The status of insecticide resistance of Anopheles coluzzii on the islands of São Tomé and Príncipe, after 20 years of malaria vector control

doi:10.21203/rs.3.rs-4993272/v1

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Research Article

The status of insecticide resistance of Anopheles coluzzii on the islands of São Tomé and Príncipe, after 20 years of malaria vector control

https://doi.org/10.21203/rs.3.rs-4993272/v1

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Background

Insecticide based malaria vector control has been implemented on the islands of São Tomé and Príncipe (STP) for more than 20 years. During this period malaria incidence was significantly reduced to pre-elimination levels. While cases remained low through 2015, there has been a steady increase in cases since that time, challenging the country’s commitment of achieving malaria elimination by 2025. To better understand the reasons for increasing malaria cases, we characterized levels and underlying mechanisms of insecticide resistance in local Anopheles coluzzii populations.

Methods

Mosquito larval collections were performed in the rainy and dry seasons, between 2022 and 2024, in two localities in São Tomé and one in Príncipe. Susceptibility to permethrin, α-cypermethrin, pirimiphos-methyl and DDT was assessed using WHO bioassays and protocols. Intensity of resistance and reversal by PBO pre-exposure were determined for pyrethroid insecticides. The kdr locus was genotyped by PCR assays in subsamples of the mosquitoes tested.

Results

Anopheles coluzzii populations were fully susceptible to pirimiphos-methyl but high levels of resistance to pyrethroids and DDT were detected, particularly in São Tomé rainy season collections. Increasing the pyrethroid and DDT dosages to 5\(\:\times\:\) and 10\(\:\times\:\) did not restore full susceptibility in all populations. Pre-exposure to PBO resulted in partial reversal of the resistance phenotype suggesting the presence of cytochrome P450 oxidase-mediated metabolic resistance. The L1014F knockdown resistance mutation was present in An. coluzzii on both islands but at much higher frequency in São Tomé where it was associated with the resistant phenotype.

Conclusions

Future vector control interventions should consider the use of non-pyrethroid insecticides or combination with synergists to overcome the high levels of pyrethroid resistance. Alternative control methods not dependent on the use of insecticides should be additionally implemented to achieve malaria elimination in STP.

Malaria

Anopheles coluzzii

insecticide resistance

kdr mutations

São Tomé and Príncipe

Several attempts to reduce the malaria burden on the Islands of São Tomé and Príncipe (STP) have been made throughout the years. In the early 1980’s, a malaria eradication program using biannual indoor residual spraying (IRS) of dichloro-diphenyl-trichloroethane (DDT) and weekly prophylaxis with chloroquine reduced malaria prevalence from 19.2–0.6% by 1982 [1]. After abrupt cessation of this malaria control program, malaria incidence rose to epidemic proportions in 1985 to 1986 with high case-mortality rates that were attributed to combinations of financial constraints and vector resistance to DDT [1]. High caseloads continued exacerbated by chloroquine resistance [2].

Starting in 2004, scale-up interventions designed to achieve millennium development goals for malaria were implemented in STP. These included IRS with α-cypermethrin, pyrethroid impregnated long lasting insecticidal nets (LLIN), artemisinin-amodiaquine and artemether-lumefantrine as first-line and second-line drugs for treatment of uncomplicated malaria, and intermittent preventive therapy with sulfadoxine-pyrimethamine [3]. An evaluation after three years of this scaled-up intervention demonstrated remarkable success by decreasing patient consultations, hospitalizations and deaths by more than 85%, 80%, and 95%, respectively, in all age groups, resulting in an overall parasite prevalence of 2.1% and leading Príncipe island to pre-elimination levels [3, 4].

