Giant-scale (Section III) analysis of broflanilide 50WP (VECTRON™ T500) for indoor residual spraying for malaria vector management in Northeast Tanzania: research protocol for a two-arm, non-inferiority, cluster-randomised neighborhood trial | BMC Infectious Ailments

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Study area and cluster selection

This study will be carried out in Muheza District, Tanga Region, Northeast Tanzania (Fig. 1). The population of Muheza District consists primarily of subsistence farmers [24]. The region has a tropical climate with a long rainy season from March to June and a short rainy season from October to December. Most houses within Muheza District are constructed from mud or cement with a thatched or iron roof. In 2012, surveys conducted in Muheza District found that 83% of households owned at least one ITN (insecticide-treated net), and there have been two LLIN distribution campaigns carried out since then [25]. Malaria transmission occurs all year round with two peaks in transmission following the two rainy seasons. The main malaria vectors in Muheza District are An. gambiae s.s., An. arabiensis and An. funestus s.s. [25]. Wild An. gambiae s.l. from Muheza District are resistant to pyrethroids [26].

Fig. 1

Study area A location of Muheza District in Northeast Tanzania; B proposed clusters in Muheza District. The maps of Tanzania and Muheza district were produced using QGIS 3.22 software [44] and overlaid on Google Earth satellite images by the study investigator (NJM). Using QGIS 3.22 software [44], a one kilometre transect from the mid-point to the perimeter of each village was digitized to produce the shape files for the clusters in the Muheza map

Study design

A matched cluster randomized controlled non-inferiority study design will be used as IRS in community-level intervention. The study monitoring period will be for 12 months post-intervention or beyond this date if residual efficacy on house walls indicates continuing effectiveness. Baseline data collection on vector densities will commence in the six months preceding the randomisation and pair matching of the clusters. At the onset of the long rainy season, one round of IRS will be done which will be followed by monthly vector density monitoring and in-situ cone bioassays. Baseline household enumeration and social-cultural and acceptability measurements will be conducted before IRS.

Power calculation, estimation of effect size and cluster selection

Sample size calculations will be based on the study primary endpoint i.e., indoor density of Anopheles mosquitoes. An estimated mean of 3.0 female Anopheles per light trap per night will be used, referencing data from recent studies in Tanzania [27]. A paired design will be used in which clusters will be matched on baseline indoor density of Anopheles mosquitoes. Previous data suggests a within-pair coefficient of variation of 0.23, but a conservative value of 0.3 will be used in our calculations.

The trial is a non-inferiority design, intended to show that spraying the intervention product, VECTRON™ T500, will not result in higher malaria vector densities, by a prespecified non-inferiority margin of 50%, than those per trap per night in the reference arm sprayed with Fludora® Fusion. Using the methods described in Hayes and Moulton [28] and the assumptions listed above, it was calculated that 8 clusters per treatment arm (16 in total) will be needed for the 50% margin to have 80% power to demonstrate non-inferiority. The calculations were based on 49 trap nights per cluster. There was very limited statistical advantage in increasing trap-nights beyond 50. This number of clusters considers the loss of degrees of freedom caused by matching.

Sixteen clusters, containing 75–200 households per cluster (HHs), will be selected for spraying. Core areas of clusters should be at least 1 km apart to prevent infiltration of mosquitoes from other villages. All consenting HHs in all 16 clusters will be sprayed. Approximately 1200–3200 households will be sprayed (~ 75–200 HHs/cluster in 16 clusters).

Although a minimum of 49 households is required per cluster according to the sample size calculations, a larger hamlet will be recruited for logistical reasons, as a proportion of households may refuse to accept the IRS spraying and others may be absent during spraying or lost during follow-up.

During baseline vector density monitoring, 20 clusters will be included, to improve the pair-matching post-intervention. The four clusters with greatest variance will not be included in the post-intervention monitoring but will still receive the IRS treatment. The 16 selected clusters will be pair-matched based on vector densities and randomly allocated to the two trial arms: one arm (8 clusters) will be sprayed with the novel intervention (VECTRON™ T500) and the other arm (8 clusters), the reference product (Fludora® Fusion). All possible randomisations will be stratified on ITN coverage and wall surface to ensure minimum difference of 15% or less in mean ITN coverage and mean ratio of cement houses to mud houses per arm. From all randomisations that meet these criteria, one will be selected at random.

