- Malaria is caused by five species of Plasmodium parasites that are transmitted between humans and female mosquitos. The parasites have complex life cycles involving sexual reproduction in mosquitos and asexual reproduction in human liver and blood cells. - While treatments and preventive measures exist, malaria continues to infect over one third of the global population. Researchers are working to develop vaccines by identifying the molecular interactions between parasite ligands and host receptors involved in invasion and infection. - The parasites invade and reside within human red blood cells, where they slowly digest the cell contents for growth. They can evade the immune system by hiding within the red blood cell. Understanding the specific molecular interactions is key to developing better drugs and vaccines.

1. Biology of the parasite
Malarial vaccines? Think host receptor ligand interactions
Malaria comprises 5 species that infect man: Plasmodium vivax, malariae, falciparum,
ovale, and knowlesi. P. knowlesi is a species found in SE Asia and infects macaques. Some
macaque to human transmissions have occurred. All of the species have the same life cycle
stages; they mainly differ in timing of stages and degree of parasitemia. There are other
differences that are unique to them, for example, only Plasmodium falciparum causes cerebral
infections. Only P. vivax and P. ovale have a hypnozoite stage in the liver which allows the
parasite to lie dormant and the disease can relapse in infected persons. Recrudescence can occur
with P. malariae and P. falciparum due to low persisting infected rbcs in endemic areas. There
are basically two phases of the life cycle, a sexual phase that occurs in the female mosquitos'
stomach and the asexual phase that occurs in humans (3,4).
A vaccine against any parasite does not exist. Today despite all of the therapies
preventive measures etc. that exist to combat malaria, malaria still affects one third of the
population (1,2,3,4,5). Research has made significant progress into malaria by identifying some
of the exact parasite ligand interactions with host receptors that is the foundation for vaccine
research (1,2,5). Essentially the search is on to identify the Achilles heel of Plasmodium spp.
and once found therapies can be designed to block infection or at least curtail growth. Sterilizing
immunity may not be achievable but like HIV-1 infection, slower growth of the parasite will
cause less pathology (1,2, 5). In an immune competent host, an individual can eventually
recover (3,4). Despite the complex life cycles of parasitic infections, there are certain surface
receptors of the host and of the parasite that have been identified. Investigators are searching for
the molecular mechanisms of parasite invasion mostly involving the merozite stage though there
are vaccine trials that have been conducted affecting the sporozoite stage which is the
exoerythrocyte stage of infection when liver tissue and liver cells are first colonized
(1,2,5). When these liver cells are lysed, merozoites are released into the bloodstream
(3,4). Some researchers are focusing on this stage of infection. Since malaria is caused by a
parasite that is a eukaryotic multi-celled organism, the parasite can do more crafty things that
bacteria cannot do. The malarial parasite can switch genes off and on genetically by epigenetic

2. mechanisms (2). Malarial parasites can use alternate invasion mechanisms, not just one to
initially invade the erythrocyte (1,2,5). There are variations in proteins among the 4 main
species of Plasmodium but there are some which are common to all and these are the ones that
are singled out for further research. Amazing advances have been made in the last years using
videomicroscopy to time the sequence of infection in human rbcs (5). Rbc receptors have been
identified by using enzymes such as neuraminidase and trypsin and chymotrypsin and identifying
the rbc receptor that plasmodium would use to infect by using for example null cells and cells
with the glycophorin A B or C protein of rbcs (1,2). The results of such studies can be seen in
the attachments of this post (1,5). The first attachment depicts host and parasite corresponding
membrane proteins (1). The parasite has two different predominant types of cell surface
proteins, reticulocyte binding protein homologue and EBA, erythrocyte binding antigens
(1,2,5). The host has glycophorins A, B, C, complement receptor 1 and basigin on the rbc
surface. An AVEXIS assay (avidity based extracellular interaction screen) was developed that
showed that host protein basignin was the receptor for PfRh5 (parasite reticulocyte binding
protein homologue)(1). This particular parasite protein is common to all species and essential to
the invasive process. Knock down studies using stem cells are being explored (1). The second
figure attached shows different ways to identify the rbc receptor assays to identify the specific
receptors Plasmodium use to invade the rbc (1). Live microscopic imaging has shown how
molecularly the parasite re-orients itself upon entry of the rbc and ion fluxes, particularly of
calcium can be visualized around the parasite and within the cell seen in the last attachment
(5). The most recent review cited breaks down the invasion of the parasite into rbcs into 4
stages: 1. weak rbc binding using the MSP-1 complex 2. strong rbc binding via the alternative
pathway of cell entry 3. Pre-tight junction formation requiring the parasite's PfRh5 protein and 4.
The formation of tight junctions, invasion and resealing of the membrane which requires the
AMA-1 protein of the parasite and the RON (rhoptry neck protein) as depicted in the third
attachment (5).
The strange thing about malaria is that there is no eosinophilia because the parasite hides
itself within the rbc by hiding within a vacuole in the rbc; it slowly digests cytoplasmic material
for its own growth and generates hemozoin a hemoglobin once thought to be the Achilles heel of
Plasmodium (3,4). It is still studied as a drug target as it is a crystallized form of heme which
would otherwise be toxic to the parasite in the rbc (3,4). Studying at the molecular level the

