WHO cone bioassay boards with or without holes: Relevance for bioassay outcomes in long-lasting insecticidal net studies

doi:10.21203/rs.3.rs-1651515/v1

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WHO cone bioassay boards with or without holes: Relevance for bioassay outcomes in long-lasting insecticidal net studies

https://doi.org/10.21203/rs.3.rs-1651515/v1

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Background: The World Health Organization (WHO) cone bioassay is a key method used to evaluate the bioefficacy of long-lasting insecticidal nets (LLINs) used for vector control. These tests also play an important role in LLIN product prequalification and longitudinal monitoring. Standardization of these assays is therefore important. While many parameters for WHO cone bioassays are defined in the respective WHO guidelines, others are not. One of these undefined parameters is the exact configuration of the bioassay boards. In cone bioassays, LLIN samples are pinned onto a bioassay board for testing. Anecdotal evidence suggests that bioassay boards with holes behind the LLIN samples lead to greater exposure to insecticide, as the mosquitoes are ‘forced to stand on the net material’. This may increase the key assay outcomes of 60 min knockdown (KD60) and 24h mortality (M24). Here, we test this hypothesis in two facilities using two fully susceptible mosquito strains.

Methods: WHO cone bioassays were performed using bioassay boards with holes and boards without holes in parallel following WHO guidelines. Five brands of LLINs with four new and unwashed samples per brand were used (total n=20). Knock-down (KD) was recorded in 10 min intervals within the first hour after exposure and mortality was recorded at 24 h. Assays with Anopheles farauti were done at the Papua New Guinea Institute of Medical Research (PNGIMR) and assays with Aedes aegypti were done at James Cook University, Australia.

Results: Results varied not only with bioassay board configuration but also with mosquito strain. In particular, with An. farauti we observed significantly higher M24 when boards with holes were used, while this was not observed with Ae. aegypti. WHO cone bioassay results were systematically biased between the two facilities such that the use of An. farauti at PNGIMR predicted higher KD60 and M24.

Conclusion: The present study highlights the need for further harmonization of WHO cone bioassay methodology. Parameters such as bioassay board configuration and mosquito species systematically affect the observations, which impedes generalizability of WHO cone bioassay outcomes.

Cone bioassay

long lasting insecticidal nets

LLIN

pyrethroid

bioassay board

holes

mosquito

Anopheles

Aedes

Long lasting insecticidal nets (LLIN) are a key vector control tool for the prevention of malaria in endemic countries. They are estimated to have saved millions of lives [1, 2]. LLIN procurement volumes have continuously increased since widespread mass distributions began in the early 2000s. Annual LLIN deliveries are now approaching 300 million pieces, while the cost per net has more than halved in the last 10 years [3]. The increasing demand for cheap LLINs, the rapid spread of pyrethroid resistance and competition for LLIN market share between manufacturers has diversified the product palette in recent years [4–6]. Alongside much needed progress (e.g., with new and innovative active ingredient formulations), this increased market complexity also led to challenges for LLIN product quality assurance, and the risk and occurrence of substandard products [4, 7–12]. Recipient countries and donors increasingly recognize that LLINs are not just ‘mosquito nets’ but complex public health commodities that require stringent quality assurance and performance monitoring [4, 12, 13].

This issue is not new and the WHO published guidelines for testing LLINs, initially in 2005 [14]. In 2013, these guidelines were updated and augmented with additional guidance for monitoring the durability of LLINs under ‘operational conditions’ [15]. The declared aims of the WHO guidelines are (1) to provide specific standardized procedures for testing of LLINs and (2) to harmonize standard testing procedures [15].

Tests for LLIN bioefficacy under laboratory conditions, i.e., the ability of LLIN products to kill mosquitoes in cone bioassays, are an integral part of the WHO testing guidelines and play a crucial role in the LLIN product prequalification process and, increasingly, in post market surveillance [4, 7, 12, 16–18]. Given that the market fate of LLIN products hinges on their performance in these bioassays, it is important that they are harmonized and transparent [4, 13].

