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Pyrethroid-resistant malaria vector Anopheles gambiae restored susceptibility after pre-exposure to piperonyl-butoxide: results from country-wide insecticide resistance monitoring in Tanzania, 2023

Malaria Journal volume 23, Article number: 395 (2024) Cite this article

Abstract

Background

Effective vector control interventions, notably insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are indispensable for malaria control in Tanzania and elsewhere. However, the emergence of widespread insecticide resistance threatens the efficacy of these interventions. Monitoring of insecticide resistance is, therefore, critical for the selection and assessment of the programmatic impact of insecticide-based interventions.

Methods

The study was conducted country-wide across 22 sentinel districts of Tanzania between May and July 2023 using standard World Health Organization susceptibility test with 1×, 5×, and 10× of deltamethrin, permethrin, and alpha-cypermethrin and discriminating concentrations of 0.25% pirimiphos-methyl. Synergist assays were conducted to explore the underlying mechanisms of the observed phenotypic pyrethroid-resistant mosquitoes. Three- to five-day-old wild adult females in the first filiar generation of Anopheles gambiae sensu lato (s.l.) were used for the susceptibility bioassays.

Results

Anopheles gambiae s.l. were resistant to all pyrethroids at the discriminating dose in most sentinel districts except in Rorya, which remains fully susceptible, and Ushetu, which remains susceptible to deltamethrin but not permethrin. In 5 sites (Bukombe, Ukerewe, Kilwa, Kibondo, and Kakonko), the An. gambiae s.l. species exhibited strong resistance to pyrethroids surviving the 10 X concentrations (mortality rate < 98%). However, they remained fully susceptible to pirimiphos-methyl in almost all the sites except in Kibondo and Shinyanga. Likewise, there was full restoration to susceptibility to pyrethroid following pre-exposure of An. gambiae s.l. to piperonyl-butoxide (PBO) in 13 out of 16 sites. The 3 sites that exhibited partial restoration include Kakonko, Tandahimba, and Newala.

Conclusion

The evidence of widespread pyrethroid resistance of the major malaria vector justifies the decision made by the Tanzania National Malaria Control Programme to transition to PBO-based ITNs. Without this switch, the gains achieved in malaria control could be compromised. Equally important, the lack of full restoration to susceptibility observed in three sentinel districts upon pre-exposure to PBO merits close monitoring, as there could be other underlying resistance mechanisms besides oxidase metabolic resistance.

Background

The widespread use of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) along with improved case management has led to a significant reduction of the malaria burden across sub-Saharan Africa (SSA) since 2000 [1]. Most of the decline has been attributed by vector control interventions, with ITNs and IRS accounting for significant contributions, 68% and 13% respectively, to cases averted (663 million clinical cases) since 2000 [1]. These critical vector control interventions rely on a few available classes of insecticides, particularly pyrethroids, which have been vulnerable to malaria vector resistance [2]. Despite the lack of strong epidemiological evidence on the association between insecticide resistance and increased malaria infections [3], insecticide resistance threatens the gains in malaria control over the past two decades. It is, therefore, essential to monitor insecticide resistance to guide the selection of effective insecticide(s) for use in ITNs and/or IRS and to assess the public health impact of insecticide-based programmatic interventions.

Resistance to pyrethroids, a common insecticide class used for impregnation of ITNs, is widespread across sub-Saharan African countries [4,5,6,7,8,9,10]. While resistance to pyrethroids is increasing and spreading among populations of malaria vectors, the resistance to non-pyrethroid insecticides, like pirimiphos-methyl and bendiocarb, is less common [7, 11]. Neonicotinoids, such as clothianidin, currently face minimal resistance and offer an effective alternative for IRS [12]. Different insecticide resistance mechanisms in malaria vectors have been established [13,14,15]. These mechanisms include: (1) target-site mutations which are caused by non-synonymous mutations affecting the neuronal proteins targeted by insecticides, preventing their binding; (2) metabolic resistance, characterized by changes in insect enzyme systems leading to rapid detoxification or sequestration of insecticides; and (3) biochemical cuticular alterations that reduce the amount of insecticide penetrating the insect [15, 16].

