Attrition, physical integrity and insecticidal activity of long-lasting insecticidal nets in sub-Saharan Africa and modelling of their impact on vectorial capacity.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention and control intervention in many parts of sub-Saharan Africa. While LLINs are expected to last at least 3 years under normal use conditions, they can lose effectiveness because they fall out of use, are discarded, repurposed, physically damaged, or lose insecticidal activity. The contributions of these different interrelated factors to durability of nets and their protection against malaria have been unclear. METHODS: Starting in 2009, LLIN durability studies were conducted in seven countries in Africa over 5 years. WHO-recommended measures of attrition, LLIN use, insecticidal activity, and physical integrity were recorded for eight different net brands. These data were combined with analyses of experimental hut data on feeding inhibition and killing effects of LLINs on both susceptible and pyrethroid resistant malaria vectors to estimate the protection against malaria transmission-in terms of vectorial capacity (VC)-provided by each net cohort over time. Impact on VC was then compared in hypothetical scenarios where one durability outcome measure was set at the best possible level while keeping the others at the observed levels. RESULTS: There was more variability in decay of protection over time by country than by net brand for three measures of durability (ratios of variance components 4.6, 4.4, and 1.8 times for LLIN survival, use, and integrity, respectively). In some countries, LLIN attrition was slow, but use declined rapidly. Non-use of LLINs generally had more effect on LLIN impact on VC than did attrition, hole formation, or insecticide loss. CONCLUSIONS: There is much more variation in LLIN durability among countries than among net brands. Low levels of use may have a larger impact on effectiveness than does variation in attrition or LLIN degradation. The estimated entomological effects of chemical decay are relatively small, with physical decay probably more important as a driver of attrition and non-use than as a direct cause of loss of effect. Efforts to maximize LLIN impact in operational settings should focus on increasing LLIN usage, including through improvements in LLIN physical integrity. Further research is needed to understand household decisions related to LLIN use, including the influence of net durability and the presence of other nets in the household.

Evaluation of the long-term efficacy of K-Othrine® PolyZone on three surfaces against laboratory reared Anopheles gambiae in semi-field conditions.

BACKGROUND: In this semi-field study, a new polymer-enhanced deltamethrin formulation, K-Othrine® PolyZone, was compared to a standard deltamethrin product for residual activity against a susceptible strain of laboratory-reared Anopheles gambiae using standard WHO cone bioassays. METHODS: Residual insecticide efficacy was recorded after exposure to metal, cement and wood panels maintained in experimental huts in sub-tropical environmental conditions in north central Florida, USA, and panels stored in a climate controlled chamber located at the Centers for Disease Control and Prevention, Georgia, USA. CONCLUSIONS: K-Othrine® PolyZone demonstrated 100% control on metal and cement panels 1 year post application and > 80% control on wood panels up to 6 mo. The new formulation should be considered for use in indoor residual spray programmes requiring long-term control of malaria vectors.

Insecticide resistance in Anopheles arabiensis from Ethiopia (2012-2016): a nationwide study for insecticide resistance monitoring.

BACKGROUND: Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) remain the cornerstones of malaria vector control. However, the development of insecticide resistance and its implications for operational failure of preventative strategies are of concern. The aim of this study was to characterize insecticide resistance among Anopheles arabiensis populations in Ethiopia and describe temporal and spatial patterns of resistance between 2012 and 2016. METHODS: Between 2012 and 2016, resistance status of An. arabiensis was assessed annually during the long rainy seasons in study sites from seven of the nine regions in Ethiopia. Insecticide resistance levels were measured with WHO susceptibility tests and CDC bottle bioassays using insecticides from four chemical classes (organochlorines, pyrethroids, organophosphates and carbamates), with minor variations in insecticides tested and assays conducted between years. In selected sites, CDC synergist assays were performed by pre-exposing mosquitoes to piperonyl butoxide (PBO). In 2015 and 2016, mosquitoes from DDT and deltamethrin bioassays were randomly selected, identified to species-level and screened for knockdown resistance (kdr) by PCR. RESULTS: Intense resistance to DDT and pyrethroids was pervasive across Ethiopia, consistent with historic use of DDT for IRS and concomitant increases in insecticide-treated net coverage over the last 15 years. Longitudinal resistance trends to malathion, bendiocarb, propoxur and pirimiphos-methyl corresponded to shifts in the national insecticide policy. By 2016, resistance to the latter two insecticides had emerged, with the potential to jeopardize future long-term effectiveness of vector control activities in these areas. Between 2015 and 2016, the West African (L1014F) kdr allele was detected in 74.1% (n = 686/926) of specimens, with frequencies ranging from 31 to 100% and 33 to 100% in survivors from DDT and deltamethrin bioassays, respectively. Restoration of mosquito susceptibility, following pre-exposure to PBO, along with a lack of association between kdr allele frequency and An. arabiensis mortality rate, both indicate metabolic and target-site mutation mechanisms are contributing to insecticide resistance. CONCLUSIONS: Data generated by this study will strengthen the National Malaria Control Programme's insecticide resistance management strategy to safeguard continued efficacy of IRS and other malaria control methods in Ethiopia.