Bioefficacy tests usually involve the exposure of living mosquitoes to LLIN product material, and thus, even in their simplest form, these tests require a complex experimental setup including the maintenance of a mosquito colony [19, 20].

The simplest bioassay recommended to evaluate LLIN products for bioefficacy is the WHO cone bioassay [15, 21]. WHO guidelines outline in detail how cone bioassays should be performed, providing important parameters such as sample size, number of mosquitoes to be exposed, number of replicates to be performed, exposure time, and acceptable temperature and humidity ranges. However, other key parameters that may also affect the key cone bioassay endpoints of 60 min knockdown (KD60) and 24h mortality (M24) are either undefined or only partially defined by WHO. An obvious example for this is that the mosquito strain to be used in WHO cone bioassays is currently not further specified. The WHO guidelines require ‘susceptible’ (female) Anopheles mosquitoes [15, 21]. Yet, it is evident that ‘susceptibility’ does not mean the same (or even similar) response of different mosquito strains to standardized exposure to insecticides. In other words, using different mosquito strains in bioassays is synonymous with systematic bias [22].

This situation can be likened to the known species-dependent mosquito responses observed in WHO tube bioassays used for insecticide resistance monitoring and the recognized need for species-specific, WHO-recommended, discriminatory insecticide concentrations to be used in these assays [22, 23]. It is thus surprising that species-specific guidance for each prequalified LLIN product is lacking, as the declared priorities of harmonization and standardization of bioassays would clearly require this level of rigor.

Besides the use of different mosquito strains, other important bioassay parameters are also left open for interpretation or are not standardized even if the relevant guidelines are explicit. As a result, testing laboratories may be inclined to ‘optimize’ these undefined or less stringently enforced bioassay parameters to achieve the highest bioefficacy outcomes (i.e., high KD60 and M24). This stands in contrast to the overarching aim of ‘harmonization’ and ‘standardization’ as it may further increase systematic bias between testing facilities and lead to situations where LLIN products may routinely ‘pass’ testing criteria in some settings but not in others.

One such undefined (or partially defined) set of parameters is the configuration of the bioassay board to which the LLIN samples are pinned for the duration of the test. While this may seem trivial, even here multiple parameters may crucially influence bioassay results. For example, while WHO guidelines state that these boards should be placed at a 45 degree angle, Owusu et al., 2016, compared different angles for positioning of the cone bioassay board set up and found that mosquitoes (pyrethroid susceptible An. gambiae Kisumu-1 and pyrethroid resistant An. stephensi STI) spent more time on the nets at a 60 degree angle [24]. As a result, some studies are now reconfiguring this experimental detail in order to maximize the measured bioefficacy indicators of M24 and KD60 [20, 25]. Another such modification is the use of bioassay boards with circular holes i.e., the material behind the tested sample being removed, again with the intention to ‘maximize exposure’ and thus increase bioefficacy endpoints (Fig. 1). While WHO guidelines do not specify whether this should be done or not and we are not aware of studies having systematically quantified the resulting effect. Several laboratories are conducting WHO cone bioassays on boards with holes cut [20, 24] while others are not [12, 20].

Therefore, in this study, we investigated whether circular holes in the bioassay boards intended to ‘force the mosquitoes to stand on the net surface’ (as shown in Fig. 1) lead to systematic bias on the KD60 and M24 key bioassay endpoints.

Study Locations

The study was conducted at two facilities (i) the Vector-borne Diseases Unit of the PNG Institute of Medical Research (PNGIMR), using fully pyrethroid susceptible Anopheles farauti colony mosquitoes and (ii) the Mosquito Research Facility at James Cook University (JCU), using fully susceptible Aedes aegypti mosquitoes.