Target site mutations are well known, and their genotyping provides useful information to track malaria vector resistance in the field [17, 18]. Pyrethroids and the organochlorine dichlorodiphenyltrichloroethane (DDT) share the same target site, the voltage-gated sodium channel (VGSC), while carbamates and organophosphates are acetylcholinesterase inhibitors [17, 19]. The VGSC is affected by knock-down resistance (kdr) mutations and involves either a leucine to phenylalanine substitution at position 995 formerly known as 1014 (L995F) or leucine to serine substitution at the same codon (L995S) [20]. The former was first identified in West Africa and, therefore, named the ‘kdr-west’ mutation, while the latter was first identified in East Africa, and named ‘kdr-east’[15]. These mutations are now widely geographically distributed [4, 21, 22]. Co-occurrence of ‘kdr-east’ and ‘kdr-west’ is becoming increasingly common [6, 23, 24]. The targets of both organophosphate and carbamate insecticides are the acetylcholinesterases which include the ace-1 gene affected by a glycine to serine substitution at position 119 (Ace1-G119S) in Anopheles mosquitoes [25]. Metabolic resistance involves enzymes from three major families, namely: the cytochrome P450-monooxygenases, carboxylesterases, and glutathione S-transferases (GSTs). The increased enzymatic activity, which is often caused by gene over-expression through structural modifications, leads to enhanced insecticide metabolism [26].

The Global Plan for Insecticide Resistance Management (GPIRM) [27] and the Global Vector Control Response [28], provide frameworks for establishing effective vector control programs despite the occurrence of resistance. These two global strategies stress the need for coordinated and intensive entomological surveillance that includes monitoring and managing insecticide resistance to protect the efficacy of the few available insecticides used for public health. Responding to these global strategies, Tanzania developed its own Insecticide Resistance Monitoring and Management Plan (IRMMP) in 2016 [29]. Among other priorities, it emphasizes the need for annual monitoring of insecticide resistance nationwide, to inform the programmatic impact of insecticide-based interventions, and for timely choice of effective vector control strategies. Annual insecticide resistance monitoring has been ongoing in Tanzania's mainland since 1999, and evidence from this platform has contributed to changes in practice/policy related to the programmatic use of insecticide-based interventions in malaria vector control. These changes include the replacement of pyrethroid-based IRS (ICON) to carbamate-based IRS (bendiocarb) in 2012, and bendiocarb-based IRS to organophosphate-based IRS (pirimiphos-methyl, Actellic 300CS) in 2015, and later organophosphate-based IRS to neonicotinoid-based IRS (clothianidin, SumiShield® 50WG, and Fudora Fusion) in 2019 [7, 8, 11, 30, 31]. To inform vector control programming in the Tanzania mainland, an assessment of the insecticide resistance profile of Anopheles gambiae sensu lato (s.l.) using WHO discriminating concentrations, intensity assays, synergist assays, and analysis of underlying mechanisms of resistance were conducted in 22 sentinel districts distributed across diverse epidemiological and ecological settings of Tanzania mainland during 2023.

Methods

Study design and sampling sites

The survey design was a cross-sectional, country-wide survey, with insecticide resistance of the primary malaria vector An. gambiae s.l. monitored at 22 pre-established sentinel sites between May and July 2023. Sentinel site selection was based on criteria that included: malaria endemicity (nationally-representative variation in malaria endemicity), high coverage of vector control interventions, demonstrated presence or absence of insecticide resistance during previous surveys, districts bordering other countries, especially if the neighbouring country has reported resistance, population density (urban vs rural), and site accessibility and history of wide pesticide use in agriculture as described elsewhere [7, 30].