In 2021, STP was selected by the WHO as a priority country for malaria elimination under the E-2025 initiative [5]. This meant reinforced financial investment and technical support commitments to improve vector control with measures such as i) rotational use of active ingredients (α-cypermethrin, malathion, pirimiphos-methyl and clothianidin) for IRS, ii) distribution of new generation LLIN impregnated with α-cypermethrin and, more recently a mosaic of α-cypermethrin and piperonyl butoxide (PBO), and iii) widespread larval control with Bacillus thuringiensis israelensis. In a report from the Ministry of Health, malaria incidence and mortality decreased a further 47% from 2010 to 2015 (unpublished data), but since then overall malaria incidence has remained constant [6]. In the first 12 weeks of 2024, official data from the National Malaria Elimination Program show a doubling of cases compared to the same 12 weeks in 2022 and 2023. Because of this increase, the STP Ministry of Health has expressed challenges precluding the achievement of malaria elimination by 2025, including a lower compliance of residents with preventative measures such as allowing IRS access and LLIN use and possibly, insecticide resistance.

Presently, the sole vector of malaria in STP is the forest cytoform of Anopheles coluzzii [7–9]. In contrast to mainland populations, this mosquito displays marked exophagic, exophilic and zoophilic behaviors, possibly due to environmental conditions (e.g. house construction) and the effect of indoors-based vector control [10–12]. Anopheles coustani, the only other anopheline in the archipelago, is restricted to the Island of São Tomé, where it is much less common than An. coluzzii and its status as a vector of malaria is currently unknown [7, 8].

An important consideration challenging malaria control is the issue of insecticide resistance. Several countries in Africa have reported resistance to the four classes of insecticides (pyrethroids, organophosphates, carbamates and organochlorines) mostly used in public health in at least one malaria vector species and high intensity resistance to pyrethroids has been detected more frequently in west Africa than in other subregions [13]. Two point mutations at position 1014 of the voltage-gated sodium channel gene, resulting in the substitution of a leucine residue by a phenylalanine (L1014F or kdr-w) or a serine (L1014S or kdr-e) are considered primary mutations conferring resistance to pyrethroids and DDT [14, 15]. Haplotype data suggests that kdr-w arose multiple times in Anopheles gambiae s.s. and underwent a selective sweep across populations [16–18]. This mutation introgressed into An. coluzzii through hybridization with An. gambiae s.s. in specific locations and then selectively swept across West and Central African populations [19–21]. The kdr-e mutation has also selectively swept through populations of both species but also may have arisen independently [16, 17]. In addition to target-site mutations, insecticide resistance is also governed by metabolic mechanisms involving detoxifying enzyme superfamilies [22]. Of these, cytochrome P450 oxidases play a central role in resistance to pyrethroids [23], the major insecticide class used for bed net treatment.

In this study, we investigated current and past knockdown resistance genotypic frequencies and levels of insecticide resistance in An. coluzzii from STP in order to: (1) assess the status of resistance to pyrethroid and non-pyrethroid insecticides; (2) determine mechanisms underlying the resistance phenotypes, if present.

Mosquito collections

Larval collections were performed by standard methods using dippers and pipette in two localities of São Tomé island and one in Príncipe island (Fig. S1, Supplementary Materials). In São Tomé, collections were carried out in Água Grande district and Ribeira Afonso in July-September 2022 (dry season) and again in May-June 2023 (rainy season). The district of Água Grande includes the capital city of the country (São Tomé) and is characterized as an urban setting where over 60% of the ca. 220,000 inhabitants of São Tomé Island live [24, 25]. The village of Ribeira Afonso harbors ca. 2,000 inhabitants living mainly from fishery and subsistence agriculture. In Príncipe, collections took place in St. António in July 2023 (dry season) and January 2024 (rainy season). This is the only urban center of the island, where more than 80% of the ca. 9,000 human inhabitants of this island live.

Field collected larvae were reared to adults in trays containing water taken from the collection site and fed daily with ca. 10 mg of Koi fish food (Doctors Foster and Smith Koi Staple Diet, Rhinelander, WI USA). Water levels in the trays were kept constant by adding mineral water. Pupae were picked daily, and every three the pupae were placed in a screen cage (30 \(\:\times\:\:\)30\(\:\:\times\:\) 30 cm), in an insectary set at 27ºC, 80% relative humidity and 12:12 h light: dark cycle. Emerged adults were provided constant access to 10% sucrose solution until 12 h before insecticide bioassay.

