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      Preventing Childhood Malaria in Africa by Protecting Adults from Mosquitoes with Insecticide-Treated Nets


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          Malaria prevention in Africa merits particular attention as the world strives toward a better life for the poorest. Insecticide-treated nets (ITNs) represent a practical means to prevent malaria in Africa, so scaling up coverage to at least 80% of young children and pregnant women by 2010 is integral to the Millennium Development Goals (MDG). Targeting individual protection to vulnerable groups is an accepted priority, but community-level impacts of broader population coverage are largely ignored even though they may be just as important. We therefore estimated coverage thresholds for entire populations at which individual- and community-level protection are equivalent, representing rational targets for ITN coverage beyond vulnerable groups.

          Methods and Findings

          Using field-parameterized malaria transmission models, we show that high (80% use) but exclusively targeted coverage of young children and pregnant women (representing <20% of the population) will deliver limited protection and equity for these vulnerable groups. In contrast, relatively modest coverage (35%–65% use, with this threshold depending on ecological scenario and net quality) of all adults and children, rather than just vulnerable groups, can achieve equitable community-wide benefits equivalent to or greater than personal protection.


          Coverage of entire populations will be required to accomplish large reductions of the malaria burden in Africa. While coverage of vulnerable groups should still be prioritized, the equitable and communal benefits of wide-scale ITN use by older children and adults should be explicitly promoted and evaluated by national malaria control programmes. ITN use by the majority of entire populations could protect all children in such communities, even those not actually covered by achieving existing personal protection targets of the MDG, Roll Back Malaria Partnership, or the US President's Malaria Initiative.


          Based on malaria transmission models, Gerry Killeen and colleagues suggest that, while coverage of pregnant women and children should still be prioritized, wide-scale communal use of insecticide-treated bed nets would provide considerable benefit to vulnerable groups and should be promoted and evaluated in the field.

          Editors' Summary


          Malaria—a parasitic disease common in tropical and subtropical countries—causes about a million deaths every year, mainly among young children and pregnant women living in sub-Saharan Africa. The parasite responsible for most of these deaths is Plasmodium falciparum. Like all malaria parasites, it has a complex life cycle, part of which takes place inside mosquitoes. When a malaria-carrying mosquito bites a person (usually at night), parasites enter the human blood stream and travel to the liver where they reproduce before invading red blood cells. Here, they multiply again before bursting out and infecting more red blood cells as well as causing a high fever and sometimes damaging the nervous system, liver, and kidneys. When a mosquito bites someone who is infected, it often picks up parasites in its blood meal (mosquitoes need mammalian blood for successful reproduction), thus completing the malarial transmission cycle.

          Why Was This Study Done?

          One way to break this cycle and reduce malarial transmission is to decrease the number of contacts between people and mosquitoes by encouraging people to sleep under insecticide-treated nets (ITNs). Field studies have shown that ITN use reduces deaths from malaria, so the Roll Back Malaria Partnership, the United Nations Millennium Development Goals, and the US President's Malaria Initiative have set a target of at least 80% use of ITNs by young children and pregnant women (the people most vulnerable to malaria) by 2010. But would broader population coverage with ITNs interrupt the malarial transmission cycle more effectively? Might the wider use of ITNs (which both directly kill mosquitoes and repel them so that mosquitoes have to travel farther to get the blood they need to reproduce) be a more effective way to reduce local mosquito numbers and, consequently, human–mosquito contacts and malarial transmission? In this study, the researchers used mathematical modeling to ask how much community-wide coverage with ITNs is needed to protect vulnerable individuals against malaria without them personally using an ITN.

          What Did the Researchers Do and Find?

          The researchers developed a model for the transmission of the malaria parasite using data collected in Tanzanian villages where malaria is common, and used it to investigate how different patterns of ITN use might affect the individual protection of ITN users and the communal protection of nonusers. High (80%) coverage targeted at young children and pregnant women (less than 20% of the population) provided limited but valuable protection to these vulnerable individuals. However, the model predicted that a similar degree of community-wide protection would result if 35% of the whole population slept under ITNs provided there was no nonhuman blood supply for the mosquitoes. In other words, the vulnerable individuals in the population received the same degree of protection from this intervention as they would have got from personally using an ITN. If an alternative blood supply for the mosquitoes (for example, cattle) was included in the model, just over half of the population needed to sleep under ITNs to provide the same degree of community-wide protection as targeted ITN use.