The distribution and malaria endemicity levels [32] of the study sentinel districts across the country are as shown in Fig. 1. Field activities were preceded with a 1-week training workshop to equip data collectors (comprised of field entomology technicians, laboratory scientists, and public health entomology scientists recruited from different partner research institutions in Tanzania) with basic knowledge of the larval collection and standard protocols for conducting insecticide resistance monitoring, morphological identification of malaria vector species, field larval sampling and rearing, specimen preservation, and transportation of samples to the central molecular laboratory. Participants also received hands-on training on the use of an electronic system for data collection. The training was essential to harmonize the protocol and accuracy of species identification to limit testing of non-targeted Anopheline mosquitoes. Data was recorded electronically using generic schema, an informatics system that was developed at Ifakara Health Institute (IHI) [33], that allows any entomological data to be stored in four entomology-specific data tables with predefined relational linkages, enabling rapid, even automated synthesis and analysis of data. Raw data were uploaded to the server from tablets via a cellular network.

Fig. 1
figure 1

Map of Tanzania mainland showing the spatial distribution of the selected 22 sentinel districts based on malaria endemicity for insecticide resistance monitoring in 2023

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Field larvae collection and rearing

The standard dipping approach was used to collect the immature stages of the Anopheles mosquito species using a 350 ml dipper [34]. Before placing larvae/pupae into containers, debris and predators were removed by sieving. Larvae/pupae were then placed into loosely closed plastic bottles arranged tightly inside a bucket and/or cooler box and transported to a rearing station in an improvised field insectary. To limit larvae mortality, the caps of the plastic bottles were loosely closed to enable aeration, and bottles were closely packed during transportation. Larvae and pupae were collected from various types of breeding habitats within each sentinel site, including cultivated agricultural areas, streams, ponds, and puddles. Collected larvae and pupae from each sentinel site were then pooled together for rearing. The geographic coordinates of each sampling site were recorded using calibrated tablets. The larvae were fed with TetraMin® fish food. All pupated larvae were transferred into shallow plastic bowls using Pasteur pipettes. These samples were then placed in appropriately labeled cages for adult emergence. Upon emergence, the adults were fed on a 10% sugar solution.

WHO insecticide susceptibility testing

Susceptibility tests were carried out using the WHO’s test kits for adult mosquitoes [35], comprising insecticide-impregnated test papers, non-impregnated papers for control, and plastic tubes marked red for exposure and green for holding. Three- to five-day-old female F1 generation An. gambiae s.l., were tested using standard WHO insecticide susceptibility procedures with four replicates per insecticide type of 20–25 wild adult female mosquitoes per test tube. Mosquitoes were exposed to papers impregnated with the WHO-recommended discriminating concentrations (DC) of deltamethrin (0.05%), alphacypermethrin (0.05%), permethrin (0.75%), and pirimiphos-methyl (0.25%) for 60 min (WHO, 2022a). At the end of the exposure period, mosquitoes were transferred into holding tubes lined with untreated papers by gently blowing them through the open space between the exposure and holding tubes. A cotton ball soaked in 10% sugar was placed on top of the holding tube to avoid mosquito death by starvation. The mortality was scored 24 h post-exposure. Both impregnated and control papers were obtained from the Universiti Sains Malaysia.

The susceptibility status was evaluated based on the WHO criteria: 98–100% mortality indicates susceptibility; 90%–97% mortality indicates suspect resistance and therefore, requires additional confirmation; and less than 90% mortality indicates resistance [35]. When control mortality between 5 and 20% was recorded, the mean observed mortality was corrected using Abbott’s formula [36]. All tested mosquitoes were preserved in 1.5 ml Eppendorf tubes containing silica and transported to the IHI branch at Ifakara, Morogoro for further laboratory analyses, including molecular species identification and detection of resistance mechanisms.

