This study investigated permethrin resistance in malaria vectors in southern Malawi. Over 700 mosquitoes were collected from 9 sites near Majete Wildlife Reserve. Bioassays on over 500 mosquitoes found 78.4% mortality in Anopheles arabiensis exposed to permethrin, suggesting resistance, but 97.3% mortality in An. quadriannulatus. Genetic testing of over 250 mosquitoes found no evidence of the kdr mutations Vgsc-1014S or Vgsc-1014F, suggesting other resistance mechanisms may be involved. The study highlights the need for ongoing vector surveillance to guide malaria control efforts.

1. RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
Malaria vector control strategies largely consist of the use of long-lasting
insecticidal nets (LLINs) and indoor residual spraying. Scaling-up of these
interventions has led to increasing selection pressure, with growing
evidence of resistant phenotypes in some of the major malaria vector
species. Permethrin in one of the chemicals used widely in LLINs, and
belongs to the only chemical class (pyrethroids) approved for use in LLINs.
INTRODUCTION
Study site and sampling procedure
• Anopheles larvae were collected from December 2015 through
February 2016.
• In total, 9 larval habitats were sampled (Figure 1).
METHODS
Figure 2. Work flow. We collected 1360 Anopheles mosquitoes, out of
which 225 were used as negative controls and 502 (i.e. 241 An. gambiae
s.l.) were exposed to permethrin during bioassays. In total 5 sets of
bioassays were performed, with 1 assay failing to meet WHO control
standards (WHO, 2013). An addition of 12 unexposed An. gambiae sl.
were added to the 241 An. gambiae s.l to run molecular tests.
CONCLUSION
Figure 1. Study area. All sites were near Majete Wildlife Reserve in
Chikwawa District. The district is 70m above sea level and experiences a
tropical climate with average temperature of 26°C, a wet season from
November to April and annual rainfall of approximately 770mm.
DISCUSSION
• We observed some evidence of permethrin resistance in An.
arabiensis, but none in An. quadriannulatus. This might be attributed
by the general endophilic and anthrophilic behavior of the
mosquitoes .
• Despite recording resistant phenotypes with the WHO susceptible
bioassays, we found no evidence of Vgsc-1014S nor Vgsc-1014F
mutations among the genotyped samples, suggesting other potential
resistance mechanisms.
• In a similar study in South Africa (Mouatcho et al., 2009), analysis
using enzyme assays showed elevated levels of monooxygenase that
correlated with the permethrin bioassay data, suggesting increased
metabolism of the insecticide as the mechanism of resistance.
REFERENCE
1. Bass, C., Nikou, D., Donnelly, M. J., Williamson, M. S., Ranson, H., Ball, A., … Field,
L. M. (2007). Detection of knockdown resistance (kdr) mutations in Anopheles
:gambiae a comparison of two new high-throughput assays with existing
methods. Malaria Journal, 6, 111. http://doi.org/10.1186/1475-2875-6-111
2. Mouatcho, J. C., Munhenga, G., Hargreaves, K., Coetzee, M., & Koekemoer, L.
L.(2009). Pyrethroid resistance in a major African malaria vector Anopheles
arabiensis from Mamfene, northern KwaZulu-Natal, South Africa in VecNet
Digital Library [Vecnet]. Retrieved August 9, 2016, from
https://dl.vecnet.org/catalog/q524k044t
3. Scott, J. A., Brogdon, W. G., & Collins, F. H. (1993). Identification of single
specimens of the Anopheles gambiae complex by the polymerase chain
reaction. The American Journal of Tropical Medicine and Hygiene, 49(4), 520–
529
4. WHO. (2013). WHO | Test procedures for insecticide resistance monitoring in
malaria vector mosquitoes. Retrieved August 9, 2016, from
http://www.who.int/malaria/publications/atoz/9789241505154/en/
ACKNOWLEDGEMENT
The Majete Malaria Project , Liverpool School of Tropical Medicine (vector group),
Malaria Alert Centre, College of Medicine, University of Malaria and for the research
mentorship received from Prof. Martin J. Donnelly, Dr Rob McCann, Dr Anja Terlouw, Dr
Themba Mzilahowa and Dr Standwell Nkoma. This project was fully funded by the
Malaria Capacity Development Consortium - thanks for the career development
opportunity, this can’t go unnoticed.