Sensitisation, community and household consent

A meeting with district authorities (District Medical Officer, District Executive Director and District Commissioner) will be held at the beginning of the project to present the objectives and research activities. Meetings with community representatives and hamlet leaders will encourage adherence of inhabitants.

Community consent will be obtained from village leaders, individual verbal and written consent will be obtained from family heads and household members. All households within each community will be offered IRS.

The risks and benefits of participation will be made known and the opportunity to withdraw will be explained without loss of other benefits. Individuals already using LLINs will be encouraged to continue usage. Informed consent will be read, and consent forms signed and dated by participants. One copy of the form will be given to the household to keep for personal records, and one copy will be kept in the project files. If a participant is unable to sign the consent form, a fingerprint will be taken.

When the information is read to the family, they will be given a consent form, translated into Swahili, to read before signing. The form has been designed to give full disclosure in a format that is easy to comprehend. The language in the form is simple and aimed at a primary education level to ensure every member of the target group will be able to understand it. The field worker will be trained to ask whether the household understands all information in the form, or whether any sections need clarification.

Enumeration of consenting households

Mapping/census of the study area will be conducted in the baseline period to update maps and add new households to the database. The census will record (a) the Global Positioning System (GPS) coordinates; (b) head of household; (c) number of occupants (adults and children); (d) number of bedrooms and total number of rooms; (e) number of sleeping places; (f) number and type of LLINs; (g) predominant wall material; and (h) predominant roof material.

Every building within each hamlet will be mapped using a GPS handheld unit (Garmin Legend e-trex) and ExpertGPS v3.8 (TopoGrafix) software. Cluster boundaries, health facilities and other landmarks will also be geo-located. Each house will be assigned a unique 8-digit household identification (ID) consisting of 3-digit village code, 2-digit hamlet code and 3-digit house code.

Indoor residual spraying and quality control

Consenting households HHs in the intervention arm will be sprayed with VECTRON™ T500 (100 mg of AI/m2), those in the reference arm will receive IRS with Fludora® Fusion (200 mg/m2). Prospective spray operators will undergo a medical examination; those with underlying health problems will be excluded. Pregnant women will also be excluded. Spray operators recruited from the clusters will be trained according to WHO guidelines. A total of 100 spray operators will be recruited to conduct the IRS campaign, 5 from each cluster. All sleeping and living rooms in consenting houses will be sprayed, whilst kitchens and storerooms will be excluded. All walls will be sprayed and ceilings will also be sprayed, if accessible. Furniture will be removed from houses and inhabitants will be asked to remain outside during spraying and for 2–3 h afterwards.

Spray operators will be provided with personal protective equipment and clothing and health checks will be performed on them before and after spraying. If operators experience any adverse events, they will receive free medical care and the effects will be documented.

Chemical analysis of the insecticide dosages applied to the walls will be carried out as a quality control of the IRS application process. Whatman® No. 1 filter papers will be attached to the walls of HHs before spraying and then removed post-spray. Four filter papers will be used per HH on different walls, in 20 randomly selected HHs per cluster.

Intervention monitoring

Vector density

CDC light traps hung inside houses next to occupied LLINs will be used to collect mosquitoes for species identification, sporozoite ELISA, and characterization of DNA-based resistance mechanisms. Four operatives will be allocated with 5 light traps each and given responsibility for trapping in 4 of the 16 clusters. In each cluster in each month there will be 3 rounds of light trap collections spaced over 3 weeks in 15 randomly selected HHs. Therefore, in each treatment arm of 8 clusters there will be 120 light trap nights in each month. All households in each cluster will be re-randomised at each monthly surveillance point. Therefore, in each cluster there will be 180 light-trap nights spread across the 12-month post intervention period, from which non-inferiority will be determined. All mosquitoes collected will be identified morphologically to genus and species complex level (An. gambiae s.l., An. funestus s.l. and Culex spp.). Sub-samples taken from each cluster each month will be identified to species level using molecular diagnostic assays; numbers and proportions identified will depend on the total numbers caught.

Phenotypic insecticide resistance testing

WHO bottle bioassays with 1 h exposure [29] will be used to determine phenotypic resistance of wild Anopheles mosquitoes to broflanilide (6 µg/bottle), deltamethrin (12.5 µg/bottle) and clothianidin (90 µg/bottle). Drawing on CDC methods regarding the coating of Wheaton bottles [30], the above-mentioned diagnostic concentrations of these insecticides have been shown to kill 100% of insecticide susceptible mosquitoes. Broflanilide will be coated together with MERO 800 ppm/bottle. Insecticide solutions will be prepared from technical grade material dissolved in acetone.

