Selection for chloroquine-sensitive Plasmodium falciparum by wild Anopheles arabiensis in Southern Zambia
Department of Molecular Microbiology & Immunology, Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health
615 N Wolfe Street, Baltimore, MD 21205
The Malaria Institute at Macha
PO Box 630166, Choma
Background: The emergence of parasite drug resistance, especially Plasmodium falciparum, persists as a major obstacle for malaria control and elimination. To develop effective public health containment strategies, a clear understanding of factors that govern the emergence and spread of resistant parasites in the field is important. The current study documents selection for chloroquine-sensitive malaria parasites by wild Anopheles arabiensis in southern Zambia. Methods: In a 2,000-sq km region, mosquitoes were collected from human sleeping rooms using pyrethrum spray catches during the 2006 malaria transmission season. After morphological examination and molecular confirmation, vector mosquitoes were dissected to separate head and thorax from the abdominal section, followed by PCR screening for P. falciparum infection. Human residents of all ages were tested for P. falciparum parasitaemia by microscopy and PCR. Plasmodium falciparum infections were genotyped at the chloroquine resistance-conferring amino acid codon 76 of the PfCRT gene, using PCR and restriction enzyme digestion. Results: In the human population there was nearly 90% prevalence of the chloroquine-resistant PfCRT K76T mutant, with no significant differences in polymorphism among smear-positive and smear-negative (submicroscopic) infections (p = 0.323, n = 128). However, infections in both abdominal and salivary gland phases of the An. arabiensis vector exhibited wild type K76-bearing parasites with up to 9X higher odds (OR (95% CI): 9 (3.7-20.2), p < 0.0005, n = 125), despite having been acquired from humans within a few weeks. Conclusions: Anopheles arabiensis selects for wild-type K76-bearing P. falciparum during both abdominal and salivary gland phases of parasite development. The rapid vectorial selection, also recently seen with antifolate resistance, is evidence for parasite fitness cost in the mosquito, and may underpin regional heterogeneity in the emergence, spread and waning of drug resistance. Understanding the nature and direction of vector selection could be instrumental for rational curtailment of the spread of drug resistance in integrated malaria control and elimination programmes.
The emergence and spread of parasite drug resistance,
especially Plasmodium falciparum, continues to pose a
major obstacle for malaria control and elimination [1,2].
Plasmodium falciparum has repeatedly proven ability for
mounting resistance to any anti-malarial drug regimen
upon wider use, including current ones [2-7]. To avert
potential resurgence owing to treatments that are no
longer effective, not only are new anti-malarials needed,
but also public health strategies that will minimize or
delay drug resistance escalation [8,9].
There are only a few strategies that have been rationalized
and adopted to date, namely, avoidance of sub-therapeutic
doses [10,11], combination therapy, especially with
artemisinin and its derivatives , and focused screening and
treatment, which was recently started in Southeast Asia in an
effort to stem the global spread of emerging P. falciparum
tolerance to artemisinin . So far P. falciparum evidently
evades all these approaches [14,15].
To develop effective strategies for containment of drug
resistance, an understanding of the epidemiological factors
that govern the emergence and spread of resistant parasites
in the field is paramount. Theoretical arguments [12,16],
although sometimes conflicting [13,14], and a review ,
have posited an association between malaria transmission
control and levels of P. falciparum drug resistance.
Meanwhile, field studies have shown an unexpected but decisive
association between mosquito control and the prevalence
of P. falciparum drug resistance [17,18]. The
prevalence of P. falciparum dihydrofolate reductase (DHFR)
wild-type (antifolate drug-sensitive) alleles increased in
Tanzania when insecticide-treated nets (ITNs) were
implemented . In an area of Zimbabwe, the introduction
of indoor residual insecticide spraying (IRS) was associated
with a four-fold reduction in the odds of chloroquine
therapeutic failure after four years , despite concurrent drug
pressure. Cessation of the IRS was linked to a four-fold
rebound in odds of chloroquine resistance after another
subsequent four years . In contrast, Shah et al. found
no significant relationship between sustained ITN use and
the prevalence of chloroquine or antifolate drug resistance
in western Kenya .
What remains unclear is why drug resistance associates
with mosquito control in some areas but not others.
