Incremental impact upon malaria transmission of supplementing pyrethroid-impregnated long-lasting insecticidal nets with indoor residual spraying using pyrethroids or the organophosphate, pirimiphos methyl

The study was conducted in the predominantly rural districts of Luangwa and Nyimba, located in Lusaka and Eastern provinces, respectively, of the Republic of Zambia (Fig. 1).

These districts have perennial transmission of Plasmodium falciparum, with the overwhelmingly predominant vector being An. funestus, which mediates a mean entomological inoculation rate (EIR) for non-users of LLINs of approximately 70 infectious bites per unprotected person per year [34]. The district of Luangwa (3468 sq km) is located 350–500 m above sea level, 325 km southeast of Lusaka, the capital city of Zambia. It has a population of approximately 27,560 residents, with an annual growth rate of 2.9 % [35]. The main economic activities in the district are fishing and agriculture. Nyimba is a larger district (10,943 sq km), with a population of 108,637 inhabitants and an annual growth rate of 3.4 % [35]. The district is located 400–1200 m above sea level, 350 km east of Lusaka. Agriculture is the predominant economic activity in Nyimba district.

In each district, seven clusters of approximately 165 households were selected and enrolled in the study to participate in longitudinal parasite surveys [36]. Of these, 15 households in each cluster were selected and enrolled at the discretion of the community health worker (CHW), so that they were geographically distributed across the cluster, for participation in monthly entomological observations, with the exception of Luangwa High School, where 30 households were enrolled. Both parasitological and entomological assessments were conducted continuously from January 2011 to March 2013 in Luangwa and from April 2011 to March 2013 in Nyimba district in all clusters.

The pyrethroid deltamethrin [wettable granule (WG) formulation] was sprayed in all consenting households at the four southernmost clusters in Luangwa in October 2010, immediately before that year’s rainy season and initiation of this study. During the study period, three other selected IRS insecticide treatments [capsule suspension (CS) formulation of the pyrethroid lambdacyhalothrin, as well as the emulsifiable concentrate (EC) and CS formulations of the organophosphate pirimiphos methyl (PM)] were randomly allocated to clusters in advance of each rainy season. In practice this randomized allocation was not strictly adhered to by the implementation agencies in the two districts (District Medical Office (DMO) in Luangwa and Abt Associates under the supervision of the DMO in Nyimba), thus resulting in a quasi-randomized study design, described cartographically in Fig. 1. The parasitological and entomological surveys were conducted by paid CHWs as previously described [34, 36] and summarized below. In the south of Luangwa district, between October and November 2010, clusters 4, 5, 6, and 7 received pyrethroid-based IRS with deltamethrin (K-Othrine WG^® 250, Bayer Environmental Science, South Africa) as described in Fig. 1a. Subsequently, the organophosphate PM was introduced as an alternative insecticide for IRS in a response to detection of resistance to pyrethroids in the primary vector, An. funestus in Luangwa district [37‐39]. The only formulation of PM that was available at the time was the relatively short-lived [33, 40] EC formulation (Actellic^® EC, Syngenta Crop Protection AG, Switzerland). This formulation was sprayed during the months of October and November 2011, in clusters 2, 4, 5 in Luangwa and 9, 11, 13 in Nyimba, while IRS with pyrethroid lambdacyhalothrin (Icon^® 10 CS formulation, Syngenta Crop Protection AG, Switzerland) was applied in only two of the four clusters in the south of Luangwa district which had been sprayed with deltamethrin the previous year, specifically in clusters 6 and 7 (Fig. 1b). The following year, in November 2012, the longer-lasting microencapsulated formulation of PM (Actellic^® 300CS, Syngenta Crop Protection AG, South Africa) was applied in clusters 8, 9, 10, 12, and 14, all of which were in Nyimba district (Fig. 1c). In February 2013, IRS in Luangwa district was implemented with PM-EC in clusters 2, 4 and 5 while clusters 6 and 7 received the CS formulation of lambdacyhalothrin (Fig. 1d).