Origin Of Llins

Five groups of LLINs were included in the present study as shown in Table 1. All were unused and unwashed, and obtained in original packaging. LLINs with manufacturing year, 2019 and 2020, were sampled from deliveries for mass distribution immediately upon arrival in PNG. These were requested to be tested for bioefficacy by the National Malaria Control Program and Rotarians Against Malaria PNG who implement LLIN mass distributions in the country. Unused PermaNet® 2.0 manufactured in 2012 was obtained from the Madang Provincial Health Authority. Two groups of PermaNet® 2.0 samples were included as they exhibit substantially different bioefficacy as shown in previous studies [12, 26]. More details about the individual LLIN samples are provided in Additional File 1.

Table 1

LLIN samples included in the present study.
N	Net Brand	Material	Active Ingredient	Denier	Manufacture Year
4	PermaNet® 2.0	Polyester	1.8 g/kg Deltamethrin	75	2012
4	PermaNet® 2.0	Polyester	1.8 g/kg Deltamethrin	75	2019
4	Interceptor®	Polyester	1.8 g/kg Deltamethrin	100	2020
4	Yorkool® LN	Polyester	5 g/kg alpha-Cypermethrin	75	2019
4	SafeNet®	Polyester	5 g/kg alpha-Cypermethrin	100	2019 & 2020

Two sets of 5 net pieces (25 cm x 25 cm) were cut from adjacent positions on the four sides and the roof of each of the LLIN samples, resulting in a total of 100 pairs. From each of the 100 pairs, one piece was retained at PNGIMR and one piece was sent to JCU. Net pieces were wrapped individually in aluminum foil, placed inside zip lock plastic bags and stored at 4°C until processed.

Who Cone Bioassay

Cone bioassays with LLINs were conducted according to WHO guidelines [15] using fully susceptible An. farauti s.s mosquitoes [12] at PNGIMR and fully susceptible Ae. aegypti mosquitoes at JCU. The experimental set up of the WHO cone bioassays is shown in Fig. 1. Two identical bioassay boards (dimensions 900 x 600 x 5mm) were prepared from clear acrylic. The boards were divided into two sections and three sets of four holes (same diameter as the bioassay cones’ diameter) were cut in one of the sections.

Cone bioassays were performed on a board ‘without holes’ (as per WHO guidelines) and ‘with holes’ in parallel (Fig. 1). A net piece was placed directly over the ‘holes’ and four bioassay cones were attached to it using small magnets. On board sections ‘without holes’, an A4 piece of paper was placed between the board and the net piece and, likewise, four cones were attached to it using magnets. The boards were set up against a wall at a 45° degree angle [24] before mosquitoes were introduced into the bioassay cones. Cone bioassays for of each LLIN were performed on both the ‘with hole’ and ‘without hole’ configurations of the bioassay boards. Boards were washed with unscented soap and water, rinsed and dried in the sun in between assays.

Using an aspirator, 5 insecticide susceptible, non-blood-fed, 2–5 day old female Anopheles farauti (PNGIMR) or Aedes aegypti (JCU) mosquitoes were introduced into a cone and a cotton ball was used to plug the hole. Mosquitoes were exposed to the net pieces for 3 min (timed individually for each cone), after which they were gently transferred from the cones to a holding cup screened with untreated netting and provided access to 10% sugar solution via a soaked piece of cotton wool placed on top of the netting. Knockdown was recorded at 10, 20, 30, 40, 50 and 60 min after exposure. Mortality was recorded at 24 h after exposure. Mosquitoes were scored as ‘alive’ if they were able to both stand upright and fly in a coordinated manner; knocked-down if it could not stand (e.g. had one or two legs), could not fly in a coordinated manner (e.g., taking off briefly but falling down immediately) and as dead if it was immobile, and showed no signs of life. An untreated polyester net was used as control. Cone bioassays at PNGIMR were performed under laboratory conditions with around 28.5 ± 2.7°C and 65 ± 9% relative humidity, whereas at JCU temperature was around 26.0 +/-2.5 degrees with a relative humidity of around 75%+/-11%.

Data analysis

Data were analyzed using Microsoft Excel 2016 (Microsoft Inc.) and GraphPad Prism 9.3.1 (GraphPad Software). The main endpoints of WHO cone bioassays are KD60 and M24, however, we also compared knockdown kinetics as an additional endpoint. WHO standard bioefficacy criteria for cone bioassays for pyrethroid LLIN are ≥ 95% KD60 and/or ≥ 80% M24.

