Field- and laboratory-based studies on correlates of Chlamydia trachomatis transmission by Musca sorbens: Determinants of fly-eye contact and investigations into fly carriage of elementary bodies.

Musca sorbens (Diptera: Muscidae) flies are thought to be vectors of the blinding eye disease trachoma, carrying the bacterium Chlamydia trachomatis (Ct) between the eyes of individuals. While their role as vectors has been convincingly demonstrated via randomised controlled trials in The Gambia, studies of fly-borne trachoma transmission remain scant and as such our understanding of their ability to transmit Ct remains poor. We examined fly-eye contact and caught eye-seeking flies from 494 individuals (79% aged ≤9 years) in Oromia, Ethiopia. Ct-carrying flies (harbouring Ct DNA) were found to cluster spatially in and nearby to households in which at least one resident had Ct infection. Fly-eye contact was positively associated with the presence of trachoma (disease), lower human body weight and increased human body temperature. Studies of laboratory-reared M. sorbens indicated that Ct is found both externally and internally following feeds on Ct culture, with scanning electron microscopy revealing how Ct bodies can cling to fly hairs (setae). Testing for Ct on field-caught M. sorbens found fly 'bodies' (thorax, wings and abdomen) to consistently test positive for Ct while legs and heads were infrequently Ct-positive. These studies strongly support the role of M. sorbens as vectors of trachoma and highlight the need for improved understanding of fly-borne trachoma transmission dynamics and vector competence.

Evaluation of the efficacy of insecticide-treated scarves to protect children from the trachoma vector Musca sorbens (Diptera: Muscidae): A phase II randomised controlled trial in Oromia, Ethiopia.

Background: The eye-seeking fly Musca sorbens can act as a vector for ocular Chlamydia trachomatis, causing trachoma, yet there has been very little research on control measures. We investigated whether insect repellent products, specifically insecticide-treated clothing, could provide personal protection to the user from eye-seeking flies. Methods: We first conducted a series of phase I laboratory studies to inform our choice of field intervention. We then conducted a phase II randomised controlled trial testing the efficacy of permethrin-treated scarves (PTS) in reducing fly-face contact in Oromia, Ethiopia. Children aged 4-10 years in full health and with no known adverse reactions to permethrin or other insecticides were allocated to either arm using restricted randomisation. Intervention arm children wore Insect Shield® versatile wraps (as PTS) for 28 days. The primary outcomes, fly-eye, -nose and -mouth contact, were assessed on the first day (0/30/60/180 minutes), on day 7 and on day 28. All participants present per timepoint were included in analyses. This trial was registered with ClinicalTrials.gov (NCT03813069). Findings: Participants were recruited to the field trial between 29/10/2019 and 01/11/2019, 58 were randomised to test or control arm. More fly (-eye, -nose and -mouth) contacts were observed in the PTS arm at baseline. After adjusting for baseline contact rates, across all timepoints there was a 35% decrease in fly-eye contacts in the PTS relative to control arm (rate ratio [RR] 0.65, 95% CI 0.52-0.83). Similar cross-timepoint reductions were seen for fly-nose and fly-mouth contacts (RR 0.69, 95% CI 0.51-0.92 and RR 0.79, 95% CI 0.62-1.01, respectively). All children were included on day 0. Two in the control arm were absent on day 7, one left the study and four were excluded from analysis at day 28. No adverse events occurred in the trial. Interpretation: Musca sorbens flies are sufficiently repelled by PTS to reduce fly-eye contacts for the wearer, thus possibly reducing the risk of trachoma transmission. Permethrin-treated scarves may therefore an alternative to insecticide space spraying for protection from these flies. Funding: Wellcome Trust.

Towards an odour-baited trap to control Musca sorbens, the putative vector of trachoma.

Musca sorbens is a synanthropic filth fly that aggressively attacks people to feed from mucous membranes of the eyes, nose or mouth, from open sores, or from sweat. It has long been suspected that this fly contributes to the transmission of eye infections, particularly trachoma, and recent work has added to the evidence base that M. sorbens is a trachoma vector in Ethiopia. There are few options to control M. sorbens, largely due to a lack of evidence. Space spraying with insecticides is effective, but an environmentally sound and long-term sustainable solution would be better, for example, mass trapping. We tested commercially available and homemade trap types in a pilot (laboratory) study and three field studies. A homemade design, built from a bucket and two empty water bottles, baited with a commercially available lure, The Buzz, was found to be most effective. This trap caught 3848 M. sorbens over 26 trap 'events' (3- or 4-day periods); mean/median per 24 h 43.6 (standard deviation 137.10)/2.25 (IQR 0.25-12.67). The Buzz lure is cheap and effective for 4 weeks, and trap components cheap and locally available. Further studies are needed to understand the impact of this trap on local fly populations and the local transmission of trachoma.

