Semi-field assessment of the BG-Malaria trap for monitoring the African malaria vector, Anopheles arabiensis

Odour-baited technologies are increasingly considered for effective monitoring of mosquito populations and for the evaluation of vector control interventions. The BG-Malaria trap (BGM), which is an upside-down variant of the widely used BG-Sentinel trap (BGS), has been demonstrated to be effective to sample the Brazilian malaria vector, Anopheles darlingi. We evaluated the BGM as an improved method for sampling the African malaria vectors, Anopheles arabiensis. Experiments were conducted inside a large semi-field cage to compare trapping efficiencies of BGM and BGS traps, both baited with the synthetic attractant, Ifakara blend, supplemented with CO2. We then compared BGMs baited with either of four synthetic mosquito lures, Ifakara blend, Mbita blend, BG-lure or CO2, and an unbaited BGM. Lastly, we compared BGMs baited with the Ifakara blend dispensed via either nylon strips, BG cartridges (attractant-infused microcapsules encased in cylindrical plastic cartridge) or BG sachets (attractant-infused microcapsules encased in plastic sachets). All tests were conducted between 6P.M. and 7A.M., with 200–600 laboratory-reared An. arabiensis released nightly in the test chamber. The median number of An. arabiensis caught by the BGM per night was 83, IQR:(73.5–97.75), demonstrating clear superiority over BGS (median catch = 32.5 (25.25–37.5)). Compared to unbaited controls, BGMs baited with Mbita blend caught most mosquitoes (45 (29.5–70.25)), followed by BGMs baited with CO2 (42.5 (27.5–64)), Ifakara blend (31 (9.25–41.25)) and BG lure (16 (4–22)). BGM caught 51 (29.5–72.25) mosquitoes/night, when the attractants were dispensed using BG-Cartridges, compared to BG-Sachet (29.5 (24.75–40.5)), and nylon strips (27 (19.25–38.25)), in all cases being significantly superior to unbaited controls (p < 000.1). The findings demonstrate potential of the BGM as a sampling tool for African malaria vectors over the standard BGS trap. Its efficacy can be optimized by selecting appropriate odour baits and odour-dispensing systems

Sub-lethal aquatic doses of pyriproxyfen may increase pyrethroid resistance in malaria mosquitoes.

BACKGROUND: Pyriproxyfen (PPF), an insect growth hormone mimic is widely used as a larvicide and in some second-generation bed nets, where it is combined with pyrethroids to improve impact. It has also been evaluated as a candidate for auto-dissemination by adult mosquitoes to control Aedes and Anopheles species. We examined whether PPF added to larval habitats of pyrethroid-resistant malaria vectors can modulate levels of resistance among emergent adult mosquitoes. METHODOLOGY: Third-instar larvae of pyrethroid-resistant Anopheles arabiensis (both laboratory-reared and field-collected) were reared in different PPF concentrations, between 1×10-9 milligrams active ingredient per litre of water (mgAI/L) and 1×10-4 mgAI/L, or no PPF at all. Emergent adults escaping these sub-lethal exposures were tested using WHO-standard susceptibility assays on pyrethroids (0.75% permethrin and 0.05% deltamethrin), carbamates (0.1% bendiocarb) and organochlorides (4% DDT). Biochemical basis of pyrethroid resistance was investigated by pre-exposure to 4% PBO. Bio-efficacies of long-lasting insecticide-treated nets, Olyset® and PermaNet 2.0 were also examined against adult mosquitoes with or without previous aquatic exposure to PPF. RESULTS: Addition of sub-lethal doses of PPF to larval habitats of pyrethroid-resistant An. arabiensis, consistently resulted in significantly reduced mortalities of emergent adults when exposed to pyrethroids, but not to bendiocarb or DDT. Mortality rates after exposure to Olyset® nets, but not PermaNet 2.0 were also reduced following aquatic exposures to PPF. Pre-exposure to PBO followed by permethrin or deltamethrin resulted in significant increases in mortality, compared to either insecticide alone. CONCLUSIONS: Partially-resistant mosquitoes exposed to sub-lethal aquatic concentrations of PPF may become more resistant to pyrethroids than they already are without such pre-exposures. Studies should be conducted to examine whether field applications of PPF, either by larviciding or other means actually exacerbates pyrethroid-resistance in areas where signs of such resistance already exist in wild the vector populations. The studies should also investigate mechanisms underlying such magnification of resistance, and how this may impact the potential of PPF-based interventions in areas with pyrethroid resistance

Evaluation of a simple polytetrafluoroethylene (PTFE)-based membrane for blood-feeding of malaria and dengue fever vectors in the laboratory