          What Do These Findings Mean?

          Although the use of ITNs by vulnerable groups should remain a priority, these findings suggest that the wide-scale ITN use by the entire population should also be promoted. The use of ITNs by about half the population, predict the researchers, could protect all the young children in that population, even those who did not sleep under a net. As with all mathematical models, the accuracy of this prediction depends on the assumptions and data incorporated into the model. So before recommending community-wide use of ITNs, the actual level of communal protection provided by increased ITN coverage must be measured by rigorously evaluating ongoing national programmes. If such surveillance data confirm this model's predictions, wide coverage with ITNs might do more for public health in Africa than previously thought, suggest the researchers, provided the financial and logistical challenges associated with achieving high ITN coverage in this poor region of the world can be solved.

          Additional Information.

          Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040229.

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          Most cited references99

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          The global distribution of clinical episodes of Plasmodium falciparum malaria.

          Interest in mapping the global distribution of malaria is motivated by a need to define populations at risk for appropriate resource allocation and to provide a robust framework for evaluating its global economic impact. Comparison of older and more recent malaria maps shows how the disease has been geographically restricted, but it remains entrenched in poor areas of the world with climates suitable for transmission. Here we provide an empirical approach to estimating the number of clinical events caused by Plasmodium falciparum worldwide, by using a combination of epidemiological, geographical and demographic data. We estimate that there were 515 (range 300-660) million episodes of clinical P. falciparum malaria in 2002. These global estimates are up to 50% higher than those reported by the World Health Organization (WHO) and 200% higher for areas outside Africa, reflecting the WHO's reliance upon passive national reporting for these countries. Without an informed understanding of the cartography of malaria risk, the global extent of clinical disease caused by P. falciparum will continue to be underestimated.
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            Heterogeneities in the transmission of infectious agents: implications for the design of control programs.

            From an analysis of the distributions of measures of transmission rates among hosts, we identify an empirical relationship suggesting that, typically, 20% of the host population contributes at least 80% of the net transmission potential, as measured by the basic reproduction number, R0. This is an example of a statistical pattern known as the 20/80 rule. The rule applies to a variety of disease systems, including vector-borne parasites and sexually transmitted pathogens. The rule implies that control programs targeted at the "core" 20% group are potentially highly effective and, conversely, that programs that fail to reach all of this group will be much less effective than expected in reducing levels of infection in the population as a whole.
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              Reduced Efficacy of Insecticide-treated Nets and Indoor Residual Spraying for Malaria Control in Pyrethroid Resistance Area, Benin