Insecticide resistance intensity testing

Any resistance phenotypes detected using the discriminating concentrations were further assessed for intensity. This was done by exposing subsequent mosquito samples from the same target vector population to substantially higher concentrations of the respective insecticides. The WHO susceptibility test papers using 5X and 10X the discriminating concentrations in a stepwise manner were used to assess the intensity of resistance to provide information on the range of resistance phenotypes present in a target vector population and their potential operational significance. Similar procedures as to those described under “WHO insecticide susceptibility testing” were used. The intensity of resistance was evaluated based on the WHO criteria: low-intensity resistance means mosquito mortality is < 90% after exposure to 1× and ≥ 98% after exposure to 5×; moderate-intensity resistance means mosquito mortality is < 90% after exposure to 1× and < 98% after exposure to 5× and is ≥ 98% at 10×, and high-intensity resistance means mosquito mortality is < 90% after exposure to the 1× and < 98% after exposure both to 5× and 10× [35].

PBO synergist bioassays

PBO synergist tests were conducted at sentinel sites where mosquitoes were resistant to pyrethroid and stratified into two groups depending on the type of ITNs that were distributed in the area (Olyset net, which is composed of permethrin, vs. Permanet net 2.0, which is composed of deltamethrin) to ascertain the involvement of mixed function oxidases in the observed phenotypic resistance. This was carried out from 16 of 22 preselected sentinel sites (eight sites exposed mosquitoes to PBO plus permethrin and the other eight sites exposed mosquitoes to PBO plus deltamethrin). Similarly, as above, female adult mosquitoes aged three to five days were pre-exposed to 4% PBO paper for one hour and subsequently exposed to either 0.75% permethrin or 0.05% deltamethrin. Two controls were used during this experiment: control one consisted of mosquitoes exposed to clean papers without insecticides or PBO and control two consisted of mosquitoes exposed to papers treated with PBO only [37]. The number of mosquitoes tested for each insecticide varied between 200 to 400. Mortalities were assessed after exposure. The PBO synergized group was compared to the un-synergized group 24 h post-exposure. This comparison was used to evaluate the potential role of cytochrome P450 genes in the observed resistance.

Molecular species identification and detection of resistance mechanisms

After susceptibility testing, An. gambiae s.l. mosquitoes were individually stored and transported from the field to the central laboratory at IHI-Ifakara, Morogoro Branch. Molecular analyses were then performed to identify sibling species of An. gambiae s.l. and to detect resistance mechanisms, including target site mutations (kdr) and metabolic resistance. A sub-sample of 200 mosquitoes was selected from each site for these analyses. This sub-sample consisted of resistant individuals from each pyrethroid insecticide type (permethrin, alphacypermethrin, and deltamethrin) at concentrations of 1×, 5×, and 10×, as well as individuals exposed to pirimiphos-methyl. Additionally, 10% of the 200 individuals were susceptible to mosquitoes, following WHO guidelines [35]. Before processing for polymerase chain reactions (PCR), all mosquitoes were morphologically identified using a standard identification key [38]. Sibling species of An. gambiae s.l. were separated by using conventional PCR by Wilkins et al. [39]. The detection of kdr L995F/L995S and ace-1 G119S alleles was conducted following the methods of Martinez-Torres et al. [18] and Weill et al. [40], respectively.

Statistical analyses

Percent mortality and 95% confidence intervals for the WHO susceptibility tests were calculated by binomial exact method using STATA software 14 (Stata Corp LP, College Station, TX, USA).

Abbott’s formula was used to correct observed mortality when mortality in the control samples was between 5 and 20%[36]. Calculations of the L995F/ L995S/ace-1 G119S mutation frequency was done using the following formula: F(kdr) = 2A + B/2n, with A the number of homozygotes, B the number of heterozygotes, and n the total number of specimens analysed.

Ethical considerations

This study received ethical approval from both the Ifakara Health Institute Ethical Review Board (IHI/IRB/No:14–2023) and the Medical Research Coordinating Committee (MRCC) of the National Institute for Medical Research (NIMR) in Tanzania (NIMR/HQ/R.8a/Vol. IX/4354). Permission for entry to the respective sentinel districts was obtained from the President's Office, Regional Administration, and Local Government Tanzania (PO-RALG) and relevant local government authorities. Districts and village leaders were informed of the study procedure including the benefits. Verbal informed consent was obtained from village leaders where mosquito larvae were collected.

