Monitoring of Artemisinin Combination

Therapy in Igombe, Tanzania.

WRITTEN REPORT

Medicine program, degree project (30 hp)

By Johanna Andersson Supervisor: Göte Swedberg Local supervisors in Tanzania: Erasmus Kamugisha & Karol Maro

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Table of Contents

Acknowledgements ......................................................................................................................... 2

1 Abstract ........................................................................................................................................ 2

2 Swedish summary / Svensk sammanfattning ................................................................................ 3

3 Background .................................................................................................................................. 3

3.1 Malaria .................................................................................................................................. 3

3.2 Artemisinin ............................................................................................................................ 4

3.3 Emerging resistance............................................................................................................... 5

3.4 How to find resistance ........................................................................................................... 6

3.5 Genetic markers for resistance ............................................................................................... 7

3.5.1 PfMDR1 and PfCRT ....................................................................................................... 7

3.5.2 Cambodian news ............................................................................................................ 9

3.6 Igombe ................................................................................................................................ 10

4 Methods ..................................................................................................................................... 11

4.1 Study area and design .......................................................................................................... 11

4.2 Recruitment of patients ........................................................................................................ 11

4.3 Ethics approval .................................................................................................................... 11

4.4 Treatment of patients and follow-up .................................................................................... 11

4.5 Molecular analysis ............................................................................................................... 12

4.5.1 DNA extraction ............................................................................................................. 12

4.5.2 DNA amplification and gel electrophoresis ................................................................... 12

4.5.3 DNA sequencing ........................................................................................................... 13

5 Results ....................................................................................................................................... 13

6 Discussion .................................................................................................................................. 16

References .................................................................................................................................... 19

Appendix ........................................................................................................................................ 1

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Acknowledgements

I would like to express my special appreciation and thanks to my supervisor Dr. Göte Swedberg.

Thank you so much for arranging this opportunity for me to go to Tanzania and for always being

patient answering questions and helping out in the laboratory. Thank you also to my two local

supervisors in Mwanza Erasmus Kamugisha and Karol Maro. You not only assisted me in my

studies and work there but you also helped me to create a home in your wonderful country. Finally I

would like to express my love and gratefulness to my two friends and fellow students joining me to

Mwanza; Maria Sjögren and Emelie Lund. Thank you for an amazing trip and a wonderful

adventure!

1 Abstract

The development of Plasmodium falciparum parasites resistant to artemisinin (ART), so far only

reported present in south-east Asia, poses a big threat towards the malaria affected part of the world.

Since we do not yet know how artemisinins work or exactly what constitutes ART resistance, an

exact way to monitor its spread is difficult. Some genes have been proposed as molecular markers

for a decreased ART suspectibility when containing certain polymorphisms. Among these are the

genes coding for P. falciparum chloroquine resistance transporter (PfCRT), P. falciparum

multidrug resistance 1 (PfMDR1) and now most recently the gene located on chromosome 13 that

coding for the K13 kelch protein, also known as the ‘K13 propeller’. In this study data from 38

malaria patients in Igombe, Tanzania, treated with artemisinin-lumefanterine (AL) combination

therapy, was used to determine the efficacy of AL treatment. Also, for the first time in this area, the

prevalence of variation inside the K13 domain in the malaria parasites was quantified. Patients’

clinical status and parasite blood levels were followed up for three days and blood samples were

collected for DNA sequencing. Results proved AL has a high efficacy in the area, curing all patients

within three days. Furthermore, one out of 34 patients carried a possible polymorphism in its K13

nucleotide sequence. The results are consistent with the clinical situation in Tanzania, where ART

combination therapy today has a good effect and resistance is not present, however, it is not possible

to say if this polymorphism is linked to ART resistance. Further studies are needed to find reliable

molecular markers to monitor ART resistance globally.

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2 Swedish summary / Svensk sammanfattning

Plasmodium falciparum är den dödligaste av de olika parasitarterna som orsakar malaria.