An Operational Framework for Insecticide Resistance Management Planning.

Arthropod vectors transmit organisms that cause many emerging and reemerging diseases, and their control is reliant mainly on the use of chemical insecticides. Only a few classes of insecticides are available for public health use, and the increased spread of insecticide resistance is a major threat to sustainable disease control. The primary strategy for mitigating the detrimental effects of insecticide resistance is the development of an insecticide resistance management plan. However, few examples exist to show how to implement such plans programmatically. We describe the formulation and implementation of a resistance management plan for mosquito vectors of human disease in Zambia. We also discuss challenges, steps taken to address the challenges, and directions for the future.

A longitudinal study of the durability of long-lasting insecticidal nets in Zambia.

BACKGROUND: A key goal of malaria control is to achieve universal access to, and use of, long-lasting insecticidal nets (LLINs) among people at risk for malaria. Quantifying the number of LLINs needed to achieve and maintain universal coverage requires knowing when nets need replacement. Longitudinal studies have observed physical deterioration in LLINs well before the assumed net lifespan of 3 years. The objective of this study was to describe attrition, physical integrity and insecticide persistence of LLINs over time to assist with better quantification of nets needing replacement. METHODS: 999 LLINs distributed in 2011 in two highly endemic provinces in Zambia were randomly selected, and were enrolled at 12 months old. LLINs were followed every 6 months up to 30 months of age. Holes were counted and measured (finger, fist, and head method) and a proportional hole index (pHI) was calculated. Households were surveyed about net care and repair and if applicable, reasons for attrition. Functional survival was defined as nets with a pHI <643 and present for follow-up. At 12 and 24 months of age, 74 LLINs were randomly selected for examination of insecticidal activity and content using bioassay and chemical analysis methods previously described by the World Health Organization (WHO). RESULTS: A total of 999 LLINs were enrolled; 505 deltamethrin-treated polyester nets and 494 permethrin-treated polyethylene nets. With 74 used to examine insecticide activity, 925 were available for full follow-up. At 30 months, 325 (33 %) LLINs remained. Net attrition was primarily due to disposal (29 %). Presence of repairs and use over a reed mat were significantly associated with larger pHIs. By 30 months, only 56 % of remaining nets met criteria for functional survival. A shorter functional survival was associated with having been washed. At 24 months, nets had reduced insecticidal activity (57 % met WHO minimal criteria) and content (5 % met WHO target insecticide content). CONCLUSIONS: The median functional survival time for LLINs observed the study was 2.5-3 years and insecticide activity and content were markedly decreased by 2 years. A better measure of net survival incorporating insecticidal field effectiveness, net physical integrity, and attrition is needed.

Long-lasting insecticidal nets in Zambia: a cross-sectional analysis of net integrity and insecticide content.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a mainstay of malaria prevention in Africa. More LLINs are available now than in any time previously due to increases in funding for malaria control. LLINs are expected to last three to five years before they need to be replaced. Reports of nets lasting less than three years are frequent in Zambia, which, if true, will increase the number of LLINs needed to maintain universal coverage. METHODS: This study collected nets distributed during mass distribution campaigns. One net was collected from each participating home in 12 districts in 2010 and all nets were examined for holes. One household member was surveyed about net use and care. RESULTS: The study collected 713 polyester nets with a median age of 31 months (range 27-44 months, interquartile (IQR) range: 29-36 months), median number of holes was 17 (IQR: 5-33), and median total hole size was 88.3 sq cm (IQR: 14.5-360.4). The median total number of holes did differ by age of the net, from 27-44 months, but not in a linear fashion. The difference in the number of holes in the newest and oldest nets was not statistically significant. The mean deltamethrin level for all nets was 23 mg/sq m (≥8 mg/sq m is considered effective). There was a larger total hole area in the lower half of the nets (repeat measures ANOVA, F=228.43, df=2, p<0.0001) compared to the upper half and roof of the net. Only 8.7% of nets had evidence of repairs. CONCLUSIONS: At 27-30 months, LLINs already had a large total hole surface area that was equivalent to the oldest nets observed. Nets were often tucked under reed mats which may explain the finding that the largest hole area was found in the lower half of the net. Studies need to be conducted prospectively to determine when physical deterioration occurs and why nets are discarded. Re-enforcing the lower half of the sides of LLINs may help decrease holes.