3. interaction of specific molecules of host and parasite origin is the key to developing better
vaccines and drugs. Hopefully, malaria can be eradicated and controlled as it has been in the
USA and more temperate climes (3,4).
1. Bei, A.K., & Duraisingh, M.T. (2012) Functional analysis of erythrocyte determinants of
Plasmodium infection. International Journal for Parasitology 42, 575-582.
2. Cortes, A. (2008) Switching Plasmodium falciparum genes on and off for erythrocyte
invasion. 24, (11), 517-524.
3. Katz, Michael, M.D., Despommier, Dickson, D., & Gwadz, Robert W. PhD. (1982) Parasitic
diseases. Springer-Verlag: New York, NY.
4. Mahon, C.R., Lehman, D.C. & Mansuelis, G. (2015) Textbook of diagnostic microbiology (5th
ed.). Maryland Heights, Mo: Elsevier.
5. Weiss, G.E., Crabb, B.S., & Gilson, P.R. (2015) Overlaying molecular and temporal aspects
of malaria parasite infection. Trends in Parasitology 1-12. Retrieved from
http://dx.doi.org/10.1016/j.pt.2015.12.007
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EpidemiologyofMalaria
Malaria is a disease of antiquity-for at least probably many thousands of years. Five
hundred years before Christ, Hippocrates classified the fevers’ timing as quotidian, daily, tertian,
every 2 days, and quartan, every 3 days. Although there were no statistics or epidemiological
studies done in ancient times, the ancient Greeks and Romans must have understood somehow
the connection between malaria and swamps. Without knowing anything about the biology
behind the disease, the ancient Greeks and Romans used the practice of draining swamps for
building construction and farming which is precisely what needs to be done to decrease the

4. incidence of any mosquito (vector) caused disease like malaria or West Nile virus or yellow
fever virus or the most recent problem of Zika virus infection. So inadvertently, the Romans’
and Greeks’ practice of draining swamps actually decreased the incidence of malaria because by
depriving the mosquito of its breeding grounds, less mosquitos were available to continue the
sexual life cycle of the malarial parasite. My old parasitology text also talks about the
derivation of the name mal (bad) aria (air) and paludisme (palus=marsh) (Plasmodium) from
ancient times. It wasn’t until 1898 that Ronald Ross a British physician described the malaria
parasites’ sexual life cycle in the mosquito and thereby proved Laveran’s hypothesis that malaria
was caused by a parasite. Laveran and Ross were awarded the Nobel Prize in 1902.3
Malaria is endemic in tropical areas but of the 5 species of Plasmodium the endemic
areas are slightly different, though sometimes overlapping. Plasmodium falciparum is the most
common species on the African continent. 1, 5 This malarial species is the most prodigious
merozoite producer in red blood cells. Forty thousand merozoites can be released from a single
infected red cell and it is this hematogenous spread that is so terrific. Plasmodium malariae
sporozoites incubate for 12-16 days compared to falciparum’s 5-7 days in the rbc and only
produces 2000 merozoites per infected cell. Cerebral involvement can occur only with the
falciparum species of parasite.3 Plasmodium vivax is the most widespread species outside of
Africa. The WHO notes that though roughly half the world’s population is at risk to develop
malaria, disease incidence worldwide and morbidity/mortality rates are slightly declining each
year due to prophylaxis of travelers to endemic areas and control of infection with anti-malarial
drugs. 1,5
Strangely enough, malaria used to be a huge problem in the Southeastern United
States. In fact the government entity that was created for its primary purpose of addressing the
problem of malaria in the southeast is called today, despite many name changes in its earliest
days, the Centers for Disease Control and Prevention.4 The CDC along with WHO are the
primary resource for epidemiological data on the disease for malaria and many other diseases. In
2012 more than half a million people died from malaria and children are the most susceptible to
severe disease and death. Pregnant women are also very susceptible as well because of the
dampening of the immune system due to progesterone, the hormone of pregnancy.1, 5