Results

Susceptibility status of An. gambiae to insecticides

Anopheles gambiae s.l. were resistant to alpha-cypermethrin (mortality rate < 90%) across all 21 sentinel sites where it was tested. These malaria vectors were also resistant to deltamethrin in 17/22 sites (except Rorya and Ushetu where they remained susceptible and in Igunga, Karagwe, and Nyasa where resistance needs to be confirmed). A similar resistance pattern was observed to permethrin in 19/22 sites (except Rorya where mosquitoes were susceptible, but in Igunga and Kibondo their resistance needs to be confirmed) (Table 1). However, the species were fully susceptible to pirimiphos-methyl in 18/22 sites (except in Kibondo and Shinyanga with confirmed resistance; and Ngara where resistance needs to be confirmed) (Table 1).

Table 1 Susceptibility status of Anopheles gambiae s.l. to the WHO- discriminating concentrations of Alphacypermethrin (0.05%), Deltamethrin (0.05%), Permethrin (0.75%) and Pirimiphos-methyl (0.25%)
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Intensity of insecticide resistance

Anopheles gambiae s.l. showed low to moderate levels of resistance to permethrin and deltamethrin (mortality rate < 98% at X5 the DC) but were all susceptible to high level of intensity (mortality rate > 98% at X10 the DC—Figs. 2 and 3). However, Ukerewe, Kilwa, and Bukombe exhibited high levels of resistance to alphacypermethrin (mortality rate < 98% at X10 the DC-Fig. 4).

Fig. 2
figure 2

Percentage mortality of wild female An. gambiae s.l. local populations to different concentrations of permethrin in sentinel districts of mainland Tanzania. The red line at 98% mortality indicates any mortality above that is susceptible and any mortality below that represent either suspect (90–97%) or confirmed resistance (90%)

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Fig. 3
figure 3

Percentage mortality of wild female An. gambiae s.l. local populations to different concentrations of deltamethrin in sentinel districts in mainland Tanzania. The red line at 98% mortality indicates any mortality above that is susceptible and any mortality below that represents either suspect (90–97%) or confirmed resistance (90%)

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Fig. 4
figure 4

Percentage mortality of wild female An. gambiae s.l. local populations to different concentrations of alphacypermethrin in sentinel districts of mainland Tanzania. The red line at 98% mortality indicates any mortality above that is susceptible and any mortality below that represents either suspect (90–97%) or confirmed resistance (90%)

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Synergist tests with PBO

Encouragingly, there was full restoration of susceptibility to permethrin and deltamethrin following pre-exposure of An. gambiae s.l. to PBO (mortality rate of > 98%) in almost all the sentinel sites except Kakonko, Tandahimba, and Newala districts (Fig. 5A, B).

Fig. 5
figure 5

Comparison of mortality rates of An. gambiae s.l. exposed to deltamethrin (0.05%) (A) and permethrin (0.75%) (B), alone and in combination with PBO, per site. The red line at 98% mortality indicates any mortality above that is susceptible and any mortality below that represents either suspect (90–97%) or confirmed resistance (90%)

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Species composition within the Anopheles gambiae complex

A total of 4,400 wild An. gambiae were subjected to molecular analysis by PCR to identify species composition within the An. gambiae complex. Of these 4, 341 (98.7%) specimens were successfully amplified, but 59 (1.3%) specimens which morphologically resembled An. gambiae did not amplified. Of those that were amplified, 3883 (89.5%) were Anopheles arabiensis, 455 (10.5%) Anopheles gambiae sensu stricto (s.s.), 2 (0.05%) Anopheles merus, and 1 (0.02%) Anopheles quadriannualatus. The non-amplified Anopheles were subsequently re-analysed using specific primers for Anopheles rufipes and Anopheles maculipalpis. Of the 59 initially non-amplified samples, 11 (0.2%) were identified as An. rufipes and 1 (0.02%) as An. maculipalpis. The remaining unamplified specimens (47) were exposed to PCR-specific primer for Anopheles stephensi, but could still not amplify. These unamplified specimens have been stored for further investigation. Figure 6 shows the breakdown of the species composition of An. gambiae s.l. across sentinel districts.

