Study Site
The field evaluation of the LLINs was conducted in the East African style experimental huts, located at Pasua (S03˚22.764’ E03˚720.793’), in Lower Moshi adjacent to Lower Moshi rice irrigation scheme (Figure 1). The irrigation scheme is getting water from the Rau river catchment, providing reliable breeding site for local malaria vectors, Anopheles arabiensis. The An. arabiensis in this area has moderate pyrethroid resistance due to elevated levels of both mixed function oxidases and β-esterases [12,13].
The GLP procedures
This study adhered to the following procedures to ensure GLP compliance, summarized in the study flow Figure 2:
Pre-study meetings
Before study initiation, a series of communication meetings with the sponsor were conducted to ensure; contract agreements (evaluation terms) are approved and signed, agreement on shipping of test items, type of reference nets, documentations (Safety Data Sheet, Certificate of Analysis), agreement to submit study plan to WHO PQT-VC for review, evaluation in accordance to shared Standard Operating Procedures (SOPs), WHOPES guidelines and GLP standards, confirmation of regeneration time for the test items, identification of GLP certified test sites for chemical analysis, key information for the study plan, timely submission of the study plan draft to sponsor and review of the study at testing site.
Parallel to this, a meeting was held at the Test Facility by the Test Facility Manager to appoint a responsible Study Director (SD)for this study. The Test Facility Management ensured that the facility had sufficient space, infrastructure, man power and materials to accommodate the study.
Identifying subcontractor for HPLC analysis
The Test Facility (TF) identified and shared with the sponsor (manufacturer); names, quotes and address of the two identified GLP compliant test sites for conducting the High-performance liquid chromatography (HPLC) for chemical analysis. One test site, BioGenius GmbH (Germany) was chosen for doing chemical analysis, based on the availability of Test Site (TS) to do the work that fits our agreed timeline, GLP certification, and cost of the quote.
Contracts
The contracts were established and signed covering: definitions, terms, performance of evaluation and reporting, confidentiality of test items, intellectual property rights, publication, payments, liability, force majeure, miscellaneous and sections for signing both parties. A similar but separate contract was also signed between Test Facility and the Test site. Along with the contract, the budget and quotes were submitted to the sponsor.
Study plan and Implementation plan
Standard format for study plan [14,15] was used with considerations to the OECD GLP guidelines [16] to establish a study plan that describe the aim, test items, test systems, experimental design and statistical analyses for the study. The established study plan was used to inform all participants on the study requirements and the timing of events to ensure that these are fulfilled to the mutual satisfaction of all parties. The study plan was also shared with the WHO PQT-VC for review and recommendations. Implementation plan was established to indicate a cascade of events in a chronological order to meet study objectives. Both implementatin and study plans were prepared by the TF and shared with sponsor and TS forreviewand agreement before actual implementation.
Study opening and the master schedule
Once all contracts, study plans and quotes were reviewed (with inputs from WHO PQT-VC, sponsor and test site) and signed,a study specific folder was opened to document all descriptive and prescriptive information related to the study, and a study was given a unique code. All the data collection forms and other forms related to this study were using this unique code.
During monthly master schedule meetings, the study was introduced to the senior management for record in the ‘’master schedule’’, which is a meeting to record the phases and dates on which various studies pass through a series of phases from planning until archiving [17] (Figure 3). In this meeting, study director, Test Facility Management, project manager and data manager. At the first master schedule meeting, number and type of forms were communicated to data manager to establish a necessary database for data entry.
Study initiation and experimental phases
Once the WHO PQT-VC recommendations were incorporated and considered in study plan, the study plan was signed by study director, Test Facility Manager, Test site Manager, TF Quality Assurance (QA) Manager and the TS QA Manager. A kick-off meeting was conducted at the TF, involving all the study participants. In that meeting, Study Director (SD) introduced the study, test items, test systems and the overview of the relationship between the TF and TS and the study plan was explained in detail. Experimental date was announced, all data collection forms that were to be used, relevant SOPs for this study, Safety Data Sheets (SDS) were discussed.
Study risk assessment and mitigation
Study plan was submitted to the Health and Safety officer to identify any potential risk associated with conducting the study and to recommend measures to be taken in order to minimise health risks and to guide study personnel in the study procedures. In Tanzania,the risk assessment is a requirement by law [18].
Quality assurance auditing for study plan and identification of critical phases
To ensure that, the procedures being conducted were of quality, and the results are trusted, the lead Quality Assurance (QA) Manager from the TF and the QA Manager from the TS were involved for the study plan auditing. The identified non-conformances were corrected by the Study Director and the Project Manager. Furthermore, the Quality Assurance Manager, Project Manager and Study Director met and discussed the critical phases that would be most appropriate for QA audits. It was agreed that the net washing, should be first to be audited since it impacts directly on the test items. The 10th wash was chosen for auditing because there would be sufficient records as well as observing the practical aspect of washing according to SOPs. Another element of the experimental phase that was audited was cone bioassays because they directly assess test items. The aspects of hut trials were agreed for auditing include, rotation of treatments, collection of mosquitoes and scoring of primary outcome measures. The critical phases were identified by Test Site QA and Principal Investigator and was included in the audit report. At the TS, it was agreed that the experimental determination of alpha-cypermethrin content in long-lasting insecticidal must be audited.
