5.2%). Feeding rates were no different among mosquitoes collected indoors or outdoors, or among mosquitoes from clusters with 2 or 3 ASBs per eligible structure. Similarly, there were no correlations observed between feeding rates and the average number of ASB stations per hectare or with weekly rainfall amounts. Conclusions Anopheles funestus and An. gambiae vector populations in Western Province, Zambia readily fed from the prototype Sarabi v1.1.1 ASB sugar bait station. Observed feeding rates are in line with those thought to be required for ATSB stations to achieve reductions in malaria transmission when used in combination with conventional control methods (IRS or LLIN). These results supported the decision to implement a large-scale, epidemiological cluster randomized controlled trial of ATSB in Zambia, deploying 2 ATSB stations per eligible structure.

Tick-borne pathogens are an emerging public health threat worldwide. However, information on tick-borne viruses is scanty in sub-Saharan Africa. Here, by RT-PCR, 363 ticks (Amblyomma, Hyalomma and Rhipicephalus) in the Namwala and Livingstone districts of Zambia were screened for tick-borne phleboviruses (TBPVs). TBPVs (L gene) were detected in 19 (5.2%) Rhipicephalus ticks in Namwala. All the detected TBPVs were Shibuyunji viruses. Phylogenetically, they were closely related to American dog tick phlebovirus. This study highlights the possible role of Rhipicephalus ticks as the main host of Shibuyunji virus and suggests that these viruses may be present outside the area where they were initially discovered.

In the recent past, sub-Saharan Africa has not escaped the devastating effects of avian influenza virus (AIV) in poultry and wild birds. This systematic review describes the prevalence, spatiotemporal distribution, and virus subtypes detected in domestic and wild birds for the past two decades (2000–2019). We collected data from three electronic databases, PubMed, SpringerLink electronic journals and African Journals Online, using the Preferred Reporting Items for Systematic reviews and Meta-Analyses protocol. A total of 1656 articles were reviewed, from which 68 were selected. An overall prevalence of 3.0% AIV in birds was observed. The prevalence varied between regions and ranged from 1.1% to 7.1%. The Kruskal–Wallis and Wilcoxon signed-rank sum test showed no significant difference in the prevalence of AIV across regions, χ2(3) = 5.237, p = 0.1553 and seasons, T = 820, z = −1.244, p = 0.2136. Nineteen hemagglutinin/neuraminidase subtype combinations were detected during the reviewed period, with southern Africa recording more diverse AIV subtypes than other regions. The most detected subtype was H5N1, followed by H9N2, H5N2, H5N8 and H6N2. Whilst these predominant subtypes were mostly detected in domestic poultry, H1N6, H3N6, H4N6, H4N8, H9N1 and H11N9 were exclusively detected in wild birds. Meanwhile, H5N1, H5N2 and H5N8 were detected in both wild and domestic birds suggesting circulation of these subtypes among wild and domestic birds. Our findings provide critical information on the eco-epidemiology of AIVs that can be used to improve surveillance strategies for the prevention and control of avian influenza in sub-Saharan Africa.

Background Understanding the composition, host feeding patterns, and infection status of all potential vectors is essential to understand changing disease transmission dynamics and inform future vector control strategies . This study assessed anopheline species composition, abundance, distribution, and host feeding preferences in Southern Province, Zambia to identify potential contributors to residual malaria transmission beyond primary vectors. Methods Adult mosquitoes were collected along a 100 km transect between the districts of Pemba, Monze and Gwembe in Southern Province, Zambia, an area of low malaria transmission. Mosquitoes were sampled from 12 randomly selected households at 5 km intervals along the transect. Centers for Disease Control and Prevention light traps were used to collect adult mosquitoes indoors and outdoors. Mosquitoes were identified using morphological keys and molecular approaches. A multiplexed PCR assay was utilized to identify mammalian hosts and Plasmodium falciparum (Pf) sporozoite infection was determined using an enzyme linked immunosorbent assay. Results A total of 5697 female anopheline mosquitoes were collected from both indoor and outdoor traps, revealing a diverse range of species. Nine species of Anopheles mosquitoes were identified, dominated by Anopheles rufipes (n = 1527, 26.8%), and An. pretoriensis (n = 1427, 25.1%). Host blood meal analysis of 401 visibly blood-fed mosquitoes revealed that Anopheles species in the study area fed primarily on goats (n = 176; 62.5%), humans (n = 55; 19.4%) and cows (n = 29; 10.2%). The human blood index across all captured anophelines was estimated at 0.22. Human blood meals were detected in secondary malaria vectors, with notably high HBI values of An. pretoriensis and An. rufipes mosquitoes captured outdoors near goat pens. Out of the 5697 female anophelines tested for sporozoite infectivity, one An. rufipes specimen was positive for P. falciparum. Conclusion This study demonstrates that malaria transmission risk in the surveyed areas may extend beyond well-recognized primary vectors. Species that are not well studied, often exophagic and assumed to be zoophagic, were found to have fed on humans and the study showed some preliminary evidence of P. falciparum infectivity. These findings indicate that peri-domestic human activity may sustain residual transmission despite high Insecticidal-Treated Nets and indoor residual spraying coverage.