5. The parasite infects the actual red blood cell and other species of Plasmodium can infect
non-human primates. We know there has been a strong selection pressure upon the human red
blood cell because we see the results of selection pressure on human populations’ genetic allele
frequencies in tropical and subtropical regions where the mosquitos’ life cycle is longer and
temperatures are optimal for the mosquitos’ larvae and also for the survival of the parasite during
its many different stages. We know that the sickle cell trait is a result of a selection pressure
upon the human red blood cell to create an unhospitable environment for the asexual stage of the
malarial parasites’ trophozoite, sporozoite and merozoite stages in primates. The Duffy antigen
which Plasmodium vivax uses to enter the red blood cell is lacking on most West
Africans. Also, my text notes that in 1982, there was speculation that the cell mediated immune
damping by the malarial parasite enhanced the probability that a person would become
susceptible to the EBV virus infection and Burkitt lymphoma which is most prevalent in Africa.3
Professor Adrian Hill of Oxford University who first noted that certain HLA haplotypes of West
Africans protect against malaria on a scale equivalent to the sickle cell trait is leading the
malarial vaccine efforts of today. His nature paper in 1991 was a seminal paper for immunology
and HLA antigen research. It was the first to note that HLA haplotype had anything to do with
protection against disease.2
Malaria is also endemic in India and my dated parasitology textbook mentions that there
was speculation that both G6P and thalassemia alelles increased in frequency as resistance to the
malarial parasite. References to these statements would be the WHO, CDC and Katz 1982
textbook of mine.
1. CDC (2016) Malaria worldwide. Retrieved from
http://www.cdc.gov/malaria/malaria_worldwide/index.html
2. Hill,A.V.S.,Allsopp,C.E.M.,Kwiatkowski,D.,Anstey,N.M.,Twumasi,P.,Rowe,P.A.…& Greenwood,
B.M. (1991) Common WestAfrican HLA antigensareassociated with protection fromsevere malaria.
3. Katz,Michael,M.D., Despommier,Dickson,D.,&Gwadz,RobertW. PhD. (1982) Parasiticdiseases.
Springer-Verlag:NewYork,NY.