An. gambiae s.l. and An. funestus s.l. will be collected as larvae from breeding sites within the study area and reared in the insectary. Progeny of these wild An. gambiae s.l. and An. funestus s.l. will then be exposed to insecticide according to standard operating procedures [29]. Knockdown will be recorded for all insecticides after 1 h and mortality will be recorded after 24 h for deltamethrin, and 72 h for broflanilide and clothianidin. Assays will be conducted at three timepoints: before IRS, at 3 months post-spraying, and at the end of the field study (12 months post-spraying) to detect whether there have been any changes in insecticide susceptibility in wild Anopheles populations resulting from exposure to the IRS. WHO bioassays will also be performed with An. gambiae s.s. Kisumu (pyrethroid-susceptible strain) and An. gambiae s.s. Zeneti or Muleba-Kis (pyrethroid-resistant strains) colonies for comparison and quality control.

Genotypic insecticide resistance monitoring

For individual mosquitoes, morphologically identified as An. gambiae s.l. [31], DNA will be extracted from legs/wings and used for species identification and detection of L1014S-kdr, L1014F-kdr and G119S-ace1 [17, 32, 33]. Sporozoite detection (CSP-ELISA) will be performed on a subset of PCR-confirmed An. gambiae s.s. and An. arabiensis [34]. Separate pools of 5 individuals, PCR-confirmed as either An. gambiae s.s. or An. arabiensis, will be prepared and RNA extracted. cDNA will be synthesized and metabolic gene expression measured relative to susceptible An. gambiae s.s. and An. arabiensis colony strains; CYP6M2, CYP6P3, CYP6P4, CYP66Z3, CYP9J5 and CYP9K1 will be measured in An. gambiae s.s. and CYP6AA1, CYP6AA2, CYP6P15P, CYP6P1, CYP6AD1, COE_4679, CYP6P3, CYP6P4 and 15,786 (ester hydrolase) in An. arabiensis [35].

For individual mosquitoes, morphologically identified as An. funestus s.l. [31], DNA will be extracted from legs/wings and used for species identification and detection of L119F-GSTe2, CYP6P9a and CYP6P9b. Sporozoite detection (CSP-ELISA) will be performed on a subset of PCR-confirmed An. funestus s.s. Separate pools of 5 individuals, PCR-confirmed as An. funestus s.s., will be prepared and RNA extracted [31, 36,37,38,39]. cDNA will be synthesized and metabolic gene expression measured relative to a susceptible An. funestus s.s. colony strain; CYP6N1, CYP6M7, CYP6M1 and CYP6Z3 will be measured [36].

IRS residual activity

WHO cone bioassays on IRS treated walls of houses will be used to assess residual activity in both trial arms at monthly intervals until the end of the trial 12 month post-IRS intervention [40]. Plastic WHO cones will be fixed to four walls per house placed in a diagonal arrangement across the room, and one additional cone will be fixed to an outside untreated wall to act as a control. Of the four selected households per cluster, two will be constructed out of cement and the other two out of mud, to compare substrate materials. To determine IRS product efficacy both pyrethroid susceptible and pyrethroid resistant strains will be used. In each cone test, 10 An. gambiae s.s. mosquitoes [40] will be aspirated into each cone and exposed for 30 min. After exposure, mosquitoes will be held in paper cups with cotton wool soaked in 10% glucose solution. Immediate knockdown and delayed mortality at 24, 48 and 72-h will be scored. Susceptible and resistant mosquitoes will be tested in the same room on alternate days according to availability. The pyrethroid resistant An. gambiae s.s. Zeneti strain possesses the L1014S-kdr mutation and elevated oxidases levels conferring metabolic resistance. If wild An. gambiae s.s. are in short supply, adult F1 of An. funestus s.l. or An. gambiae s.s. Muleba-kis will be laboratory-reared for monthly residual bioassays. With 4 test houses per cluster, and 4 cone tests per house, up to 160 susceptible and 160 resistant mosquitoes will be tested per cluster per month.

Operational safety and community acceptance

Safety

The health status of spray men will be examined before and after spraying by qualified medical staff. Spray men will be asked to complete a questionnaire immediately after spraying has finished and then one month later to determine what, if any, adverse events they may have experienced and at what frequency. Similar questionnaires on adverse events will also be distributed in the community after spraying. Study participants will also be encouraged to contact members of the study team if they experience health issues that they believe are related to the IRS.

