Efficacy of Metarhizium anisopliae, Isolate ICIPE 7, against Anopheles arabiensis, Glossina fuscipes, and Rhipicephalus spp.

Arthropod vectors are responsible for a multitude of human and animal diseases affecting poor communities in sub-Saharan Africa. Their control still relies on chemical agents, despite growing evidence of insecticide resistance and environmental health concerns. Biorational agents, such as the entomopathogenic fungus Metarhizium anisopliae, might be an alternative for vector control. Recently, the M. anisopliae isolate ICIPE 7 has been developed into a commercial product in Kenya for control of ticks on cattle. We were interested in assessing the potential of controlling not only ticks but also disease-transmitting mosquitoes and tsetse flies using cattle as blood hosts, with the aim of developing a product for integrated vector management. Laboratory bioassays were carried out with M. anisopliae, isolate ICIPE 7 and isolate ICIPE 30, to compare efficacy against laboratory-reared Anopheles arabiensis. ICIPE 7 was further tested against wild Glossina fuscipes and Rhipicephalus spp. Dose-response tests were implemented, period of mosquito exposure was evaluated for effects on time to death, and the number of spores attached to exposed vectors was assessed. Exposure to 109 spores/mL of ICIPE 7 for 10 min resulted in a similar mortality of An. arabiensis as exposure to ICIPE 30, albeit at a slower rate (12 vs. 8 days). The same ICIPE 7 concentration also resulted in mortalities of tsetse flies (LT50: 16 days), tick nymphs (LT50: 11 days), and adult ticks (LT50: 20 days). Mosquito mortality was dose-dependent, with decreasing LT50 of 8 days at a concentration of 106 spores/mL to 6 days at 1010 spores/mL. Exposure period did not modulate the outcome, 1 min of exposure still resulted in mortality, and spore attachment to vectors was dose-dependent. The laboratory bioassays confirmed that ICIPE 7 has the potential to infect and cause mortality to the three exposed arthropods, though at slower rate, thus requiring further validation under field conditions.

Tungiasis among children in Kenya is associated with poor nutrition status, absenteeism, poor school performance and high impact on quality of life.

Tungiasis is a highly neglected tropical skin disease caused by the sand flea, Tunga penetrans. The flea burrows into the skin inducing a strong inflammatory response, leading to pain and mobility restrictions with potential impacts on quality of life. Few countries implement control efforts and there are few data on the impact of the disease to support policy decisions. We conducted a survey to determine the impact of tungiasis among primary school children across nine counties of Kenya. A total of 10,600 pupils aged 8 to 14 years were randomly selected from 97 primary schools and examined for tungiasis. For 81 cases and 578 randomly selected controls, anthropometric measurements were made, and school attendance and exam scores were collected from school records. Of those with tungiasis, 73 were interviewed regarding their quality of life using a tungiasis-specific instrument. Mixed effect ordered logistic and linear models were used to assess associations between disease status and impact variables. Compared to uninfected pupils, those with tungiasis had lower weight-for-age z-scores (adjusted ß -0.41, 95% CI: -0.75-0.06, p = 0.020), missed more days of school the previous term (adjusted Incidence Rate Ratio: 1.49, 95% CI: 1.01-2.21, p = 0.046) and were less likely to receive a high score in mathematics (aOR 0.18, 95% CI: 0.08-0.40, p<0.001) and other subjects. Pupils with severe disease (clinical score >10) were four times more likely to experience severe pain than those with mild disease (OR 3.96, 95% CI: 1.35-11.64, p = 0.012) and a higher impact on their quality of life than those with mild disease (aOR 3.57, 95% CI: 1.17-10.8, p = 0.025) when adjusted for covariates. This study has demonstrated tungiasis has a considerable impact on children's lives and academic achievement. This indicates the need for integrated disease management for school-aged children to protect their physical and cognitive development and their future prospects.

Identifying Potential Determinants of Faecal Contamination on Domestic Floors in Three Settings in Rural Kenya: A Mixed Methods Analysis.