Data management, Software validation, and accuracy checks
The study director discussed with the data management team the forms to be used in this study for net cone bioassays, mosquito collection, test system biometrics, bottle bioassays and mosquito packing. The data entry procedure, databases preparation, computers and software validation SOPs, comparison and accuracy check plans for the study were reviewed and discussed.
Record of procedures
In this GLP study, the record of procedures, a form that keeps track of specific actions that involve the test items in the experimental procedures was used throughout the study duration (May 2019 to December 2019). Recorded experimental procedures include: cutting net pieces, washing nets, cutting holes on nets, collection of test systems during hut trial, scoring of immediate and delayed mortality, packing of test systems, cutting of net pieces for bioassays, conduct of bioassays, scoring of bioassay outcome measures, shipment of test items and archiving of test items. In the record of procedures information on equipment used, test items, test systems, technician initials and date were recorded. Sequence of events, equipment used and staff qualification involved in the experimental phase were among essential records that were used by our external auditor, South African National Accreditation System (SANAS).
Final report
The final report was written and signed by SD, lead QA, TFM and sponsor.
Archiving
At the end of the study, the original study folder, containing; the study plan, schedule of activities, correspondence with sponsors, amendments & deviations, records of procedures, the raw data, ancillary study data, analysis print-outs and final reports, was archived at the test facility for a period of five years in accordance with internal SOPs after agreement with the sponsor. Also, one unwashed unused net of each treatment was retained in the Test Facility and archived for one year.
All used or expired test items were disposed with the sponsor’s consent at the conclusion of the study. The dried carcasses of the test systems analysed in the molecular laboratory are being archived for a period of five years in accordance with internal SOPs.
Waste disposal
All waste generated in the course of the study were disposed in accordance to the OECD GLP document number 19 [19], the Test Facility waste disposal SOPs and recommendations from the National Environmental Management Council of Tanzania (NEMC).
Experimental evaluation of SafeNet® and SafeNet NF® LLIN
Test items
The test items for the current study were SafeNet®and the ‘SafeNet NF®’ LLINs; Piperonyl butoxide (PBO) and alphacypermethrin (ACM). The LLINs are polyester nets coated with alphacypermethrin. The difference between the two candidate nets is the physical characteristics, with SafeNet NF® having wider mesh, lower grams per square meter (GSM), higher denier, lower bursting strength, Table 1.
Technical grade insecticides for supplementary bioassays
The working solutions of Piperonyl butoxide (PBO, batch number 8615500) and alphacypermethrin (ACM, batch numbers 5823400 and 8592500) were prepared from technical grade received from a commercial supplier (Chem Service Inc. West Chester PA, USA). The purity of the first sample was reported as 98.4%±0.5%, and the second sample as 99.5%±0.5%, by an ISO-certified testing facility.
The purity of this test item was reported as 98.3% ±0.5% as indicated on the certificate of analysis by an ISO-certified testing facility. The certificates of analysis were considered sufficient verification of integrity and quality of the technical grade insecticides.
Characterization of test systems
Bottle bioassays, biometric tests, and molecular assays were conducted to characterize mosquitoes that were used for the experimental hut trial and laboratory bioassays.
Test systems for experimental hut trial: The wild population of An. arabiensis in Lower Moshi were caught at larvae stage and reared to adults for use in bottle bioassays. In total, 300 female unfed 2-5day old adult mosquitoes were tested against alphacypermethrin with the 1x and 5x diagnostic dose bottle bioassays as per CDC guidelines [20], and 150 in the pre-exposure bottle bioassay (including 50 for the controls).
Test systems for cone bioassays: An. gambiae s.s. Kisumu, a fully-susceptible strain was used. Unfed 2-5d old adult females from the insectary were characterised in terms of body weight, wing length, resistance status (phenotypic and genotypic) and species identification, during the experimental phase of the study.
A total of 88 An.gambiae s.s. (Kisumu strain) mosquitoes were tested for species identification and kdr E genotype using quantitative real time qPCR technique. DNA was extracted from the Anopheles spp using the modified chelex extraction method by Walsh [27]. Identification of the members of the An.gambiae s.l. species complex was performed using the Taqman 3-plex assay of Bass et al [22]. Detection of kdr mutations was performed using the Taqman assay method [21]. A separate sample of 100 An. gambiae s.s. Kisumu were used for the biometric characterisation of the colony following the modified methods by Yeap [23] and Nasci [24].
Washing and preparation of LLINs for field trial
Whole nets were washed in accordance with WHOPES guidelines [25]. In brief, each net was washed in Savon de Marseilles soap solution for 10 minutes: 3 minutes stirring, 4 minutes soaking, then another 3 minutes stirring. This was followed by 2 rinse cycles of the same duration with tap water only. The mean water pH was 6 for all washes. The mean water hardness was 50.4 parts per million (ppm) and always within the WHOPES limit of ≤ 89 ppm. Seven nets of each treatment (Table 1) were washed for use in the hut study, and one additional net was washed and retained/‘held back’ (HB), which is not used in the hut study (Table 2). All nets used in the experimental hut study had 30 holes (4x4 cm) cut in them to simulate the conditions of a torn used net.