              During the last decade, pyrethroid-treated mosquito nets have become the main method of malaria prevention in many malaria-endemic African countries ( 1 , 2 ). In a few notable exceptions, usually those with a more developed health infrastructure, such as South Africa, a longstanding practice of applying indoor residual spraying (IRS) has been successful ( 3 ). The 2 approaches to malaria prevention, insecticide-treated nets (ITNs) and spraying (IRS), are not mutually exclusive, and in malaria-endemic areas where ITN coverage is still limited, the feasibility of introducing IRS to reduce transmission is being considered, for example, by the President’s Initiative Fund ( 4 ). Trials of IRS and ITNs have shown that in areas with pyrethroid-susceptible Anopheles gambiae the effectiveness of the 2 methods in controlling malaria does not differ ( 5 ). This comparability may not hold true for areas with pyrethroid-resistant populations. In southern Africa, for example, IRS with pyrethroid failed to control pyrethroid-resistant An. funestus and necessitated a switch to an alternative class of insecticide to which there was no resistance ( 6 ). During the last decade, pyrethroid resistance caused by the kdr mechanism has become widespread in An. gambiae in West Africa and is common in some areas ( 7 ). Whether kdr undermines the effectiveness of ITN in areas of high prevalence is unclear. An early experimental hut trial of ITNs in Côte d’Ivoire demonstrated a survival advantage of homozygotes for kdr resistance ( 8 ), whereas subsequent hut trials in adjacent resistant and susceptible populations showed no apparent difference in the effectiveness of ITNs between the 2 localities ( 9 ). Village randomized trials in Côte d’Ivoire showed that ITNs continued to prevent malaria despite a vector population that was kdr resistant ( 10 ). Whether kdr would undermine the effectiveness of IRS in the same way as resistance due to oxidases did against An. funestus in southern Africa ( 6 ) is unknown. To assess the practicability of applying IRS with pyrethroid in West Africa, we need to examine the effectiveness of this approach against a kdr-resistant population of An. gambiae is important. To get a clearer understanding of the influence of kdr resistance on the effectiveness of ITN, further experimental hut trials of ITNs against kdr-resistant populations need to be conducted. We describe 2 experimental hut trials in Benin. One compares the impact of IRS and ITN against a kd- resistant population in the southern part of the country; the other compares IRS and ITNs against a pyrethroid-resistant population several hundred kilometers to the north. Material and Methods Study Sites Ladji is a large village on the outskirts of Cotonou, the capital of Benin. The village floods during the rainy season. An. gambiae comprises the Mopti (M) cytotype and shows resistance to pyrethroids and DDT; kdr is present at high frequency ( 11 ). The nuisance mosquito Culex quinquefasciatus is also present and shows resistance to pyrethroids. Five experimental huts belonging to the Centre de Recherche Entomologique de Cotonou (CREC) are situated in the village. Malanville is in northern Benin, 800 km from Cotonou, in an irrigated rice-growing valley. The local An. gambiae comprises the M cytotype, but the kdr gene is almost absent and mosquitoes are susceptible to lambdacyalothrin and deltamethrin. Six experimental huts are present at Malanville. Experimental Huts The treated nets, residual spray treatments, and their respective untreated controls were evaluated in 4 experimental huts at each field site. Experimental huts are specially designed to test vector control product against freely entering mosquitoes under natural but controlled conditions. Huts were typical of the region. Each was made from concrete bricks, with a corrugated iron roof and a ceiling of thick polyethylene sheeting lined with hessian sackcloth on the interior surface, and each was built on a concrete base surrounded by a water-filled moat to exclude ants ( 12 ). Mosquito access was through 4 window slits, constructed from pieces of plywood fixed at an angle to create a funnel with a 1-cm gap, present on 3 sides of the huts. Mosquitoes had to fly upward to enter through the gaps and downwards to exit; this precluded or limited exodus through the aperture and enabled us to account for most entering mosquitoes. A veranda trap projected from the back wall of each hut. Movement of mosquitoes between a room and the veranda was unimpeded. Mosquito Net Treatments The nets were made of white, 100-denier polyester (SiamDutch Mosquito Netting Co., Bangkok, Thailand). Nets measured 2.0-m long, 1.6-m wide, and 1.8-m tall and had a surface area of 16.9 m2. To simulate badly torn nets, 80 holes, each measuring 2 × 2 cm, were cut in the sides and ends of each net. Insecticides used were formulations of lambdacyhalothrin (Icon, Syngenta, Switzerland): lambdacyhalothrin 2.5% CS, a microencapsulated suspension designed for ITNs, and lambdacyhalothrin 10% WP, a wettable powder designed for IRS. The lambdacyhalothin application rates of 18 mg/m2 