Observational evidence suggests that household floors may be an important domain for the transmission of enteric and parasitic infections. However, little work has been done to investigate how household floors can become contaminated with human and animal faeces. This study uses a mixed methods approach to postulate the proximal and distal determinants of household floor contamination with faeces in groups of rural villages in 3 counties in Kenya (Bungoma, Kwale and Narok). Quantitative data was collected through a household census and analysed descriptively and using mixed effects logistic regression models. Qualitative data was collected through unstructured observations of daily routines and in-depth interviews. These data were analysed thematically with case memos produced for routine activities that were hypothesised to be determinants of floor contamination. Possible proximal determinants of floor contamination included; (1) animal contact with floors; (2) child faeces disposal, and; (3) floor cleaning routines. Distal determinants are suggested to be rooted in the socioeconomic, environmental, and cultural context in which households were located and included; (1) the type and number of animals owned by households; (2) presence/absence of dedicated shelters for housing animals at night, which impacted whether sleeping or cooking areas were exposed to animals; (3) Accessibility of inside spaces to poultry and other roaming animals; (4) ownership of an improved floor; (5) ability of animals to access neighbours compounds; (6) seasonal changes in weather. These results will be of use in identifying the contexts in which faecal contamination of domestic floors may be contributing towards transmission of enteric and parasitic infections and in designing effective interventions to prevent this exposure.

Willingness to accept and participate in a Microsporidia MB-based mosquito release strategy: a community-based rapid assessment in western Kenya.

BACKGROUND: Microsporidia MB, an endosymbiont naturally found in Anopheles mosquitoes inhibits transmission of Plasmodium and is a promising candidate for a transmission-blocking strategy that may involve mosquito release. A rapid assessment was carried out to develop insight into sociodemographic factors, public health concerns, and malaria awareness, management, and prevention practices with the willingness to accept and participate in Microsporidia MB-based transmission-blocking strategy to develop an informed stakeholder engagement process. METHODS: The assessment consisted of a survey conducted in two communities in western Kenya that involved administering a questionnaire consisting of structured, semi-structured, and open questions to 8108 household heads. RESULTS: There was an overall high level of willingness to accept (81%) and participate in the implementation of the strategy (96%). Although the willingness to accept was similar in both communities, Ombeyi community was more willing to participate (OR 22, 95% CI 13-36). Women were less willing to accept (OR 0.8, 95% CI 0.7-0.9) compared to men due to fear of increased mosquito bites near homes. Household heads with incomplete primary education were more willing to accept (OR 1.6, 95% CI 01.2-2.2) compared to those educated to primary level or higher. Perceiving malaria as a moderate or low public health issue was also associated with a lower willingness to accept and participate. Experience of > 3 malaria cases in the family over the last six months and knowledge that malaria is transmitted by only mosquito bites, increased the willingness to accept but reduced the willingness to participate. Awareness of malaria control methods based on mosquitoes that cannot transmit malaria increases the willingness to participate. CONCLUSION: The study showed a high level of willingness to accept and participate in a Microsporidia MB-based strategy in the community, which is influenced by several factors such as community, disease risk perception, gender, education level, knowledge, and experience of malaria. Further research will need to focus on understanding the concerns of women, educated, and employed community members, and factors that contribute to the lower disease risk perception. This improved understanding will lead to the development of an effective communication strategy.

Identification of tungiasis infection hotspots with a low-cost, high-throughput method for extracting Tunga penetrans (Siphonaptera) off-host stages from soil samples-An observational study.

BACKGROUND: The sand flea, Tunga penetrans, is the cause of a severely neglected parasitic skin disease (tungiasis) in the tropics and has received little attention from entomologists to understand its transmission ecology. Like all fleas, T. penetrans has environmental off-host stages presenting a constant source of reinfection. We adapted the Berlese-Tullgren funnel method using heat from light bulbs to extract off-host stages from soil samples to identify the major development sites within rural households in Kenya and Uganda. METHODS AND FINDINGS: Simple, low-cost units of multiple funnels were designed to allow the extraction of >60 soil samples in parallel. We calibrated the method by investigating the impact of different bulb wattage and extraction time on resulting abundance and quality of off-host stages. A cross-sectional field survey was conducted in 49 tungiasis affected households. A total of 238 soil samples from indoor and outdoor living spaces were collected and extracted. Associations between environmental factors, household member infection status and the presence and abundance of off-host stages in the soil samples were explored using generalized models. The impact of heat (bulb wattage) and time (hours) on the efficiency of extraction was demonstrated and, through a stepwise approach, standard operating conditions defined that consistently resulted in the recovery of 75% (95% CI 63-85%) of all present off-host stages from any given soil sample. To extract off-host stages alive, potentially for consecutive laboratory bioassays, a low wattage (15-25 W) and short extraction time (4 h) will be required. The odds of finding off-host stages in indoor samples were 3.7-fold higher than in outdoor samples (95% CI 1.8-7.7). For every one larva outdoors, four (95% CI 1.3-12.7) larvae were found indoors. We collected 67% of all off-host specimen from indoor sleeping locations and the presence of off-host stages in these locations was strongly associated with an infected person sleeping in the room (OR 10.5 95% CI 3.6-28.4). CONCLUSION: The indoor sleeping areas are the transmission hotspots for tungiasis in rural homes in Kenya and Uganda and can be targeted for disease control and prevention measures. The soil extraction methods can be used as a simple tool for monitoring direct impact of such interventions.