A Comparison of Carbon Dioxide Sources for Mosquito Capture in Centers for Disease Control and Prevention Light Traps on the Florida Gulf Coast (1).

Traditional sources of carbon dioxide (CO2), dry ice, and compressed gas, were tested against 3 combinations of food-grade reagents known to generate CO2using a compact, lightweight generator delivery system with Centers for Disease Control and Prevention light traps. Three 6 × 6 Latin square trials were completed near the Florida Gulf Coast in the Lower Suwannee Wildlife Refuge during the summer of 2013, collecting a total of 31,632 female mosquitoes. Treatments included dry ice, compressed CO2gas, a control trap (no CO2), citric acid + sodium bicarbonate, vinegar + sodium bicarbonate, and yeast + sugar. Decreasing order of trap collections (treatment mean number of mosquitoes per trap night ± standard error) were dry ice 773.5 (± 110.1) > compressed gas 440.7 (± 42.3) > citric acid + sodium bicarbonate 197.6 (± 30.4), yeast + sugar 153.6 (± 27.4) > vinegar + sodium bicarbonate 109.6 (± 16.2) > control 82.4 (± 14.0). A 2-way Kruskal-Wallis analysis by treatment, site, and treatment × site interaction identified significant differences between all treatments. Although dry ice and compressed CO2gas collected significantly more mosquitoes than other combinations (P < 0.05), use of citric acid and sodium bicarbonate or yeast and sugar greatly outperformed unbaited traps and offer a good alternative to dry ice and compressed gas in areas where these agents are not readily available or are difficult to obtain due to logistical constraints. An inexpensive, portable CO2generator for use with food-grade reagents is described.

Optimizing Collection of Anopheles gambiae s.s. (Diptera: Culicidae) in Biogents Sentinel Traps.

Surveillance of malaria vectors in Africa is most often accomplished using CDC-type light traps or human landing catches (HLCs). Over the past 30 yr, a variety of commercial and experimental mosquito traps have been developed for residential mosquito control or for improved surveillance of disease vector species, including the BG Sentinel (BGS) trap. To optimize collection of Anopheles gambiae Giles using this trap, BGS traps were modified with an opening (vent) added to the trap base to decrease exhaust airflow. Four traps configurations were tested with colony-reared host-seeking female An. gambiae in free-flying laboratory enclosures. Six attractant treatments (three attractants: BG-Lure, Limburger cheese, and a blank, with and without CO2) were tested concurrently. Across all trap-attractant combinations, significantly more mosquitoes (P < 0.05) were collected in standard, unmodified traps set upright (mean: 10.0) or upside down (9.8) than vented traps, whether upright (5.9) or upside down (7.0). The BG-Lure + CO2 and Limburger Cheese + CO2 bait combinations were more attractive than the other attractants tested alone. All attractant combinations collected significantly more mosquitoes than unbaited traps. Field studies are needed to determine if BG-Lure + CO2- or Limburger cheese + CO2-baited BGS traps are comparable with HLCs in collecting host-seeking An. gambiae.

SR450 And Superhawk XP Applications Of Bacillus thuringiensis israelensis Against Culex quinquefasciatus.

Sprayer comparisons and larval morality assays were conducted following SR450 backpack mist blower and Superhawk XP thermal fogger applications of Vectobac® WDG Bacillus thuringiensis israelensis (Bti) against Culex quinquefasciatus. Bacillus thuringiensis israelensis was applied at maximum label rate in a 232.26-m(2) field plot located in north-central Florida with containers placed at 2 heights (ground level and 1.52 m above ground) on stakes positioned 3.04, 6.09, 9.14, 12.19, and 15.24 m from the spray line. Results indicated that there was no significant (P > 0.05) difference in 24- and 48-h larval mortality between the 2 sprayers or between the 2 heights. There was significant difference (P < 0.05) among the 5 rows, with mortality continuously decreasing with increasing distance from sprayer. Both sprayers provided on average >70% larval mortality 3.04-9.14 m from the spray line, and <60% mortality at 12.19 and 15.24 m. The data suggest that the SR450 and Superhawk XP may be comparable sprayers for use with Bti to control mosquito larvae.