Utvecklingen av falciparumstammar resistenta mot ett av de mest använda malarialäkemedlen,

artemisinin (ART), utgör ett stort hot mot den malariadrabbade delen av vår värld. Hittills har dessa

bara rapporterats funna i sydöstra Asien. Ett sätt att övervaka förekomst och spridning av resistens

är att finna små förändringar i parasiters genom, genetiska polymorfismer, som kan kopplas till en

försämrad läkemedelskänslighet. Några möjliga sådana polymorfismer har redan föreslagits för

artemisininresistens i gener så som de som kodar för P. falciparum chloroquine resistance

transporter (PfCRT), P. falciparum multidrug resistance 1 (PfMDR1) och nu nyligen den genen

belägen på kromosom 13 som kodar för det så kallade K13-kelchproteinet. I denna studie utförd i

Igombe, Tanzania, har 38 malariapatienter som erhållit behandling med artemisinin-lumefanterine

(AL), följts för att undersöka behandlingens effektivitet samt förekomsten av polymorfismer i K13-

domänen i parasiternas genom. Patienternas kliniska tillstånd samt parasitnivåer i blodet har följts

upp i tre dagar och blodprover har tagits för gensekvensering. Resultaten visar att AL har fortsatt

hög effektivitet i området samt att en av 34 patienter bar på en parasitstam med en möjlig

polymorfism i K13 domänen. Då förändringar i K13-domänen hittills bara kopplats till

artemisininresistens i Asien, kan man inte dra några vidare slutsatser av dessa resultat. Vidare

studier bör göras för att finna en pålitlig och globalt användbar genmarkör för ART-resistens för att

kunna följa dess utveckling.

3 Background

3.1 Malaria

Although increased prevention and control measures have reduced the malaria mortality rate in the

recent decades, it still remains the most important parasite disease worldwide. Among the five

species of malaria parasites that infect human, Plasmodium falciparum is the one causing practically

all deaths. In 2012 about 3.4 billion people lived in malaria endemic areas. The same year there

were about 207 million malaria cases and the number of deaths it caused was estimated to be 627

000. Among the deaths, around 90 % occurred in Sub-Saharan Africa and most of them were among

children under the age of five years [1]. A falciparum infection has a good prognosis if diagnosed at

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an early stage and when effective chemotherapy is administered, but left untreated its mortality rate

is high. Today the World Health Organization (WHO) recommends artemisinin combination therapy

(ACT) as the first-line treatment for uncomplicated malaria caused by Plasmodium falciparum.

3.2 Artemisinin

Artemisinin (ART) was discovered by Chinese scientists during the Vietnam War in 1971 when a

new treatment for chloroquine resistant malaria was badly needed [2]. Thereafter, many potent

derivates have been produced including arthemeter (ATM), artesunate and artemotil. Despite being

in use ever since, nobody has been able to determine their mechanism of action. The different

models proposed, as well as evidence both supporting and opposing them, have been summarized in

a review article by Ding and colleagues include interference with the heme-detoxification pathway;

induction of alkylation of translationally controlled tumor protein; inhibition of the

sarco/endoplasmatic reticulum membrane calcium transporting ATPase 6; and interference with

mitochondrial function [3].

ART compounds have broad stage specificity and compared to other anti-malarials, a more effective

elimination of the young asexual circulating ring stage parasites, inhibiting these from further

maturation and sequestration (the adherence of infected erythrocytes containing late developmental

stages of the parasite to the endothelium of capillaries and venules). This, while also killing the

mature blood-stage parasites. Altogether, this makes their total parasite elimination extremely fast,

achieving a 10 000-fold reduction in parasite number per asexual cycle [4]. Most patients have

cleared their peripheral parasitemia by day 3 (~72 h) after the start of the treatment [5].

Given in monotherapy, ART demands at least six-seven days therapy duration to act as curative.

Since in practice it is often used shorter than this, recrudescence (the reappearance of parasites that

have escaped treatment) and reinfection is common. ART derivatives have therefore been combined

with longer lasting partner drugs, which include lumefantrine (in combination with ATM globally

the most widely used ACT), amodiaquine, mefloquine, sulfadoxine–pyrimethamine, piperaquine

and pyronaridine.

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3.3 Emerging resistance

The first proof of parasite ART resistance was reported from western Cambodia in 2008 [6]. In a

study conducted there between 2006 and 2007, four out of 60 patients treated with artesunate during

seven days had parasites reemerging within 28 days after start of the treatment (a criterium of

resistance). Two of them had prolonged parasite-clearance time (135 and 95 hours compared to

median of 52.2 h among the other cured patients) in spite of adequate plasma drug concentrations.

In vitro test analyzing dihydroartemisinin (the active metabolite of all ART compounds)

susceptibility also showed a half-maximum inhibitor concentration (IC50) notably higher than

normal within these subjects.

