Study arms
This study consists of two arms evaluating the durability of DuraNet® Plus compared to DuraNet® A description of the two ITN types is provided below:
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DuraNet® Plus is WHO prequalified pyrethroid-PBO mixture 150 dernier net made of monofilament high density polyethylene (HDPE). It is incorporated with a mixture of alpha-cypermethrin at 6.0 g of active ingredient/kg and Piperonyl Butoxide (PBO) at 2.2 g of active ingredient/kg. It has a mesh size of 20 holes/cm2 and a fabric weight of 45g/m2.
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DuraNet® is a WHO prequalified standard pyrethroid-only 150 dernier net also made of mono-filament high density polyethylene (HDPE). It is incorporated with alpha-cypermethrin at 5.8 g of active ingredient/kg. It has a mesh size of 20 holes/cm2 and a fabric weight of 45g/m2. DuraNet® will serve as the reference net against which DuraNet® Plus will be compared.
DuraNet® Plus and DuraNet® are manufactured by Shobikaa Impex pvt Ltd (Karur, Tamil Nadu 639006, and India).
Study setting
The study will be conducted in selected malaria endemic villages in 3 different countries across sub-Saharan Africa: Benin, Cameroon and Tanzania. The countries have been selected to provide a suitable representation in the different regions in the area, West (Benin), Central (Cameroon) and East Africa Tanzania). Villages have been selected based on road accessibility, willingness to participate, availability of enough households, history of high ITN usage and proximity to the laboratory and insectary facilities of the participating institutional partners. A description of study area chosen for each country is provided in Table 1 below:
Table 1
Description of study sites in Benin, Cameroon and Tanzania
Characteristics | Benin | Cameroon | Tanzania |
Study villages | Agonkanme, Zantata Centre, Doutin and Agondokpoe | Ekoumeyeck and Agonfeme | Kibaoni, Magila, Misongeni and Ubembe |
Estimated population size | ~ 11,000 | ~ 6000 | ~ 7000 |
District | Zakpota District in Zou Department | Mbalmayo Districts in Central Region | Muheza district in Tanga Region |
Geographical Coordinates | Latitudes 7° 13′ 49″ North Longitudes 2° 12′ 55″ East | Latitude: 3° 31′ 12″ North, Longitude: 11° 30′ 44″ East | Latitude: 5° 10' 0.012" South. Longitude: 38° 46' 59.988" East |
Climate | Tropical humid climate. Annual rainfall 1003.4mm and temperature of 27.1°C. | Tropical savannah climate with wet and dry season. Annual rainfall 2402.8mm and temperature 26.5°C | Tropical savannah climate with wet and dry season Annual rainfall 293.6mm and temperature 23.7°C |
Main vector population | Anopheles gambiae sl. resistant to pyrethroids | Anopheles gambiae sl. and Anopheles funestus resistant to pyrethroids | Anopheles gambiae ss. and Anopheles funestus resistant to pyrethroids |
Typical economical activities | Agriculture and livestock. | Agriculture, fishing and livestock. | Agriculture, and livestock. |
Study design outline
The study will be conducted in a minimum of 1800 households in each country to be randomly assigned to the two study arms. Nets will be distributed to each household and followed prospectively. Before net distribution, a baseline census will be carried out in the selected study villages to collect demographic information and socio-economic characteristics of the participating households. This will be followed by a net distribution campaign after which a series of follow-up surveys will be performed to assess adverse events, ITN survivorship, fabric integrity, insecticidal efficacy and chemical content over a period of 36 months. Figure 1 provides a summary of the surveys and the different studies to be performed at each timepoint.
Randomisation, blinding and sample size calculations
Households will be the unit of randomisation of this study. Participating households will be blinded to the type of ITN they received while data collectors will be blinded to the allocation of households to the study arms. Study nets (DuraNet® Plus and DuraNet®) will be identical in appearance. Two cohorts of nets of each ITN type will be followed; cohort 1 to assess ITN survivorship, attrition and Fabric integrity and cohort 2 for nets to be withdrawn for assessment of bioefficacy and insecticide content. A sample size of 510 households per study arm was found sufficient to detect a 7 points difference in ITN functional survival rate over 3 years between households receiving DuraNet® Plus and those receiving DuraNet® with 80% power, assuming a baseline 3-year survival rate of 25%. This sample size was increased by 100 households to account for loss to follow up over time. To account for nets to be withdrawn for bioefficacy studies, hut trials and insecticidal content, an additional 310 households per arm will be included in the study, hence a total of 920 households will be recruited per study arm at each site. It is estimated that about 3 nets will be required per household therefore the study will require 2,760 nets of each ITN type at each study site.
