Study area and sample collection
At the inception of the Bioko Island Malaria Control Project (BIMCP) in 2004, island-wide indoor residual spraying (IRS) was the main bi-annual vector control activity undertaken on the Island [16]. Bed nets, as a major intervention community-level strategy, were introduced in late 2007 and early 2008 through a mass distribution campaign of LLINs (PermaNet 2.0®, Vestergaard-Frandsen) impregnated with deltamethrin. The distribution was carried out by the Equatorial Guinea National Malaria Control Program (NMCP), with the support of the BIMCP. As such, a total of 111,301 LLINs were distributed and installed through door-to-door direct hang-up by teams of community-based Equatoguinean volunteers, under the supervision of the Spanish Red Cross. From the beginning of the project, the BIMCP has conducted annual Malaria Indicators Surveys (MIS), within 18 sentinel sites on the Island, as part of its evaluation plan to measure the impact of the project's interventions. Hence, between February 25th and March 31st 2012, MIS data was used to re-visit households that indicated having LLINs during the survey to obtain LLIN samples (Fig. 1; map showing the sentinel sites) to investigate their insecticidal activity and insecticide concentration.
These sites were selected because of the entomological monitoring and vector control activities that were being conducted within them. Only households located within the 18 sentinel sites that had received LLINs from the BIMCP during the 2007/2008 mass distribution campaign, and through antenatal care (ANC) distributions for the next four years, were eligible for the sampling process. Using the project's household mapping system, households within the sentinel sites indicating ownership of LLINs during the 2011 MIS were randomly selected [17]. A total of 356 LLINs were collected in March 2012, from 235 randomly selected households, each time replacing collected nets with new ones. Additional information was recorded at the household level and on the LLINs, including the sentinel site in which the house belonged, unique house identification number, the duration of net usage (i.e., age), net manufacturer information, net expiry date, and batch number.
Following WHO guidelines, the physical integrity of the 356 PermaNet 2.0® LLINs collected were assessed at the BIMCP laboratory. A subset of 130 LLINs were selected to assess the physical integrity of the nets, the insecticidal activity and the insecticide content; these were divided into five groups according to the period of their use: Group 1, 0 - 11 months (n = 20); Group 2, 12 - 23 months (n = 24); Group 3, 24 - 35 months (n = 31); Group 4, 36 - 47 months (n = 28); and Group 5, 48 - 52 months (n = 27).
Analysis of the insecticide content and insecticidal activity of LLINs
The LLINs were divided into five sections (the 'roof,' two wide and two narrow sides), with three tests carried out per section of every net (replicates). The results of all the tests for the five sections were averaged to determine the insecticide content for the net as a whole. Deltamethrin content was assessed using the colorimetric Test Kit [15], quantified using high-performance liquid chromatography with diode array detection (HPLC-DAD), while the insecticidal activity was assessed using cone bioassays [10].
a) High-performance liquid chromatography determination of insecticide concentration
b) Colorimetric analysis determination of insecticide content
The ‘proof of principal’ colorimetric Test Kit used here comprises of three tubes containing Reagent A, B, and D, and a fourth tube C to carry out the test. Reagent A (25 mM potassium hydroxide in analytical grade ethanol) is added to B (para nitrobenzaldehyde 6.25 mM plus 2,3,5 triphenyltetrazolium chloride 20 mM in analytical grade ethanol) in tube C and shaken to mix the two reagents. Glacial acetic acid (10%) in analytical grade ethanol (solution D) was added to stop the reaction after 10 mins. The resultant depth of the pink colour produced gives an indication of the amount of deltamethrin detected in the test. Calibrating solutions containing known concentrations of deltamethrin (between 0 and 100 mg/mL) were used for the colorimetric Test Kit to visually determine the amount of deltamethrin detected. Neat ethanol was used as a control blank (no colour). A concentration of <15 mg/m2 was advised by the LSHTM in house vector control experts (Rowland R and Lines J, pers. Comm.) as the cut off value for failed nets versus nets that pass (i.e., will be effective against the mosquitoes).
This Test Kit was applied to two subsets of randomly selected LLINs, one set on Bioko Island and the second set at the LSHTM. The test is based upon the detection of cyanide ions released from type II pyrethroids and can be performed in situ. On Bioko Island, the insecticide content of 20 LLINs was tested in situ, i.e., while hanging in place at a household (Fig. 2), before collection and replacement with new nets. The colorimetric test was applied to all five sections for each LLIN, from which the insecticide content for the net as a whole was calculated.
The reliability of the colour produced in the colorimetric Test Kit [15] was assessed at LSHTM. From the 130 LLINs collected from households on Bioko Island, a subset of 20 LLINs were randomly selected across the observed range of deltamethrin concentrations. The colorimetric test was performed on each section of the LLINs following the same protocol used on the Bioko Island in situ tests, as per the instructions on the kit. After 10 minutes, the colour reaction was stopped and recorded using a digital camera (Pentax™, Optio S7). The colour produced was quantified by measuring the red, green, and blue (RGB) colour components using the digital picture-processing program Photoshop [19]. The RGB values (0 to 255) were combined, and log-transformed to determine the colour value. The colour value was then compared to the test sample's corresponding deltamethrin concentration, determined using HPLC-DAD [18]. A similar approach has been used in other colour reaction analyses [20].
c) Bioassay determination of the insecticidal activity
The bio-efficacy of the LLINs was evaluated for a subset of 50 LLINs from the 130 LLINs, representing 38% of the analysed nets. The residual insecticide activity was assessed at the BIMCP laboratory using the WHO cone tests [21] with susceptible Anopheles mosquitoes reared at the laboratory. Three to five-day old unfed female mosquitoes were used for the tests. WHO cones were used to expose the mosquitoes to the net samples (25cm x 25cm) from four of the five sections of the net; the front was excluded per WHO guidelines. Mosquito mortality was recorded 24 hours after exposure. A negative control with an untreated net sample was used, where observed mortalities were within the 5-20% range, and mortalities were adjusted using Abbott's formula [21]. The insectary had a temperature-controlled environment between 26 ± 2°C and humidity between 75% and 90%.