for ITNs and 30 mg/m2 for IRS were within the ranges recommended by the manufacturer. Indoor residual treatments were applied with a hand-operated compression sprayer equipped with a flat fan nozzle. The cement walls and sackcloth ceilings were sprayed uniformly after masking the veranda and window slits with protective coverings. The control hut was sprayed with water only. The treated huts were left for 1 week before evaluations were started. Sleepers and Mosquito Collections Preliminary experiments showed the huts to be evenly attractive to mosquitoes. The treatments were randomly allocated to the 4 experimental huts at each site. The main trials were conducted from April to June 2005 at the Ladji site and from September to November 2005 at the Malanville site. Eight adult men employed by CREC slept overnight in the huts and collected mosquitoes from the huts in the mornings. Informed consent to participate in the study was given beforehand, and chemoprophylaxis was provided during the trial. Ethical approval was granted by the London School of Hygiene and Tropical Medicine (LSHTM) and Benin national ethics committees. The trial ran for 50 nights for 8 weeks at each site. The sleepers were rotated between huts to correct for possible variation in individual attractiveness. Each morning, mosquitoes were collected from the floors, walls, and ceilings of rooms, verandas, and nets with aspirators and torches. Mosquitoes were identified and scored as blood-fed or unfed and dead or live. Live mosquitoes were held in netted plastic cups and supplied with 10% honey solution for 24 h before delayed death was recorded. Male mosquitoes were not recorded. The entomologic impact of each treatment on mosquitoes was expressed relative to the control in terms of the following: deterrence, the proportional reduction in the number of mosquitoes entering a treated hut relative to that entering the control hut; induced exophily, the proportion of mosquitoes collected from the veranda trap of the treatment hut relative to the proportion in the veranda of the control hut; blood-feeding inhibition, the reduction in blood-feeding rate relative to the control hut; and mortality, the proportions of mosquitoes found dead in the hut at the time of collection and after a 24-h holding period. If a treatment deters a considerable number of mosquitoes from entering the hut, the values given by proportion blood-feeding or proportion killed in the treatment hut may underestimate the full personal protective effect and overestimate the full insecticidal efficacy of the treatment. The personal protective effect of a treatment is better described by the reduction in the number of blood-fed mosquitoes in the treatment hut relative to the number blood-fed in the control hut: % personal protection = 100 (Bu – Bt)/Bu where Bu = is the total number of blood-fed mosquitoes in the untreated control huts and Bt is the total number blood-fed mosquitoes in the huts with insecticide treatment. The overall insecticidal effect of a treatment needs to take into account that a considerable number of mosquitoes might be deterred from entering the hut and hence not be killed by the treatment. A mass killing effect is desirable to reduce transmission. The overall insecticidal effect of a treatment relative to the number of mosquitoes that would ordinarily enter an untreated hut can be estimated by using the following formula and expressed as a percentage: Overall insecticidal effect (%) = 100 (Kt – Ku)/(Tu – Ku) where Kt is the number killed in the treated hut, Ku is the number dying in the untreated control hut, and Tu is the total number collected from the control hut. Residual Activity of Insecticide Treatments To evaluate residual activity, World Health Organization (WHO) cone bioassays were undertaken monthly in the Ladji huts and bimonthly in the Malanville huts with a laboratory-susceptible strain of An. gambiae (Kisumu). An. gambiae females, 3–5 days old, were exposed within the cones to nets for 3 min or to sprayed walls and ceilings for 30 min. Approximately 50 mosquitoes in 5 replicates of 10 mosquitoes were tested on each substrate. Honey solution was provided during the 24-h holding period, and the temperature was kept at 25°C. Biochemical Assays Biochemical tests on individual mosquitoes were conducted to determine the activity of mixed function oxidases and nonspecific esterases present in pyrethroid-resistant and -susceptible samples of An. gambiae from the Ladji and Malanville sites. Tests were conducted on 3-day-old adult females (initially collected as larvae) in microtiter plates ( 13 ). Susceptible (Kisumu) and pyrethroid-resistant (Vkper) An. gambiae served as controls. Genotyping of An. gambiae was carried out to assess kdr frequency at both field stations ( 14 ). Adult Bioassay Data To determine whether a stronger pyrethroid resistance mechanism was present in the Ladji population than in the standard kdr strain Vkper, bioassays with 0.05% lambdacyalothrin-treated papers (18 mg/m2) were conducted in WHO resistance test kits by using a range of exposure times on batches of 25 unfed An. gambiae females 