6. 4. Regis,Ed.(1998) Virusground zero:Stalking the killer viruseswith the CentersforDisease Control.
GalleryBooks:NewYork,NY.
5. WHO (2015) Fact sheetNo.94. retrievedfromhttp://www.who.int/mediacentre/factsheets/fs094/en/
Lab diagnosis
Current molecular methods of malaria detection
Malaria has been a plague of mankind for many thousands of years, and scientific
findings of the 20th and 21st centuries have revealed its intricacies and complexities. The
eradication of small pox and polio were difficult enough for 2 rather simple viruses with only
one strain and 3 strains respectively. To eradicate malaria from the face of the earth seems
monumental given its complex life cycle in different hosts and its widespread prevalence
throughout the globe for over thousands of years. The World Health Organization (WHO) has
had on its agenda the eradication of malaria and today there is much research being done to
develop tests that are more sensitive and specific and easy to use in low-resource settings (LSR)
(3, 4). WHO has set the stage for developing diagnostics for the developing world; they must be
ASSURED: Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free,
and Deliverable to end users (3, 4). Also, researchers are focusing on trying to identify
asymptomatic carriers by using molecular methods (1,2,3,4). For areas that had been endemic
for malaria, it would be wise to monitor epidemiologically the carrier rate among the population
and molecular methods can detect malarial parasitemia better than microscopy (2). The most
widespread molecular PCR based method uses primers to the parasite's 18S RNA which is
highly conserved among the Plasmodium species (2).
One of these methods seems to meet the ASSURED model standards and serves as a
prototype of tests to come (3). This crafty test uses a thermos bottle and lid and calcium oxide
coupled within an EPCM (engineered phase change material). The device is named NINA (non-
instrumented nucleic acid amplification) and thus meets the “E” and “D” recommendations of
WHO; it requires no electricity and relies on physical chemistries of CaO2 and the EPCM to
maintain the proper temperature for the assay. The creators of this device compared its
temperature calibration and performance to another instrument and also showed that in the
isothermal LAMP amplification procedure they used, the results were comparable to widely

7. commercially used Perkin Elmer instrumentation (3). The cost for the device is only a few
dollars!
Many field practices for screening various diseases in LRS rely on capillary finger prick
samples which are too low a volume to accurately quantify parasite burden. Another test often
used is dried blood spots on filter paper but the small volumes used will detect high levels of
parasitemia but not low levels. A French and British group of researchers monitoring areas of
Southeast Asia for malaria endemicity showed that taking venipuncture samples, spinning the
blood cells to a pellet, carefully removing the buffy coat and extracting DNA from the RBC
pellet gives a more sensitive and accurate account of true levels of parasitemia. They used a
quantitative PCR approach to measure these levels and compared their assay to other
methods. Their assay could detect 20 parasites per ml in whole blood (2).
Finally, a British tropical medicine group in Uganda has compared LAMP, nested PCR,
and microscopy detection of malarial parasites and have found that the LAMP methodology
compares well with other methods. The false negatives, very few, were likely due to procedural
errors in that the actual amount of parasitemia was well above the limit of detection (LOD) for
the particular assays. The staff on site had only 2 days of training which is remarkable in that
most PCR methods are highly technical and contamination by DNA can easily be detected
leading to false positive results. These newer NAT, nucleic acid tests like LAMP are greatly
simplifying the process and require less skilled staff (1).
It is important to know, in an area where malaria has been seemingly eradicated, where
malarial parasites lurk in the blood stream of asymptomatic patients, to know geographically
where hot spots for a potential outbreak are (1,2). Especially now that mosquitoes are becoming
resistant to the insecticides and the vector is now thriving in areas that saw reductions in disease
burden, keeping track of malaria reservoirs as a part of surveillance is key to disease control (1,2,
4). Molecular methods in some instances will surpass the traditional microscopy method of
detection (1,2,3,4).
1. Hopkins, H. Gonzalez, I.J., Polley, S.D., Angutoko, P., Ategeka, J., Asiimwe, C., Agaba, B.,
Kyabayinze, D.J., Sutherland, C.J., Perkins, M.D., & Bell, D. (2013) Highly sensitive detection