Fig. 6
figure 6

Percentage distribution of Anopheles gambiae s.l. sibling species in 22 sentinel districts. Footnote: One An. quadrannulatus was identified in Bariadi, and two An. merus was identified in Kilwa

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Insecticide resistance mechanisms

Knock down resistance (kdr-L995S/L995F) mutations

Mosquitoes from all the sentinel districts were screened for kdr-east (L995S) and kdr-west (L995F). kdr-east (L995S) was detected in mosquitoes from 21 districts (95.5%) out of 22 sentinel districts with an overall allelic frequency of 46% in An. arabiensis. The kdr-west (L995F) was detected in mosquitoes from 20 districts (90.9%) out of 22 districts with an overall allelic frequency of 35% in An. arabiensis (Table 2). Similarly, both kdr-east (L995S) and kdr-west (L995F) were detected in An. gambiae s.s. with overall allelic frequencies of 44% and 25% respectively (Table 3). There was no variation detected for the species-specific association of kdr-east (L995S) and kdr-west (L995F) for either An. arabiensis or An. gambiae s.s. (p = 0.8). Some individual mosquitoes had mixed kdr- containing both kdr-east (L995S) and kdr-west (L995F) mutations.

Table 2 Distribution and allelic frequencies of kdr-east (L995S) and kdr-west (L995F) mutations among the An. gambiae s.s. in the 9 sentinel districts where An. gambiae s.s. were found
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Table 3 Distribution and allelic frequencies of kdr-east (L995S) and kdr-west (L995F) mutations among the An. arabiensis in the 22 sentinel districts
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Ace-1R (119S) mutation

A total of 4533 mosquito specimens were screened for the presence of the ace-1R (119S) point mutation. Of these, 4189 were An. arabiensis and 344 were An. gambiae s.s. The overall frequency of ace-1R (119S) mutation among An. arabiensis and An. gambiae s.s. samples were 1.0% and 0.58%, respectively. The ace-1R (119S) mutation detected in An. arabiensis was at low frequencies in all 12 sites, at frequency ranging from 0.25% to–3.47% (Table 4). Unlike An. arabiensis, the ace-1R (119S) mutation for the An. gambiae s.s. were only detected in two sites, Kakonko and Ukerewe, with allelic frequency of 8.33% and 2.34%, respectively (Table 4). Generally, the frequency of ace-1R (119S) mutation remains low despite the wide use of organophosphate and carbamates in the agricultural sector.

Table 4 Distribution and allelic frequencies of ace-1R (119S) mutations among An. arabiensis and An. gambiae s.s. in the 12 sentinel districts registered with this resistance gene
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Discussion

Of the sibling species of An. gambiae s.l., An. arabiensis appears to be the predominant malaria vector across sentinel sites in Tanzania. This means that the resistance profile discussed in this report largely reflects this sibling species. This predominant malaria vector species exhibits a range of low to high resistance intensity to pyrethroids, except in one sentinel district, which was fully susceptible to pyrethroid. Anopheles arabiensis also were largely susceptible to pirimiphos-methyl (an insecticide commonly used for IRS) across sentinel districts. kdr-east and kdr-west are in high frequencies and widely distributed geographically. Encouragingly, despite wide resistance to pyrethroids, this species exhibited full restoration to susceptibility following pre-exposure to PBO in almost all sites except 3 of the 16 sites.

The dominance of An. arabiensis relative to its sibling species across sentinel districts supports recent findings from the Tanzania NMCP’s country-wide malaria vector entomological survey [41]. Following widespread use of standard pyrethroid-based ITNs in Tanzania, and elsewhere in the region, the proportion of An. arabiensis has been progressively increasing relative to its sister species An. gambiae s.s. [42,43,44], which was previously the main malaria vector in Tanzania, and across SSA [45,46,47]. This is because the use of standard ITNs has been effective against An. gambiae s.s. due to its behaviour of feeding predominantly indoor and later at night, thus increasing its vulnerability to fatal contact with ITNs [48]. Despite the predominance of An. arabiensis, across sentinel sites, An. gambiae s.s. still exist in appreciable proportion in some settings in the northwest and southeast of Tanzania. This persistence merits attention, and it could be linked to consistently low utilization of ITNs, a common observation in urban settings like Dar es Salaam [49] where this species has remained prevalent [50, 51].