Five pieces 30x30 cm were cut from nets held back (one net for each treatment arm) before and after they were washed, 10 pieces in total per net (HB0-HB9). At the conclusion of the hut trial, hut used (HU) nets were returned to the Test Facility and five pieces (HU1-HU5) were cut from one net from each treatment arm, chosen randomly. In all instances, pieces were cut from pre-defined positions on the nets. Pieces were wrapped in aluminium foil, and kept in a fridge in test room 2 at 5±3°C until needed for assays.
Preparation of bottle bioassay working solutions
Four bottles of ACM at 12.5 µg/mL were prepared for testing in a single test, and four bottles of ACM at 60 µg/mL for a separate assay test. The bottles were coated evenly following CDC Bottle Bioassay guideline [16]. Four additional Wheaton bottles were coated with 1mL acetone only; these were used as the negative controls. The PBO bottles were prepared in the same way, from which a dilution in acetone to 25 µg/mL was prepared. One mL of this dilution was used to coat each of 3 Wheaton bottles.
All stock and working solutions were used within 24 h of preparation. Stock solutions were diluted immediately to create the working solutions, which were immediately used to coat the bottles. Likewise, once treated the bottles were used within 5 days.
Running the experimental hut trial
The experimental hut study was conducted from June 2019 to August 2019 at Pasua Field Station in seven huts, with treatment arms as indicated in Table 2.
In brief, treatments and baits ( cows used to attract mosquitoes into experimental huts) were randomized using a 7X7 Latin square design (https://www.dcode.fr/latin-square; accessed 20 May 2018). The treatments were rotated weekly, with daily rotation of candidate nets of the same treatements, while the cows were rotated daily. Cows were kept inside the experimental huts for 12 hrs (from 6:30pm to 6:30am), and collection of mosquitoes was conducted in the morning. Mosquitoes were collected daily from the window exit traps, verandahs, room and inside the net from each of the 7 huts. Both live and dead mosquitoes were collected in separate cups for live, blood fed, unfed, dead and their point of location recorded. All mosquitoes caught alive were kept in paper cups provided with glucose solution 10%. Mortality was scored 24 h later.
Supplementary cone bioassays
Cone bioassays for pieces cut from hut trial’s used and unused net were conducted in accordance to the standard WHOPES guidelines [26]. In brief, 2 replicates for each of the 5 pieces per net were run with 5 mosquitoes per cone, making a total of 50 mosquitoes for each net representing each treatment arm. Negative control pieces were tested alongside the test items and reference item. Any replicates for which control mortality exceeded 10% at 24 h were not analysed, and further replicates were carried out to replace those that were excluded. For all bioassays, there was a minimum of one hour holding period pre-exposure and a 3-minute exposure time. After exposure, mosquitoes were released into holding cups and provided with 10% glucose solution, 60 minutes knockdown and 24h mortality were recorded.
Supplementary bottle bioassays
The CDC Bottle Bioassay guideline [20] was followed for both a) the ACM-only biaossays and b) the PBO pre-exposure bottle bioassays. In brief, in the ACM-only assays mosquitoes were exposed directly to ACM 1x or 5x (4 bottle of each concentration, 25±3 mosquitoes/bottle). In the PBO pre-exposure assays mosquitoes were exposed to acetone and PBO (25 µg/mL) for 1 hour, then held in cages for 1 hour before exposure to ACM 1x (4 bottles, 25±3 mosquitoes/bottle) and acetone (2 bottles, 25±3 mosquitoes/bottle).
During the 30 min exposure to ACM or acetone, knockdown was recorded at 0 minutes and then every 5 minutes until 30 minutes. After 30 minutes, mosquitoes were removed from the bottles, transferred back into the holding cups, and provided with glucose solution 10%. At 24h post-exposure mosquitoes were scored as dead or alive.
Data analysis
The number of mosquitoes per bottle and cones were within the acceptable range of 25±3 and 5±1 mosquito respectively. The control mortality for cones and bottle bioassays were all less than 10%, therefore mortality in the insecticide treatment groups were not adjusted.
The 95% confidence intervals for proportionate data were calculated from the proportion of observations of interest (number knockdown/number dead), the total observed, assuming α = 0.05, using the following formula:
Double entry, comparison checks and accuracy checks on all the datasets were carried out in MS Access 2016. All datasets were transferred into Stata I/C v11.0 (StataCorp LLC, Texas USA) statistical software using Stat Transfer v8.0. Graphs were created in MS Excel 2016.
The data for the free-flying mosquito collections in the hut trial were analysed using logistic regression for grouped data with odds ratio output, adjusting for effects of hut and sleeper. Statistics were run using Stata/IC version 11.1 (StataCorp LP College Station TX77845, USA). The main outcomes from the experimental huts were: deterrence, blood-feeding inhibition, and mortality. The 2 sample t-test was used to compare the mortality data between candidate net and reference nets at both 0 and 20 times washes for the pieces derived from hut used nets.