2–5 days of age. One hundred mosquitoes per exposure period were tested. Deaths were scored 24 h later. Log-time mortality curves were generated, and lethal time to kill 50% (LT50), estimated by using probit analysis. Data Analysis Proportional data from the hut trial (exophily, blood-feeding, deaths) were analyzed by using logistic regression (STATA 6 software, Stata Corporation, College Station, TX, USA). Deterrence rates were analyzed by comparing the number of mosquitoes entering each hut by using the Wilcoxon rank sum test. Biochemical activity was analyzed with Kruskal–Wallis and Wilcoxon rank sum tests. The level of resistance to lambdacyalothrin in insecticide bioassays was analyzed by using probit analysis. Results Insecticide Residual Activity Residual activity on ITN as measured by cone bioassay tests showed no decline during the 8 weeks of the trial. Activity of the IRS wettable powder formulation on sackcloth and cement showed a decline in performance by week 4. This trend continued until the end of the trial (Table 1). Table 1 Residual activity of lambdacyalothrin (insecticide)-treated nets (ITNs) and indoor residual spraying over 3 mo in experimental huts, Malanville and Ladji field stations* When and where substrate tested ITNs at 18 mg/m2 Indoor residual spraying at 30 mg/m2 Sides + top of net Ceiling Walls No. tested % Corrected mortality No. tested % Corrected mortality No. tested % Corrected mortality Wk 0 Malanville 77 100 33 100 60 100 Ladji 51 100 30 93.3 54 100 Wk 2 Ladji 52 100 22 100 41 100 Wk 4 Ladji 54 100 21 52.4 47 42.5 Wk 6 Ladji 57 100 25 80.0 45 31.1 Wk 8 Malanville 52 100 29 41.4 54 2.6 Ladji 44 97.7 8 25.0 39 18.5 *As determined by using World Health Organization cone bioassays and susceptible Anopheles gambiae (Kisumu). Efficacy of Treatments in Huts Over the 2-month trial, 1,395 An. gambiae, 3,070 Cx. quinquefasciatus, and small numbers of Mansonia uniformis, An. pharoensis, and Aedes aegypti were collected at Ladji. At Malanvile, 1,523 An. gambiae, 2,804 Mansonia sp., and smaller numbers of An. funestus and Ae. aegypti were collected. Only the malaria vector An. gambiae and the nuisance mosquito Cx. quinquefasciatus were analyzed further. Fewer An. gambiae entered the ITN- and IRS-treated huts than the respective control huts. The treatment induced reduction in hut entry was more evident in the resistance area than in the susceptible area (Table 2). The proportion deterred at each site did not differ between ITN or ITS treatments. Table 2 Experimental hut results of lambdacyhalothrin (insecticide)-treated nets (ITNs) and indoor residual spraying (IRS) against Anopheles gambiae, Ladji (pyrethroid resistance) and Malanville (pyrethroid susceptibility) field stations* Ladji (pyrethroid resistance) Malanville (pyrethroid susceptibility) ITN Untreated net Lambdacyhalothrin 18 mg/m2 Untreated net Lambdacyhalothrin 18 mg/m2 Total collected 689a 386b 363a 267b Deterred, % _ 44.0 ._ 26.4 Exiting, % (CI) 25.0†
(21.7–28.2) 29.0†
(24.5–33.5) 36.1
(31.1–41.0) 46.8‡
(40.8–52.8) Blood-fed, % (CI) 82.0†
(79.1–84.9) 82.1†
(78.3–85.9) 77.7†
(73.4–81.9) 3.0‡
(0.9–5.0) Blood-feeding inhibition, % _ 0 _ 96.1 Personal protection, % (no. bloodfed) – (572) 44.6 (317) – (282) 97.2 (8) % Dead (CI) 13.6† (11.1–16.2) 29.8‡ 25.2–34.4) 3.6† (1.7–5.5) 98.5‡ (97.0–99.9) Insecticidal effect, % (no. dead) – (94) 3.0 (115) – (13) 68.9 (263) IRS Unsprayed hut Lambdacyhalothrin 30 mg/m2 Unsprayed hut Lambdacyhalothrin 30 mg/m2 Total collected 203† 117‡ 498† 395‡ Deterred, % _ 42.4 _ 20.7 Exiting, % (CI) 45.8† (38.9–52.7) 58.1† (49.2–67.1) 54.4† (50.0–58.8) 63.3† (58.5–68.0) Blood-fed, % (CI) 87.7† (83.2–92.2) 73.5‡ (65.5–81.5) 93.8† (91.6–95.9) 69.6‡ (65.1–74.2) Blood-feeding inhibition, % _ 16.2 _ 25.8 Personal protection, % (no. bloodfed) – (178) 51.7 (86) – (467) 41.1 (275) Dead, % (CI) 12.3† (7.8–16.8) 30.8† (22.4–39.1) 1.4† (0.4–2.4) 72.1‡ (67.7–76.6) Insecticidal effect, % (no. dead) – (25) 5.4 (36) – (7) 55.8 (285) *For each untreated–treated pair, values not sharing the same superscript are significantly different at the 5% level.. CI, 95% confidence interval. The untreated net was little or no barrier to blood-feeding of An. gambiae at either field site owing to the large number of holes cut in each net. Treating the holed net with pyrethroid led to a 96% reduction in the number of mosquitoes blood-feeding at the susceptible site (Malanville) but to no reduction in blood-feeding at the resistant site (Ladji). Inhibition of blood-feeding by IRS at either the resistant or susceptible site was limited (Table 2). Natural mortality of An. gambiae occurred in both types of control huts but was notably higher at Ladji than at Malanville. Both modes of treatment were highly insecticidal at Malanville: ITNs treated with 18 mg/m2 lambdacyhalothrin killed 99%, and IRS applied at 30 mg/m2 killed 72% of An. gambiae that entered the huts. At Ladji, the proportions of An. gambiae killed in either the ITN- or IRS-treated hut did not exceed 30% (Table 2). The proportion of An. gambiae collected from the veranda traps in the mornings was greater at Malanville than at Ladji and greater