8. of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated
amplification kit in a remote clinic in Uganda. Journal of Infectious Disease 208, 645-52.
2. Imwong, M., Hanchana, S., Malleret, B., Renia, L., Day, N.P.J., Dondorp, A., Nosten, F.,
Snounou, G., & White, N.J. (2014) High-throughput ultrasensitive molecular techniques for
quantifying low-density malaria parasitemias. Journal of Clinical Microbiology 52 (9), 3303-
3309.
3. Labarre, P., Hawkins, K.R., Gerlach, J., Wilmoth, J., Beddoe, A., Singleton, J., Boyle, D., &
Weigl, B. (2011) A simple inexpensive device for nucleic acid amplification without electricity
toward instrument free molecular diagnostics in low-resource settings. PLoS One 6, (5), 1-8.
4. Oriero, E.C., Jacobs, J., Van Geertruyden, J-P., Nwakanma, D., & D’Alessandro, U. (2014)
Molecular isothermal tests for field diagnosis of malaria and their potential contribution to
malaria elimination. Journal of Antimicrobial Chemotherapy 70, 2-13.
Medical Treatment
Malarial Vaccine Dreams and Alternative, Imaginative Hypothetical Possibilities
Malaria and HIV-1 research has stirred many minds to imagine new alternatives and try
new things. They may not seem similar but they are in regard to vaccine development. It is
doubtful that sterilizing immunity will be achieved by vaccines with either the parasite or the
reverse transcriptase containing human immunodeficiency virus. The stakes are high and the
brightest minds are on it. Professor Adrian Hill of Oxford University has studied malaria for
many years and recently in 2013 published in PLOSOne, a Phase Ib trial on the safety and
immunogenicity of an adenoviral and vaccinia viral vectored vaccine against the Plasmodium
falciparum malarial parasite (4). Though the data are preliminary, it seems that a potent immune
response is possible; they recorded more than 2000 IF gamma producing CD 8+ T cells on
average in their small population tested. They do remark that improvements need to be made but
two years later has published several articles in Vaccine. In the 2015 paper Professor Hill and
colleagues resorted to microarray and genome analysis and found that IF gamma pathway
induced genes were found in the supposedly immune protected persons they tested. Their
population was taken from previously vaccinated persons from 2 vaccine strategies (1). “These

9. include a prime-boost regimen with RTS,S/AS02A and modified vaccinia virus Ankara (MVA)
expressing the CSP (circumsporozoite) antigen, and a DNA-prime, MVA-boost regimen
expressing the METRAP antigens.” (1) TRAP stands for thrombospondin-related adhesive
protein; ME-TRAP comprises 17 epitopes from 6 pre-erythrocytic P. falciparum antigens fused
to TRAP at the N terminus. The epitopes are derived from B cells and T cells: 14 CD8+ T cell
epitopes, 1 CD4+ epitope and 2 B cell epitopes. There were 11 unvaccinated control subjects in
the U.K. There were 12 persons in the CSP vaccinated group, and 8 in the ME-TRAP vaccinated
group. One third of the CSP vaccinated persons demonstrated sterilizing immunity when
challenged-REALLY-with mosquitos engineered to have chloroquine sensitive genes while one
eighth of the ME-TRAP vaccinated persons demonstrated no parasites after 21 or more days
(1)! All vaccinated subjects showed a delay in observed parasitemia relative to the control
subjects. The data from their study showed up regulation of JAK2 STAT1 pathways that are
also induced when IF gamma is expressed and also IL-13. Genes in the proteasome degradation
pathway were upregulated (PSME2, PSMB9, PSMB6, and PSMA4). Seventy seven percent of
the genes upregulated in the CSP vaccinated subjects were also upregulated in the ME-TRAP
vaccinated subjects which is somewhat surprising considering that the vaccines used different
antigens. CD74 and LAP3 were also induced as a result of engaging IF gamma production upon
vaccination. These studies utilized PBMCs, peripheral blood mononuclear cells (1). Overall a
fascinating study using cutting edge technologies. Adrian Hill’s 2011 review of malarial
vaccines is worth the read despite knowing some of his own results years later.
Another group from Johns Hopkins, Institut Pasteur and the University of Heidelberg
make the startling discovery that everyone assumes when a mosquito takes a blood meal,
parasites or viruses are injected immediately into the bloodstream but in actuality, there are 13
phases when the parasite is either actively or passively moving. Which proteins are involved in
these movement strategies should be targeted either with drug therapies or antibodies
(5). Possibly a topical cream could be used as a host defense (5)!
Finally, a paper by P. Duffy (NIH), not the hemophiliac patient for whom the Fya or Fyb
rbc antigens are named after, and Jean Langhorne of the Francis Crick Institute in London
published a review article in the Journal of Experimental Medicine about malaria host and