Further studies from Cambodia and Thailand have added evidence supporting emerging ART

resistance by showing prolonged clearance rates. Between 2007 and 2008, parasite clearance rates

from 40 patients from either site were compared [7]. Cambodian patients treated with artesunate at a

dose of 4 mg/kg had a median parasite clearance time of 84 hours compared to 48 hours in

Thailand. However, the parasites showing a slow clearance rate did not show any increased

susceptibility in conventional in vitro resistance testing, nor was there any correlation with

previously studied drug resistance genetic markers. The clinical effectiveness of oral ACT was

confirmed since cure rates remained around 95%. Also, after observing malaria patients treated with

various ACT's between the years 2001-2010, an increase in parasite clearance half-life was found in

Thailand (2.6 h to 3.7 h) while steadily high mean dito (5.5 h) was seen in Cambodia [8]. The

proportion of variation in parasite clearance attributed to parasite genetics also increased from 30%

to 66%. A larger longitudinal study from the northwest border of Thailand summarized artesunate-

mefloquine treatment in 3264 malaria cases between the years 1995-2007. It showed a similar result

with remaining high clinical efficacy, but slowly increasing parasite clearance times which also

increased the risk of gametocytemia [9]. Finally, ART resistant falciparum malaria has been

reported from southern Burma [10].

When chloroquine resistance emerged in the same area in Asia in the 50's and 60's and then reached

Africa in the early 80’s, it caused millions of deaths [11]. Evidence has been proposed that

resistance to sulfadoxine- pyrimethamine, the next drug of choice after chloroquine resistance had

emerged, also arrived to Africa from south-east Asia [12]. Should ART resistance develop in Asia, it

is likely to once again spread to Africa where its consequences surely would be devastating.

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3.4 How to find resistance

Because ACTs have the ability to quickly reduce the patient's clinical symptoms associated with

parasitemia, many patients stop taking them as soon as they feel better. This opens up for treatment

failure. Monotherapy with artemisinins are associated with a high rate of recrudescences, however

this is not the same thing as resistance and it is not necessarily caused by it. It is also hard to

distinguish recrudescence from reinfection if polymerase chain reaction (PCR) is not used. By PCR

it is possible to sequence the parasites’ DNA and differing one primarily infecting clone from

another reinfecting one. Only treatment failure is therefore no sign of ART resistance since this can

be a result of incomplete duration of treatment, variations in population immunity and metabolism

(i.e. spleen function), variations in drug quality and reinfection. And finally, since the ART is used

in combination with a partner drug, resistance patterns are even more complex. Due to lack of

available in vitro methods or good molecular markers, WHO has defined artemisinin resistance

upon clinical and parasitological outcomes observed during routine therapeutic efficacy studies of

ACTs and clinical trials of artesunate monotherapy. The definition can be viewed in Table 1.

Artemisinin resistance:

• an increase in parasite clearance time, as evidenced by ≥ 10% of cases with parasites detectable on

day 3 after treatment with an ACT (suspected resistance)

or

• treatment failure after treatment with an oral artemisinin-based monotherapy with

adequate antimalarial blood concentration, as evidenced by the persistence of

parasites for 7 days, or the presence of parasites at day 3 and recrudescence within

28 days (confirmed resistance) [13].

Table 1. WHO definition of artemisinin resistance.

Parasite clearance time (the time between the start of administration of anti-malarial treatment until

parasites are no longer detectable in the peripheral blood film) is the most robust and used method

to measure therapy response but it is not precise and affected by several factors besides drug

susceptibility [14]. Comparing parasite clearance data from over 18 000 patients suffering from

falciparum malaria in 25 different countries, the factors associated with a prolonged clearance rate

in vivo were be established [5]. The most prominent were high parasite density on admission, low

level of malaria transmission in the area and a less effective partner-drug to the ART compound in

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the ACT. The study confirmed that ART resistance is highly unlikely if the proportion of patients

who have a positive smear result on day 3 is <3% (with pre-treatment parasite densities of <100,000

parasites/mL, given the currently recommended 3-day ACT). Therefor the day 3 blood slide was

proposed as a measurement for detecting decreased susceptibility in vivo.

To analyze the heritability in parasite clearance half-life - that is the proportion of variance in a trait

that is explained by genetics - trait variation can be compared between parasites that are identical

across the genome but have infected different patients. The blood samples and clearance data from

efficacy trials of ART based therapy in western Cambodia in 2007–2008 were analyzed in such a

way. Results linked a substantial proportion (56%–58%) of the variation in clearance rate to parasite

genetics which could be seen as proof of slowing clearance rate being a parasite trait [15].