BACKGROUND: Controlled blood-feeding is essential for maintaining laboratory colonies of disease-transmitting mosquitoes and investigating pathogen transmission. We evaluated a low-cost artificial feeding (AF) method, as an alternative to direct human feeding (DHF), commonly used in mosquito laboratories. METHODS: We applied thinly-stretched pieces of polytetrafluoroethylene (PTFE) membranes cut from locally available seal tape (i.e. plumbers tape, commonly used for sealing pipe threads in gasworks or waterworks). Approximately 4 ml of bovine blood was placed on the bottom surfaces of inverted Styrofoam cups and then the PTFE membranes were thinly stretched over the surfaces. The cups were filled with boiled water to keep the blood warm (~37 degrees C), and held over netting cages containing 3-4 day-old inseminated adults of female Aedes aegypti, Anopheles gambiae (s.s.) or Anopheles arabiensis. Blood-feeding success, fecundity and survival of mosquitoes maintained by this system were compared against DHF. RESULTS: Aedes aegypti achieved 100% feeding success on both AF and DHF, and also similar fecundity rates (13.1 +/- 1.7 and 12.8 +/- 1.0 eggs/mosquito respectively; P > 0.05). An. arabiensis had slightly lower feeding success on AF (85.83 +/- 16.28%) than DHF (98.83 +/- 2.29%) though these were not statistically different (P > 0.05), and also comparable fecundity between AF (8.82 +/- 7.02) and DHF (8.02 +/- 5.81). Similarly, for An. gambiae (s.s.), we observed a marginal difference in feeding success between AF (86.00 +/- 10.86%) and DHF (98.92 +/- 2.65%), but similar fecundity by either method. Compared to DHF, mosquitoes fed using AF survived a similar number of days [Hazard Ratios (HR) for Ae. aegypti = 0.99 (0.75-1.34), P > 0.05; An. arabiensis = 0.96 (0.75-1.22), P > 0.05; and An. gambiae (s.s.) = 1.03 (0.79-1.35), P > 0.05]. CONCLUSIONS: Mosquitoes fed via this simple AF method had similar feeding success, fecundity and longevity. The method could potentially be used for laboratory colonization of mosquitoes, where DHF is unfeasible. If improved (e.g. minimizing temperature fluctuations), the approach could possibly also support studies where vectors are artificially infected with blood-borne pathogens

'We spray and walk away': wall modifications decrease the impact of indoor residual spray campaigns through reductions in post-spray coverage

Malaria prevalence has significantly reduced since 2000, largely due to the scale-up of vector control interventions, mainly indoor residual spraying (IRS) and long-lasting insecticide-treated nets (LLINs). Given their success, these tools remain the frontline interventions in the fight against malaria. Their effectiveness relies on three key ingredients: the intervention, the mosquito vector and the end-user. Regarding the intervention, factors such as the insecticide active ingredient(s) used and the durability and/or bio-efficacy of the tool over time are critical. For the vectors, these factors include biting and resting behaviours and the susceptibility to insecticides. Finally, the end-users need to accept and properly use the intervention. Whilst human attitude and behaviour towards LLINs are well-documented both during and after distribution, only initial coverage is monitored for IRS and in a few geographic settings the residual efficacy of the used product. Here, the historical evidence on end-users modifying their wall surfaces post-spraying is presented, a behaviour that has the potential to reduce actual IRS coverage, effectiveness and impact, as fewer people are truly protected. Therefore, clear guidelines on how to monitor IRS acceptability and/or coverage, both before, during and after spraying, are urgently needed as part of the Monitoring and Evaluation of malaria programmes

Using Stable Isotopes of Carbon and Nitrogen to Mark Wild Populations of Anopheles and Aedes Mosquitoes in South-Eastern Tanzania.