Active monthly parasitological surveys were coupled with questionnaires recording clinical symptoms of illness, as well as access and utilization of preventive measures such as LLINs, IRS and intermittent preventive therapy (IPT), between January 2011 and May 2013, spanning a period of approximately 29 months, as described previously [36]. These surveys were conducted by paid CHWs who made active monthly visits to households that consented to participate in the study. In between active visits, study participants who developed symptoms were encouraged to seek care through passively offered diagnosis and treatment services, either from the CHWs at their place of residence or at the nearest health facility. The rapid diagnostic test used in the study was manufactured by ICT Diagnostics to detect circulating P. falciparum histidine-rich protein-2 antigen (ICT Malaria P.f. cassette test). All participants that were found positive for antigenaemia, which was presumed equivalent to infection, were treated with artemether-lumefantrine as per national malaria diagnosis and treatment policy [41]. In both the active and passive visits, all participants found to be negative for malaria infection but febrile or had any other complaints, were referred to the nearest health facility.

The monthly mosquito collections were conducted by paid CHWs using Centres for Disease Control and Prevention light traps (LT) and Ifakara tent traps (ITT) between January 2010 and April 2013, spanning a period of approximately 28 months, as described previously [34].

Fifteen well-distributed houses were semi-arbitrarily selected for mosquito trapping in each housing cluster, with the exception of Luangwa High School which had 30 houses due to the availability of an extra CHW involved in mosquito trapping. Each house was visited once per month for mosquito trapping using both the LT and ITT, on a consistent date of the month which was pre-agreed with each consenting household head [34]. In each consenting household, the LTs were placed at the foot end of an occupied sleeping space covered with an LLIN, hanging approximately 1.5 m above the floor. An ITT was placed immediately outside, approximately 5 m away from the house where the LT was installed and was occupied by an adult male volunteer from the same household. All the mosquito traps were set up in the evenings and collection of the captured mosquitoes was done in the early morning by aspiration. All the collected mosquitoes were initially sorted in the field to genus level by the CHWs, based on crude taxonomic features and then stored over silica until they were collected on a monthly basis and transported to a central laboratory at the National Malaria Control Centre (NMCC) for further detailed examination. Additional morphological identification of Anopheles to species group or complex [42] was conducted at the central laboratory of the NMCC in Lusaka. Polymerase chain reaction (PCR) for the identification of species within the An. funestus group [43] or An. gambiae complex [44] were conducted on selected samples in the NMCC laboratory.

A team of trained entomological technicians from the NMCC periodically collected samples from the study sites to ascertain the susceptibility of the mosquitoes to different classes of insecticides, as background descriptive data to support appropriate interpretation of apparent impacts of various supplementary IRS treatments upon the vector population. In Luangwa district, mosquitoes were collected from cluster 2 from 2010 to 2013. However, in Nyimba district, collections were done over 3 years in different clusters (cluster 14 in 2011, cluster 9 in 2012 and cluster 13 in 2013). Adult mosquitoes were either collected while attacking humans by human landing catch (HLC) or by using pack aspirators for the indoor wall-resting mosquitoes. These were collected in cups covered with a netting material and placed in cooler box for transportation to the NMCC insectary where individual female An. funestus mosquitoes where allowed to feed on mouse blood so they could lay eggs that were then reared into F1 generation mosquitoes. Standard WHO susceptibility tests using insecticide-impregnated papers with discriminatory dosages of two pyrethroids (deltamethrin 0.05 % and lambdacyhalothrin 0.05 %), a carbamate (bendiocarb 0.1 %), an organophosphate (malathion 0.4 %) and an organochlorine (DDT 4 %) were carried out on 2–5 day-old F1 An. funestus mosquitoes. Control papers were impregnated with oil as directed by the WHO protocol [45]. Knock-down and mortality rates after 1 and 24 h post-exposure periods were recorded.