3.5 Genetic markers for resistance

3.5.1 PfMDR1 and PfCRT

Many studies have performed genotyping of single nucleotide polymorphisms (SNPs) to see if there

is any special signature SNP pattern that is positively selected for by, or associated with, a slower

parasite clearing rate. A SNP is a DNA sequence variation occurring when a single nucleotide - A,

T, C or G - in the genome differs between members of a biological species or between paired

chromosomes. Identification of the genetic basis of resistance would provide tools for molecular

surveillance, facilitating efforts to contain a developing resistance. Several genes have been

associated with alterations in parasite in vitro susceptibility to artemisinins. Among these are the

genes encoding for the P. falciparum chloroquine resistance transporter (PfCRT) and the P.

falciparum multidrug resistance 1 (PfMDR1) transporter. These are transporter proteins in the

digestive vacuole membrane, a specialized proteolytic organelle wherein hemoglobin is degraded

and detoxified to produce aminoacids used for parasite growth and maturation.

Mutations in PfCRT have been shown to confer chloroquine resistance [16]. The gene differs

widely between different geographical regions but all mutant haplotypes share the same PfCRT

mutations K76T and A220S suggesting these are the ones most important for chloroquine resistance

[17]. Studies show that alterations in PfCRT can also be associated with changes in susceptibility to

other antimalarials including quinine, monodesethylamodiaquine (the primary metabolite of

amodiaquine), halofantrine, lumafentrine and finally ART [16]. Various PfCRT mutant alleles

introduced by allelic exchange in parasite clones and associated with chloroquine resistance induced

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an increased (ca 2.5-4-fold) sensitivity to ART and dehydroartemisinin in vitro [17]. Additionally,

other PfCRT polymorphisms have showed a 2-fold decrease in ART sensitivity [18].

Mutations within the gene encoding PfMDR1, also known as P-glycoprotein homologue-1, have

been shown to confer chloroquine resistance, whereas the mutation alone is not enough to produce

such resistance. This suggests that the mutation might help to modulate and enhance chloroquine

resistance alternatively compensate for physiological changes due to PfCRT mutations [19-20].

How different PfMDR1 polymorphisms and mutations have been seen to influence on artesunate,

ART and dehydroartemisinin susceptibility in vitro is summarized in a review by Ding et al [3].

Some polymorphisms such as the N86Y and the Y184F are associated with slightly increased

sensitivity to the three compounds. Others, such as the S1034C and N1042D were associated with

an either increased or decreased dito. There are also several in vitro studies indicating that an

increased gene copy number of PfMDR1 may significantly decrease parasite susceptibility to ARTs

and vice versa that a decreased gene copy number can increase artemisinin susceptibility.

Artemether-lumefanterine (AL) is known to induce expression of several SNPs; most prevalent are

PfMDR1 N86, 184F and D1246 and the PfCRT K76 alleles [21-23]. Results also indicate that usage

of AL can increase prevalence of wild type genotypes of the PfCRT and PfMDR1 genes wherein

mutations are associated with chloroquine resistance.

Retrospective studies from Malawi, the first country in Africa where chloroquine was replaced by

other first-line treatments due to increasing treatment failure, aimed to determine whether

withdrawal of chloroquine could lead to reemergence of chloroquine sensitivity [24]. The

prevalence of the PfCRT 76T molecular marker for chloroquine-resistant Plasmodium falciparum

malaria was measured. Results showed a prominent decrease in prevalence of the chloroquine-

resistant PfCRT genotype from 85% in 1992 to 13% in 2000. Further studies aiming to trace these

parasites showed that they likely represent a reexpansion of the susceptible parasites that survived

in the population despite widespread drug pressure in the region [25]. The resurgence of susceptible

parasites is best explained by a fitness cost of drug resistance that allows surviving susceptible

organisms to outcompete resistant organisms in the absence of drug pressure. For example, signs of

lesser virulence, in this case the inability to cause a symptomatic infection with fever, have been

observed among the PfCRT and PfMDR1 mutant parasites compared to their wild-type relatives

[26].