House Screening Reduces Exposure to Indoor Host-Seeking and Biting Malaria Vectors: Evidence from Rural South-East Zambia.

This study evaluated the impact of combining house screens with long-lasting insecticidal nets (LLINs) on mosquito host-seeking, resting, and biting behavior. Intervention houses received house screens and LLINs, while control houses received only LLINs. Centre for Disease Control light traps, pyrethrum spray collections and human landing catches were used to assess the densities of indoor and outdoor host-seeking, indoor resting, and biting behavior of malaria vectors in 15 sentinel houses per study arm per sampling method. The protective efficacy of screens and LLINs was estimated through entomological inoculation rates (EIRs). There were 68% fewer indoor host-seeking Anopheles funestus (RR = 0.32, 95% CI 0.20-0.51, p < 0.05) and 63% fewer An. arabiensis (RR = 0.37, 95% CI 0.22-0.61, p < 0.05) in screened houses than unscreened houses. There was a significantly higher indoor biting rate for unscreened houses (6.75 bites/person/h [b/p/h]) than for screened houses (0 b/p/h) (χ2 = 6.67, df = 1, p < 0.05). The estimated indoor EIR in unscreened houses was 2.91 infectious bites/person/six months, higher than that in screened houses (1.88 infectious bites/person/six months). Closing eaves and screening doors and windows has the potential to reduce indoor densities of malaria vectors and malaria transmission.

Myth or truth: investigation of the jumping ability of Tunga penetrans (Siphonaptera: Tungidae).

Female sand fleas (Tunga penetrans Linnaeus, 1758, Siphonaptera: Tungidae) cause a severe parasitic skin disease known as tungiasis. T. penetrans is a small flea, measuring less than 1 mm in length. The females of this species burrow into the skin of human and animal hosts and mostly affect the feet. This has led to the anecdotal assumption that T. penetrans, unlike its relatives in the Siphonaptera family, would have a limited jumping ability potentially not reaching higher body parts. However, there is no data supporting this. This study evaluated the jumping capabilities of T. penetrans for height and distance using sticky tapes. The vertical jump of the female T. penetrans ranged from 4.5 to 100 mm with a mean of 40 mm whereas the vertical jump of the male T. penetrans ranged from 1.2 to 138 mm with a mean of 46 mm. The horizontal jump of the female T. penetrans ranged from 18 to 138 mm with a mean of 64 mm and that of the male ranged from 9 to 251 mm with a mean of 80 mm. Based on the literature, fleas of various species have been described as jumping vertically 50-100 times their size and horizontally 5-100 times their size. In this respect, sand fleas appear to have equal expert jumping abilities to their relatives. Their aggregation on people's feet is not likely a result of their poor jumping ability but might be an adaptation to the host's behavior which would require further investigations.

Developing low-cost house floors to control tungiasis in Kenya - a feasibility study.

CONTEXT: Tungiasis is a neglected tropical skin disease endemic in resource-poor communities. It is caused by the penetration of the female sand flea, Tunga penetrans, into the skin causing immense pain, itching, difficulty walking, sleeping and concentrating on school or work. Infection is associated with living in a house with unsealed earthen house floors. METHODS: This feasibility study used a community-based co-creation approach to develop and test simple, locally appropriate, and affordable flooring solutions to create a sealed, washable floor for the prevention of tungiasis. Locally used techniques were explored and compared in small slab trials. The floor with best strength and lowest cost was pilot trialed in 12 households with tungiasis cases to assess its durability and costs, feasibility of installation in existing local houses using local masons and explore community perceptions. Disease outcomes were measured to estimate potential impact. RESULTS: It was feasible to build the capacity of a community-based organization to conduct research, develop a low-cost floor and conduct a pilot trial. The optimal low-cost floor was stabilized local subsoil with cement at a 1:9 ratio, installed as a 5 cm depth slab. A sealed floor was associated with a lower mean infection intensity among infected children than in control households (aIRR 0.53, 95%CI 0.29-0.97) when adjusted for covariates. The cost of the new floor was US$3/m2 compared to $10 for a concrete floor. Beneficiaries reported the floor made their lives much easier, enabled them to keep clean and children to do their schoolwork and eat while sitting on the floor. Challenges encountered indicate future studies would need intensive mentoring of masons to ensure the floor is properly installed and households supervised to ensure the floor is properly cured. CONCLUSION: This study provided promising evidence that retrofitting simple cement-stabilised soil floors with locally available materials is a feasible option for tungiasis control and can be implemented through training of community-based organisations. Disease outcome data is promising and suggests that a definitive trial is warranted. Data generated will inform the design of a fully powered randomized trial combined with behaviour change communications. TRIAL REGISTRATION: ISRCTN 62801024 (retrospective 07.07.2023).