BACKGROUND Marking wild mosquitoes is important for understanding their ecology, behaviours and role in disease transmission. Traditional insect marking techniques include using fluorescent dyes, protein labels, radioactive labels and tags, but such techniques have various limitations; notably low marker retention and inability to mark wild mosquitoes at source. Stable isotopes are gaining wide spread use for non-invasive marking of arthropods, permitting greater understanding of mosquito dispersal and responses to interventions. We describe here a simple technique for marking naturally-breeding malaria and dengue vectors using stable isotopes of nitrogen (15N) and carbon (13C), and describe potential field applications. METHODS We created man-made aquatic mosquito habitats and added either 15N-labelled potassium nitrate or 13C-labelled glucose, leaving non-adulterated habitats as controls. We then allowed wild mosquitoes to lay eggs in these habitats and monitored their development in situ. Pupae were collected promptly as they appeared and kept in netting cages. Emergent adults (in pools of ~4 mosquitoes/pool) and individually stored pupae were desiccated and analysed using Isotope Ratio Mass Spectrometry (IRMS). FINDINGS Anopheles gambiae s.l and Aedes spp. from enriched 13C and enriched 15N larval habitats had significantly higher isotopic levels than controls (P = 0.005), and both isotopes produced sufficient distinction between marked and unmarked mosquitoes. Mean δ15N for enriched females and males were 275.6±65.1 and 248.0±54.6, while mean δ15N in controls were 2.1±0.1 and 3.9±1.7 respectively. Similarly, mean δ13C for enriched females and males were 36.08±5.28 and 38.5±6.86, compared to -4.3±0.2 and -7.9±3.6 in controls respectively. Mean δ15N and δ13C was significantly higher in any pool containing at least one enriched mosquito compared to pools with all unenriched mosquitoes, P<0.001. In all cases, there were variations in standardized isotopic ratios between mosquito species. CONCLUSION Enrichment of semi-natural mosquito larval habitats with stable isotopes of nitrogen and carbon resulted in effective marking of Anopheles and Aedes mosquitoes colonizing these habitats. This approach can significantly enhance studies on mosquito eco-physiology, dispersal, pathogen transmission and responses to control measures

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Evidence of the absence of Human African Trypanosomiasis in two Northern Districts of Uganda: analyses of cattle, pigs and tsetse flies for the presence of Trypanosoma brucei gambiense

Background Large-scale control of sleeping sickness has led to a decline in the number of cases of Gambian human African trypanosomiasis (g-HAT) to <2000/year. However, achieving complete and lasting interruption of transmission may be difficult because animals may act as reservoir hosts for T. b. gambiense. Our study aims to update our understanding of T. b. gambiense in local vectors and domestic animals of N.W. Uganda. Methods We collected blood from 2896 cattle and 400 pigs and In addition, 6664 tsetse underwent microscopical examination for the presence of trypanosomes. Trypanosoma species were identified in tsetse from a subsample of 2184 using PCR. Primers specific for T. brucei s.l. and for T. brucei sub-species were used to screen cattle, pig and tsetse samples. Results In total, 39/2,088 (1.9%; 95% CI=1.9-2.5) cattle, 25/400 (6.3%; 95% CI=4.1-9.1) pigs and 40/2,184 (1.8%; 95% CI=1.3-2.5) tsetse, were positive for T. brucei s.l.. Of these samples 24 cattle (61.5%), 15 pig (60%) and 25 tsetse (62.5%) samples had sufficient DNA to be screened using the T. brucei sub-species PCR. Further analysis found no cattle or pigs positive for T. b. gambiense, however, 17/40 of the tsetse samples produced a band suggestive of T. b. gambiense. When three of these 17 PCR products were sequenced the sequences were markedly different to T. b. gambiense, indicating that these flies were not infected with T. b. gambiense. Conclusion The lack of T. b. gambiense positives in cattle, pigs and tsetse accords with the low prevalence of g-HAT in the human population. We found no evidence that livestock are acting as reservoir hosts. However, this study highlights the limitations of current methods of detecting and identifying T. b. gambiense which relies on a single copy-gene to discriminate between the different sub-species of T. brucei s.l

Apparent density, trypanosome infection rates and host preference of tsetse flies in the sleeping sickness endemic focus of northwestern Uganda

BACKGROUND: African trypanosomiasis, caused by protozoa of the genus Trypanosoma and transmitted by the tsetse fly, is a serious parasitic disease of humans and animals. Reliable data on the vector distribution, feeding preference and the trypanosome species they carry is pertinent to planning sustainable control strategies. METHODOLOGY: We deployed 109 biconical traps in 10 villages in two districts of northwestern Uganda to obtain information on the apparent density, trypanosome infection status and blood meal sources of tsetse flies. A subset (272) of the collected samples was analyzed for detection of trypanosomes species and sub-species using a nested PCR protocol based on primers amplifying the Internal Transcribed Spacer (ITS) region of ribosomal DNA. 34 blood-engorged adult tsetse midguts were analyzed for blood meal sources by sequencing of the mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) genes. RESULTS: We captured a total of 622 Glossina fuscipes fuscipes tsetse flies (269 males and 353 females) in the two districts with apparent density (AD) ranging from 0.6 to 3.7 flies/trap/day (FTD). 10.7% (29/272) of the flies were infected with one or more trypanosome species. Infection rate was not significantly associated with district of origin (Generalized linear model (GLM), χ(2) = 0.018, P = 0.895, df = 1, n = 272) and sex of the fly (χ2 = 1.723, P = 0.189, df = 1, n = 272). However, trypanosome infection was highly significantly associated with the fly’s age based on wing fray category (χ(2) = 22.374, P < 0.001, df = 1, n = 272), being higher among the very old than the young tsetse. Nested PCR revealed several species of trypanosomes: T. vivax (6.62%), T. congolense (2.57%), T. brucei and T. simiae each at 0.73%. Blood meal analyses revealed five principal vertebrate hosts, namely, cattle (Bos taurus), humans (Homo sapiens), Nile monitor lizard (Varanus niloticus), African mud turtle (Pelusios chapini) and the African Savanna elephant (Loxodonta africana). CONCLUSION: We found an infection rate of 10.8% in the tsetse sampled, with all infections attributed to trypanosome species that are causative agents for AAT. However, more verification of this finding using large-scale passive and active screening of human and tsetse samples should be done. Cattle and humans appear to be the most important tsetse hosts in the region and should be considered in the design of control interventions