Background The primary vector control interventions in Zambia are long-lasting insecticidal nets and indoor residual spraying. Challenges with these interventions include insecticide resistance and the outdoor biting and resting behaviours of many Anopheles mosquitoes. Therefore, new vector control tools targeting additional mosquito behaviours are needed to interrupt transmission. Attractive targeted sugar bait (ATSB) stations, which exploit the sugar feeding behaviours of mosquitoes, may help in this role. This study evaluated the residual laboratory bioefficacy of Westham prototype ATSB® Sarabi v.1.2.1 Bait Station (Westham Ltd., Hod-Hasharon, Israel) in killing malaria vectors in Western Province, Zambia, during the first year of a large cluster randomized phase-III trial (Clinical Trials.gov Identifier: NCT04800055). Methods This was a repeat cross-sectional study conducted within three districts, Nkeyema, Kaoma, and Luampa, in Western Province, Zambia. The study was conducted in 12 intervention clusters among the 70 trial clusters (35 interventions, 35 controls) between December 2021 and June 2022. Twelve undamaged bait stations installed on the outer walls of households were collected monthly (one per cluster per month) for bioassays utilizing adult female and male Anopheles gambiae sensu stricto (Kisumu strain) mosquitoes from a laboratory colony. Results A total of 84 field-deployed ATSB stations were collected, and 71 ultimately met the study inclusion criteria for remaining in good condition. Field-deployed stations that remained in good condition (intact, non-depleted of bait, and free of dirt as well as mold) retained high levels of bioefficacy (mean induced mortality of 95.3% in males, 71.3% in females, 83.9% combined total) over seven months in the field but did induce lower mortality rates than non-deployed ATSB stations (mean induced mortality of 96.4% in males, 87.0% in females, 91.4% combined total). There was relatively little variation in corrected mortality rates between monthly rounds for those ATSB stations that had been deployed to the field. Conclusion While field-deployed ATSB stations induced lower mortality rates than non-deployed ATSB stations, these stations nonetheless retained relatively high and stable levels of bioefficacy across the 7-month malaria transmission season. While overall mean mosquito mortality rates exceeded 80%, mean mortality rates for females were 24 percentage points lower than among males and these differences merit attention and further evaluation in future studies. The duration of deployment was not associated with lower bioefficacy. Westham prototype ATSB stations can still retain bioefficacy even after deployment in the field for 7 months, provided they do not meet predetermined criteria for replacement.

Animal rabies remains underreported in low-income countries, hindering effective control. This scoping review aimed to map reported animal rabies cases, identify key reservoir species, and assess gaps in surveillance coverage in mainland Tanzania from 1993 to 2023. Specifically, it addressed the distribution of cases, species involved, and the extent of surveillance coverage during this period. Literature searches in PubMed, Google Scholar, and Science Direct were screened using Rayyan. Twenty articles published between 1993 and 2023 reported 7319 animal rabies cases across the Northern Zone (NZ), Southeastern Zone (SEZ), and Coastal Zone (CZ). In the NZ, domestic dogs accounted for most cases (5387), followed by jackals (225), cats (77), livestock (311), and various wildlife species including African wild dogs, bat-eared foxes, lions, cheetahs, and striped hyenas. Additionally, 102 cases involved unidentified animals. In SEZ, domestic dogs (588) were the primary source, followed by jackals (262), hyenas (8), cats (10), honey badgers (5), and leopards (2). In CZ, domestic dogs accounted for 94 cases. The findings confirm domestic dogs as the main rabies reservoir, highlighting the need for strengthened surveillance and control. The role of wildlife in rabies maintenance and spillover remains poorly understood and warrants further investigation, especially in enzootic hotspots.