To summarize proportion data (Figs. 2 and 3), mean proportions and 95% confidence intervals of proportions were used. To compare population proportions between boards with holes and without holes, or between facilities (mosquito species), two-sample Z tests of proportions were used. For correlation data, we used non-parametric correlation statistics (Spearman) correlation.

Effect of holes in bioassay boards on knockdown and mortality rates

Results obtained with WHO cone bioassays using bioassay board configurations with holes and without holes and using either An. farauti or Ae. aegypti mosquitoes are summarized for each LLIN type and overall in Table 2. The table shows the percent KD60 and percent M24 for all mosquitoes tested with each net type (i.e., the tests for individual nets and net panels combined).

Table 2

**Summary of test results using bioassay boards with and without holes**. Results are presented as proportions (in %) and their 95% confidence intervals (in parentheses). Resulting KD60 and M24 for the different bioassay board configurations were compared using Z-tests of proportions.
KD60	Ae. aegypti		An. farauti
	holes	no holes	holes	no holes
PermaNet 2.0 (2012)	98.8% (95.6–99.9)	99.4% (96.5–99.9)	98.7% (93.2–100.0)	100% (95.3–100)
PermaNet 2.0 (2019)	17.1% (11.6–23.9)	20% (14.1–27.0)	33.8% (23.6–45.2)	47.5% (36.2–59.0)
Interceptor	45.0% (36.6–53.6)	53.2% (44.6 to 61.7)	74.1% (63.1–83.2)	68.4% (56.9–78.4)
Yorkool	64.4% (56.9–71.5)	77.8% (70.7–84.2)**	83.8% (73.8–91.1)	53.2% (41.6–64.5)***
SafeNet	74.3% (67.2–80.6)	78.8% (72.1–84.5)	82.1% (71.7–89.8)	80.3% (69.9–88.3)
Average	60.7% ( 57.3–64.1)	66.4% (63.0–69.7)*	74.4% (69.8–78.6)	69.7% (64.9–74.2)
M24	Ae. aegypti		An. farauti
	holes	no holes	holes	no holes
PermaNet 2.0 (2012)	68.1% (60.3 to 75.3)	66.2% (58.3–73.4)	98.7% (93.2–100.0)	100% (95.3–100)
PermaNet 2.0 (2019)	12.7% (7.9–18.9)	12.5% (7.8–18.6)	39.8% (28.1–50.3)	30% (20.3–41.3)
Interceptor	19.3% (13.1–26.8)	30.2% (22.7–38.6)*	39.5% (28.8–51.0)	29.1% (19.4–40.4)
Yorkool	27.7% (21.2–34.9)	39.6% (32.0–47.7)*	68.8% (57.4–78.7)	39.2% (28.4–50.9)**
SafeNet	47.4% (39.8–55.1)	57.0% (49.4–64.4)	56.4% (44.7–67.6)	43.1% (32.2–54.7)
Average	35.6% (32.3–39.0)	41.7% (38.2–45.2)*	60.3% (53.3–65.1)	48.0% (43.0–53.0)**
0.01 < p < 0.05; 0.001 < p < 0.01;**p < 0.001.

Interestingly there were slightly opposite tendencies in the effect that the holes in the bioassay boards had on WHO cone bioassay endpoints, depending on the mosquito species used. For Aedes aegypti, we observed a small (around 5%), and mildly statistically significant (0.01 < p < 0.05, Z-test to compare proportions) decrease in observed KD60 and M24 when boards with holes were used. For Anopheles farauti, we observed a moderate increase in M24 of around 12%; p = 0.002) when bioassay boards with holes were used.