Evaluating RNAlater® as a preservative for using near-infrared spectroscopy to predict Anopheles gambiae age and species.

BACKGROUND: Mosquito age and species identification is a crucial determinant of the efficacy of vector control programmes. Near-infrared spectroscopy (NIRS) has previously been applied successfully to rapidly, non-destructively, and simultaneously determine the age and species of freshly anesthetized African malaria vectors from the Anopheles gambiae s.l. species complex: An. gambiae s. s. and Anopheles arabiensis. However, this has only been achieved on freshly-collected specimens and future applications will require samples to be preserved between field collections and scanning by NIRS. In this study, a sample preservation method (RNAlater(®)) was evaluated for mosquito age and species identification by NIRS against scans of fresh samples. METHODS: Two strains of An. gambiae s.s. (CDC and G3) and two strains of An. arabiensis (Dongola, KGB) were reared in the laboratory while the third strain of An. arabiensis (Ifakara) was reared in a semi-field system. All mosquitoes were scanned when fresh and rescanned after preservation in RNAlater(®) for several weeks. Age and species identification was determined using a cross-validation. RESULTS: The mean accuracy obtained for predicting the age of young (<7 days) or old (≥ 7 days) of all fresh (n = 633) and all preserved (n = 691) mosquito samples using the cross-validation technique was 83% and 90%, respectively. For species identification, accuracies were 82% for fresh against 80% for RNAlater(®) preserved. For both analyses, preserving mosquitoes in RNAlater(®) was associated with a highly significant reduction in the likelihood of a misclassification of mosquitoes as young or old using NIRS. Important to note is that the costs for preserving mosquito specimens with RNAlater(®) ranges from 3-13 cents per insect depending on the size of the tube used and the number of specimens pooled in one tube. CONCLUSION: RNAlater(®) can be used to preserve mosquitoes for subsequent scanning and analysis by NIRS to determine their age and species with minimal costs and with accuracy similar to that achieved from fresh insects. Cold storage availability allows samples to be stored longer than a week after field collection. Further study to develop robust calibrations applicable to other strains from diverse ecological settings is recommended.

Detection of broadly distributed sodium channel alleles characteristic of insect pyrethroid resistance in West Nile virus vector Culex pipiens complex mosquitoes in the United States.

West Nile virus (WNV) has emerged as a health threat to the North American population since its initial outbreak in New York City in 1999. Culex (Culex) pipiens complex mosquitoes have been considered to play the primary role in the enzootic maintenance and transmission of WNV in North America. The voltage-gated sodium channel (NaCh) gene contains pyrethroid resistance-associated mutations in the coding region in many insect species. However, the knowledge of potential NaCh mutations was minimal in Culex. Seeking pyrethroid resistance alleles in Culex, we evaluated a transect along the east coast of the United States with an NaCh-based genotyping tool that amplified a portion of the transcribed sequence containing kdr mutations and the intron immediately downstream of the mutation site. Three genotypes that are typically associated with pyrethroid resistance in insects have been identified in Culex pipiens complex mosquitoes in this study: susceptible wild type kds, the classical knock-down resistance Leu --> Phe mutation (Phe/kdr), and a second resistance mechanism, a Leu --> Ser mutation (Ser/kdr). Moreover, we observed heterozygotic individual mosquitoes possessing both kdr alleles. Results of this study advance our knowledge of the potential for pyrethroid insecticide resistance among the populations of Cx. pipiens complex in the United States.

Use of vector diagnostics during military deployments: recent experience in Iraq and Afghanistan.

Vector-borne diseases such as malaria, dengue, and leishmaniasis are a threat to military forces deployed outside of the United States. The availability of specific information on the vector-borne disease threat (e.g., presence or absence of a specific disease agent, temporal and geographic distribution of competent vectors, and vector infection rates) allows for effective implementation of appropriate measures to protect our deployed military forces. Vector diagnostics can provide critical, real-time information crucial to establishing effective vector prevention/control programs. In this article we provide an overview of current vector diagnostic capabilities, evaluate the use of vector diagnostics in Operation Enduring Freedom and Operation Iraqi Freedom, and discuss the concept of operations under which vector diagnostics are employed.

Determination of oseltamivir quality by colorimetric and liquid chromatographic methods.