Neurocognitive and mental health outcomes in children with tungiasis: a cross-sectional study in rural Kenya and Uganda.

BACKGROUND: Tungiasis, a neglected tropical parasitosis, disproportionately affects children. Few empirical studies have reported neurocognitive and mental health outcomes of children with ectoparasitic skin diseases like tungiasis. Pathophysiology of tungiasis suggests it could detrimentally affect cognition and behaviour. This study pioneered the investigation of neurocognitive and mental health outcomes in children with tungiasis. METHODS: This was a multi-site cross-sectional study including 454 quasi-randomly sampled school-children aged 8-14 from 48 randomly selected schools in two counties in Kenya and a district in Uganda. The participants were stratified into infected and uninfected based on the presence of tungiasis. The infected were further classified into mild and severe infection groups based on the intensity of the infection. Adapted, validated, and standardized measures of cognition and mental health such as Raven Matrices and Child Behaviour Checklist were used to collect data. Statistical tests including a multilevel, generalized mixed-effects linear models with family link set to identity were used to compare the scores of uninfected and infected children and to identify other potential risk factors for neurocognitive and behavioural outcomes. RESULTS: When adjusted for covariates, mild infection was associated with lower scores in literacy [adjusted ß(aß) = - 8.9; 95% confidence interval (CI) - 17.2, - 0.6], language (aß = - 1.7; 95% CI - 3.2, - 0.3), cognitive flexibility (aß = - 6.1; 95% CI - 10.4, - 1.7) and working memory (aß = - 0.3; 95% CI - 0.6, - 0.1). Severe infection was associated with lower scores in literacy (aß = - 11.0; 95% CI - 19.3, - 2.8), response inhibition, (aß = - 2.2; 95% CI - 4.2, - 0.2), fine motor control (aß = - 0.7; 95% CI - 1.1, - 0.4) and numeracy (aß = - 3; 95% CI - 5.5, - 0.4). CONCLUSIONS: This study provides first evidence that tungiasis is associated with poor neurocognitive functioning in children. Since tungiasis is a chronic disease with frequent reinfections, such negative effects may potentially impair their development and life achievements.

National prevalence and risk factors for tungiasis in Kenya.

BACKGROUND: Tungiasis is a highly neglected tropical skin disease caused by the sand flea, Tunga penetrans, the female of which burrows into the skin, causing pain and itching. The disease occurs throughout South America and sub-Saharan Africa but there are few systematic data on national disease burdens. The tungiasis research community is keen to develop survey methods to fill this gap. Here we used a school-based, thorough examination method to determine the prevalence and risk factors for tungiasis in Kenya. METHODS: We conducted the first nationally representative survey of tungiasis, including nine counties covering the major ecological zones of Kenya. A stratified multistage random sampling was used to select 22 primary schools from each of the nine counties and to select up to 114 pupils aged 8 to 14 years in each school. Pupils were examined thoroughly for tungiasis. Two surveys were conducted, the first between May and July 2021 and the second between October 2021 and April 2023 when pupils were also interviewed for risk factors. Mixed effect logistic regression models were used to test associations of independent variables with tungiasis using the school as a random effect. RESULTS: The overall prevalence of tungiasis in the first survey was 1.35% [95% confidence interval (CI): 1.15-1.59%], and 0.89% in the second survey. The prevalence ranged from 0.08% (95% CI: 0.01-0.59%) in Taita Taveta county to 3.24% (95% CI: 2.35-4.44%) in Kajiado county. Tungiasis infection was associated with county of residence, male sex [adjusted odds ratio (aOR) = 2.01, 95% CI: 1.52-2.67], and lower age (aOR = 0.81, 95% CI: 0.75-0.88). For the first time we demonstrate an association with attending public schools rather than private schools (aOR = 5.62, 95% CI: 1.20-26.22) and lower socioeconomic status (aOR = 0.10, 95% CI: 0.03-0.33). Using a rapid screening method of the top of feet only, would have missed 62.9% of all cases, 78.9% of mild cases and 20.0% of severe cases. CONCLUSIONS: Tungiasis is widely but heterogeneously distributed across Kenya. School-based surveys offer an efficient strategy for mapping tungiasis distribution.