The high frequency and intensity of pyrethroid resistance observed is consistent with previous findings [8]. This might be attributed to increased insecticide pressure driven by the cumulative effect of the wide use of standard ITNs over a long period, in the setting of heavy reliance on pyrethroids in Tanzania’s agriculture sector [8, 52,53,54,55]. This widespread resistance threatens malaria control efforts [56], despite the lack of clarity regarding the relative contribution of resistance versus other ITN-related factors to the stalled decline of malaria cases and deaths, globally [3]. While standard pyrethroid-based ITNs may continue to provide direct personal protection due to the physical barrier provided by the ITN [57, 58], in this area with wide-spread pyrethroid resistance, the community-level effect [59] may be hugely attenuated or negligible, and consequently, might not have a significant impact on reducing human malaria infection [60]. This is because of the minimized impact on reducing mosquito survival, which allows continued malaria transmission [61].

The full restoration of pyrethroid susceptibility following pre-exposure to PBO in most sites in this study suggests that cytochrome P450 monooxygenases may be playing a more significant role in the observed pyrethroid resistance phenotype than kdr. PBO increases the effectiveness of pyrethroids by inhibiting the cytochrome P450-monooxygenase-mediated metabolic enzyme defense system within the mosquito [62,63,64]. The restoration of pyrethroid susceptibility across almost all sentinel sites in Tanzania is encouraging and strongly supports the Tanzania NMCP’s decision to transition to PBO-based ITNs. Recent trials of new-generation nets (PBO- and/or chlorfenapyr-based ITNs) in Tanzania and elsewhere demonstrate significant reductions in human malaria infections relative to standard ITNs [65, 66]. These findings serve as a basis for justifying the prioritization of new-generation nets in settings with widespread pyrethroid resistance, as it offers an approach to tackling insecticide resistance to sustain malaria control gains and/or accelerate toward subnational malaria elimination [67]. Nevertheless, the lack of full restoration to susceptibility upon pre-exposure to PBO detected in a few sites merits consideration and close monitoring. At present, it remains unclear whether this lack of restoration was just by chance or driven by other underlying mechanisms of resistance.

Knockdown resistance mechanisms were identified in almost all sentinel districts. Both the L995S (kdr-east) and L995F (kdr-west) mutations were identified at high allelic frequencies across the country. The occurrence of L995F (kdr-west) mutations is becoming increasingly common in Tanzania, following its first detection in 2014 [6] and subsequent increase in frequency, geographic distribution, and co-occurrence with L995S (kdr-east) [8]. Of note, kdr is now widely considered to be a fixed allele, highlighting the importance of other markers like ace-1, which can better elucidate the drivers of resistance. Alternatively, the interaction of the kdr mutation and the overexpression of detoxification enzymes could strengthen the intensity of pyrethroid resistance, as demonstrated in Burkina Faso with Anopheles coluzzii [68].

This study had limitations. The cross-sectional design which represents one-time data collection limited seasonal representation, which could impact insecticide exposure-based responses in the mosquitoes. It also tested only one species complex, An. gambiae s.l., despite the presence of other malaria vector species, including Anopheles funestus, in different parts of the country. Despite these limitations, the study provides useful information for the selection and implementation of high-impact insecticide-based malaria interventions.

