Insecticide susceptibility status of Anopheles gambiae s.l. mosquitoes and the efficacy of a piperonyl butoxide (PBO) synergist in northern Namibia
Authors: Joseph Rosalia N. , Mwema Tabeth , Eiseb Seth J. , Maliti Deodatus V. , Tambo Munyaradzi , Iitula Iitula , Eloff Lydia , Lukubwe Ophilia , Smith-Gueye Cara , Vajda Elodie , Tatarsky Allison , Katokele Stark T. , Uusiku Petrina N. , Walusimbi Dennis , Ogoma Sheila B. , Mumbengegwi Davis R. , Lobo Neil F. 
Published: October 2023

Abstract
Background
Malaria continues to pose a significant public health challenge in northern regions of Namibia, while southern regions remain malaria-free. The country has employed indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) with pyrethroids as key vector control measures. LLINs have demonstrated effectiveness in reducing malaria transmission. However, pyrethroid resistance in Anopheles mosquitoes may compromise the efficacy of these interventions. To address this challenge, the WHO recommends the use of piperonyl butoxide (PBO) LLINs in areas with confirmed mixed function oxidase (MFO)-mediated pyrethroid resistance.
Methods
This study, conducted as part of the National Vector-borne Disease Control Program's annual entomological surveillance, assessed the susceptibility of An. gambiae s.l. mosquitoes to WHO tube bioassays with 4% DDT and 0.05% deltamethrin insecticides. The Anopheles mosquitoes that underwent the insecticide resistance assays were both morphologically and molecularly identified. Additionally, the study explored the potential of novel pyrethroid piperonyl butoxide (PBO) LLINs, designed to enhance mortality in pyrethroid-resistant mosquitoes. This was done by simultaneously exposing mosquitoes to deltamethrin (0.05%) alone, PBO (4%) + deltamethrin(0.05%), and PBO alone.
Results
In northern Namibia, PCR species diagnostic tests revealed that An. gambiae sensu stricto (62%) was more prevalent than An. arabiensis (38%). WHO insecticide resistance assays confirmed resistance to deltamethrin 0.05% in the Oshikoto, Kunene, and Kavango West regions, with mortality rates of 79%, 86%, and 67%, respectively. Additionally, reduced susceptibility to deltamethrin was observed in An. gambiae s.s. populations in the Omusati (97% mortality) and Ohangwena (94% mortality) regions. While An. gambiaes.s. remained fully susceptible to DDT in most regions, reduced susceptibility was recorded in Kavango West, with 91% mortality. In contrast, An. arabiensis displayed resistance to deltamethrin 0.05% in Oshikoto (82% mortality) and reduced susceptibility in Kavango West (96% mortality) but full susceptibility (100% mortality) in other regions. Notably, there was a 4% reduction in susceptibility to DDT in An. arabiensis from the Kavango West region, while those from other regions remained fully susceptible (100% mortality).
Subsequently, molecular analysis of a subsample from PBO synergist assays in 2020 demonstrated a high proportion of An. arabiensis (84.4% in Oshana; 73.6% in Oshikoto), along with non-amplifiers(15.2% in Oshana; 26.4% in Oshikoto). A single specimen of An. quadriannulatus was captured in Oshana. Deltamethrin resistance was consistently observed in An. gambiae s.l. populations across all sites in both 2020 and 2021. In 2020, deltamethrin induced mortality rates of 93.3% in Oshana and 95.0% in Oshikoto. In 2021, mortality rates ranged from 70.0% to 95.0% in Otjozondjupa, Oshikoto, Kavango East, Kavango West, and Zambezi. Following pre-exposure to the PBO synergist, deltamethrinsusceptibility was fully restored, with 100.0% mortality observed at all sites during assays conducted in both 2020 and 2021.
Conclusions
The study underscores the importance of ongoing entomological surveillance and routine monitoring of malaria vectors' susceptibilities to insecticides in northern Namibia, along with appropriate responses based on evidence gathered. Pyrethroid resistance was identified in An. gambiae s.s. and An. arabiensis in the Kavango West, Kunene, and Oshikoto regions and reduced susceptibility to DDT in the Kavango West. These findings suggest potential challenges for IRS and LLINs using pyrethroids and could impede malaria elimination efforts. Resistance data resulted in evaluating PBO to combat potential reductions in intervention efficacy. Consequent data highlights the promise of pyrethroid-PBO LLINs in addressing resistance issues in the region. Further species-specific testing is recommended for a comprehensive understanding of insecticide resistance dynamics.