Evaluation of the solar-powered Silver Bullet 2.1 (Lumin 8) light trap for sampling malaria vectors in western Kenya.

BACKGROUND: Centers for Disease Control and Prevention (CDC) light traps are widely used for sampling mosquitoes. However, this trap, manufactured in the USA, poses challenges for use in sub-Saharan Africa due to procurement costs and shipping time. Traps that are equally efficient than the CDC light trap, but which are amenable for use in remote African settings and made in Africa, are desirable to improve local vector surveillance. This study evaluated a novel solar-powered light trap made in South Africa (Silver Bullet trap; SB), for its efficiency in malaria vector sampling in western Kenya. METHODS: Large cage (173.7 m3) experiments and field evaluations were conducted to compare the CDC-incandescent light trap (CDC-iLT), CDC-UV fluorescent tube light trap (CDC-UV), SB with white diodes (SB-White) and SB with UV diodes (SB-UV) for sampling Anopheles mosquitoes. Field assessments were done indoors and outdoors following a Latin square design. The wavelengths and absolute spectral irradiance of traps were compared using spectrometry. RESULTS: The odds of catching a released Anopheles in the large cage experiments with the SB-UV under ambient conditions in the presence of a CDC-iLT in the same system was three times higher than what would have been expected when the two traps were equally attractive (odds ratio (OR) 3.2, 95% confidence interval CI 2.8-3.7, P < 0.01)). However, when the white light diode was used in the SB trap, it could not compete with the CDC-iLT (OR 0.56, 95% CI 0.48-0.66, p < 0.01) when the two traps were provided as choices in a closed system. In the field, the CDC and Silver Bullet traps were equally effective in mosquito sampling. Irrespective of manufacturer, traps emitting UV light performed better than white or incandescent light for indoor sampling, collecting two times more Anopheles funestus sensu lato (s.l.) (RR 2.5; 95% CI 1.7-3.8) and Anopheles gambiae s.l. (RR 2.5; 95% 1.7-3.6). Outdoor collections were lower than indoor collections and similar for all light sources and traps. CONCLUSIONS: The solar-powered SB trap compared well with the CDC trap in the field and presents a promising new surveillance device especially when charging on mains electricity is challenging in remote settings.

A randomized, double-blind placebo-control study assessing the protective efficacy of an odour-based 'push-pull' malaria vector control strategy in reducing human-vector contact.

Novel malaria vector control strategies targeting the odour-orientation of mosquitoes during host-seeking, such as 'attract-and-kill' or 'push-and-pull', have been suggested as complementary tools to indoor residual spraying and long-lasting insecticidal nets. These would be particularly beneficial if they can target vectors in the peri-domestic space where people are unprotected by traditional interventions. A randomized double-blind placebo-control study was implemented in western Kenya to evaluate: a 'push' intervention (spatial repellent) using transfluthrin-treated fabric strips positioned at open eave gaps of houses; a 'pull' intervention placing an odour-baited mosquito trap at a 5 m distance from a house; the combined 'push-pull' package; and the control where houses contained all elements but without active ingredients. Treatments were rotated through 12 houses in a randomized-block design. Outdoor biting was estimated using human landing catches, and indoor mosquito densities using light-traps. None of the interventions provided any protection from outdoor biting malaria vectors. The 'push' reduced indoor vector densities dominated by Anopheles funestus by around two thirds. The 'pull' device did not add any benefit. In the light of the high Anopheles arabiensis biting densities outdoors in the study location, the search for efficient outdoor protection and effective pull components needs to continue.

Characterization of tungiasis infection and morbidity using thermography in Kenya revealed higher disease burden during COVID-19 school closures.