Conclusions

This study strengthens previous evidence of widespread pyrethroid resistance against the predominant malaria vector in Tanzania. The full restoration of susceptibility to pyrethroids upon pre-exposure to PBO, irrespective of the spread of the kdr in this population, suggests that metabolic resistance mechanisms could be the major underlying mechanism of resistance determining the observed resistance phenotype in this setting. Therefore, prioritizing the timely deployment of new types of ITNs, including PBO synergists or dual-active ingredients, could effectively address this pyrethroid resistance mechanism in settings like Tanzania, where national malaria programs wish to sustain the gains in malaria control and accelerate the journey towards malaria elimination. To this end, continued close monitoring in the few sites without full susceptibility restoration is essential to ascertain the presence of other resistance mechanisms besides oxidase metabolic resistance.

Availability of data and materials

No datasets were generated or analysed during the current study.

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Acknowledgements

We are grateful to the invaluable contributions and support from various people and institutions. We are grateful to the Ministry of Health (MOH) through National Malaria Control Program (NMCP) for their collaboration and logistic support. We would like to extend our sincere gratitude to PO-RALG for providing us the guidance and permit for the entry to the local government. We are also thankful to the District Executive Directors and District Medical Officers from the respective study districts for their invaluable co-operation in allowing their District Vector Control Officers/Health Officers to participate and support the resistance monitoring activities

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention or the U.S. Agency for International Development.

Funding

This study was made possible by the generous financial support of the American people through the U.S. President’s Malaria Initiative (PMI) under the terms of USAID/PSI Cooperative Agreement number 72062122CA0008 with subcontract awarded to Ifakara Health Institute. Part of the study, especially, the electronic data and management system and both analysis and development of the manuscript was jointly supported by UKRI-Medical Research Council (under the African Research Leaders Award number MR/T008873/1) and the UK Foreign, Commonwealth & Development office (FCDO) under the MRC/FCDO concordant agreement which is also part of the EDCTP2 programme supported by the European Union. SI.

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  1. Bilali Kabula and Yeromin P. Mlacha have contributed equally to this manuscript.

Authors and Affiliations

  1. Amani Medical Research Centre, National Institute for Medical Research, Muheza, Tanzania

    Bilali Kabula

  2. Environmental Health and Ecological Science Department, Ifakara Health Institute, Mikocheni, Dar es Salaam, Tanzania

    Yeromin P. Mlacha, Samson Kiware, James S. Michael, Victor Mero, Said Abbasi & Nicodem J. Govella

  3. U.S. President’s Malaria Initiative, US Agency for International Development, Dar es Salaam, Tanzania

    Naomi Serbantez

  4. National Malaria Control Programme, Ministry of Health, Community Development, Gender, Elderly and Children, Dodoma, Tanzania

    Samwel L. Nhiga & Charles D. Mwalimu

  5. University of Dar es Salaam, College of Information and Communication Technologies, Department of Electronics & Telecommunication Engineering, P.O. Box 33335, Dar es Salaam, Tanzania

    Sigsbert Mkude & Nicodem J. Govella

  6. PMI Dhibiti (Control) Malaria Project, Population Services International, Dar es Salaam, Tanzania

    James S. Michael

  7. U.S. President’s Malaria Initiative, US Centers for Disease Control and Prevention, Dar es Salaam, Tanzania

    Sarah-Blythe Ballard

  8. U.S. President’s Malaria Initiative, US Centers for Disease Control and Prevention, Atlanta, USA

    Adeline Chan

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Contributions

NJG, SM, NC, AC, SB, CDM, SLN conceived the study. NJG, YM, BK designed the study. NJG, YM, BK, SK, SA, and JM, implemented the study and data analytical support. KB, YM drafted the manuscript with support from NJG. NJG, NS, SB, and AC reviewed and edited the draft manuscript. All authors reviewed and approved the final version of the manuscript.

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Correspondence to Yeromin P. Mlacha.

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Kabula, B., Mlacha, Y.P., Serbantez, N. et al. Pyrethroid-resistant malaria vector Anopheles gambiae restored susceptibility after pre-exposure to piperonyl-butoxide: results from country-wide insecticide resistance monitoring in Tanzania, 2023. Malar J 23, 395 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12936-024-05211-7

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Malaria Journal

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