10. parasite interactions known to date. The article was published in February of this year and
somewhat echoes Professor Adrian Hill’s findings (3).
1. Dunachie, S., Berthoud, T., Hill, A.V.S., & Fletcher, H.A. (2015) Transcriptional changes
induced by candidate malaria vaccines and correlation with protection against malaria in a
human challenge model. Vaccine 33, 5321-5331.
2. Hill, A.V.S. (2011) Vaccines against malaria. Trans. R. Soc. B, 366, 2806-2814.
3. Langhorne, J., & Duffy, P.E. (2016) Expanding the antimalarial toolkit: targeting host-parasite
interactions. J Ex Med 213 (2), 143-153.
4. Ogwang, C., Afolabi, M., Kimani, D., Jagne, Y.J., Sheehy, S.H., Bliss, C.M., et al. (2013) Safety
and immunogenicity of heterologous prime-boost immunization with Plasmodium falciparum
malaria candidate vaccines, ChAd63 ME-TRAP and MVA-TRAP, in healthy Gambian and
Kenyan adults. PLOSOne 8 (3), 1-11.
Government and Society
SP-IPTi in East Africa
IPTp, intermittent preventive therapy in pregnancy, was first implemented by WHO as
universal recommendations for pregnant women in the beginning of this century (around 2004)
(7.8). WHO, the World Health Organization, has not changed its policy from 2012 despite a lot
of evidence of the changing landscape of drug resistances in people and also insecticide
resistance in the mosquito that harbors the parasite (7, 8). Clinical trials monitoring birth
weights of infants, monitoring infant parasitemia and vital statistics along with vital signs have
noted that even in the face of quintuple genetic mutations in the malarial parasite rendering it
resistant to the drug therapy widely used, sulfadoxine/pyrimethamine, some protection is
afforded to mother and infant (1, 2, 3). The effectiveness is waning however in preventing
infections, though it is unsure if the virulence has declined somewhat. Despite WHO’s
recommendations, the African Medical and Research Foundation (AMREF) in Kenya has noted
in their article in the Malaria Journal in 2013 that there are many, many obstacles to be overcome
before 100 percent of households have: insecticide treated nets, that all pregnant women have

11. easy access to healthcare and medicines, that treatments are affordable, that the countries
involved have the resources, infrastructure, and funding, that healthcare workers are not burned
out and are more incentivized to adhere to WHO standards and educated about them
(5). Another therapy, mefloquine, was tried in a clinical trial and compared to SP,
sulfadoxine/pyrimethamine, and found to be somewhat superior slightly in preventing clinical
malaria cases but was also more toxic, causing vomiting and dizziness and other adverse
events. Therefore they concluded that the current WHO recommendations should remain as they
are for IPTp for pregnant women (3).
These drug resistances in the circulating parasites and circulating mosquito vectors are
being closely monitored by a few groups worldwide. Cally Roper of the London School of
Hygiene and Tropical medicine and her colleague Inbarani Naidoo of the Malaria Research
Programme of South Africa are some of the few who are mapping year by year the resistances
seen in published reports of the 5 different mutations which render the parasite resistant to
SP. Although WHO is still recommending IPTp for pregnant women and their newborns, for
standard treatment in areas where resistance is highly prevalent, SP is not recommended as
therapy. It is hoped that by releasing the drug selection pressure and using other anti-parasite
medications, the problem of drug resistance will wane somewhat, or at least not increase (4). A
map of dhps K540E is attached which shows > 50% prevalence of this drug resistance mutation
in East African countries (4). The international entity that monitors drug resistance is the
WorldWide Antimalarial Resistance Network (WWARN) (6). Two years later in a Trends in
Parasitology paper, Cally Roper and her colleagues note that it is unknown how IPTp selects for
drug resistances but warn that in East Africa where >50% of parasites recovered have the Pfdhps
K540E mutation, the population served is more than likely vulnerable to infection and its
consequences. Parts of Tanzania and Ethiopia harbor parasite with the quintuple mutation in the
Pfdhps gene (6). They remark that given the high resistances within the gene in East Africa, the
WHO recommendation for infants in their SP-IPTi should be reevaluated given that infants’
immune systems cannot control a parasitemia the same way that their mothers could (6).

