Background
Strong commitment from international agencies and home governments to reduce the burden of malaria in sub-Saharan Africa has led to a major scale-up of vector control measures and increased access to effective anti-malarial treatment, and it is reported that malaria is on the wane in several African countries [
1]. However a major menace is threatening the present achievements. Resistance to pyrethroid insecticide is spreading rapidly across Africa and could reduce the impact of our two most successful malaria prevention interventions - indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) [
2‐
4]. The two main mechanisms responsible for pyrethroid resistance are target site insensitivity, known as knock down resistance
kdr, and metabolic resistance due to elevated levels of detoxifying enzymes [
2].
Kdr is caused by mutations to the sodium channel, a leucine to phenylalanine change first observed in West Africa [
5] and a leucine to serine mutation observed in East Africa [
6]. Recently a new mutation in the sodium channel conferring additional resistance to DDT and permethrin as been reported associated with the
kdr-west mutation [
7].
To reduce the malaria burden in Tanzania, the National Malaria Control Programme (NMCP) is increasing the coverage of LLINs and IRS. Over the last 10 years LLINs have been distributed initially by targeting the most vulnerable groups, pregnant women and children aged under five, through discounted vouchers issued at antenatal clinics [
8], and then by a national, free LLIN distribution campaign in 2010 [
9] which was extended to the general population though a universal coverage LLIN distribution campaign in 2011. IRS with pyrethroid was initiated in Kagera region, situated on the western shore of Lake Victoria, in 2006 with support from the President’s Malaria Initiative and extended to all Lake Zone in 2011.
Previous surveys conducted in Tanzania showed little or no resistance to DDT and pyrethroids in the
Anopheles gambiae s.l population [
10‐
12]. A study carried out in 2009/2010 showed no resistance to deltamethrin and DDT in Muleba [
10], north-western district of Tanzania, where the present study is conducted. In this paper, the insecticide resistance status of the malaria vectors
An. gambiae s.s and
Anopheles arabiensis to insecticides in use for IRS (lambdacyhalothrin and bendiocarb) and LLIN (permethrin and deltamethrin) is presented. The prevalence of the
kdr mutation was also investigated.
Results
Light trap collection
Seven rounds of light trap collection were completed in the 40 villages between April and December 2011 and a total of 5,762
An. gambiae s.l females were caught. The majority of mosquitoes (92%) were collected from 13 villages situated in the south-west of the district (Figure
1). A subsample of 2,346 was identified to species. The weighted proportion showed a ratio of 80% (95% CI: 75%-84%)
An. gambiae s.s to 20% (95% CI: 16%-25%)
An.arabiensis.
WHO resistance tests
Anopheles gambiae s.s accounted for 96.0% and An. arabiensis for 3.7% of the An. gambiae s.l tested for resistance (N = 901). Anopheles arabiensis was detected only in four sites, and made up 7.2% of An. gambiae s.l in Kishuro and 3.4% in Kikagate in surveys done in May, and 2.6% in Kyamorwa and 18.2% in Kiteme in the surveys done in November. All 100% An. arabiensis were killed in tests with, lambdacyhalothrin (n = 11), deltamethrin (n = 2), DDT (n = 10) and bendiocarb (n = 2), and 50% (4/8) in tests with the pyrethroid permethrin.
Anopheles gambiae s.s mortality to lambdacyhalothin 0.05% test papers ranged from 8% to 40% (Table
1) across the 11 villages sampled, indicating a high frequency of resistance in the area and some variation in resistance frequency by cluster. Mosquitoes tested in two villages, Kyarmorwa and Kikagate, showed a reduction in percentage mortality between May and November 2011 (from 34% to 8% in Kyarmorwa and from 40% to 26% in Kikagate). The resistance to lambdacyhalothrin extended to permethrin and deltamethrin (Table
2) and also showed cross-resistance to DDT with mortality ranging from 13% to 40% between villages. Similar temporal trends between surveys were observed to DDT as to lambdacyhothrin, indicating cross-resistance between DDT and pyrethroids.