Mosquito species had a pronounced small-to-medium systematic impact on the WHO cone bioassay outcomes, and using pyrethroid susceptible Ae. aegypti resulted in systematically lower KD60 (63.5% vs 72.0%, Cohen’s h = 0.18, p < 0.0001) and M24 (38.6% vs 54.2%, Cohen’s h = 0.31, p < 0.0001) as compared to using pyrethroid susceptible An. farauti. Interestingly, this effect was most prominent and apparent across all products when bioassay boards with holes were used. In this group (bioassay boards with holes), M24 for An. farauti was much higher (35.6% vs 60.3%, Cohen’s h = 0.5, P < 0.0001), and KD60 was also significantly increased (p = P < 0.0001) as compared to Ae. aegypti. The results are graphically presented in Fig. 2.

Figure 3 shows the knock down kinetics in the 60 minutes after exposure as measured in 10 min intervals for the two bioassays board configurations (with and without holes) and the two mosquito species. Some LLIN types seemed to invoke larger differences between board configurations and/or species than others did. However, with the exception of Yorkool LN (An. farauti, without holes) and PermaNet 2.0 2012 (An. farauti, without holes), the general pattern of Ae. aegypti resulting in consistently lower KD values is evident.

Figure 4 shows a correlation analysis for KD60 and M24 endpoints between mosquito species. The figure again illustrates that for An. farauti, bioassays resulted in systematically higher KD60 and M24 outcomes and that this effect was more pronounced when bioassay boards with holes were used, whereas for Ae. aegypti overall KD60 and M24 were lower and the differences caused by the bioassay configuration were smaller (i.e., the points for bioassays conducted on boards with no holes are closer to the line of identity).

Overall, our results demonstrate that pyrethroid susceptible An. farauti are more sensitive to bioassay board configuration changes as compared to susceptible Ae. aegypti, and that the combination of bioassay board configuration and mosquito strain can result in quite substantial systematic differences in the key WHO cone bioassay outcomes of KD60 and M24. For example, in our study, overall M24 in the bioassay board configuration group of ‘with holes’, was 36% for Ae aegypti but 60% for An. farauti (p < 0.0001).

WHO cone bioassays are used to evaluate and compare LLIN products, with the intention to ‘harmonize testing procedures in order to generate data for registration and labelling’ [15]. Our results demonstrate that this desired standardization is currently not being achieved because important parameters are not defined in the current testing guidelines [15]. Bioassay outcomes can currently only be interpreted taking into consideration the mosquito species that was used and other important parameters such as the configuration of the bioassay boards. As a consequence, it is questionable if WHO cone bioassays in their current form should be used to evaluate products against fixed, universal bioefficacy performance thresholds such as 95% KD60 and 80% M24 across different settings.

To us, this does not mean that cone bioassays conducted in different settings are not equally meaningful and reproducible, but as testing laboratories use different mosquito strains and different bioassay configurations, often with the intention to maximize exposure (and thus, bioefficacy endpoints) the outcomes obtained in different settings are bound to be systematically biased.

While variation of factors that can easily be standardized such as bioassay board configuration could be avoided, other factors are much harder to standardize, given the complexity and number of parameters involved. In addition, some settings may simply not be able to use specific mosquito strains due to regulatory restrictions. As such, it seems very unlikely that full standardization of WHO cone bioassays is achievable or desirable across settings. A better option may be to enable harmonization between settings, by conducting dedicated multicentre studies to establish baseline parameters defining the systematic bias between settings. We have recently conducted such a study between PNGIMR and the Ifakara Health Institute [20].

Other WHO documents e.g., those used for insecticide resistance monitoring provide mosquito species-specific guidance [22]. However, since other factors such as temperature [27] have been reported to potentially influence bioassay outcomes, it is questionable if mosquito species-specific bioefficacy thresholds are the solution to the harmonization/standardization problem. In fact, we cannot really exclude the possibility that what we are attributing to species specific differences in the present study may in part be due to other differences in parameters that cannot easily be controlled for between settings.