We developed a colorimetric and chromatographic assay for oseltamivir to assess the authenticity of Tamiflu (F. Hoffmann-La Roche Ltd., Basel, Switzerland) because of a growing concern about counterfeit oseltamivir. The colorimetric assay is quantitative and relies on an extractable colored ion-pair complex of oseltamivir with Congo red or bromochlorophenol blue. The reverse-phase chromatographic assay uses an alkaline mobile phase with UV detection. Both methods were evaluated for variability and selectivity and subsequently applied to batches of oseltamivir products acquired through the Internet. The Congo red test showed greater assay sensitivity, linearity, and accuracy. Colorimetric and chromatographic analysis showed all batches of oseltamivir product were within +/-15% of the stated amount of active ingredient.

Locally acquired mosquito-transmitted malaria: a guide for investigations in the United States.

Recent outbreaks of locally acquired mosquito-transmitted malaria in the United States demonstrate the continued risk for reintroduction of the disease. Since 1957, when CDC's Malaria Branch started conducting malaria surveillance, 63 outbreaks have occurred, constituting 156 cases (annual range: 1-32) that were a result of locally acquired mosquitoborne transmission. This report describes the steps that should be taken to 1) investigate a case that might have been acquired locally, 2) prevent a small focus of malaria cases from becoming a source of sustained transmission, and 3) inform clinicians regarding the process of an investigation so they can effectively address concerns and questions from patients. Although these locally acquired mosquito-transmitted outbreaks frequently involve only a limited number of infected persons, they frequently raise concerns in the community and require substantial public health resources. For example, as a result of the most recent local outbreak of eight malaria cases in Florida in 2003, reverse 911 telephone calls (a community notification system) were made to approximately 300,000 residents; insect repellent, postcards, flyers, and posters in multiple languages were distributed; public announcements were made through the media and to schools and homeless shelters; and notifications were sent to local hospitals and physicians to inform residents of that community. When a local health department investigates a potential locally acquired mosquito-transmitted case, the systematic inquiry should include epidemiologic, environmental, and laboratory components. Local and state health departments inquiring about the proper approach to investigate and control a potential locally acquired case frequently request urgent assistance and tools from CDC. This report provides a starting point for such investigations to local and state health departments by providing them with the tools necessary to initiate an investigation.

Malaria vector incrimination in three rural riverine villages in the Brazilian Amazon.

Vector incrimination studies were conducted from April 2003 to February 2005 at three riverine villages 1.5 km to 7.0 km apart, along the Matapi River, Amapa State, Brazil. A total of 113,117 mosquitoes were collected and placed in pools of

Identification of blood meal source and infection with Trypanosoma cruzi of Chagas disease vectors using a multiplex cytochrome b polymerase chain reaction assay.

Long-term control of triatomine bugs in Chagas endemic regions will depend on a full understanding of vector-parasite-host interactions. Herein we describe a cytochrome b multiplex polymerase chain reaction (PCR)-based strategy for blood meal source identification in bug foregut contents. This technique discriminates human from animal blood, and has been tested in five Triatoma species from México. Host identification has been validated for human, four rodent species, two bat species, dog, rabbit, sheep, and opossum. In addition, Trypanosoma cruzi can be identified simultaneously using S34/S67-specific kinetoplast DNA primers. Both host and parasite identification were possible as long as 10 weeks after bug feeding, and in samples stored up to 6 years. The blood meal identification procedure described here represents a powerful tool for large-scale studies identifying the biological, ecological, and environmental variables associated with Chagas disease transmission.

Evidence for transmission of Plasmodium vivax among a duffy antigen negative population in Western Kenya.

We present evidence that a parasite with characteristics of Plasmodium vivax is being transmitted among Duffy blood group-negative inhabitants of Kenya. Thirty-two of 4,901 Anopheles gambiae and An. funestus (0.65%) collected in Nyanza Province were ELISA positive for the P. vivax circumsporozoite protein VK 247. All positives were found late in the rainy season, when An. funestus predominated, and disproportionately many were found at a single village. A P. vivax specific sequence of the SSU rRNA gene was amplified from three of six ELISA-positive mosquitoes. Erythrocytes from 31 children, including 9 microscopically diagnosed as infected with P. vivax, were negative by flow cytometry for the Fy3 or Fy6 epitopes, which indicate Duffy blood group expression. A DNA fragment specific for the C terminus of the gene for P. vivax merozoite surface protein 1 (MSP-1) was amplified from the blood of four of these children and subsequently sequenced from two.

