Contact bioassay in tandem with HPLC analysis of the active ingredients in LLINs revealed hidden possible causes of the unabated rise in malaria cases

Background The World Malaria Report 2021 revealed that Nigeria accounted for 27% of 241 million malaria cases worldwide and 31% of 602,000 malaria deaths. This WHO report is in sharp contrast to the ambitious goal of the National Malaria Strategic Plan (NMSP) 2014-2020, which aimed to transition Nigeria from malaria control to malaria elimination status by 2020. In this study, we combined contact bioassay with HPLC analysis to investigate how the end users’ treatment of Long-Lasting Insecticide Treated Nets (LLINs) inadvertently contributes to the unabated rise in malaria cases. Methods We randomly selected a few LLINs used under normal household conditions in Port-Harcourt and one on sale in Lagos, Nigeria. The continued potency of these LLINs to protect the users against malaria vectors, assuming the local vectors are insecticide-susceptible, was evaluated by exposing laboratory-susceptible Anopheles gambia Kisumu strain to the nets according to the WHO cone bioassay protocol. After the exposure, the active ingredients (AI) in the LLINs were extracted and analysed using reverse phase HPLC to establish the quantity of the AIs and correlate it to bio-efficacy. The AI loss per wash was also computed based on the information provided by the users. Results The labels on the LLINs revealed them to be Royal Sentry, PermaNet 2.0 and DuraNet. The bio-efficacy of these LLINs under household usage is less than two years. AI lost per wash was higher by 2.5-fold in LLIN made of polyethylene nets than polyester nets: 0.35g/Kg/wash and 0.14g/Kg/wash, respectively. The AI lost per wash increased by 31% for net exposed longer to the harsh environmental conditions of this region. The LLIN (ParmaNet) purchased in the open market has the highest AI concentration but only achieved 70.62% mortality, far below the acceptable standard. Conclusion The AI in the LLIN bought from the open market was the highest, but of poor quality. This bed net was a counterfeit product, not approved by WHOPES. More importantly, this study revealed that field examination of LLINs under normal household use should be examined per region for policy makers to have informed knowledge of the duration of the bio-efficacy of the LLINs being distributed. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript * NMSP : National Malaria Strategic Plan LLINs : Long-Lasting Insecticide Treated Nets IRS : Indoor Residual Spraying WHOPES : World Health Organization Pesticide Evaluation Scheme PBO : piperonyl butoxide AI : active insecticide KD : Knockdown LOD : level of detection LOQ : level of quantification (LOQ) NAFDAC : National Agency for Food and Drug Administration and Control