in the huts with untreated nets than in the unsprayed control huts. Relative to the controls, lambdacyalothrin-treated nets and IRS induced little or no exophily of the pyrethroid-resistant An. gambiae into the verandas of the Ladji huts, despite high survival rate of mosquitoes in huts. At Malanville, pyrethroid-induced exophily by ITN or IRS hut was not evident and may have been obscured by the high death rates among the mosquitoes. The personal protection derived from ITN was almost 100% in the susceptible area. Despite the low mortality rate and high rate of blood-feeding observed with ITN in the resistance area, the level of personal protection there was almost 50% because of the deterrent effect of lambdacyhalothin on mosquito entry into huts. The personal protective effect of IRS was low in both areas, and IRS was no barrier to blood-feeding. The overall insecticidal effect of pyrethroid-treated nets and IRS was negligible in the resistance area ( 55.8%). Table 3 breaks down the mortality data into 2-week blocks. Mortality associated with IRS treatments decreased week by week at both sites but started at a lower rate at the Ladji site because of the expression of resistance. Mortality associated with ITN treatments also showed a downward trend over time at Ladji but not at Malanville, where mosquitoes showed high susceptibility throughout the study. Table 3 Mortality rate of free-flying, naturally entering mosquitoes in huts, first 8 weeks of trial Ladji (pyrethroid-resistant Anopheles gambiae) Malanville (pyrethroid-susceptible An. gambiae) ITN IRS ITN IRS Wk No. % Corrected mortality No. % Corrected mortality No. % Corrected mortality No. % Corrected mortality 1–2 41 43.2 15 53.3 67 100 91 100 3–4 83 50.5 42 47.6 93 100 108 88.7 5–6 209 28.7 39 24.2 54 92.6 78 57.8 7–8 53 5.7 21 23.8 53 98.8 118 39.0 *ITN, insecticide-treated net; IRS, indoor residual spraying. Both ITN and IRS treatments at Ladji showed poor efficacy against Cx. quinquefasciatus (this species was not encountered in Malanville). Insecticide-induced deterrence was greater for ITN than for IRS (Table 4). Neither method killed many Culex nor stimulated repellency into verandas. The IRS treatment produced an unusually high level of blood-feeding inhibition. Table 4 Experimental hut results of lambdacyalothrin (insecticide)-treated nets (ITNs) and indoor residual spraying (IRS) against Culex quinquefasciatus, Ladji (pyrethroid resistance) field station* Results Treatments ITNs IRS Untreated net Lambdacyhalothrin 18 mg/m2 Unsprayed hut Lambdacyhalothrin 30 mg/m2 Total entered 845 598 858 769 Deterred, % _ 29.2 _ 10.4 Exiting, % (CI) 29.8 (26.7–32.9) 35.9 (32.1–39.8) 52.7 (49.3–56.0) 54.6 (51.1–58.1) Blood-fed, % (CI) 62.8 (59.6–56.1) 59.5 (55.6–63.5) 85.1 (82.7–87.5) 42.9 (39.4–46.4) Blood-feeding inhibition – NS – 49.6 Personal protection, % (no. blood-fed – (531) 33.1 (355) – (730) 54.8 (330) Dead, % (CI) 4.3 (2.9–5.6) 8.5 (6.3–10.8) 3.4 (2.2–4.6) 16.3 (13.7–18.9) Insecticidal effect, % (no. dead) – (36) 1.9 (51) – (29) 11.6 (125) *CI, 95% confidence interval. Biochemical Assays and kdr Genotyping An. gambiae from Ladji expressed a significantly higher level of oxidase activity than the standard susceptible (Kisumu) and the laboratory kdr (Vkper) strains, which had a similar level of oxidase activity. However, the pyrethroid-susceptible strain from Malanville showed a level of oxidase activity that was not significantly different from that of the Ladji strain. This finding would appear to rule out any contribution from oxidases to the pyrethroid resistance observed in An. gambiae from Ladji. The level of α-esterase activity in An. gambiae from Ladji was significantly higher than that expressed in Malanville or Kisumu strains, whereas the level of β-esterase activity in Ladji, Vkper, and Kisumu strains was similar and clearly played no part in resistance (Table 5). Overall, the mean level of esterase activity at Malanville was significantly lower than that of the susceptible reference strain (p 95%, a degree of personal protection associated with ITNs and IRS was still evident (45%–50%) relative to the untreated net or unsprayed hut owing to a partial deterrent effect of treatments on entry of mosquitoes rather than to any inhibition of blood-feeding once the insects were inside the huts. Indeed, on entering the huts, most mosquitoes did go on to blood-feed, and the deliberately holed ITN was no barrier to resistant mosquitoes. By contrast, in northern Benin, only 4% of the insecticide-susceptible mosquitoes that entered the hut fed through the holed ITN. The loss of personal protection and loss of mosquito mortality associated with resistance would presumably combine to make ITNs unattractive from the perspective of both the individual user and the malaria control manager. Incision of 80 holes per net is the standard for ITN trials in West Africa ( 8 , 9 , 12 ), and such nets have given a degree of personal protection in earlier trials. An ITN with no or few holes might be expected to give some protection against resistant mosquitoes from Ladji, but there were insufficient huts available to test