Escalation of Pyrethroid Resistance in the Malaria Vector Anopheles funestus Induces a Loss of Efficacy of Piperonyl Butoxide-Based Insecticide-Treated Nets in Mozambique

Abstract

Background: Insecticide resistance poses a serious threat to insecticide-based interventions in Africa. There is a fear that resistance escalation could jeopardize malaria control efforts. Monitoring of cases of aggravation of resistance intensity and its impact on the efficacy of control tools is crucial to predict consequences of resistance.

Methods: The resistance levels of an Anopheles funestus population from Palmeira, southern Mozambique, were characterized and their impact on the efficacy of various insecticide-treated nets established.

Results: A dramatic loss of efficacy of all long-lasting insecticidal nets (LLINs), including piperonyl butoxide (PBO)-based nets (Olyset Plus), was observed. This An. funestus population consistently (2016, 2017, and 2018) exhibited a high degree of pyrethroid resistance. Molecular analyses revealed that this resistance escalation was associated with a massive overexpression of the duplicated cytochrome P450 genes CYP6P9a and CYP6P9b, and also the fixation of the resistance CYP6P9a_R allele in this population in 2016 (100%) in contrast to 2002 (5%). However, the low recovery of susceptibility after PBO synergist assay suggests that other resistance mechanisms could be involved.

Conclusions: The loss of efficacy of pyrethroid-based LLINs with and without PBO is a concern for the effectiveness of insecticide-based interventions, and action should be taken to prevent the spread of such super-resistance.

Keywords: An. funestus; Mozambique; cytochrome P450; insecticide resistance; long-lasting insecticidal nets; malaria; metabolic resistance; vector control.

Figure 1.

Bioefficacy of different commercial long-lasting insecticidal nets against Anopheles funestus sensu stricto in Palmeira, Mozambique, in 2016 (A), 2017 (B), and 2018 (C). Error bars represent standard error of the mean.

Figure 2.

Susceptibility profiles of an Anopheles funestus population from Palmeira, southern Mozambique. A, Susceptibility profile in 2016 for females only. B, Susceptibility profile in 2017 for both males and females. C, Synergist assay with piperonyl butoxide (n = 4). D, Time-point mortality rates for permethrin with estimation of the median lethal time (LT₅₀) at 1 hour 45 minutes. Error bars represent standard error of the mean. Abbreviations: DDT, dichlorodiphenyltrichloroethane; PBO, piperonyl butoxide.

Figure 3.

Exploration of the molecular basis of the escalation of pyrethroid resistance in Anopheles funestus. A, Comparative gene expression of the cytochrome P450 genes CYP6P9a and CYP6P9b in Mozambique comparatively to other African regions. Error bars represent standard error of the mean. B, Allele frequency of the CYP6P9a pyrethroid resistance marker in southern Mozambique populations from 2002, 2016, and 2017, showing a fixation of the CYP6P9a_R allele. C, Distribution of the genotypes of resistance markers in Palmeira including CYP6P9a_R, L119F-GSTe2, N485I-Ace-1, and A296S-RDL. Abbreviations: CYP, cytochrome P450; MOZ, Mozambique.