Bioassays

Insecticide susceptibility to permethrin (0.75%), α-cypermethrin (0.05%), pirimiphos-methyl (0.25%) and dichloro-diphenyl-trichloroethane (DDT) (4%) was tested by WHO tube tests and protocols [26, 27]. A minimum of 100 three to five days-old non-blood fed females were exposed for one hour in WHO tubes with treated paper (diagnostic dose of each insecticide) along with 50 control specimens in WHO tubes with control papers (insecticide carrier). Mortality was recorded 24 h after exposure. When mortality in control tubes was between 5% and 20%, mortality in treated tubes was corrected using Abbott’s formula. The population was deemed resistant if mortality was below 90%, suspected resistant if mortality was between 90% and 98%, and susceptible if mortality was above 98% [26].

Whenever resistance was detected for pyrethroids (i.e. permethrin and α-cypermethrin) intensity of resistance was tested by exposing mosquitoes to 5\(\:\times\:\) and 10\(\:\times\:\) the diagnostic dose [26]. In addition, presence of metabolic resistance mediated by monooxygenase enzymes was assessed by exposing mosquitoes to the synergist piperonyl butoxide (PBO) prior to insecticide exposure, following the WHO (2022) [28] protocol. PBO restores susceptibility by inhibiting the monooxygenases of the resistant mosquito.

Genotyping of the knockdown resistance locus

Genomic DNA was extracted from pyrethroid susceptible, resistant and control individual mosquitoes using the peqGOLD Blood and Tissue DNA Mini Kit (VWR Avantor, Radnor PA, USA). Mosquitoes were identified to species using an established PCR-RFLP assay targeting species-specific polymorphisms at the intergenic spacer of the ribosomal DNA [29]. Reactions were performed on 10µL volume containing 1µL (~ 1.4 ng) of DNA template.

For genotyping of the kdr locus, two allele-specific PCR assays were employed using the primers described by Martinez-Torres et al. [14] for the L1014F kdr-w mutation, and Ranson et al. [15] for the L1014S kdr-e mutation. Each PCR reaction was made in a volume of 15µL containing 1µL (~ 1.4 ng) of DNA template, 0.1mM of primers Agd1 and Agd2, 0.4mM of primers Agd3 (or Agd5) and Agd4, and 1\(\:\times\:\) Taq Plus master Mix (VWR Avantor, Radnor PA). The PCR cycling conditions consisted of an initial step of 5 min at 94ºC followed by 35 cycles each with 30 s at 94ºC, 40s at 48ºC (Agd3) or 55ºC (Agd5) and 40s at 72ºC, and a final 10 min extension at 72ºC. Amplified products were visualized under UV light after separation by electrophoresis in 1.5% agarose gels stained with GreenSafe Premium (NZYTech, Lisbon, Portugal).

Historical dataset

In addition to the data collected in 2022–2024, we present the results of two surveys carried out in Água Grande District, in April 2003 (rainy season) and September 2004 (dry season). WHO bioassays and procedures [30] were used to test permethrin (0.75%) and DDT (4%) at the diagnostic doses only. Non-blood fed females were used in the tests, but these mosquitoes were collected by landing catches performed with WHO aspirators between 20.00 and 00.00 h, so that mosquito age was not controlled in the assays. Subsamples of tested mosquitoes were molecularly identified to species and genotyped for the kdr locus as described above.

Data analysis

When mortality rates were between 90% and 98% a replicate of the diagnostic exposures was performed [26], and the value considered was the simple mean between replicates. Significant differences between groups were assessed by chi-squared tests on contingency tables or by the non-parametric equivalent Fisher’s exact test.

The results of susceptibility assays conducted at diagnostic doses for the four insecticides tested are shown in Table 1. In all tests, there was 100% mortality to pirimiphos-methyl indicating full susceptibility to this insecticide. In contrast, resistance to DDT and pyrethroids was found in most cases with mortality rates < 90%. In general, susceptibility was higher in Príncipe island (St. António) when compared with the two localities of São Tomé island. There was also a consistent trend for higher levels of susceptibility during the dry season when compared to the rainy season. These seasonal differences were significant in all cases where data was available for comparison (Table 1).