An independent IRS human safety assessment for VECTRON™ T500 will be conducted by Environmental Resources Management (ERM) according to the terms of the ‘WHO Generic risk assessment model for indoor residual spraying of insecticides’ [41]. According to the risk assessment report, the insecticide is non-mutagenic and non-genotoxic, shows low levels of mammalian toxicity, and at the application rate proposed (100 mg/m2) poses acceptable risk of exposure to professional spray operators and to adult residents sleeping in treated houses; this being about 30-fold and 100-fold lower than the acceptable daily intake, respectively, using the WHO model [42].

Acceptability

Questionnaires on the ease of insecticide application will be given to all spray operators while questionnaires on community acceptability will be given to a random sample of two clusters per study arm immediately post-spraying and then repeated with the same sub-sample after 6 months.

Focus group discussions (FGDs) will be carried out with a subsample of community participants (8 participants in each FGD) in two randomly selected clusters in each arm. These will be performed six months post-intervention. Within these discussions, factors such as perceived adverse events, benefits, supplementary usage of nets and other means of mosquito protection will be explored and discussed.

Data analysis

Data analysis will be carried out using NVivo software for qualitative data and Stata (Version 16) for quantitative data. For the FGD data, emerging themes will be assessed using the NVivo software. Count and binary data will be analysed using Generalised Linear Models. Data analysis will be carried out using cluster level summaries since random effects models perform poorly in a matched design with fewer than 20 clusters per arm [28]. For the primary endpoint of mosquito density, the ratio of densities in each matched pair will be calculated and inference will be based on a paired t-test. Adjustment will be made for time post-spraying, LLIN use and wall type using the two-stage method described by Hayes and Moulton [28]. The secondary outcomes of EIR, sporozoite rate, estimated number of infective bites per person per year, and residual bioefficacy will be analysed in the same way.

Data management

Household data collected during the census and during entomological data monthly surveillance will be captured in electronic forms on smartphones installed with Open Data Kit (ODK) collect. The data will be stored on a secure server located at PAMVERC (Pan African malaria vector research consortium) at NIMR/KCMC/LSHTM. All data management and analyses will be done using Stata software.

Standard operating procedures (SOP) for data collection will be developed and field study staff trained. QC will be conducted by a supervisor who will monitor field staff for completeness and internal consistency of responses. Data collected on paper forms (mosquito identification, insecticide resistance results) will be double entered by two data clerks.

All data will be uploaded into a secure server. Data will be stored encrypted and access to the data will be restricted only to authorised study investigators and data management staff. A unique identifier number will be given to each household to safeguard confidentiality.

Upon completion of the study, electronic files will be stored on a server and also copied to encrypted USB. Electronic data and paper source records will be retained for a minimum of 10 years.

Oversight and monitoring

A Trial Steering Committee (TSC) will be established to provide study oversight. The TSC members will be independent of the trial and its institutions and have the necessary expertise to monitor study progress and participant safety. Although the risk to study participants is considered minimal from either of the study IRS products, their safety experiences will be documented.

Ethics, informed content and dissemination

This study has been approved by the ethical review committee of the Ministry of Health in Tanzania (NIMR/HQ/R.8a/VOL.IX/3520), the institutional review board of LSHTM (N° 22,459). The study is registered on clinicaltrials.gov (NCT05150808, registered on 26 November 2021). The study will be conducted according to the Declaration of Helsinki and the International Guidelines for Ethical Review of Epidemiological Studies. The protocol of the study has been reviewed by the independent Expert Scientific Advisory Committee of the IVCC and by the WHO Prequalification Vector Control Team (WHO PQT/VCT).

Before any project activities, villages and hamlet leaders and local health staff will be invited to sensitisation sessions. Community health workers within each cluster will be fully informed as to the aims of the study and will be on hand to answer day-to-day questions. The consent form will be in Swahili and indicate the purpose of the study, procedures, risks, and benefits of the intervention. All participation is voluntary.

Findings of the study will be shared with international policymakers such as WHO PQT/VCT and WHO Vector Control Advisory Group (VCAG). The project will involve the engagement of national, local, and community authorities and leaders. The findings will be published in peer reviewed journals and shared with the product manufacturer, project funders and presented at the annual meeting of the WHO Roll Back Malaria Vector Control Working Group.

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