Detecting extra-ocular Chlamydia trachomatis in a trachoma-endemic community in Ethiopia: Identifying potential routes of transmission.

BACKGROUND: Trachoma elimination efforts are hampered by limited understanding of Chlamydia trachomatis (Ct) transmission routes. Here we aimed to detect Ct DNA at non-ocular sites and on eye-seeking flies. METHODS: A population-based household survey was conducted in Oromia Region, Ethiopia. Ocular and non-ocular (faces, hands, clothing, water containers and sleeping surfaces) swabs were collected from all individuals. Flies were caught from faces of children. Flies, ocular swabs and non-ocular swabs were tested for Ct by quantitative PCR. RESULTS: In total, 1220 individuals in 247 households were assessed. Active trachoma (trachomatous inflammation-follicular) and ocular Ct were detected in 10% and 2% of all-ages, and 21% and 3% of 1-9-year-olds, respectively. Ct was detected in 12% (95% CI:8-15%) of tested non-ocular swabs from ocular-positive households, but in none of the non-ocular swabs from ocular-negative households. Ct was detected on 24% (95% CI:18-32%) of flies from ocular-positive households and 3% (95% CI:1-6%) of flies from ocular-negative households. CONCLUSION: Ct DNA was detected on hands, faces and clothing of individuals living in ocular-positive households suggesting that this might be a route of transmission within Ct infected households. In addition, we detected Ct on flies from ocular-positive households and occasionally in ocular-negative households suggesting that flies might be a vector for transmission within and between Ct infected and uninfected households. These potential transmission routes may need to be simultaneously addressed to suppress transmission.

Responses of the putative trachoma vector, Musca sorbens, to volatile semiochemicals from human faeces.

The putative vector of trachoma, Musca sorbens, prefers to lay its eggs on human faeces on the ground. This study sought to determine whether M. sorbens females were attracted to volatile odours from human faeces in preference to odours from the faeces of other animals, and to determine whether specific volatile semiochemicals mediate selection of the faeces. Traps baited with the faeces of humans and local domestic animals were used to catch flies at two trachoma-endemic locations in The Gambia and one in Ethiopia. At all locations, traps baited with faeces caught more female M. sorbens than control traps baited with soil, and human faeces was the most successful bait compared with soil (mean rate ratios 44.40, 61.40, 10.50 [P<0.001]; 8.17 for child faeces [P = 0.004]). Odours from human faeces were sampled by air entrainment, then extracts of the volatiles were tested by coupled gas chromatography-electroantennography with laboratory-reared female M. sorbens. Twelve compounds were electrophysiologically active and tentatively identified by coupled mass spectrometry-gas chromatography, these included cresol, indole, 2-methylpropanoic acid, butanoic acid, pentanoic acid and hexanoic acid. It is possible that some of these volatiles govern the strong attraction of M. sorbens flies to human faeces. If so, a synthetic blend of these chemicals, at the correct ratios, may prove to be a highly attractive lure. This could be used in odour-baited traps for monitoring or control of this species in trachoma-endemic regions.

Molecular quantification of Plasmodium parasite density from the blood retained in used RDTs.

Most malaria-endemic countries are heavily reliant upon rapid diagnostic tests (RDT) for malaria case identification and treatment. RDT previously used for malaria diagnosis can subsequently be used for molecular assays, including qualitative assessment of parasite species present or the carriage of resistance markers, because parasite DNA can be extracted from the blood inside the RDT which remains preserved on the internal components. However, the quantification of parasite density has not previously been possible from used RDT. In this study, blood samples were collected from school-age children in Western Kenya, in the form of both dried blood spots on Whatman filter paper, and the blood spot that is dropped into rapid diagnostic tests during use. Having first validated a robotic DNA extraction method, the parasite density was determined from both types of sample by duplex qPCR, and across a range of densities. The methods showed good agreement. The preservation of both parasite and human DNA on the nitrocellulose membrane inside the RDT was stable even after more than one year's storage. This presents a useful opportunity for researchers or clinicians wishing to gain greater information about the parasite populations that are being studied, without significant investment of resources.

Plasmodium-associated changes in human odor attract mosquitoes.