this idea. These experimental hut results from southern Benin stand in contrast to results from an area of Côte d’Ivoire (Yaokoffikro) that had a comparable frequency of kdr (78%) to that of Ladji (83%) ( 15 ) and where lambdacyhalothrin-treated nets and other ITN showed continuing efficacy, with mortality rates of 45%–68% ( 8 , 16 – 19 ). We sought evidence that other resistance mechanisms than kdr might be contributing to the reduced efficacy of pyrethroids at Ladji. Metabolic resistance due to mixed function oxidases (MFO) has, for example, undermined attempts at malaria control with deltamethrin residual spraying in southern Africa caused by An. funestus ( 6 ), and elevated MFO activity in a strain of An. gambiae from Cameroon reduced the efficacy of permethrin-treated netting in laboratory tests ( 20 ). The combined elevated activity of MFOs, glutathione S–transferase, and esterases resulted in a failure of the S. Mexican IRS program against An. albimanus ( 21 ). Our examination of enzymatic activity in An. gambiae showed no evidence that MFO activity is any greater in mosquitoes from Ladji than in mosquitoes from Malanville, nor did esterase activity differ between Ladji and Vkper (kdr) strains. Thus, there was no evidence of metabolic resistance enhancing the resistance already caused by kdr in mosquitoes from Ladji. Lambdacyhalothin bioassay tests showed no evidence of resistance level differing between Ladji and Vkper strains, and we conclude that metabolic mechanisms made no contribution to the observations in Ladji. In East Africa a different type of kdr based on a leucine-to-serine mutation, which confers resistance to permethrin and DDT ( 22 ), has been detected in several countries. However, no mosquitoes of this genotype were detected in tests on samples of An. gambiae from Ladji ( 23 ). The complete absence of efficacy of lambdacyalothrin against Cx. quinquefasciatus in Ladji merely confirms earlier findings involving other types of pyrethroid in experimental huts in West Africa ( 6 , 9 , 16 , 18 ). The contribution of kdr to pyrethroid resistance in An. gambiae needs to be reappraised. While lambdacyhalothin-treated nets (reported here) and permethrin-treated nets reported earlier ( 24 ) were less effective in hut trials in the kdr area of Benin (Ladji) than in a corresponding area of Côte d’Ivoire (Yaokoffikro), pyrethroid-treated nets were more effective in the susceptible area of Benin (Malanville) than in the corresponding susceptible area of Côte d’Ivoire (M’Be) ( 9 ) for reasons that are presently unknown. Other differences between the biology of An. gambiae from Côte d’Ivoire and Benin exist. Ivorian An. gambiae with kdr is mainly of the S molecular form, whereas Beninoise An. gambiae is of the M form (V. Corbel, unpub. data). M and S forms differ in ecologic distribution and habitat. While mosquitoes of the M form with kdr might behave differently from those of the S form with kdr when exposed to pyrethroids, this is mere speculation. Moreover, the M form in Malanville showed higher vulnerability to ITN than did the corresponding S form in Côte d’Ivoire, a finding that seems at odds with a behavioral hypothesis. Our study provides persuasive evidence that pyrethroid resistance in Benin is capable of undermining control measures based on ITN. Nor is there reassurance to be taken from IRS, and any attempt to switch vector control strategy would seem doomed to fail. Whereas the earlier phase 3 malaria control trials of ITN in Côte d’Ivoire showed continuing effectiveness despite kdr at high levels ( 10 ), our phase 2 results from Benin give no grounds for optimism. However, only phase 3 can provide a definitive answer. Further phase 3 trials using pyrethroid-treated nets and IRS need to be undertaken in Benin in an area of pyrethroid resistance. The normal practice with phase 3 is to aim at complete community coverage. Coverage in real life is usually less than total, and the danger with the type of pyrethroid resistance found in Benin is that at lower levels of coverage the important mass protective effect of ITNs ( 25 , 26 ) may be lost and transmission may continue unabated among those who do not have ITNs. To establish whether this is true, phase 3 trials on resistant mosquito populations should ideally set the coverage level at <100%. If it is considered unacceptable to deny a section of the trial population access to ITNs, an alternative but much less rigorous approach would be to monitor malaria incidence among users and non-users of long-lasting insecticide nets (LLIN) during the proposed scaling up of LLIN coverage in Benin currently being considered. Pyrethroid resistance in Benin is far from homogeneous, and LLIN should give good protection wherever mosquito populations are susceptible. Use of LLIN should be encouraged but scale-up of treated nets may ultimately select for further resistance. The need to develop alternative insecticides to replace or supplement pyrethroids on nets is urgent and should be put on a par with the seeking of new antimalarial drugs or vaccines that have received far greater attention and resources in recent years.