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Similar studies performed in the Democratic Republic of Congo, Kenya and Tanzania, where

chloroquine was withdrawn and replaced with ACTs in 2002, 1998 and 2001 respectively, also

show a decline in the prevalence of the chloroquine resistance PfCRT and PfMDR mutants and a

return of wild-type/sensitive strains [27-29; 23].

3.5.2 Cambodian news

Recent clinical trials of artesunate efficacy conducted by Takala-Harrison et al. in Cambodia

examined genotypes for signatures of positive selection and association with parasite clearance

profiles, and both parasite clearance half-life and clearance time following artesunate treatment

were found to be heritable. 8079 SNPs were examined through genome-wide association and four

of these, one on chromosome 10 (MAL10-688956), two on 13 (MAL13-1718319 and MAL13-

1719976) and one on 14, were significantly associated with delayed parasite clearance [30].

Interestingly all the three SNPs on chromosomes 10 and 13 lie in or near genes involved in the same

DNA damage-tolerance pathway used for postreplication repair.

Also using genome-wide association, Cheeseman and colleagues [31] examined SNP patterns in

parasites in Cambodia, Thailand and Laos to determine which ones were under strong positive

selection. Also here, a region on chromosome 13 showed a strong positive selection as well as an

evident association with prolonged parasite clearing rates.

Ariey et al. [32] applied an alternative method to study molecular markers. Here ART sensitive

parasite clones were in vitro exposed to ART intermittently during five years. Then genome

sequencing was used to compare the surviving parasites clones with parasites from clinical trials in

Cambodia showing clinically varying ART susceptibility. The study, published in January 2014,

showed one gene was found to strongly correlate with in vitro resistance, clinically prolonged

clearance rates and the spread of decreased susceptibility among parasites in different Cambodian

provinces. The gene, PF3D7_1343700 kelch propeller domain (‘K13-propeller’), is located within

one of the SNP regions on chromosome 13 mentioned by Takala-Harrison and close to the region

under positive selection mentioned by Cheeseman, and it encodes a kelch protein called K13. Kelch

proteins are a widespread group of proteins involved in a variety of protein–protein interactions,

and the normal function of K13 is not yet known. However this polymorphism could serve as a

useful molecular marker for tracking the emergence and spread of ART resistant P. falciparum.

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3.6 Igombe

Tanzania introduced AL as first-line treatment country-wide in the last quarter of 2006, replacing

sulfadoxine-pyrimethamine (SP) that had been equally introduced in 2001 as first-line treatment due

to the increased chloroquine resistance [33-34]. Studies of AL treatment efficacy in uncomplicated

falciparum malaria in East Africa so far only show good result with high curing rates and few

treatment failures [35-36]. However evidence of a continuous selection of molecular markers

associated with artemether-lumefantrine tolerance/resistance within the parasites has been seen. In a

study analyzing dried blood spots collected during six consecutive studies from children with

uncomplicated falciparum malaria in the Bagamoyo District, Tanzania, showed a statistically

significant yearly increase of polymorphisms in PfMDR1 N86, 184F, D1246 and PfCRT K76

between 2006–2011 from 14% to 61%, 14% to 35%, 54% to 85% and 49% to 85% respectively

[37].

This study, performed in Igombe in northern Tanzania, aims to establish the efficacy of artemisinin-

lumefanterine treatment as well as the prevalence of drug resistance markers within falciparum

parasites causing uncomplicated malaria infection. A similar study was performed in the same area

by E. Kamugisha et al. between 2010 and 2011 [38]. 103 patients were followed up for 28 days. The

AL efficacy proved high with a mean parasite clearance rate at 34.7 h. When examining drug

resistance molecular markers, prevalence of parasites carrying wild type alleles in PfCRT 76 K and

PfMDR1 86N was high compared to other studies previously done in Tanzania which might

indicate return of chloroquine sensitive parasites either due to proper control of chloroquine or

selection of wild type due to AL treatment. It also showed a high frequency of mutations in PfCRT

and PfMDR1 among the reinfections and a pattern in molecular markers indicating sulfadoxine-

pyrimmethamine resistance. In this study however, we will focus on the K13 domain to see if it is

variable or not in the area. Much variability in the gene could possibly indicate a developing drug

resistance. It is part of a larger study planned by Erasmus Kamugisha at Weill-Bugando University

College of Health Sciences, Mwanza, concerning the efficacy of artemisinin and the prevalence of

molecular markers associated with ART resistance.