To estimate proportions of human exposure to An. funestus bites and malaria transmission that occurs indoors and outdoors, HLCs were conducted both indoors and outdoors by a team of trained entomological technicians from the NMCC in Lusaka and these were complemented by cross-sectional questionnaire surveys of when residents went indoors for the night, went to sleep, awoke in the morning, and left the house in the morning, as previous described [39], again as background descriptive data to support appropriate interpretation of apparent impacts of various supplementary IRS treatments upon the vector population and malaria transmission. Trained CHWs conducted HLC from 18.00 to 06.00 h, with the exception of the previously described 2010 studies where the starting time was 19.00 and finished at 07.00. The 2010 and 2011 HLC surveys were conducted in cluster 4 (Chisobe and Nyamumba villages of Luangwa district) as part of a trap effectiveness study [38], while those conducted in 2012 and 2013, where part of the quality assurance surveys were conducted in 13 clusters as part of a subsequent effectiveness assessment for a community-based trapping scheme [34]. Mosquitoes were collected for 45 min per hour to allow a 15-min break for rest and refreshment for the collectors. Each hourly collection were labelled and kept for identification to genus and species as described above. The proportion of time that residents spent outdoors and indoors, as well as asleep in bed, was estimated directly from answers to questionnaires during a cross-sectional household survey in April 2010 in Luangwa district, in which people indicated the time they usually went indoors and when they went to the bed as well as when they arose in the morning and when they left their houses [39].

The CHW Malaria Register data describing rapid diagnostic test (RDT) results associated with questionnaire responses were double entered into Excel^®, verified, reconciled, and then cleaned following descriptive frequency analysis of the distributions of values for each variable. All entomological data were single entered, verified and cleaned prior to analysis. All statistical analyses were accomplished using SPSS version 20 (IBM) and R version 2.14.1, augmented with the lattice, Matrix and LME4 packages.

Previous analyses of these data collected by CHWs have demonstrated that diagnostic positivity (DP) for malaria infection, expressed as the proportion of RDT-tested individuals who were found to be positive, was a extremely powerful indicator of malaria risk that allowed numerous important epidemiological phenomena to be clearly illustrated [36]. It also proved to be a more consistent and robust indicator of geographic and temporal variation than absolute numbers of malaria infections detected, presumably because variations in CHW service utilization rates, as well as RDT and ACT availability, occur in both the nominator and denominator of DP [46], and was therefore treated as the primary epidemiological outcome used for statistical analysis of the effects of various IRS treatments, rather than incidence in terms of detected events per number of participants per unit time.

Four sequential time period categories, based on the integer number of months since the most recent spray round was completed were created for all the IRS treatments: 1–3, 4–6 and 7–12 months since beginning of the last spray round started, as well as a fifth category combining areas that had not yet received spraying during the study period and those for which the last spray round began more than 12 months ago, which was treated as the reference value. Generalized linear mixed models (GLMMs) were fitted to evaluate the association between observed malaria infection risk among human residents and the various IRS treatments applied. Malaria infection status was treated as the binary dependent, with use of an LLIN, having slept in a house that had been treated with IRS in the previous 6 months and the categorized cluster-wide IRS treatments as the independent variables of primary interest. Age category (<1, 1–4, 5–10, 11–14, 15–24, 25–44 and >45 years of age), sex, season (hot and wet from December to April, cool and dry from May to August, and hot and dry from September to November), number of previous RDTs conducted per individual and geographical location (cluster) were also included as independent variables of secondary interest (all categorical except for number of RDTs) while random effects to capture variance associated with nuisance variables of no direct interest were also included in the model (the individual identity number nested within the CHW catchment nested within the study cluster, as well as date of participant contact). IPT use was not included in the final model as an independent variable because it had no apparent effect on malaria infection prevalence (P = 0.8633). Further, in order to test for and quantify incremental impact of PM IRS as a supplement to LLINs, relative to LLINs supplemented with pyrethroid-based IRS, both pyrethroid formulations were represented by a single treatment variable, coding the same periods of months since before spraying. Similarly, in order to test for and quantify the incremental impact of the CS formulation of PM, relative to the EC formulation of the same active ingredient, as well as the two pyrethroid formulations, an additional variable was created which combined any previous treatment with any of the latter three formulations in the reference group. In all cases, incremental protective efficacy (IPE) was calculated as the complement of the odds ratio (OR) estimated directly by these GLMMs (IPE = 1−OR).