Field evaluation of the BG-Malaria trap for monitoring malaria vectors in rural Tanzanian villages.

BG-Malaria (BGM) trap is a simple adaptation of the widely-used BG-Sentinel trap (BGS). It is proven to be highly effective for trapping the Brazilian malaria vector, Anopheles darlingi, in field conditions, and the African vector, Anopheles arabiensis, under controlled semi-field environments, but has not been field-tested in Africa. Here, we validated the BGM for field sampling of malaria vectors in south-eastern Tanzania. Using a series of Latin-Square experiments conducted nightly (6pm-7am) in rural villages, we compared mosquito catches between BGM, BGS and human landing catches (HLC). We also compared BGMs baited with different attractants (Ifakara-blend, Mbita-blend, BG-Lure and CO2). Lastly, we tested BGMs baited with Ifakara-blend from three odour-dispensing methods (BG-Cartridge, BG-Sachet and Nylon strips). One-tenth of the field-collected female Anopheles gambiae s.l. and Anopheles funestus were dissected to assess parity. BGM captured more An. gambiae s.l. than BGS (p < 0.001), but HLC caught more than either trap (p < 0.001). However, BGM captured more An. funestus than HLC. Proportions of parous An. gambiae s.l. and An. funestus consistently exceeded 50%, with no significant difference between methods. While the dominant species caught by HLC was An. gambiae s.l. (56.0%), followed by Culex spp. (33.1%) and Mansonia spp. (6.0%), the BGM caught mostly Culex (81.6%), followed by An. gambiae s.l. (10.6%) and Mansonia (5.8%). The attractant-baited BGMs were all significantly superior to un-baited controls (p < 0.001), although no difference was found between the specific attractants. The BG-Sachet was the most efficient dispenser for capturing An. gambiae s.l. (14.5(2.75-42.50) mosquitoes/trap/night), followed by BG-Cartridge (7.5(1.75-26.25)). The BGM caught more mosquitoes than BGS in field-settings, but sampled similar species diversity and physiological states as BGS. The physiological states of malaria vectors caught in BGM and BGS were similar to those naturally attempting to bite humans (HLC). The BGM was most efficient when baited with Ifakara blend, dispensed from BG-Sachet. We conclude that though BGM traps have potential for field-sampling of host-seeking African malaria vectors with representative physiological states, both BGM and BGS predominantly caught more culicines than Anopheles, compared to HLC, which caught mostly An. gambiae s.l

Household modifications after the indoor residual spraying (IRS) campaign in Mozambique reduce the actual spray coverage and efficacy.

Indoor residual spraying of insecticides (IRS) is a key malaria vector control strategy. Whilst human attitude towards IRS is monitored before or shortly after implementation, human activities leading to the modification of insecticide-treated walls post-IRS are not. This could inadvertently reduce the protective effects of IRS. We monitored the extent of modifications to the sprayed indoor wall surfaces by household owners for six months post-IRS campaigns in two districts targeted for malaria elimination in southern Mozambique. In parallel, we assessed building of any additional rooms onto compounds, and mosquito net use. We quantified the contribution of wall modifications, added rooms, prolonged spray campaigns, and product residual efficacies on actual IRS coverage and relative mosquito bite reduction, using a mechanistic approach. Household owners continually modified insecticide-treated walls and added rooms onto compounds. Household surveys in southern Mozambique showed frequent modification of indoor walls (0-17.2% of households modified rooms monthly) and/or added rooms (0-16.2% of households added rooms monthly). Actual IRS coverage reduced from an assumed 97% to just 39% in Matutuine, but only from 96% to 91% in Boane, translating to 43% and 5.8% estimated increases in relative daily mosquito bites per person. Integrating post-IRS knowledge, attitude, and practice (KAP) surveys into programmatic evaluations to capture these modification and construction trends can help improve IRS program efficiency and product assessment