Biting behavior and Plasmodium infection rates of Anopheles arabiensis from Sille, Ethiopia.

The man-biting behavior and Plasmodium infection rates of anopheline mosquitoes were investigated in Sille, a hyperendemic malarious area in southern Ethiopia. Seven Anopheles species were identified from all night landing collections, conducted from 18:00 to 06:00h between October 2001 and August 2002. The predominant species was Anopheles arabiensis (55.8%), followed by Anopheles coustani (31.5%), Anopheles pharoensis (9.5%), Anopheles funestus (2.2%), Anopheles nili (0.5%), Anopheles marshallii (0.4%) and Anopheles demeilloni (0.2%). Dissection of A. arabiensis showed an average parous rate of 73.2%. A large proportion of the parous mosquitoes were caught biting in the latter part of the night. Malaria sporozoite rates were determined by ELISA for A. arabiensis, with 0.5% (4/796) infective with Plasmodium falciparum and 1.76% (14/796) with Plasmodium vivax; there were no mixed infections. From our small sample of sporozoite positives we found no association between biting behavior and sporozoite infection status.

Determination of Insecticidal Effect (LC50 and LC90) of Organic Fatty Acids Mixture (C8910+Silicone) Against Aedes aegypti and Aedes albopictus (Diptera: Culicidae).

Emerging and re-emerging vector-borne diseases such as chikungunya and dengue and associated Aedes vectors are expanding their historical ranges; thus, there is a need for the development of novel insecticides for use in vector control programs. The mosquito toxicity of a novel insecticide and repellent consisting of medium-chain carbon fatty acids (C8910) was examined. Determination of LC50 and LC90 was made against colony-reared Aedes aegypti (L.) and Aedes albopictus (Skuse) using probit analysis on mortality data generated by Centers for Disease Control and Prevention bottle bioassays. Six different concentrations of C8910 + silicone oil yielded an LC50 of 160.3 µg a.i/bottle (147.6-182.7) and LC90 of 282.8 (233.2-394.2) in Ae. aegypti; five concentrations yielded an LC50 of 125.4 (116.1-137.6) and LC90 of 192.5 (165.0-278.9) in Ae. albopictus. Further development of C8910 and similar compounds could provide vector control specialists novel insecticides for controlling insect disease vectors.

Short persistence of bendiocarb sprayed on pervious walls and its implication for the indoor residual spray program in Ethiopia.

BACKGROUND: With the emergence and spread of vector resistance to pyrethroids and DDT in Africa, several countries have recently switched or are considering switching to carbamates and/or organophosphates for indoor residual spraying (IRS). However, data collected on the residual life of bendiocarb used for IRS in some areas indicate shorter than expected bio-efficacy. This study evaluated the effect of pH and wall type on the residual life of the carbamates bendiocarb and propoxur as measured by the standard World Health Organization (WHO) cone bioassay test. METHODS: In phase I of this study, bendiocarb and propoxur were mixed with buffered low pH (pH 4.3) local water and non-buffered high pH (pH 8.0) local water and sprayed on two types of wall surface, mud and dung, in experimental huts. In the six month phase II study, the two insecticides were mixed with high pH local water and sprayed on four different surfaces: painted, dung, mud and mud pre-wetted with water. The residual bio-efficacy of the insecticides was assessed monthly using standard WHO cone bioassay tests. RESULTS: In phase I, bendiocarb mixed with high pH water killed more than 80% of susceptible Anopheles arabiensis mosquitoes for two months on both dung and mud surfaces. On dung surfaces, the 80% mortality threshold was achieved for three months when the bendiocarb was mixed with low pH water and four months when it was mixed with high pH water. Propoxur lasted longer than bendiocarb on dung surfaces, staying above the 80% mortality threshold for four and five months when mixed with high and low pH water, respectively. Phase II results also showed that the type of surface sprayed has a significant impact on the bio-efficacy of bendiocarb. Keeping the spray water constant at the same high pH of 8.0, bendiocarb killed 100% of exposed mosquitoes on impervious painted surfaces for the six months of the study period compared with less than one month on mud surfaces. CONCLUSIONS: Mixing the insecticides in alkaline water did not reduce the residual bio-efficacy of bendiocarb. However, bendiocarb performed much better on impervious (painted) surfaces than on porous dung or mud ones. Propoxur was less affected by wall type than was bendiocarb. Studies on the interaction between wall materials, soil, humidity, temperature and pH and the residual bio-efficacy of new and existing insecticides are recommended prior to their wide use in IRS.