                Author and article information

                Role: Academic Editor
                PLoS Med
                PLoS Medicine
                Public Library of Science (San Francisco, USA )
                July 2007
                3 July 2007
                : 4
                : 7
                [1 ] Ifakara Health Research and Development Centre, Ifakara, Morogoro, United Republic of Tanzania
                [2 ] Department of Biological and Biomedical Sciences, University of Durham, Durham, United Kingdom
                [3 ] Department of Public Health and Epidemiology, Swiss Tropical Institute, Basel, Switzerland
                [4 ] Division of Infection and Immunity, Glasgow University, Glasgow, United Kingdom
                [5 ] Division of Environmental and Evolutionary Biology, Glasgow University, Glasgow, United Kingdom
                [6 ] United States Public Health Service Commissioned Corps and Malaria Branch, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
                Princeton University, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: gkilleen@ 123456ihrdc.or.tz
                06-pmed-RA-1013R2 pmed-04-07-01
                This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
                Page count
                Pages: 13
                Research Article
                Infectious Diseases
                Public Health and Epidemiology
                Infectious Diseases
                Health Policy
                Resource Allocation and Rationing
                Public Health
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                Killeen GF, Smith TA, Ferguson HM, Mshinda H, Abdulla S, et al. (2007) Preventing childhood malaria in Africa by protecting adults from mosquitoes with insecticide-treated nets. PLoS Med 4(7): e229. doi: 10.1371/journal.pmed.0040229



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