Similarly, why does the removal of drug selection pressure
result in decisive re-emergence of sensitive parasites in
some areas [20-25] and yet other locations show slow
recovery or fixation [26-29]? A recent study suggests
that mosquito vectors may play a role in determining
field levels of drug-resistant P. falciparum  and thus
constitute an additional governing factor in drug resistance
epidemiology. This means that mosquito control may
influence parasite drug resistance indirectly, through impact on
vector species that select on drug resistance polymorphism.
Highly contrasting P. falciparum antifolate drug resistance
polymorphisms were found in human and mosquito hosts
that seemed to suggest strong selection against
pyrimethamine resistance mutants by the Anopheles arabiensis
vector . The current study documents An. arabiensis
selection on the chloroquine resistance-conferring
polymorphism of the P. falciparum chloroquine resistance
transporter (PfCRT) gene.
Study area and population
The study was based in a 2,000-sq km vicinity of the
Malaria Institute at Macha (MIAM) and Macha Mission
Hospital, a 208-bed, rural, district-level hospital in Southern
Province of Zambia. The area has an altitude of 900
1,000 m above sea level and historically experienced
hyperendemic malaria, which is transitioning to
hypoendemicity following scale-up of ITN and artemisinin
combination therapy (ACT) interventions since 2004.
The resident population is approximately 160,000 and
comprises primarily subsistence farmers of the Batonga
communities. As with the rest of Zambia, chloroquine,
the former first-line treatment for malaria for many
years, was suspended from use in 2003 and replaced
Parasite DNA sample collection
Following prior appointment with the resident
communities, whole mosquitoes were collected from human sleeping
rooms by pyrethrum spray catches in the morning from
Through prior arrangement, willing community residents
of all ages assembled at existing central meeting points,
during the afternoons of the same day as pyrethrum spray
catches, for malaria screening by microscopy as previously
described . Parasite DNA samples were simultaneously
collected as filter paper dry blood spots (DBS) from the
same finger-prick used to prepare microscopy blood films.
Parasite DNA extraction
The mosquitoes collected from sleeping rooms of the
resident population were identified morphologically, with
confirmation by sibling species complex differentiation PCR for
Anopheles gambiae s.l. and Anopheles funestus s.l. as
previously described [30,32]. Parasite DNA was extracted
separately from the mosquito abdominal and salivary gland
phase using a modified chelex protocol .
Assays for Plasmodium falciparum drug resistance alleles
Plasmodium falciparum infections were genotyped for
the chloroquine resistance-conferring K76T mutation
in the PfCRT gene using nested PCR and allele-specific
restriction enzyme digestion [31,35].
The study was approved by the Johns Hopkins IRB and
the University Of Zambia Research Ethics Committee
(UNZAREC) as part of an ongoing geographical and
demographic reconnaissance study of malaria.
Prevalence PfCRT K76T in human malaria infections
From a total of 2,278 residents screened, 128 P. falciparum
infections were genotyped for PfCRT-76 polymorphism
(Figure 1). In the humans, the prevalence of
chloroquineresistant K76T-bearing P. falciparum was virtually 90%.
No significant differences in the K76T single nucleotide
polymorphism (SNP) were observed among
microscopypositive and microscopy-negative (submicroscopic) human
malaria infections (Figure 1).
Prevalence PfCRT K76T in mosquito malaria infections
Of 753 vector mosquitoes captured from human
sleeping rooms, 745 (99%) were An. arabiensis, the remaining
8 (1%) being An. funestus. Despite having been
acquired from humans within the previous ten days, An.
arabiensis mosquito abdominal infections exhibited
more than twice the proportion of wild-type (K76-bearing)
P. falciparum that is sensitive to chloroquine (Figure 1).
The proportion of K76-bearing P. falciparum was more
than five times higher in the mosquito salivary gland
phase than in humans, despite having been acquired
from the latter within the previous few weeks defining
the natural life span of a mosquito in the wild (Figure 1).
Only one of the eight An. funestus was found positive
for P. falciparum infection, which was in the abdomen
and bore PfCRT 76 T.
Data from the present study point to a strong natural
selection for chloroquine-sensitive P. falciparum by
An. arabiensis during both the abdominal and salivary
gland phases of development. Within the short mosquito
phase of the parasite, lasting only a few weeks, P. falciparum
infections in salivary gland stage exhibited four-fold and
nine-fold higher odds of chloroquine-sensitive parasites
than abdominal and human phase infections, respectively.