Table 1

*An. coluzzii* mortality rates at diagnostic doses in samples collected in São Tomé and Príncipe
		Água Grande			Ribeira Afonso			St. António
		N	%	p	N	%	p	N	%	p
\(\:\alpha\:\)-cypermethrin	Dry	122	100.0	< 0.001	225	97.3*	< 0.001	225	92.3*	0.003
	Rainy	102	80.2		117	47.9		221	85.1*
Permethrin	Dry	242	87.6*	< 0.001	102	98.1	< 0.001	-	-	N.A.
	Rainy	109	60.6		108	73.2		104	85.6
Pirimiphos-methyl	Dry	115	100.0	N.A.	116	100.0	N.A.	111	100.0	N.A.
	Rainy	116	100.0		118	100.0		114	100.0
DDT	Dry	218	90.5*	0.007	108	100.0	< 0.001	110	99.1	1.000**
	Rainy	112	79.9		116	79.3		108	100.0
N: number of female mosquitoes exposed to the insecticide. %: percentage mortality 24 hours after insecticide exposure; average of two replicate experiments when the first exposure gave a mortality rate between 90% and 98% [34]. p: p-value of a qui-square test on contingency table with two degrees of freedom comparing dry and rainy season mortalities. * Two-sided Fisher’s test.

Increasing the dosage of pyrethroids to 5\(\:\times\:\) and 10\(\:\times\:\) the diagnostic dose resulted in increased mortality but not always sufficient to reach susceptibility levels (i.e., \(\:>\)98%) (Fig. 1). This was the case for α-cypermethrin in Água Grande and Ribeira Afonso, and for permethrin in Água Grande, where mortality at 10\(\:\times\:\) the diagnostic exposure varied between 89% and 93%. Pre-exposure to PBO significantly increased mortality to pyrethroids in samples collected in the rainy season, but complete reversal to susceptibility was only seen in Ribeira Afonso for both pyrethroids (Fig. 2). Differences between PBO + pyrethroid and pyrethroid only were non-significant for the two assays carried out in the dry season.

Figure 3 shows the results of the genotyping of the kdr locus. The L1014F (kdr-w) mutation was the only one found and it was detected in the three localities albeit at varying frequency, being highest in Água Grande, intermediate in Ribeira Afonso and lowest in St. António. There was no evident trend between seasons in kdr frequency. In Água Grande, it was higher in the rainy season (chi-square test, p = 0.024) and the opposite was found in Ribeira Afonso (chi-square test, p = 0.105). In Água Grande, in both seasons, there was a significant association between the resistance phenotype and the presence of the kdr allele (Fig. 3). This association was not so evident in Ribeira Afonso, where a marginally significant association was detected in the dry season only.

The comparison of current susceptibility levels to DDT and permethrin found in Água Grande with those recorded in 2003 and 2004, suggests a slight but steady decrease of mortality rates from 2004 onwards (Fig. S2 A, Supplementary Materials). No kdr mutations were found in 2003 (N = 303) or 2004 (N = 100) compared to the 16% and 11% frequency of the kdr-w allele found in 2022 and 2023, respectively (Fig. S2 B, Supplementary Materials).

High levels of resistance to pyrethroids were found in An. coluzzii, particularly in São Tomé island. Mortalities below 90% were obtained in exposures to 10\(\:\times\:\) the diagnostic dose. While this may be considered a predictor of operational failure, the real impact of the insecticide resistant phenotype in vector control remains uncertain [26, 31, 32]. The high levels of pyrethroid resistance found in STP may be a consequence of the intensive usage of this insecticide class for malaria control over the past 20 years in the country [3, 33, 34]. Permethrin has been used in STP since the 1990s in periodical campaigns of bed nets impregnation. Following the introduction of LLIN in 2004 two additional pyrethroids, deltamethrin and α-cypermethrin, were introduced and widely used in the country [35]. In addition, α-cypermethrin was the insecticide of choice for IRS in the National Strategic Plan to Control Malaria implemented in 2004, being used for two consecutive years [33].