BACKGROUND: Tungiasis is a neglected tropical skin disease caused by the sand flea Tunga penetrans. Female fleas penetrate the skin, particularly at the feet, and cause severe inflammation. This study aimed to characterize disease burden in two highly affected regions in Kenya, to test the use of thermography to detect tungiasis-associated inflammation and to create a new two-level classification of disease severity suitable for mapping, targeting, and monitoring interventions. METHODS: From February 2020 to April 2021, 3532 pupils age 8-14 years were quasi-randomly selected in 35 public primary schools and examined for tungiasis and associated symptoms. Of the infected pupils, 266 were quasi-randomly selected and their households visited, where an additional 1138 family members were examined. Inflammation was assessed using infra-red thermography. A Clinical score was created combining the number of locations on the feet with acute and chronic symptoms and infra-red hotspots. RESULTS: The overall prevalence of tungiasis among all the school pupils who were randomly selected during survey rounds 1 and 3 was 9.3% [95% confidence interval (CI): 8.4-10.3]. Based on mixed effects logistic models, the odds of infection with tungiasis among school pupils was three times higher in Kwale (coastal Kenya) than in Siaya [western Kenya; adjusted odds ratio (aOR) = 0.36, 95% CI: 0.18-0.74]; three times higher in males than in females (aOR = 3.0, 95% CI: 2.32-3.91) and three times lower among pupils sleeping in a house with a concrete floor (aOR = 0.32, 95% CI: 0.24-0.44). The odds of finding an infected person among the household population during surveys before the COVID-19 pandemic was a third (aOR = 0.32, 95% CI: 0.19-0.53) of that when schools were closed due to COVID-19 restrictions and approximately half (aOR = 0.44, 95% CI: 0.29-0.68) in surveys done after school re-opening (round 3). Infection intensity was positively correlated with inflammation as measured by thermography (Spearman's rho = 0.68, P < 0.001) and with the clinical score (rho = 0.86, P < 0.001). Based on the two-level classification, severe cases were associated with a threefold higher level of pain (OR = 2.99, 95% CI: 2.02-4.43) and itching (OR = 3.31, 95% CI: 2.24-4.89) than mild cases. CONCLUSIONS: Thermography was a valuable addition for assessing morbidity and the proposed two-level classification of disease severity clearly separated patients with mild and severe impacts. The burden of tungiasis was considerably higher in households surveyed during COVID-19 restrictions suggesting underlying risks are found in the home environment more than in school.

Anopheles rufipes implicated in malaria transmission both indoors and outdoors alongside Anopheles funestus and Anopheles arabiensis in rural south-east Zambia.

BACKGROUND: The primary malaria vector-control interventions, indoor residual spraying and long-lasting insecticidal nets, are effective against indoor biting and resting mosquito species. Consequently, outdoor biting and resting malaria vectors might elude the primary interventions and sustain malaria transmission. Varied vector biting and resting behaviour calls for robust entomological surveillance. This study investigated the bionomics of malaria vectors in rural south-east Zambia, focusing on species composition, their resting and host-seeking behaviour and sporozoite infection rates. METHODS: The study was conducted in Nyimba District, Zambia. Randomly selected households served as sentinel houses for monthly collection of mosquitoes indoors using CDC-light traps (CDC-LTs) and pyrethrum spray catches (PSC), and outdoors using only CDC-LTs for 12 months. Mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were further identified using molecular techniques. Plasmodium falciparum sporozoite infection was determined using sandwich enzyme-linked immunosorbent assays. RESULTS: From 304 indoor and 257 outdoor light trap-nights and 420 resting collection, 1409 female Anopheles species mosquitoes were collected and identified morphologically; An. funestus (n = 613; 43.5%), An. gambiae sensu lato (s.l.)(n = 293; 20.8%), Anopheles pretoriensis (n = 282; 20.0%), Anopheles maculipalpis (n = 130; 9.2%), Anopheles rufipes (n = 55; 3.9%), Anopheles coustani s.l. (n = 33; 2.3%), and Anopheles squamosus (n = 3, 0.2%). Anopheles funestus sensu stricto (s.s.) (n = 144; 91.1%) and Anopheles arabiensis (n = 77; 77.0%) were the dominant species within the An. funestus group and An. gambiae complex, respectively. Overall, outdoor CDC-LTs captured more Anopheles mosquitoes (mean = 2.25, 95% CI 1.22-3,28) than indoor CDC-LTs (mean = 2.13, 95% CI 1.54-2.73). Fewer resting mosquitoes were collected with PSC (mean = 0.44, 95% CI 0.24-0.63). Sporozoite infectivity rates for An. funestus, An. arabiensis and An. rufipes were 2.5%, 0.57% and 9.1%, respectively. Indoor entomological inoculation rates (EIRs) for An. funestus s.s, An. arabiensis and An. rufipes were estimated at 4.44, 1.15 and 1.20 infectious bites/person/year respectively. Outdoor EIRs for An. funestus s.s. and An. rufipes at 7.19 and 4.31 infectious bites/person/year, respectively. CONCLUSION: The findings of this study suggest that An. rufipes may play an important role in malaria transmission alongside An. funestus s.s. and An. arabiensis in the study location.

Six decades of malaria vector control in southern Africa: a review of the entomological evidence-base.