Community sensitisation and baseline census
A series of community sensitisation meetings will be held in selected villages in each study site prior to the baseline surveys to ensure community participation. The study procedure and its objectives will be explained to leaders of all participating villages to ensure their cooperation with the study team. The meetings will be conducted in the local language of each community to ensure full participation. After the community sensitisation meetings, baseline census will be conducted in all consenting households in the selected villages for each study site using a structured questionnaire. The census will provide a framework for allocation of study nets and for sampling at follow-up. The following information will be recorded: name of the village, name of the head of the household, number of household members with details of adults and children living in the house, number of nets already in the household, number of sleeping places and the global positioning system (GPS) coordinates will be recorded to assist in identifying houses during follow-up. Each house will be assigned a unique identifier which will be marked on the door of the house during the survey using indelible ink.
ITN distribution and net master lists
The baseline census data will be used to randomly allocate households to each study arm using a random number generation scheme. The study team will then visit all participating households to provide vouchers for collection of study ITNs at a central point in each village. Where possible, households will be visited door-to-door to ensure that they receive their allocated net. Each ITN will be marked with a unique identifier before being provided to the household. The unique identifier will contain the household number, a unique number for the ITN, the ITN type and the cohort (cohort 1 and 2) to which the net has been assigned. Using these unique identifiers, net master lists will be generated, and these will contain a unique identification for each study net available in each household and separated by cohort to avoid any risk of destructive sampling of nets from households in cohort 1. As described previously, nets in the cohort 1 master list will be followed for attrition and fabric integrity while nets in the cohort 2 master lists will be withdrawn (and replaced with new nets of same type) for bioefficacy and insecticidal content studies.
Follow up surveys
Follow-up for ITN hang-up and adverse events
Between 1 week and up to 1 month post-distribution, sleeping places in houses in the net sampling lists (cohort 1 and cohort 2) will be observed to ensure that study nets have been set up. Where necessary, study teams will assist participants in hanging up their nets. The nets available in the house will also be checked to ensure that they match with what is available in the net sampling list which was generated after distribution and corrections will be made as necessary. Using a questionnaire, the adverse effects related to net usage will be investigated in 30 randomly selected households in each study arm at each study site. Heads of households will be questioned on any perceived adverse events including sneezing, itching, headache, numbness, eye discharge, any unpleasant smells and nausea in line with WHO guidelines. Participants will be advised to seek medical care from the nearest health facility should the symptoms persist.
Follow-up for ITN attrition
Net attrition rate in households will be measured by recording the physical presence of the net at 6-, 12-, 24- and 36-months post ITN distribution. All households in cohort 1 (at least 750 nets/arm) will be visited and nets assessed for attrition at each timepoint by recording the physical presence/absence of each net attributed to the household. Where a net is not found, the householder will be questioned about the reason for the loss of the net e.g given away, sold, stolen, worn out and disposed of etc. Any nets which have never been used, will be recorded and excluded from the analysis. ITNs lost due to wear and tear will be considered as lost due to attrition, and those lost because they were stolen, sold or given away to others, will be considered as lost to follow-up. During these surveys, a questionnaire adapted from WHO guidelines will be used to collect data on the number of washes and washing practices for the nets found in each household.
Follow-up for ITN fabric integrity: Fabric integrity (hole index) and condition will be observed on all nets of cohort 1 at 6-, 12-, 24- and 36-months post distribution. Where the minimum number (250) of nets for fabric integrity is not attainable from the longitudinal cohort (cohort 1) due to loss, ITNs sampled destructively from the same clusters for bio-efficacy would first be examined for fabric integrity prior to the bio-efficacy studies. The survey team will inspect the nets outside in broad day light to determine the hole index using a portable frame over which the net can be draped during inspection. The nets will be returned to the family right after the inspection. Hole number and size will be assessed on each ITN panel using hole assessment sheet and classified into 4 sizes (size 1: 0.5-2cm, Size 2: 2–10 cm, Size 3: 10-25cm, and size 4: >25cm). Physical integrity will be measured as the proportionate hole index (pHI), categorized based on recommended cut-off points into “good” condition (pHI 0–64), “acceptable” condition (pHI 65–642) and “torn” (pHI 643+).