12. 1. Aponte, J.J., Schellenburg, D., Egan, A., Breckenridge, A., Carneiro, I., Critchley, J., Danquah,
I., Dodoo, A., Kobbe, R., Lell, B., May, J., Premji, Z., Sanz, S., Sevene, E., Soulaymani-
Becheikh, R., Winstansley, P., Adjei, S., Anemana, S., Chandramohan, D., Issifou, S.,
Mockenhaupt, F., Owusu-Angei, S., Greenwood, B., Grobusch, M.P., Kremsner, P.G., Macete,
E., Mshinda, H., Newman, R.D., Slutsker, L., Tanner, M., Alonso, P., & Menendez, C. (2009)
Efficacy and safety of intermittent preventive treatment with sulfadoxine-pyrimethamine for
malaria in African infants: a pooled analysis of six randomized, placebo controlled trials. Lancet
374, 1533-1542.
2. Eisele, T.P., Larsen, D.A., Anglewicz, P.A., Keating, J., Yukich, J., Bennet, A., Hutchinson, P.,
& Steketee, R.W. (2012) Malaria prevention in pregnancy, birthweight, and neonatal mortality:
a meta-analysis of 32 national cross-sectional datasets in Africa. Lancet Infec Dis 12, 942-949.
3. Gonzalez, R., Momba-Ngoma, G., Ouedraogo, S.,Kakolwa, M.A., Abdulla, S., Accrombessi, M.,
Aponte, J.J., Akerey-Diop, D., Basra, A., Briand, V., Capan, M., Cot, M., Kabanywanyi, A.M.,
Kleine, C., Kremsner, P.G., Macete, E., Mackanga, J.-R., Massougbodgi, A., Mayor, A.,
Nhacolo, A., Pahlavan, G., Ramharter, M., Ruperez, M., Sevene, E., Vala, A., Zoleko-Manego,
R., & Menendez, C. (2014) Intermittent preventive treatment of malaria in pregnancy with
mefloquine in HIV-1 negative women: a multicenter randomized controlled trial. PLOS
Medicine 11 (9), 1-17.
4. Naidoo, I., & Roper, C. (2011) Drug resistance maps to guide intermittent preventive treatment
of malaria in African infants. Parasitology 138, 1469-1479.
5. Thiam, S., Kimotho, V., & Gatongo, P. (2013) Why are IPTp coverage targets so elusive in sub-
Saharan Africa? A systematic review of health system barriers. Malaria Journal 12, 1-7.
6. Venkatesan, M., Alifrangis, M., Roper, C., & Plowe, C.V. (2013) Monitoring antifolate
resistance in intermittent preventive therapy for malaria. Trends Parasitol. 29 (10), 1-16.
7. WHO (2016) Intermittent preventive treatment in pregnancy. Retrieved from
http://www.who.int/malaria/areas/preventive_therapies/pregnancy/en/
8. WHO (2012) Updated WHO policy recommendation: intermittent preventive treatment of
malaria in pregnancy using sulfadoxine-pyrimethamine (IPTp-SP). Retrieved from
http://www.who.int/malaria/publications/atoz/who_iptp_sp_policy_recommendation/en/
Insecticide resistance in malaria bearing mosquitoes