Table 1
Mortality rates (95% confidence interval) and blood-feeding status of
Anopheles gambiae
s.l from various localities exposed to lambdacyhalothrin 0.05% in WHO resistance tests
May 2011 | Kikagate | 35 | 2 | 34% | (19–52) | 100% | 95% |
| Kyamyorwa | 230 | 12 | 40%3 | (33–46) | 70% | 100% |
Nov 2011 | Kishuro | 40 | 2 | 13% | (4–27) | 24% | 99% |
| Ngenge | 52 | 3 | 25% | (14–39) | 59% | 99% |
| Rwigembe | 42 | 2 | 31% | (18–47) | 84% | 100% |
| Buhuma | 30 | 2 | 17% | (6–35) | 78% | 100% |
| Kangoma | 105 | 6 | 8%1 | (3–14) | 40% | 96% |
| Kikagate | 142 | 7 | 8% | (4–14) | 74% | 98% |
| Kiteme | 104 | 6 | 31%2 | (22–41) | 92% | 99% |
| Kyamyorwa | 149 | 7 | 26%3 | (19–34) | 75% | 98% |
| Nyakahama | 13 | 1 | 38% | (14–68) | 77% | 100% |
| Buhanga | 36 | 2 | 22%3 | (10–39) | 86% | 98% |
| Kimbuga | 21 | 2 | 0% | (0–16) | 43% | 94% |
| Kisumu strain | 100 | 4 | 100% | (96–100) | | |
Table 2
Mortality rates of the
Anopheles gambiae
s.l field populations from various localities exposed to deltamethrin, permethrin, DDT and bendiocarb
Bendiocarb (0.1%) | May 2011 | Kyamyorwa | 112 | 6 | 100% | (97–100) | 100% |
Nov 2011 | Kyamyorwa | 106 | 7 | 84%1 | (76–90) | 78% |
| | Kangoma | 54 | 3 | 70%3 | (56–82) | 86% |
| | Kikagate | 100 | 5 | 86% | (78–92) | 65% |
| | Kiteme | 84 | 5 | 90%2 | (82–96) | 96% |
| | Kisumu strain | 61 | 4 | 97% | (89–100) | |
DDT (4%) | May 2011 | Kyamyorwa | 98 | 5 | 37% | (27–47) | 100% |
| Nov 2011 | Kyamyorwa | 99 | 5 | 13%1 | (7–21) | 88% |
| | Rwigembe | 8 | 1 | 13% | (0–53) | 36% |
| | Buhuma | 12 | 1 | 17% | (2–48) | 58% |
| | Kikagate | 20 | 1 | 35% | (15–59) | 55% |
| | Kiteme | 85 | 4 | 40%2 | (30–51) | 100% |
| | Kisumu strain | 100 | 4 | 100% | (96–100) | |
Deltamethrin (0.05%) | May 2011 | Kishuro | 20 | 1 | 70% | (46–88) | 100% |
| Kyamyorwa | 106 | 5 | 28%3 | (20–38) | 100% |
Permethrin (0.75%) | Nov 2011 | Kiteme | 98 | 5 | 11% | (6–19) | 98% |
Anopheles gambiae s.s showed reduced susceptibility to the carbamate bendiocarb in WHO resistance tests. Percentage mortality in the village Kyarmorwa was 100% in May 2011 and 84% in November 2011. Tests, done in other villages in November, produced mortality rates ranging from 70% to 90%. Checks done on the An. gambiae Kisumu susceptible strain produced 100% mortality on DDT and lambdacyhalothrin and 97% mortality with the bendiocarb test papers.
Of the
An. gambiae collected for testing, 79% were blood-fed, 14% were unfed and the remaining were gravid or semi-gravid (Table
1). There were variations in blood-feeding rates between the tests, however no differences in frequency of fed mosquitoes were observed in alive and dead mosquitoes exposed to lambdacyhalothrin (p = 0.15).
Kdr mutation
Of the 2,049 An. gambiae s.s collected by light trap and tested for the kdr east allele, 96.8% (n = 1,983) were homozygous for kdr, 3% (n = 62) were heterozygous and only 0.2% (n = 4) were homozygous for the susceptible type. All An. arabiensis tested (N = 297) were homozygous for susceptible type. There was no significant difference in genotype frequency in An. gambiae s.s between collection rounds (Chi2 = 1.2, df = 2, P = 0.55) or villages (Chi2 = 2.5, df = 2, P = 0.29).
Kdr genotype frequencies in the An. gambiae s.s collected resting in houses (later exposed to WHO resistance tests) were similar to the frequencies in the light trap collections. Of the 772 An. gambiae s.s tested 96.9% (747) were homozygous for kdr, 3.0% (24) were heterozygous and only one was wild type. None of the An. arabiensis tested (n = 31) carried the kdr east mutation. No kdr west mutation was found in any of the 176 An. gambiae s.s or An. arabiensis tested.
Allelic frequency was compared between An. gambiae s.s surviving or dying in the pyrethroid resistance tests. Because the kdr frequency was almost fixed (98%) there was no association between kdr-e allele frequency and the phenotypic resistance in WHO tests (lambdacyhalothin, permethrin, deltamethrin p-value = 1.0, DDT p-value = 0.59).
Discussion
Anopheles gambiae s.s resistance to pyrethroids and DDT was widespread throughout the study area of Muleba district in north-western Tanzania. The frequency of the kdr east approached fixation in the An. gambiae s.s population but was absent in An. arabiensis. Emerging resistance to bendiocarb was observed for the first time.
There was considerable variation in the density of mosquitoes between clusters. Greater densities of mosquitoes were to be found in the south-west. It was important to test samples of Anopheles from the north-east area to investigate resistance as a possible cause of the heterogeneity in Anopheles density between the two areas. While larger samples from the north-east were desirable, this was not possible with the sampling plan and resources available. The level of mortality recorded in the resistance tests was low and never exceed 40% in any of the clusters (and always read against a control) regardless the sample size tested and hence considerable confidence can be placed in the overall trend in resistance.