Malaria parasites (Plasmodium) can change the attractiveness of their vertebrate hosts to Anopheles vectors, leading to a greater number of vector-host contacts and increased transmission. Indeed, naturally Plasmodium-infected children have been shown to attract more mosquitoes than parasite-free children. Here, we demonstrate Plasmodium-induced increases in the attractiveness of skin odor in Kenyan children and reveal quantitative differences in the production of specific odor components in infected vs. parasite-free individuals. We found the aldehydes heptanal, octanal, and nonanal to be produced in greater amounts by infected individuals and detected by mosquito antennae. In behavioral experiments, we demonstrated that these, and other, Plasmodium-induced aldehydes enhanced the attractiveness of a synthetic odor blend mimicking "healthy" human odor. Heptanal alone increased the attractiveness of "parasite-free" natural human odor. Should the increased production of these aldehydes by Plasmodium-infected humans lead to increased mosquito biting in a natural setting, this would likely affect the transmission of malaria.

Plasmodium falciparum parasites with histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in two endemic regions of Kenya.

Deletions of the Plasmodium falciparum hrp2 and hrp3 genes can affect the performance of HRP2-based malaria rapid diagnostic tests (RDTs). Such deletions have been reported from South America, India and Eritrea. Whether these parasites are widespread in East Africa is unknown. A total of 274 samples from asymptomatic children in Mbita, western Kenya, and 61 genomic  data from Kilifi, eastern Kenya, were available for analysis. PCR-confirmed samples were investigated for the presence of pfhrp2 and pfhrp3 genes. In samples with evidence of deletion, parasite presence was confirmed by amplifying three independent genes. We failed to amplify pfhrp2 from 25 of 131 (19.1%) PCR-confirmed samples. Of these, only 8 (10%) samples were microscopic positive and were classified as pfhrp2-deleted. Eight microscopically-confirmed pfhrp2-deleted samples with intact pfhrp3 locus were positive by HRP2-based RDT. In addition, one PCR-confirmed infection showed a deletion at the pfhrp3 locus. One genomic sample lacked pfhrp2 and one lacked pfhrp3. No sample harbored parasites lacking both genes. Parasites lacking pfhrp2 are present in Kenya, but may be detectable by HRP-based RDT at higher parasitaemia, possibly due to the presence of intact pfhrp3. These findings warrant further systematic study to establish prevalence and diagnostic significance.

Odours of Plasmodium falciparum-infected participants influence mosquito-host interactions.

Malaria parasites are thought to influence mosquito attraction to human hosts, a phenomenon that may enhance parasite transmission. This is likely mediated by alterations in host odour because of its importance in mosquito host-searching behaviour. Here, we report that the human skin odour profile is affected by malaria infection. We compared the chemical composition and attractiveness to Anopheles coluzzii mosquitoes of skin odours from participants that were infected by Controlled Human Malaria Infection with Plasmodium falciparum. Skin odour composition differed between parasitologically negative and positive samples, with positive samples collected on average two days after parasites emerged from the liver into the blood, being associated with low densities of asexual parasites and the absence of gametocytes. We found a significant reduction in mosquito attraction to skin odour during infection for one experiment, but not in a second experiment, possibly due to differences in parasite strain. However, it does raise the possibility that infection can affect mosquito behaviour. Indeed, several volatile compounds were identified that can influence mosquito behaviour, including 2- and 3-methylbutanal, 3-hydroxy-2-butanone, and 6-methyl-5-hepten-2-one. To better understand the impact of our findings on Plasmodium transmission, controlled studies are needed in participants with gametocytes and higher parasite densities.

Development of the larval migration inhibition test for comparative analysis of ivermectin sensitivity in cyathostomin populations.

Cyathostomins are the most prevalent parasitic pathogens of equids worldwide. These nematodes have been controlled using broad-spectrum anthelmintics; however, cyathostomin resistance to each anthelmintic class has been reported and populations insensitive to more than one class are relatively commonplace. The faecal egg count reduction test (FECRT) is considered the most suitable method for screening anthelmintic sensitivity in horses, but is subject to variation and is relatively time-consuming to perform. Here, we describe a larval migration inhibition test (LMIT) to assess ivermectin (IVM) sensitivity in cyathostomin populations. This test measures the paralysing effect of IVM on the ability of third stage larvae (L3) to migrate through a pore mesh. When L3 from a single faecal sample were examined on multiple occasions, variation in migration was observed: this was associated with the length of time that the L3 had been stored before testing but the association was not significant. Half maximal effective concentration (EC50) values were then obtained for cyathostomin L3 from six populations of horses or donkeys that showed varying sensitivity to IVM in previous FECRTs. Larvae from populations indicated as IVM resistant by FECRT displayed significantly higher EC50 values in the LMIT than L3 from populations classified as IVM sensitive or L3 from populations that had not been previously exposed to IVM or had limited prior exposure. The analysis also showed that EC50 values obtained using L3 from animals in which IVM faecal egg count reduction (FECR) levels had been recorded as <95% were significantly higher than EC50 values obtained using L3 from animals for which FECR was measured as >95%. For one of the populations, time that had elapsed since IVM administration had an effect on the EC50 value obtained, with a longer time since treatment associated with lower EC50 values. These results indicate that the LMIT has value in discriminating IVM sensitivity amongst cyathostomin populations, but several factors were identified that need to be taken into account when executing the test and interpreting the derived data.