When comparing the two islands, pyrethroid resistance was consistently lower in Príncipe island. São Tomé island, and Água Grande district in particular, concentrates over 90% of the ca. 220.000 inhabitants of the country. Furthermore, malaria prevalence has been consistently lower in Príncipe and this region has implemented since 2023 a regime of reactive indoor spraying as opposed to the large-scale IRS employed in São Tomé [6, 7, 36]. These differences may be explained by a lower insecticide selective pressure in Príncipe when compared to São Tomé island.

The presence of the L1014F kdr mutation coupled with partial reversal of the resistant phenotype when mosquitoes were pre-exposed to PBO suggest that pyrethroid resistance in An. coluzzii from STP is mediated by both target-site and metabolic mechanisms. The inhibition of cytochrome P450 oxidase activity by PBO and the consequent increase in mortality after exposure to permethrin or α-cypermethrin suggests a role of this detoxifying enzyme superfamily in the resistance phenotype. This type of metabolic resistance has been reported in continental populations of An. coluzzii, including in nearby west African countries, many times also with the concomitant presence of kdr mutations [37–39]. Among the cytochrome P450 oxidase genes mostly associated with pyrethroid resistance in this vector species are CYP6M2, CYP6Z2 and CYP9K1 [23, 38]. Further studies involving gene expression analysis will be required to ascertain the specific genes involved in the resistance phenotype of An. coluzzii in STP.

The pyrethroid resistance-associated L1014F kdr mutation was found in both islands albeit at higher frequency in São Tomé island, where positive associations between kdr-w and the resistant phenotype were found. This result agrees with other studies that show increasing frequencies of resistance-associated alleles and the development of resistance after deployment of insecticide-based vector control [40, 41]. In STP, the kdr-w mutation was not detected in collections performed in 2003 and 2004 (present study) suggesting a recent emergence of this resistance-associated allele. The first record of the kdr-w allele was in 2010 in São Tomé island, six years after the implementation of IRS with α-cypermethrin, whereas in Príncipe this mutation was reported for the first time in 2014 [33]. However, there is no information about genotyping of kdr mutations between 2004 and 2010 and the first report showed that the kdr-w mutation was already present in 4 districts of São Tomé island in 2010 [33]. The emergence of the kdr-w allele in STP may be a result of mutational events or introgression with kdr-carrying mosquitoes that may have accidently reached the islands. Both origins, including interspecific introgression, have been previously described throughout the African continent [16–20]. Further studies involving haplotype analysis of genomic regions surrounding the kdr locus would help clarify the origin of the kdr-w allele in STP.

Haplotype diversity analysis has also shown evidence for strong positive selection of the kdr-w allele in West Africa, which may explain the rise of this allele from 0.3–54% in An. coluzzii from Ghana [18]. However, such an increase was not so evident in An. coluzzii from STP. The initial report of Chen (2019) [33] describes kdr-w frequencies of 13% in 2010, reaching a peak in 2013 with 78% and decreasing to 40% in 2016. A second report shows kdr-w frequencies between 8% and 44% in three districts of São Tomé island sampled in 2018 [42]. These values contrast with the kdr-w frequencies found in this study (2022–2024) that did not exceed 16%. A decrease in kdr-w frequency may be explained by a relaxation of the selective pressure due to the interruption of pyrethroid-based IRS campaign in 2021 when a rotation scheme with non-pyrethroid insecticides started to be employed for IRS. It may also reflect a possible increase of the outdoor biting and resting preferences of this mosquito vector on the islands leading to a lower selective pressure [10, 11, 33].