1. Malawi-Liverpool-Wellcome Trust clinical research programme, 2. University of Malawi, College of Medicine, 3. Wageningen University and Research Centre, 4.College of
Medicine Malaria Alert Centre, 5. Vector group, Liverpool School of Tropical Medicine, Liverpool, UK
Trancizeo Lipenga1, Rob McCann2, 3, Themba Mzilahowa4, Standwell Nkhoma1, 2, Martin Donnelly5
Permethrin resistance status of Anopheles gambiae sensu lato in
Chikwawa District, with investigation of a potential mechanism
AIM
• To determine phenotypic susceptibility levels of Anopheles gambiae s.l.
populations in the Majete area of Chikwawa to insecticides used in
malaria interventions and explore potential underlying molecular
mechanisms.
Table 1. Post bioassay status of An. gambiae s.l exposed to permethrin
against locality. Irrespective of locality, observed mortality after
permethrin exposure was 97.3% (36/37) in An. quadriannulatus and 78.4%
(84/116) in An. arabiensis . The lowest mortality recorded was in An.
arabiensis (52.6%) and was reported in samples collected from Masakala.
mosquito larval
collection sites
An. quadriannulatus post-
biossay status to permethrin
exposure
Total
Dead
Live
Mwalija 7 0 7
Kandeu II 1 0 1
Masakala 2 0 2
Namkantha 26 1 27
Total 36 1 37
mosquito larval
collection sites
An. arabiensis post-bioassay
status to permethrin exposure
Total
Dead Live
Mwalija 60 22 82
Kandeu II 1 0 1
Masakala 10 9 19
Namkantha 13 1 14
Total 84 32 116
species identity L1014S allele results Total
LL None
An. arabiensis 130 0 130
An. quadriannulatus 121 2 123
Total 251 2 253
species identity L1014F allele results Total
LL None
An. arabiensis 130 0 130
An. quadriannulatus 120 3 123
Total 250 3 253
Table 2. kdr results for genotyped Vgsc-1014F and Vgsc-1014S point-
mutations against An. arabiensis and An. quadriannulatus species. Over
99% (251/253) of the samples genotyped for Vgsc-1014S and Vgsc-1014F
mutation, were scored as wild-type homozygous (LL) for both L1014S and
L1014F alleles. The remaining specimens could not be scored because of
fluorescence below the set background level.
I360
total samples collected
727
samples used in bioassays
225
samples used as controls
502
permethrin exposed samples
261
other Anopheles
species i.e.
pretoriensis
241
An. gambiae s.l
species
153
validated for
interpretation
(passed set
control standards)
116
An. arabiensis samples
37
An. quadriannulatus
samples
636
samples excluded from
bioassays (moribund)
Figure 3. Spatial distribution of An. gambiae s.l in Majete area.
More An. gambiae s.l. specimens came from Mwalija (82/253), with the
least coming from Kandeu II (2/253). We collected other Anopheles species
(largely An. pretoriensis) in Chibwalizo, Maganga and Tsekela).
This study underlines the importance of routine surveillance of vector
populations being subjected to insecticides with an aim of eradicating
vectors responsible for malaria transmission. Additionally, it suggests
that other insecticide resistance pathways besides kdr are responsible for
resistance; information that will be important for planning and
monitoring malaria vector control.
RESULTS
Bioassays
• Both male and female (non-blood-fed) adult mosquitoes were used
• Bioassays were performed according to the WHO standard protocol
(WHO, 2013).
• Impregnated papers with diagnostic concentrations of 0.75%
permethrin were used.
Species Identification
• We extracted DNA from the dried mosquito specimens using nexttec™
1-Step DNA Isolation.
• Extracted DNA was used for mosquito species identification (Scott,
Brogdon, & Collins, 1993) and in the identification of M and S forms of
An. gambiae (Santolamazza et al., 2008).
kdr genotyping
• kdr genotyping of Vgsc-1014F and Vgsc-1014S mutation was
conducted using a TaqMan assay (Bass et al., 2007).
Data analysis
• We interpreted data using WHO standard protocol (WHO, 2013).
• Further descriptive analyses were carried on IBM SPSS Statistics
package.
Email: tlipenga@mlw.mw