Bioefficacy studies and chemical analysis
A separate subsample of nets belonging to cohort 2 will be randomly selected per arm for bioefficacy studies and chemical analysis. Nets of each type (DuraNet® Plus and DuraNet®) will be destructively sampled from households at 6, 12, 18-, 24-, 30- and 36-months post-distribution and replaced with new nets of the same type (brand) at each sampling point. They will be subjected to chemical analysis to monitor alphacypermethrin and PBO content and to laboratory bioassays (WHO cone bioassays and tunnel tests where necessary) and experimental hut trials to monitor entomological efficacy against mosquito vectors over time. For laboratory bioassays, net pieces measuring 30cmx30cm will be cut from each net following WHO guidelines [9]. The number of whole ITNs of each type to be subjected to each type of study at each timepoint is summarised in Table 2 below.
Table 2
Number of DuraNet® Plus and DuraNet® ITNs to be sampled for laboratory assays, chemical analysis and hut trials at each study site.
Months post ITN distribution | No of ITNs for Laboratory assays | No. of pieces per ITN | Total number of pieces for bioassays | No. of pieces for chemical analysisa | ITNs for hut trialsb |
0 | 30 | 5 | 150 | 150 | - |
6 | 30 | 4 | 120 | - | - |
12 | 30 | 4 | 120 | 120 | 54 |
18 | 30 | 4 | 120 | - | - |
24 | 30 | 4 | 120 | 120 | 54 |
30 | 30 | 4 | 120 | - | - |
36 | 50 | 4 | 200 | 200 | 54 |
Total | 230 | | 950 | 590 | 162 |
a Net piece preserved for chemical analysis will be obtained from adjacent positions on the same ITNs cut for bioassays. These net pieces would measure 30cmX30cm.
bThe hut trials will be performed only in Benin but will include nets from all study sites.
Chemical analysis
The net pieces preserved for chemical analysis at 0, 12, 24 and 36 months (590 pieces per net type) will be wrapped in Aluminum foil and stored at 40c (+/-2°c) and afterwards shipped for chemical content analysis to International Institute of Biotechnology and Toxicology (IIBAT), a GLP laboratory in India for detection of alphacypermethrin and PBO content using high performance liquid chromatography. Net pieces will be shipped within 3 months after collection.
WHO cone bioassays and tunnel tests
Cone bioassays and tunnel tests will be performed at research partner institutions in each study country (Centre de Recherche Entomologique de Cotonou in Benin, Centre for Research in Infectious Diseases, Cameroon, and Vector Control Training Centre in Tanzania). To monitor the bioefficacy of alphacypermethrin in DuraNet® Plus and DuraNet®, ITN pieces obtained from each field collected net will be tested in standard WHO cone bioassays. One hundred (100) unfed 3–5 days old mosquitoes of the susceptible Anopheles gambiae Kisumu strain will be exposed for 3 minutes to each ITN in replicates of 5 mosquitoes per cone and 2 cones per net piece. Knockdown will be recorded after 60 mins and mortality rates after 24hrs. Comparison will be made to a new unused net of each ITN type. ITNs which fail to achieve WHO efficacy criteria in cone bioassays (pooled knock-down is ≥ 95% and/or pooled 24hr mortality is ≥ 80%) will be subjected to tunnel tests. For tunnel tests, 100 unfed 5–8 days old susceptible Kisumu mosquitoes will be exposed overnight to one ITN piece from each failed net and mortality (after 24hrs) and blood-feeding inhibition rates measured following standard WHO protocols. The proportion of ITNs of each type passing criteria for alpha-cypermethrin bioefficacy in cone bioassays (≥ 95% knockdown or ≥ 80% mortality) and/or tunnel tests (≥ 80% mortality or ≥ 90% blood-feeding inhibition) will be determined for each study site. All cone bioassays will be performed at 27 ± 2°C and 75% ± 10%
To monitor the bioefficacy of PBO in DuraNet® Plus, tunnel tests will be performed using 1 net piece from ITNs withdrawn for bioassays at 12, 24 and 36 months using the pyrethroid resistant An. coluzzi Akron strain (BEI resources) for Benin and Cameroon and the pyrethroid resistant An gambiae ss Zenet strain in Tanzania. Both strains exhibit intense resistance to pyrethroids mediated by high kdr frequencies and overexpressed P450 enzymes evidenced by increased mortality with alpha-cypermethrin following PBO pre-exposure in susceptibility bioassays. They will be fully characterised prior to each round of laboratory bioassays. The proportion of DuraNet® Plus ITNs inducing ≥ 80% mortality of the pyrethroid resistant strain in tunnel tests will also be determined for each study site. Comparison will be made with a new unused DuraNet® Plus at each timepoint. All tunnel tests will run overnight between 18:00 and 09:00 at 27 ± 2°C and 75% ± 10% relative humidity.