The samples for testing were deliberately chosen from adult collections to represent natural age-structured populations. These would be a mix of young and old mosquitoes, and because the level of resistance often decreases in ageing mosquitoes [
19,
20] the proportion surviving in the tests would have been higher had, for example, F1 adults reared from larval collections been chosen for testing instead. The disadvantage of using larval collections is the limited gene pool of the collected samples, and the possible bias in resistance frequency which is much less likely to occur with adult collections. While it is possible that the adult collection was under selection from decaying pyrethroid residues in houses, the resistance frequency would still be representative of the population.
In the resistance tests with lambdacyhalothrin on mosquitoes that were identified by PCR, 23% of
An. gambiae s.s and 100% of
An. arabiensis were killed overall. This is the first time that high-level pyrethroid resistance and a high frequency of the
kdr east mutation is reported in Tanzania. A national resistance survey conducted in 2009/2010 in 12 sentinel sites indicated that resistance is starting to be detected in other parts Tanzania but not at the levels found in Muleba district of Kagera region where IRS with lambdacyhalothrin has been intensively applied between 2006 and 2011 [
10]. In the Lower Moshi agricultural zone of Kilimanjaro region, for example,
An. arabiensis is the predominant species and is resistant to permethrin (13%), attributed to elevated levels of mixed function oxidases rather than
kdr as the species is still fully susceptible to DDT [
12],
kdr east has not been recorded [
21] and
kdr west is present at very low frequency [
22]. Kagera region, however, borders on neighbouring countries and resistance findings in the present study site is more similar of Uganda [
23], Burundi [
24] and Kenya [
25] where phenotypic resistance to pyrethroids and DDT is high and
kdr east allele reported at a high frequency.
The high prevalence of pyrethroid resistance and high frequency of
kdr might be a response to selection by recurrent IRS with lambdacyhalothin since it is found nowhere else at this frequency and no other region of Tanzania has been under such intense selection pressure from pyrethroid IRS since 2006. However, it is possible that kdr present in
An. gambiae in a neighbouring area spread to Muleba by migration. It is not clear whether the resistance to bendiocarb is independent of the resistance to pyrethroids or if there is a common mechanism [
26] arising from pyrethroid selection since bendiocarb resistance was detected before it was used as IRS.
The implications of the pyrethroid resistance on the operational impact of vector control measures, particularly LLINs, are currently uncertain. In West Africa, in areas of high resistance, LLINs show reduced effectiveness against vector populations [
27,
28]. Control failure attributed to insecticide resistance has been observed after IRS campaigns in South Africa [
2,
29] and the island of Bioko [
4]. While historically in some West African countries ITN/LLINs provide some protection against
kdr resistant
Anopheles populations [
30‐
32], that situation appears to be changing with selection of additional metabolic mechanisms [
27,
28]. In Burundi, the high frequency of
kdr did not lead to a loss of efficacy of IRS [
24,
33]. In western Kenya where
kdr in
An. gambiae s.s was reaching fixation, a species shift occurred towards the more zoophilic sibling species
An. arabiensis[
25] despite
kdr being selected in
An. gambiae s.s This has been attributed to the increased use of LLIN and their continued effectiveness against
An. gambiae s.s [
34]. Unlike western Kenya and the coast of Tanzania [
35], in Muleba,
An. gambiae s.s is still the predominant species of the
gambiae complex despite several rounds of pyrethroid IRS and increased use of LLINs. The possible selection of supplementary resistance mechanisms in
An.gambiae s.s based on enhanced metabolism may explain the high prevalence of resistance and persistence of
An.gambiae s.s in our study area, as it has been reported in a different area of Kenya [
36], but this has yet to be confirmed.
Acknowledgements
The authors express their sincere thanks to the field workers and all the PAMVERC staff at Muleba for their hard work collecting the data. We wish to thank all those who participated in the study. LSHTM, KCMC and NIMR are members of the Pan African Malaria Vector Research Consortium.
This study was funded by the United States Agency for International Development under Translating Research into Action, Cooperative Agreement No. GHS-A-00-09-00015-00. This study was made possible by the support of the American people through the United States Agency for International Development (USAID). The findings of this study are the sole responsibility of LSHTM, and do not necessarily reflect the views of USAID or the United States Government.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NP was involved in the study design, supervised the implementation of the study and data collection, analysed data, drafted and revised the manuscript. JM was involved in data collection, helped to analyse the data, draft and revised the manuscript. RM was involved in the study design, implementation and supervision of the data collection and revised the manuscript. ReK, RoK and AW performed the real time PCR testing and revised the manuscript. PW was involved in the study design, supported the field work and revised the manuscript. IK, WK and FM were involved in the overall trial design, helped to draft and revised the manuscript. MR was involved in study design, interpretation of the data and revisions of the manuscript. All authors have read and approved the final version of the manuscript.