BACKGROUND: Countries in the southern Africa region have set targets for malaria elimination between 2020 and 2030. Malaria vector control is among the key strategies being implemented to achieve this goal. This paper critically reviews published entomological research over the past six decades in three frontline malaria elimination countries namely, Botswana Eswatini and Namibia, and three second-line malaria elimination countries including Mozambique, Zambia, and Zimbabwe. The objective of the review is to assess the current knowledge and highlight gaps that need further research attention to strengthen evidence-based decision-making toward malaria elimination. METHODS: Publications were searched on the PubMed engine using search terms: "(malaria vector control OR vector control OR malaria vector*) AND (Botswana OR Swaziland OR Eswatini OR Zambia OR Zimbabwe OR Mozambique)". Opinions, perspectives, reports, commentaries, retrospective analysis on secondary data protocols, policy briefs, and reviews were excluded. RESULTS: The search resulted in 718 publications with 145 eligible and included in this review for the six countries generated over six decades. The majority (139) were from three countries, namely Zambia (59) and Mozambique (48), and Zimbabwe (32) whilst scientific publications were relatively scanty from front-line malaria elimination countries, such as Namibia (2), Botswana (10) and Eswatini (4). Most of the research reported in the publications focused on vector bionomics generated mostly from Mozambique and Zambia, while information on insecticide resistance was mostly available from Mozambique. Extreme gaps were identified in reporting the impact of vector control interventions, both on vectors and disease outcomes. The literature is particularly scanty on important issues such as change of vector ecology over time and space, intervention costs, and uptake of control interventions as well as insecticide resistance. CONCLUSIONS: The review reveals a dearth of information about malaria vectors and their control, most noticeable among the frontline elimination countries: Namibia, Eswatini and Botswana. It is of paramount importance that malaria vector research capacity and routine entomological monitoring and evaluation are strengthened to enhance decision-making, considering changing vector bionomics and insecticide resistance, among other determinants of malaria vector control.

Less is more: repellent-treated fabric strips as a substitute for full screening of open eave gaps for indoor and outdoor protection from malaria mosquito bites.

BACKGROUND: Providing protection from malaria vector bites, both indoors and outdoors, is crucial to curbing malaria parasite transmission. Screening of house entry points, especially with incorporated insecticides, confers significant protection but remains a costly and labour-intensive application. Use of spatial repellents has shown promise in creating areas of protection in peri-domestic areas. METHODS: This study aimed at comparing the protection provided by transfluthrin-treated and untreated complete screens over open eave gaps with incomplete transfluthrin-treated eave strips as a potential replacement for a full screen. Human landing catches were implemented independently inside and outside an experimental hut under controlled semi-field conditions, with insectary-reared Anopheles arabiensis mosquitoes. RESULTS: The odds of a female mosquito finding a human volunteer indoors and attempting to bite were similar whether the eaves were completely open or there was an untreated fabric strip fixed around the eaves. However, when the eave gap was completely screened without insecticide, the odds of receiving a bite indoors were reduced by 70% (OR 0.30, 95% CI 0.20-0.47). Adding transfluthrin to the full screen, further increased the protection indoors, with the odds of receiving a bite reduced by 92% (0.08, 95% CI 0.04-0.16) compared to the untreated screen. Importantly, the same protection was conferred when only a narrow transfluthrin-treated fabric strip was loosely fixed around the eave gap (OR 0.07, 95% CI 0.04-0.13). The impact of the transfluthrin treatment on outdoor biting was correlated with evening temperatures during the experiments. At lower evening temperatures, a transfluthrin-treated, complete screen provided moderate and variable protection from bites (OR 0.62, 95% CI 0.37-1.03), whilst at higher evening temperatures the odds of receiving a bite outdoors was over four times lower in the presence of transfluthrin, on either a full screen (OR 0.22 95% 0.12-0.38) or a fabric strip (OR 0.25, 95% 0.15-0.42), than when no treatment was present. CONCLUSION: The findings suggest that transfluthrin-treated fabric strips can provide a substitute for complete eave screens. They are a simple, easy-to-handle tool for protecting people from malaria mosquito bites indoors and potentially around the house in climatic areas where evening and night-time temperatures are relatively high.

Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material.

Tungiasis is a neglected tropical disease caused by skin-penetrating female Tunga penetrans fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify T. penetrans immature stages and sites where they develop, diagnostic PCRs are required. However, flea larvae feed on soil organic matter rich in PCR inhibitors. Here, three DNA preparation methods, including a soil DNA kit that removes inhibitors, a simple ammonium acetate precipitation approach (AmAcet) and a crude lysate of larvae (CL), were combined with amplification by the highly processive FIREPol® Taq or the inhibitor-resistant Phusion® polymerase. Independent of the polymerase used, the frequency of successful amplification, Cq values and PCR efficacies for the low-cost CL and AmAcet methods were superior to the commercial kit for amplification of a 278 bp partial internal transcribed spacer-2 (ITS-2) and a 730 bp pan-Siphonaptera cytochrome oxidase II PCR. For the CL method combined with Phusion® polymerase, the costs were approximately 20-fold lower than for the methods based on the soil DNA kit, which is a considerable advantage in resource-poor settings. The ITS-2 PCR did not amplify Ctenocephalides felis genomic or Tunga trimammilata ITS-2 plasmid DNA, meaning it can be used to specifically identify T. penetrans.