The in vitro diagnosis of anthelmintic resistance in cyathostomins.

Cyathostomins are the primary parasitic pathogens of equids. For over 40 years, these nematodes have been controlled using broad spectrum anthelmintics. Three classes of anthelmintic are currently available for this use but, unfortunately, resistance to each of these has now been recorded in cyathostomin populations. As part of an optimal strategy to control cyathostomin infections in the field, it will be important to identify drug-resistant worms at as early a stage as possible. This objective needs to be supported by methodologies that will allow the accurate comparison of anthelmintic resistance in different nematode populations. At present, the faecal egg count reduction test is considered the most suitable method for initial screening for anthelmintic resistance in equine nematode populations. However, in its current state, this test lacks sensitivity. It is also costly and time-consuming to perform. Laboratory-based techniques, such as the egg hatch assay, larval development assay, larval migration inhibition assay and the larval feeding inhibition assay offer alternative options for assessing anthelmintic resistance in nematode populations. All of these tests have been investigated for their utility in measuring drug resistance in sheep nematode populations and some have proven useful. The egg hatch assay, larval development assay and larval migration inhibition assay have been investigated for use in measuring levels of drug resistance in equine nematode populations. However, at best, the results obtained thus far indicate that these tests require further refinement.

Targeting cattle-borne zoonoses and cattle pathogens using a novel trypanosomatid-based delivery system.

Trypanosomatid parasites are notorious for the human diseases they cause throughout Africa and South America. However, non-pathogenic trypanosomatids are also found worldwide, infecting a wide range of hosts. One example is Trypanosoma (Megatrypanum) theileri, a ubiquitous protozoan commensal of bovids, which is distributed globally. Exploiting knowledge of pathogenic trypanosomatids, we have developed Trypanosoma theileri as a novel vehicle to deliver vaccine antigens and other proteins to cattle. Conditions for the growth and transfection of T. theileri have been optimised and expressed heterologous proteins targeted for secretion or specific localisation at the cell interior or surface using trafficking signals from Trypanosoma brucei. In cattle, the engineered vehicle could establish in the context of a pre-existing natural T. theileri population, was maintained long-term and generated specific immune responses to an expressed Babesia antigen at protective levels. Building on several decades of basic research into trypanosomatid pathogens, Trypanosoma theileri offers significant potential to target multiple infections, including major cattle-borne zoonoses such as Escherichia coli, Salmonella spp., Brucella abortus and Mycobacterium spp. It also has the potential to deliver therapeutics to cattle, including the lytic factor that protects humans from cattle trypanosomiasis. This could alleviate poverty by protecting indigenous African cattle from African trypanosomiasis.

Comparison of the CDC Backpack aspirator and the Prokopack aspirator for sampling indoor- and outdoor-resting mosquitoes in southern Tanzania.

BACKGROUND: Resting mosquitoes can easily be collected using an aspirating device. The most commonly used mechanical aspirator is the CDC Backpack aspirator. Recently, a simple, and low-cost aspirator called the Prokopack has been devised and proved to have comparable performance. The following study evaluates the Prokopack aspirator compared to the CDC backpack aspirator when sampling resting mosquitoes in rural Tanzania. METHODS: Mosquitoes were sampled in- and outdoors of 48 typical rural African households using both aspirators. The aspirators were rotated between collectors and households in a randomized, Latin Square design. Outdoor collections were performed using artificial resting places (large barrel and car tyre), underneath the outdoor kitchen (kibanda) roof and from a drop-net. Data were analysed with generalized linear models. RESULTS: The number of mosquitoes collected using the CDC Backpack and the Prokopack aspirator were not significantly different both in- and outdoors (indoors p = 0.735; large barrel p = 0.867; car tyre p = 0.418; kibanda p = 0.519). The Prokopack was superior for sampling of drop-nets due to its smaller size. The number mosquitoes collected per technician was more consistent when using the Prokopack aspirator. The Prokopack was more user-friendly: technicians preferred using the it over the CDC backpack aspirator as it weighs considerably less, retains its charge for longer and is easier to manoeuvre. CONCLUSIONS: The Prokopack proved in the field to be more advantageous than the CDC Backpack aspirator. It can be self assembled using simple, low-cost and easily attainable materials. This device is a useful tool for researchers or vector-control surveillance programs operating in rural Africa, as it is far simpler and quicker than traditional means of sampling resting mosquitoes. Further longitudinal evaluations of the Prokopack aspirator versus the gold standard pyrethrum spray catch for indoor resting catches are recommended.