13. There are many drugs to treat malaria but there is only one widespread use of insecticide,
pyrethrin. IRS (indoor residual spraying) of houses or huts using pyrethrin and pyrethrin coated
bedding nets are now used in an estimated 54% of sub Saharan African households (1,4,6). The
combined use of insecticide coated nets and IRS since around 2000 has dramatically decreased
the incidence of malaria cases and deaths; malarial disease incidence and deaths decreased by
50% but in the beginning only pyrethrin was used (1,4,6). WHO is recommending that the
insecticide used for IRS be different from the pyrethrin coated bedding nets so that there is less
selection pressure for pyrethrin resistant mosquitoes (1,4,6). In instances where a country or
locale in Africa registered a dramatic increase in malarial cases if they switched to a different
insecticide the numbers of malaria cases dropped indicating a resistance to that pesticide;
evidence for this is seen in figure 4 of the Trends in Parasitology paper (4). But there are now
resistances reported to the other insecticides in use such as carbamate, DDT, and
organophosphates; in Mali and Cote d’Ivoire resistances to all 4 classes of insecticides have been
documented (4).
These preventive measures are now possibly less efficacious in areas where pyrethrin
resistant mosquito populations are breeding (1,4,6). Pyrethroid resistance prevalence is
extremely difficult to quantitate precisely because there are so many variables to contend with
such as species of mosquito, breeding and feeding habits, ecological differences at differing
geographical locations, etc. (4,6). A recent meta-analysis of the types of studies that address the
issue of insecticide resistance in the mosquito population began with 990 studies but whittled the
number down to only 60 (4). They included studies that included one of 3 testing procedures
common to mosquito borne diseases. In malarial research the tests are a cone test, tunnel test
using guinea pigs, and a field trial test harkening back to the early twentieth century days of
yellow fever research (4,5). Basically one human guinea pig agrees to sleep under a pyrethrin
coated net in a house coated with another insecticide. The mosquitoes that cause malaria feed at
night. One counts the dead mosquitoes in the next hour and 24 hours later. Whether blood fed
mosquitoes can be found in the enclosed area is also assessed (4).
There is some evidence that the netting is still protective somewhat even in areas where
pyrthroid resistant Anopheles species predominate. The literature on the issue of pyrethrin
resistance in mosquitoes is not very standardized so the authors of the meta-analysis wrote a long

14. list of tests that should be done for studies looking at mosquito morbidity and resistance to
insecticides. The authors comment on the “heterogeneity” of study designs and the need to
standardize the data (4).
The authors of the papers cited all agree that in the future, this issue of insecticide
resistance needs to be addressed and monotherapies need to be avoided (1,4,6). There is much
work done in creating netting coated with other insecticides or combinations of them and much
work is being done to develop more insecticides (1,4,6). This problem echoes the problems seen
with antibacterial resistances to TB or Staph infections (2,3). The parasites are constantly
evolving (2,3). It seems that there has been no fitness cost to the Anopheles mosquitoes who are
resistant to the different insecticides, but one wonders has that lessened the virulence of the
malarial parasite or lessened the infective dose given for that species (1,4,6)? Other studies
would need to be done to address that. Still, researchers are very worried that this insecticide
resistance will increase mosquito numbers and increase malarial disease burden and deaths and
perhaps even reverse the goals achieved in the first decade of the 21st century. That is cause for
great alarm (1,4,6).
1. Hemingway, J., Ranson, H., Magill, A., Kalaczinski, J., Fornadel, C., Gimnig, J., Coetzee, M.,
Simard, F., Roch, D.K., Hinzoumbe, C.K., Pickett, J., Schellenberg, D., Gethig, P., Hoppe, M.,
& Hamon, N. (2016) Averting a malarial disaster: will insecticide derail malaria control? Lancet
Retrieved from doi.org/10.1016/S0140-6736 (15)00417-1
2. Katz, Michael, M.D., Despommier, Dickson, D., & Gwadz, Robert W. PhD. (1982) Parasitic
diseases. Springer-Verlag: New York, NY.
3. Mahon, C.R., Lehman, D.C. & Mansuelis, G. (2015) Textbook of diagnostic microbiology (5th
ed.). Maryland Heights, Mo: Elsevier.
4. Ranson, H., & Lissenden, N. (2016) Insecticide resistance in African Anopheles mosquitoes: a
worsening situation that needs urgent action to maintain malaria control. Trends in Parasitology
32 (3), 187-196.
5. Pierce, J.R., & Writer, J.V. (2005) Yellow Jack: how yellow fever ravaged America and Walter
Reed discovered its deadly secrets. Hoboken, NJ: Wiley.

15. 6. Strode, C., Donegan, S., Garner, P., Enayati, A.E., & Hemingway, J. (2014) The impact of
pyrethroid resistance on the efficacy of insecticide-treated bed nets against African anopheline
mosquitoes: systematic review and meta-analysis. PLOS Medicine 11 (3), 1-32.