The effect of different IRS treatment regimens on densities of An. funestus species were estimated by fitting GLMMs where An. funestus densities were treated as a dependent variable with a Poisson distribution. In order to account for variance in mosquito densities by location, identities for households were nested within those villages and then nested within clusters as random effects. Similarly, nightly temporal variance in vector density was accounted for by including date as an additional random effect. The different IRS treatment regimens were coded in terms of time period since the last round of IRS application began, exactly as described above for the epidemiological primary outcomes, so that these treatments could be included as categorical independent variables with which to detect and quantify impact upon these entomological secondary outcomes. In all, the relative rate (RR) at which mosquitoes were captured was calculated as estimated directly by these GLMMs. Unfortunately, efforts to develop laboratory capacity for determining sporozoite infection status by enzyme-linked immunosorbent assay (ELISA) at NMCC were unsuccessful so neither sporozoite prevalence nor entomological inoculation rate could be assessed as additional entomological secondary outcomes.

Insecticide susceptibility assays were conducted on 2–5 day-old F1 generation An. funestus as described by the WHO standard protocol [47] using papers impregnated with deltamethrin (0.05 %), lambdacyhalothrin (0.05 %), bendiocarb (0.1 %), malathion (0.1 %) or DDT (4 %). In order to test for time trends in physiological resistance of An. funestus to pyrethroids and carbamates over time, survival status of mosquitoes exposed to these insecticides in standard WHO protocols [45] was treated as the binary outcome variable in GLMMs with year as a continuous covariate and a unique identification code for each experimental replicate as a random effect. The data were stratified into sub-sets on the basis of the insecticide class, with separate models fitted for the carbamate (bendiocarb), and the combined pyrethroids (deltamethrin and lambdacyhalothrin). The model of resistance time trends for the two pyrethroids, the identities of these two insecticides within this class were included as a categorical independent variable. No such model was fitted for either the organochlorine (DDT) or the organophosphate (Malathion) because no resistance to either insecticide was apparent.

The distribution of human exposure to An. funestus bites, and presumably malaria transmission, across different times of the night and across indoor and outdoor compartments of their living environment was calculated by weighting HLC measurements of indoor and outdoor biting rates for each hour of the night by the estimated proportion of humans indoors and outdoors during that time period, exactly as previously described [39]. These estimates of human exposure distribution across indoor and outdoor environments were calculated and presented graphically for both users and non-users of LLINs, so that the proportions of human exposure that occur indoors in the presence (π _i,n ) and absence (π _i ) of a protective LLIN could be quantified and visualized.

Prior to the study, community sensitization was conducted and permission obtained from the local community leadership. Informed consent was obtained from all study participants during all surveys and spraying activities. The study team ensured that all treatment and diagnostic protocols were adhered to and that patients requiring malaria treatment received it promptly or were referred to the nearest health facility. All participants who took part in the HLCs gave written consent to participate after being informed of the risks and benefits, and were provided with weekly prophylaxis using the nationally recommended combination drug of 100 mg Dapsone and 12.5 mg pyrimethamine (Deltaprim^®, CAPS Pharmaceuticals, Zimbabwe) so that their overall malaria risk was considered to be far lower than it would otherwise be in the course of their normal lives if they did not participate in the study [48]. All standard safety protocols for IRS application were adhered to as per national guidelines. Ethical approval was obtained from the University of Zambia, Biomedical Research Ethics Committee (Reference 004-05-09) and the Research Ethics Committee of the Liverpool School of Tropical Medicine (Approval 09.60). Authority to conduct and publish the study was also obtained from the Ministry of Health in Lusaka, Zambia.

A total population of 25,354 people centred around health facilities in the 14 clusters participated in the study and were followed up for a period of 29 months in Luangwa and 26 months in Nyimba, starting from January 2011 and April 2011, respectively. Out of these participants, 29 % (7412) were children under the age of 5 years but DP peaked in older children between the age of five and ten. The overall cluster populations ranged from 1158 to 3429. A total of 31,974 malaria infections (21.7 % DP) were identified, which translates into an incidence of nine infections per 100 person years. The study population reported a relatively high average rate of LLIN utilization of 81.7 % of questionnaire responses over the course of the study, indicating that the respondent had slept under an LLIN the previous night, while 39.2 % of participant questionnaire responses indicated that the respondent’s house had been treated by IRS in the last 6 months. During same overall study period mean DP by cluster across all age groups and other potential stratification criteria ranged from 6.4 to 41.9 % (mean = 24.5 %), with the lowest being in the southern urban cluster and the highest in the northern rural cluster (Tables 1, 2). The potential confounding effect of LLIN ownership was excluded from the final model described in Table 2 because it had no significant effect (P = 0.7584) on diagnostic positivity.