Background Anopheles squamosus is a widespread mosquito species in sub-Saharan Africa. It is a potential vector for human malaria parasites and has been found naturally infected with Plasmodium falciparum and Plasmodium vivax . Morphological identification is challenging even with pristine specimens and current molecular methods such as the use of the internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) cannot distinguish An. squamosus from morphologically similar Anopheles species . Described in the following methods is the development and validation of a new PCR assay that will reliably identify An. squamosus . Methods Multiple alignments of previously published ITS2 contig sequences in NCBI from An. squamosus and An. species 11 and 15, were used to identify candidate ITS2 regions for primer design. Six sets of primers were evaluated overall for specificity of species identification. The one set with An. squamosus species-specific amplification was tested using 78 specimens morphologically identified from Zambia and South Africa. Results A new assay consisting of a forward (ITS2-ASQ-R10, 5’-CCC TCG AAG GGT GCT GTG-3’) and reverse (ITS2-ASQ-R10 5’-AAT CCA CGG TGT GAT GGC-3’) primer reliably (> 94.9%) amplified an ITS2 fragment of 301 bp length for An. squamosus . The An. squamosus- specific primer set can be multiplexed with existing ITS2 assays frequently used for anopheline species identification. Conclusions The development of this robust PCR assay for An. squamosus is vital to accurate identification of this species in malaria vector surveillance efforts. Improved understanding of the anopheline community composition will lead to better targeted methods of vector eradication and malaria prevention. To further the validation of this ITS2 PCR assay, more species of Anopheles should be compared in addition to An. squamosus collected in different regions. To refine and optimize the PCR process with these primers, touchdown PCR can be used to increase specificity. Applying genomic tools to correctly identify An. squamosus will allow for a better understanding of their role in malaria transmission and may lead to genomic insights into what influences their behaviour, thus leading to new innovations in malaria elimination.

Background The attractive targeted sugar bait (ATSB) is a novel malaria vector control tool designed to attract and kill mosquitoes using a sugar-based bait, laced with oral toxicant. Western Province, Zambia, was one of three countries selected for a series of phase III cluster randomized controlled trials of the Westham ATSB Sarabi version 1.2. The trial sites in Kenya, Mali, and Zambia were selected to represent a range of different ecologies and malaria transmission settings across sub-Saharan Africa. This case study describes the key characteristics of the ATSB Zambia trial site to allow for interpretation of the results relative to the Kenya and Mali sites. Methods This study site characterization incorporates data from the trial baseline epidemiological and mosquito sugar feeding surveys conducted in 2021, as well as relevant literature on the study area. Results: Characterization of the trial site The trial site in Zambia was comprised of 70 trial-designed clusters in Kaoma, Nkeyema, and Luampa districts. Population settlements in the trial site were dispersed across a large geographic area with sparsely populated villages. The overall population density in the 70 study clusters was 65.7 people per square kilometre with a total site population of 122,023 people living in a geographic area that covered 1858 square kilometres. However, the study clusters were distributed over a total area of approximately 11,728 square kilometres. The region was tropical with intense and seasonal malaria transmission. An abundance of trees and other plants in the trial site were potential sources of sugar meals for malaria vectors. Fourteen Anopheles species were endemic in the site and Anopheles funestus was the dominant vector, likely accounting for around 95% of all Plasmodium falciparum malaria infections. Despite high coverage of indoor residual spraying and insecticide-treated nets, the baseline malaria prevalence during the peak malaria transmission season was 50% among people ages six months and older. Conclusion Malaria transmission remains high in Western Province, Zambia, despite coverage with vector control tools. New strategies are needed to address the drivers of malaria transmission in this region and other malaria-endemic areas in sub-Saharan Africa.

Background Rotavirus B (RVB) causes diarrhea in humans and pigs. Although various RVB strains were identified in humans and various animals globally, little is known about the epidemiology RVB infection in Africa. In this study, we attempted to examine the prevalence of RVB infection in pig populations in Zambia. Methods Metagenomic analyses were conducted on pig feces collected in Zambia to detect double stranded RNA viruses, including RVB. To clarify the prevalence of RVB infection in pig populations in Zambia, 147 fecal samples were screened for the RVB detection by RT-qPCR. Full genome sequence of a detected RVB was determined by Sanger sequencing and genetically analyzed. Results The metagenomic analyses revealed that RVB sequence reads and contigs of RVB were detected from one fecal sample collected from pigs in Zambia. RT-qPCR screening detected RVB genomes in 36.7% (54/147) of fecal samples. Among 54 positive samples, 13 were positive in non-diarrheal samples ( n  = 48, 27.1%) and 41 in diarrheal samples ( n  = 99, 41.4%). Genetic analyses demonstrated that all the segments of ZP18-18, except for VP4, had high nucleotide sequence identities (80.6–92.6%) with all other known RVB strains detected in pigs. In contrast, the VP4 sequence of ZP18-18 was highly divergent from other RVB strains (< 64.6% identities) and formed a distinct lineage in the phylogenetic tree. Notably, the VP8 subunit of the VP4 showed remarkably low amino acid identities (33.3%) to those of known RVB strains, indicating that the VP8 subunit of ZP18-18 was unique among RVB strains. According to the whole genome classification for RVB, ZP18-18 was assigned to a genotype constellation, G18-P[9]-I12-R4-C4-M4-A8-N10-T5-E4-H7 with the newly established VP4 genotype P[9]. Conclusions This current study updates the geographical distribution and the genetic diversity of RVB. Given the lack of information regarding RVB in Africa, further RVB surveillance is required to assess the potential risk to humans and animals.