Our study demonstrates that products should be evaluated against a range of mosquito species and it seems crucial to establish reference bioefficacy parameters for products in order to more generally interpret bioassay results obtained in specific settings. Reference samples and facility-specific reference bioefficacy data can help to detect bioefficacy shifts in products that are indicative of real bioefficacy changes due to e.g., changes in product specifications, as we have demonstrated occurred in LLINs delivered to Papua New Guinea [12].

It is equally important that detailed chemical reference specifications pertaining to insecticide, net material and coating (if applicable) are part of publicly available product documentation, especially for products that are funded by public donor funding. This could include e.g., reference spectra from combustion ion chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry reference spectra for each product, electron micrographs of expected product surface appearance, wash resistance profiles etc. These reference specifications should be validated routinely as part of LLIN pre-delivery inspections.

While it was not part of this study to assess individual products against WHO performance thresholds we note that only one of the 5 tested products (PermaNet 2.0 from 2012) resulted in either > 80% average M24 or > 95% average KD with the fully susceptible Anopheles strain used in the present study. In contrast, all of these products achieved 100% or near 100% KD60 and M24 when they were evaluated by WHO Pesticide Evaluation Scheme [16–18, 28, 29]. The products used in the present study were all coated polyester nets and thus belong to the same class of products. Both, Ae. aegypti and An. farauti have previously been used in studies to evaluate LLIN products [11, 12, 20, 26]. Even though WHO guidelines do not recommend the use of non-anophelines for LLIN evaluation, in reality this is routinely done [30, 31].

The fact that systematic bias is strikingly evident, even for the simplest of bioassays available to test LLIN products makes it very likely that more complex assays, such as the tunnel test and experimental huts, with many more undefined parameters (e.g., exposure time, bait animal, mosquito age, tunnel and hut configurations etc.), are even more sensitive to these issues and even less likely to predict similar results across different settings. ‘Next generation’ LLIN products that employ active ingredients (AIs) with different modes of action (such as Interceptor G2 [32]) or varying AI composition and concentrations on different net sections (such as PermaNet® 3.0 [33]), require further specific refinement of the testing guidelines that incorporate these complexities in order to enable a meaningful evaluation.

WHO cone bioassay outcomes are systematically biased depending on the pyrethroid susceptible mosquito strain employed and bioassay board configuration. Harmonization of testing procedures across different settings requires quantification of the systematic bias between different settings and setting-specific reference bioefficacy data are needed. Reference samples for each product should be retained at prequalification.

AI Active ingredient

IMR Institute of Medical Research

KD60 Knockdown after 60-minutes

LLINs Long-lasting insecticidal nets

M24 Mortality at 24-hours

PNG Papua New Guinea

WHO World Health Organization

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The data set for this study is available on request from the corresponding author.

Competing interests

The authors declare no competing interests.

Funding

SK was supported by an NHMRC Career Development Fellowship (GNT 1141441). This study was supported by an NHMRC Ideas Grant (GNT 2004390) and the Global Fund to Fight Aids Tuberculosis and Malaria.

Authors' contributions

Conceived Study: SK; Conducted Bioassays: MK, BA, NB, KK, DL; Wrote first draft: MK, SK; Data Analysis: MK, SK; Reviewed Manuscript: MK, BA, NB, KK, DL, SK.

Acknowledgement

The authors thank the PNG National Malaria Control Program (Leo Makita) and Rotarians Against Malaria PNG (Tim Freeman) for making available the LLIN samples for testing. We would also like to thank William Pomat, Moses Laman and the staff of the PNGIMR Vector-borne Diseases Unit for operational support. We thank Michael Townsendfrom JCU for support with mosquito rearing.

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No competing interests reported.

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Editorial decision: Major revision
06 Nov, 2022
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05 Nov, 2022
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Reviewers agreed at journal
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You are reading this latest preprint version

WHO cone bioassay boards with or without holes: Relevance for bioassay outcomes in long-lasting insecticidal net studies

Status:

Version 1

Abstract

Figures

Background

Methods

Study Locations

Origin Of Llins

Who Cone Bioassay

Data analysis

Results

Effect of holes in bioassay boards on knockdown and mortality rates

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1