The close associations of DP for P. falciparum malaria infection and An. funestus density, clinical symptoms of illness, and a variety of other factors of this setting are described in detail elsewhere based on the first year of data collection [34, 36]. The detailed profile of the study participants, and their survey contacts over the course of the entire study, are summarized in the context of the study design in Fig. 2.

A descriptive comparison of summarized data restricted to the period 1–6 months post-spraying demonstrates variability among study clusters not only in IRS coverage (range = 0–100 %, mean = 29.4 %) but also LLIN use (range = 6.6–100 %, mean = 68.2 %) and diagnostic positivity (range = 2.99–61.9 %, mean = 25.4 %) (Table 1; Additional file 1). Further analysis using Pearson’s correlation, revealed a positive but weak association (r² = 0.31) between IRS coverage and LLIN use, suggesting that as IRS coverage increases, so does LLIN use. However, this does not necessarily imply any causal relationship and factors which affect delivery (e.g., accessibility) and acceptance (e.g., attitudes towards malaria or mosquitoes) may well be similar for both of these vector control measures. However, there was no obvious and clear-cut effect of any particular IRS treatment in this crude descriptive comparison (Table 1; Additional file 1) so detailed regression modelling analysis was required to detect and estimate the separate impacts of these four different formulations (Table 2, Figs. 3, 4, 5).

Reported coverage of deltamethrin WG, lambdacyhalothrin CS, PM EC, and PM CS, by respondents within the first 3 months after their application in clusters to which they were assigned was 82 % (2132/2599), 61 % (2068/3384), 53 % (5909/11,078), and 69 % (2716/3913), respectively. Over the study period, DP ranged from 13.3 to 23.8 % (mean = 18.4 %), 4.5 to 22.7 % (mean = 10.3 %), 16.0 to 28.8 % (mean = 21.4 %) and 13.0 to 49.5 % (mean = 28.7 %) for clusters assigned with deltamethrin WG, lambdacyhalothrin CS, PM EC, and PM CS respectively (Tables 1, 2). As illustrated in Fig. 3, PM CS conferred the strongest initial incremental protection in the first 3 months after application (IPE [95 % CI = 0.63 [0.57, 0.69], P < 0.001), relative to LLINs alone, followed by the CS formulation of lambdacyhalothrin (IPE [95 % CI] = 0.31 [10, 47], P = 0.006), the EC formulation of PM (IPE [95 % CI] = 0.23 [0.15, 0.31], P < 0.001) and the WP formulation of deltamethrin (IPE [95 % CI] = 0.19 [−0.01, 0.35], P = 0.064). However, neither pyrethroid formulation provided any incremental protection beyond 3 months post-application, while the incremental protection provided by CS and EC formulations of PM persisted undiminished for 6 and 12 months, respectively (Fig. 3).

The first 3 months after IRS with the CS formulation of PM offered greater protection against malaria infection than IRS with pyrethroids IPE [95 % CI] = 0.51 [0.38, 0.62], P < 0.001 for LLINs + IRS with PM-CS compared to LLINs + IRS in all clusters treated with either DM-WG or LC-CS but not PM-EC (P < 0.001). The incremental protection against malaria infection by IRS with both PM formulations outlasted both pyrethroid formulations so that they both offered greater protection from 4 to 6 months post-application IPE [95 % CI] = 0.79 [0.75, 0.83], P < 0.001 for LLINs + IRS with PM-CS and IPE [95 % CI] = 0.42 [0.33, 0.48], P < 0.001 for LLINs + IRS with PM-EC, compared to LLINs + IRS with either DM-WG or LC-CS) (Fig. 4).