Another important aspect was the differences found in susceptibility levels between seasons, with a significant trend of increased resistance in the rainy season. Dry season sampling bias due to lower number of breeding sites might contribute to these differences but this would be a more plausible explanation if resistance was found to be higher in the dry season, suggesting that more siblings harboring kdr alleles (or other resistance mechanisms) had been used in bioassays. However, the differences found between seasons on kdr genotype distribution were only marginally significant in Água Grande and do not seem to explain the observed phenotypic patterns. A more plausible explanation may be attributed to seasonal differences in the physiological/nutritional status of An. coluzzii. We argue that during the rainy season the greater availability of larval habitats suitable for mosquito breeding reduces inter- and intraspecific competition leading to a higher nutritional intake and resulting in adults with an average higher percentage of body fat tissue, known to be rich in detoxifying enzymes [43, 44]. This hypothesis needs confirmation as it contrasts with previous observations that dry season mosquitoes are more resistant than rainy season ones [45, 46].

The higher resistance levels found in the dry season are generally explained by the selective pressure of pollutants in larval habitats that lead to increased expression of detoxifying enzymes such as P450 oxidases [46]. This is more evident in rural areas with marked seasonality where agriculture insecticide usage occurs mainly during the dry season. This may not be the case for São Tomé, in which the short duration of the dry season may not be sufficient to promote a high concentration of xenobiotics in larval habitats.

Regarding non-pyrethroid insecticides, resistance to DDT was also observed in São Tomé (but not in Príncipe). This agrees with the historical use of DDT during the malaria eradication program in the 1980s and the emergence of resistance to this insecticide has been considered a major reason for disruption of this program [1, 24]. This may also reflect cross-resistance, as DDT shares with pyrethroids the same active site, the voltage-gated sodium channel, and the L1014F mutation confers resistance to both insecticide classes [47, 48].

Anopheles coluzzii populations were fully susceptible to pirimiphos-methyl. This result may be explained by the fact that pirimiphos-methyl was used only in one campaign in the country (2019), and subsequently implemented in the rotation scheme, which appears to work positively to prevent resistance from arising [34]. This result contrasts to that observed for pyrethroids when widely used for consecutive years. While mechanisms of organophosphate resistance are likely to be absent from these island populations, this may also reflect lower selective pressure associated with the outdoors behavior of this mosquito in STP.

The high levels of insecticide resistance found, specifically in São Tomé island and during the rainy season, reflect the selective pressures exerted by the extensive usage of pyrethroid insecticides for malaria vector control and raises concern about the efficacy of insecticide-based measures that are being implemented for malaria control. Recently, malaria cases have been increasing at a concerning pace in STP which may reflect a reduced efficacy of pyrethroid-based vector control. When planning future vector control interventions, selection of non-pyrethroid insecticides employed in rotation, and application of vector control at specific times of the year should be taken into account. In addition, alternative non-insecticidal malaria control tools should be considered in the malaria control strategic plan of the country.

WHO

World Health Organization

IRS

Indoor residual spraying

LLIN

Long lasting insecticidal nets

DDT

dichloro-diphenyl-trichloroethane

PBO

Piperonyl butoxide

Kdr

Knockdown resistance

Bti

Bacillus thuringiensis isrealensis

PCR-RFLP

PCR-Restriction Fragment Length Polymorphism.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and additional files.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was implemented in the framework of the University of California Malaria Initiative (http://stopmalaria.org) with funding from the Open Philanthropy Project (www.openphilanthropy.org) (A20-3521-001).

Author’s contributions

JP, AJC and GCL conceived and designed the study. JP and MC managed the study. MC, CAS, JV, RO and JP did the field collections and the bioassays. GR, JL, AC, MC and JP did the laboratory analyses and ensured data quality. All authors read and approved the final manuscript and agreed on its submission.

Acknowledgments

To the people of STP for embracing our team during the development of the UCMI Project. To the entomology technicians of the Centro Nacional de Endemias and the Health District Delegacies for the support given in mosquito sampling.

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The status of insecticide resistance of Anopheles coluzzii on the islands of São Tomé and Príncipe, after 20 years of malaria vector control

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Abstract

Background

Methods

Results

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Background

Methods

Mosquito collections

Bioassays

Genotyping of the knockdown resistance locus

Historical dataset

Data analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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