Experimental hut trials
To further assess the entomological superiority of DuraNet® Plus over DuraNet®, experimental hut trials will be performed with field collected ITNs at 12-, 24- and 36-months post ITN distribution from each country at the Cove experimental hut station in Benin against wild free-flying pyrethroid-resistant Anopheles gambiae sl. Fifty-four (54) DuraNet® Plus and 54 DuraNet® nets from all three countries will be tested together in experimental huts at each annual timepoint. Comparison will be made with new unused nets of each type. The hut trials will consist of the following 9 treatments
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Untreated net – 6 replicates
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DuraNet® (new unused) – 6 replicates
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DuraNet® Plus (new unused) – 6 replicates
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DuraNet® Benin (12-, 24- or 36-months field net) – 54 replicates
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DuraNet® Cameroon (12-, 24- or 36-months field net) – 54 replicates
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DuraNet® Tanzania (12-, 24- or 36-months field net) – 54 replicates
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DuraNet® Plus Benin (12-, 24- or 36-months field net) – 54 replicates
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DuraNet® Plus Cameroon (12-, 24- or 36-months field net) – 54 replicates
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DuraNet® Plus Tanzania (12-, 24- or 36-months field net) – 54 replicates
Each trial will last 9 weeks and will involve 9 consenting human volunteers sleeping in huts from dusk to dawn 6 days of each week of the trial. Treatments will be rotated each week using a randomised Latin Square Design while sleepers will be rotated daily to control for hut position effect and sleeper attractiveness to mosquitoes respectively. On the 7th day of each week, huts will be cleaned and aired in preparation for the next rotation cycle. The trials will run for a total of 54 nights and replicate nets collected from the field will be rotated between huts on successive nights (one net per night). The efficacy of each ITN hut treatment at each timepoint will be measured in terms of the proportion of mosquitoes dead after 24 hours, proportion exiting into the veranda trap and the reduction in blood-feeding relative to the control untreated nets.
Data management and analysis
Household data collected during the census and ITN follow up surveys will be captured on electronic forms using smartphones installed with OpenDataKit (ODK) Collect in Benin and by paper forms in Cameroon and Tanzania. Data from the surveys at 12, 24, and 36 months will be used to calculate attrition, functional survival and median survival time using Kaplan–Meier estimators. For both attrition and functional survival, nets reported as given away, sold, or stolen will be excluded from the analysis. Negative binomial regression will be used to compare hole surface area between DuraNet® Plus and DuraNet®. Data on WHO bioassays and tunnel tests will be obtained from 30 nets of each product type sampled at each time point. A chi-squared test will be used to assess the proportion of nets of each ITN type passing the WHO criteria for alphacypermethrin and PBO bioefficacy based on combined cone and tunnel tests. Proportional outcomes (mortality, blood-feeding, exophily) from the experimental hut trials will be compared between DuraNet® Plus and DuraNet® at each time point using blocked logistic regression while numerical outcomes (entry) will be compared with negative binomial regression. A separate model will be fitted for each outcome and adjusted to account for variation between the huts, sleepers, weeks of the trial and the pHI category of ITNs. All analysis will be performed on STATA version 17.
Ethical considerations
Ethical approval for this study will be sought from the national ethics review committee of the Ministry of Health in Benin, Cameroon and Tanzania. Prior to any project activities, village and hamlet leaders will be invited to sensitization sessions conducted by district health officers and written informed consent will be sought from the local leader before starting data collection. Heads of households involved in the study will give informed consent prior to their participation in the study. The consent form will be written in French for Benin and Cameroon abd Swahili for Tanzania and will indicate the purpose of the study, the procedures, risks and benefits, that participation is completely voluntary, and that they may withdraw at any time. Where necessary, the consent form will be explained to them in their local language by a trained interpreter. Participants will be asked to sign the consent in duplicate, one will be kept by the project and they will keep the other. Where the individual is unable to read or write, their fingerprint will be taken, and a signature obtained from a witness to the informed consent procedure. All personal data will be anonymised prior to data processing. Written informed consent will also be obtained from all human volunteer sleepers for experimental hut trials prior to participation. Sleepers will be offered a free course of chemoprophylaxis spanning the duration of the study and 4 weeks following its completion to mitigate malaria infection risk. Approval for use of guinea pigs for tunnel tests in Benin has been obtained from the LSHTM Animal Welfare Ethics Review Board (Ref: 2020-01). In all study countries, guinea pig colonies will be maintained according to institutional standard operating procedures (SOPs) developed in line with relevant national and international regulations governing use of animals for scientific research purposes.