Evaluating the efficacy, impact, and feasibility of community-based house screening as a complementary malaria control intervention in southern Africa: a study protocol for a household randomized trial.

BACKGROUND: Concerted effort to control malaria has had a substantial impact on the transmission of the disease in the past two decades. In areas where reduced malaria transmission is being sustained through insecticide-based vector control interventions, primarily long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), non-insecticidal complementary tools will likely be needed to push towards malaria elimination. Once interruption in local disease transmission is achieved, insecticide-based measures can be scaled down gradually and eventually phased out, saving on costs of sustaining control programs and mitigating any unintended negative health and environmental impacts posed by insecticides. These non-insecticidal methods could eventually replace insecticidal methods of vector control. House screening, a non-insecticidal method, has a long history in malaria control, but is still not widely adopted in sub-Saharan Africa. This study aims to add to the evidence base for this intervention in low transmission settings by assessing the efficacy, impact, and feasibility of house screening in areas where LLINs are conventionally used for malaria control. METHODS: A two-armed, household randomized clinical trial will be conducted in Mozambique, Zambia, and Zimbabwe to evaluate whether combined the use of house screens and LLINs affords better protection against clinical malaria in children between 6 months and 13 years compared to the sole use of LLINs. Eight hundred households will be enrolled in each study area, where 400 households will be randomly assigned the intervention, house screening, and LLINs while the control households will be provided with LLINs only. Clinical malaria incidence will be estimated by actively following up one child from each household for 6 months over the malaria transmission season. Cross-sectional parasite prevalence will be estimated by testing all participating children for malaria parasites at the beginning and end of each transmission season using rapid diagnostic tests. CDC light traps and pyrethrum spray catches (PSC) will be used to sample adult mosquitoes and evaluate the impact of house screening on indoor mosquito density, species distribution, and sporozoite rates. DISCUSSION: This study will contribute epidemiological data on the impact of house screening on malaria transmission and assess the feasibility of its implementation on a programmatic scale. TRIAL REGISTRATION: ClinicalTrials.gov PACTR202008524310568 . Registered on August 11, 2020.

Grass-like plants release general volatile cues attractive for gravid Anopheles gambiae sensu stricto mosquitoes.

BACKGROUND: Understanding the ecology and behaviour of disease vectors, including the olfactory cues used to orient and select hosts and egg-laying sites, are essential for the development of novel, insecticide-free control tools. Selected graminoid plants have been shown to release volatile chemicals attracting malaria vectors; however, whether the attraction is selective to individual plants or more general across genera and families is still unclear. METHODS: To contribute to the current evidence, we implemented bioassays in two-port airflow olfactometers and in large field cages with four live graminoid plant species commonly found associated with malaria vector breeding sites in western Kenya: Cyperus rotundus and C. exaltatus of the Cyperaceae family, and Panicum repens and Cynodon dactylon of the Poaceae family. Additionally, we tested one Poaceae species, Cenchrus setaceus, not usually associated with water. The volatile compounds released in the headspace of the plants were identified using gas chromatography/mass spectrometry. RESULTS: All five plants attracted gravid vectors, with the odds of a mosquito orienting towards the choice-chamber with the plant in an olfactometer being 2-5 times higher than when no plant was present. This attraction was maintained when tested with free-flying mosquitoes over a longer distance in large field cages, though at lower strength, with the odds of attracting a female 1.5-2.5 times higher when live plants were present than when only water was present in the trap. Cyperus rotundus, previously implicated in connection with an oviposition attractant, consistently elicited the strongest response from gravid vectors. Volatiles regularly detected were limonene, ß-pinene, ß-elemene and ß-caryophyllene, among other common plant compounds previously described in association with odour-orientation of gravid and unfed malaria vectors. CONCLUSIONS: The present study confirms that gravid Anopheles gambiae sensu stricto use chemical cues released from graminoid plants to orientate. These cues are released from a variety of graminoid plant species in both the Cyperaceae and Poaceae family. Given the general nature of these cues, it appears unlikely that they are exclusively used for the location of suitable oviposition sites. The utilization of these chemical cues for attract-and-kill trapping strategies must be explored under natural conditions to investigate their efficiency when in competition with complex interacting natural cues.