Beyond 6 months post-application, LLINs plus IRS with PM-CS provided no apparent incremental protection relative to LLINs alone (P = 0.204), much less LLINs + IRS with pyrethroids (P = 0.432). However, LLINs + PM-EC continued to provide incremental protection relative to not only LLINs alone (Fig. 3), but also relative to all other LLIN + IRS treatments (IPE [95 % CI] = 0.41 [0.34, 0.48], P < 0.001). When the duration of efficacy of PM-EC was examined in further detail by breaking down the third post-spray time period into two halves, it was clear that it lasted approximately a full year because similar levels of incremental protection was confirmed for both the seven and 9 months post-spray period (IPE [95 % CI] = 0.32 [0.22, 0.40], P < 0.001) and the 10 to 12 months post-spray period (IPE [95 % CI] = 0.42 [0.31, 0.52], P < 0.001).

Comparing these two IRS formulations of PM with each other as supplements to LLINs, the CS formulation confers greater protection than the EC formulation, (IPE [95 % CI] = 53.6 [0.43, 0.66]  %, P < 0.001 from 1 to 3 months post-application and 0.64 [0.57, 0.69], P < 0.001 from 4 to 6 months post-application for the contrast between LLINs + PM-CS versus the LLIN + PM-EC as the reference group) (Fig. 5). However, once the incremental benefit of supplementing LLINs with IRS using PM-CS waned after 6 months, IRS using PM-EC proved statistically superior to all other IRS formulations as supplements to LLINs for a further 6 months, including the CS formulation of the same active ingredient (IPE [95 % CI] = 0.52 [0.21, 0.70], P < 0.001 for the contrast between LLINs + PM-EC versus LLIN + PM-CS as a reference group between seven and 12 months post-application).

Detailed description of the local mosquito fauna in the study area [34] showed that 34.5 % of all mosquitoes caught over the course of the study were identified morphologically as members of the An. funestus group, of which 96.5 % (575/596) of those which were successfully amplified by PCR, were confirmed to be An. funestus. Densities of the An. funestus group, as determined by routine morphological classification can therefore be considered quite reliable, as of An. funestus, the abundance of which is consistent with previous studies in this area [38, 39] indicating it as the overwhelmingly dominant vector of malaria in these two districts of Zambia. Therefore, subsequently in this report all mosquitoes caught from the An. funestus group are the nominate species in the strict sense.

The relative rates and the mean catches of An. funestus per IRS treatment are presented in Table 3. Relative to the times and places that had never been sprayed, or sprayed or had been sprayed >12 months previously, there were no obvious differences in the densities of An. funestus during the first 3 months post-spraying for both pyrethroid formulations (DM-WG (IPE [95 % CI] = 0.01 [−0.56, 0.37], P = 0.103) and LC-CS (IPE [95 % CI] = −0.03 [−0.88, 0.44], P = 0.195) and PM-EC (IPE [95 % CI] = −0.04 [−0.30, 0.17], P = 0.103) (Fig. 6, Table 3). However, where PM-CS was applied, mosquito densities were dramatically reduced during the same period of 3 months immediately after spraying (IPE [95 % CI] = 0.93 [0.87, 0.97], P < 0.001). Between the fourth and the sixth month after spraying with DM-WG, there was an apparent, but presumably spurious, 3-fold increase in An. funestus densities while LC-CS, PM-EC and PM-CS achieved 5-, 3- and 71-fold reductions, respectively (Table 3). However, from the seventh to 12th months after spraying, DM-WG and PM-EC had no obvious effect on the An. funestus densities, while insufficient data were available to examine the incremental impact of LC-CS or PM-CS.

From the outset of the study, An. funestus exhibited high level of resistance to both pyrethroids against which they were tested, and resistance level generally increased over the course of the study (P < 0.001). Alarming rates of resistance to the carbamate bendiocarb were also observed but these did not increase over the course of the study (P = 0.565). During this same period, there was no evidence of malathion or DDT resistance detected in the mosquito populations (Fig. 7).

Throughout the study period, humans lacking LLINs were exposed to far more bites by An. funestus indoors during the late hours of the night up to the early morning hours (Fig. 8), consistent with the known behaviour of An. funestus across the continent [42, 49]. The vast majority potential exposure to bites by this dominant vector occurred indoors at times when most individuals are asleep (Fig. 8). Even for those using an LLIN to prevent most indoor transmission, most residual human exposure to An. funestus bites and presumably malaria transmission, occurred indoors, increasing gradually from 57 % in 2010 to 71 % by 2013 (Fig. 8).