Thus, there appears to be two distinct and sequential stages
of this vectorial selection for wild type P. falciparum in
the mosquito host.
The observed data concur with earlier findings showing
An. arabiensis selection against pyrimethamine resistance
mutants  in the same area. Owing to low numbers
for An. funestus, it could not be determined whether this
vector exerts similar selection as seen in An. arabiensis.
Nonetheless, in a Tanzanian study by Temu et al. ,
An. arabiensis appeared to consistently carry a lower
proportion of PfCRT 76 T mutants than both An. funestus
and An. gambiae s.s.
The mechanistic basis for the vectorial selection
on drug resistance polymorphism is not clear from a
cross-sectional field survey. However, fitness cost is a
possible explanation. Drug resistance mutations,
including PfCRT, are known to bestow altered biological
fitness on the parasite, which has so far been reported
n = 63
n = 65
n = 62
n = 81
Figure 1 PfCRT K76T polymorphism in human and mosquito phases of infection (interspersing numbers are odds ratios (95% CI)).
in vitro, in humans [37-42] and more recently in
mosquitoes . Mosquitoes have been shown to mount
effective immune clearance of malarial infection, hence
bottlenecking parasites during mid-gut as well as
salivary gland invasion [43,44]. Presumably, less fit mutated
parasites are largely eliminated during these successive
survival-limiting stages of parasite development in the
mosquito. Mosquitoes are also the definitive host where
parasite genetic recombination takes place that can break
down resistance haplotypes.
On the other hand, mid-gut microbiota have been
reported to have direct anti-malarial properties [45-47]
that could possibly impose selection on P. falciparum in
the mosquito. Differences in gametocytogenesis [48,49]
between mutated and wild type parasites may be another
possible reason for selective parasite survival in the
vector. However, both the mid-gut microbiota and
gametocytogenesis would not explain the strong selection for
K76 observed between abdominal and salivary gland
phases of the parasite, which was similarly observed with
antifolate resistance polymorphism . Thus, a general
fitness cost of resistance in the mosquito appears the
most reasonable hypothesis.
Irrespective of the underlying mechanisms, the results
observed in the present study appear to lend further
evidence that vector mosquitoes may play an important
role in drug resistance epidemiology . It is
hypothesized that the vectorial selection contributes to
recovery of chloroquine-sensitive malaria parasites following
withdrawal of drug use, as observed initially in Hainan
Province of China  and subsequently Malawi [23,24]
and other areas, including the site of the current study
(Sialumano et al. in prep). Such natural vectorial selection
is in concert with observations by Laufer et al. that
reemerging chloroquine-sensitive P. falciparum are
preexisting diverse parasites that survived the chloroquine
era, rather than a new invading strain . Vectorial
selection on parasite drug resistance polymorphisms
would also appear to explain the field observations of
association between mosquito control and prevalence of
P. falciparum drug resistance [17,18], and may underpin
regional differences in susceptibility to the emergence and
spread of drug resistance, depending on relative survival
of mutated parasites in the local vector species. To better
understand the possible role of the vector mosquitoes in
anti-malarial drug resistance epidemiology, more field
studies and controlled laboratory experiments are needed.
Anopheles arabiensis selects for wild type (K76-bearing)
P. falciparum during the abdominal and salivary gland
phases of parasite development. The vectorial selection
is evidence for P. falciparum drug resistance fitness cost
in the mosquito and may underlie regional heterogeneity
in the emergence, spread and waning of drug resistance.
Understanding the nature and direction of vector selection
could be instrumental for rational curtailment of the spread
of drug resistance in integrated malaria control and
SM was the principal investigator who initiated, designed and ran the study,
followed by data analysis and writing of the manuscript. MS performed
laboratory assays and reviewed the manuscript. KL reviewed and contributed
to the manuscript. AS provided essential technical input and reviewed the
manuscript. PT was the overall advisor and reviewed the manuscript. All
authors read and approved the final manuscript.
The research team is grateful to the chiefs, headmen and communities of
Macha, Muchila, Mapanza and Chikanta areas for their support and
participation in the study. Harry Hamapumbu assisted tremendously in
coordination of field research activities and staff supervision. The study was
supported by the Johns Hopkins Malaria Research Institute pilot grant
system and the Southern Africa International Centres of Excellence for
Malaria Research programme under NIH/NIAID grant 1U19AI089680.