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4 Methods

4.1 Study area and design

This study was an interventional prospective single cohort study. Patients were recruited from the

health center of Igombe, a semi-urban area with a population of around 40 000 inhabitants near the

city of Mwanza, Tanzania. Here, malaria is mesoendemic, that is, transmission is seasonal under

normal rainfall conditions but in times of drought, it will decline. Rain periods in northern Tanzania

occur in October to December and in March to May.

4.2 Recruitment of patients

Patients attending the health clinic between November 2013 and January 2014 with incidence of

fever ≥ 37.5 °C or a history of fever during the previous 48 h were screened for falciparum

infections with a Paracheck ® Rapid Test for P. falciparum malaria (MRDT). If this was positive,

thin and thick blood films were obtained for P. falciparum parasite count and classification and

blood samples were collected for further DNA analysis. Patient data and exclusion criteria can be

viewed in the Appendix.

4.3 Ethics approval

Ethical approval to conduct the research was obtained from the joint CUHAS/Bugando ethics

committee and regional administration of Mwanza. All patients gave an informed consent before

being enrolled in this study. In case the patient was a minor, consent was obtained from the parent

or guardian of the child.

4.4 Treatment of patients and follow-up

Standard treatment of the admitted patients was a six-dose regimen of artemether-lumefanterine

(Coartem ®, Novartis), each tablet containing 20 mg of artemether and 120 mg of lumefanterine.

Tablets were administered twice daily during three days. A first dose was given as a direct observed

therapy, after which the patient was kept and observed for 30 minutes. Then the remaining therapy

was given to the patient to take eight hours after the first dose and then morning and evening the

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two following days. Children weighing from 5-14.9 kg received one tablet, those from 15-24.9 kg

two tablets, those from 25-34.9 kg three tablets and finally those above 35 kg four tablets. Tablets

were crushed in water for young children unable to swallow whole tablets. If a patient vomited

within 30 minutes after the first dose, a new one was administered and if repeatedly vomiting, the

patient was excluded from the study. Patients with fever were administered paracetamol.

Repeated evaluations were performed on scheduled occasions day one, two and three or in case the

patient did not feel well. At these occasions blood samples were collected on filter paper and body

temperature was measured. Also, an MRDT was taken and if this was positive, the blood was

examined in microscope for parasite counting.

4.5 Molecular analysis

All the molecular analysis was performed at the Department of Medical Biochemistry and

Microbiology at Uppsala University.

4.5.1 DNA extraction

DNA was extracted from the filter papers using Tris-EDTA (TE) buffer-based extraction. For this

purpose, only the day 1 sample was used. The Tris-EDTA buffer was composed of 10 mM Tris, pH

8.0 (Tris base plus Tris-HCl) and 0.1 mM EDTA in distilled water. A piece of filter paper 3.0 mm in

diameter was cut out and placed into a tube, and then 65 µL of TE buffer was added to each tube

and left to soak at room temperature for 1 hour. The tubes were then incubated at 50 °C for 15

minutes followed by another 15 minutes at 95 °C. During latter incubation the punches were

pressed towards the bottom of the tube a few times with a pipette tip. The liquid extracts were then

collected to new tubes and stored at -20 °C until further use.

4.5.2 DNA amplification and gel electrophoresis

The extracted DNA was amplified with nested mutation-specific polymerase chain reaction (PCR).

The following ingredients were added to a PCR tube: 2 µL of Thermo Scientific 10x Dream Taq

Green buffer, 2 µL of 1.0 mM KAPA dNTP Mix, 0.25 µL of 5 U/µL Thermo Scientific Dream Taq

DNA Polymerase, 1 µL of the forward primer and 1 µL of the reverse primer and then finally 2 µL

of parasite DNA.

The forward and reverse primer used in the first outer PCR reaction was Eurofins MWG Operon

13

K13-1 5'-CGG AGT GAC CAA ATC TGG GA-3' and Eurofins MWG Operon K13-4 5'-GGG AAT

CTG GTG GTA ACA GC-3 respectively'. Forward primer in the second nested PCR reaction was

Eurofins MWG Operon K13-2 5'-GCC AAG CTG CCA TTC ATT TG-3' AND P2 and reverse

primer was Eurofins MWG Operon K13-3 5'-GCC TTG TTG AAA GAA GCA GA-3'. The PCR

program used was the same in both of the PCR reactions and consisted of 94 °C 2 min; 30 cycles:

94 °C 30 sec, 60 °C 30 sec, 72 °C 1 min 30 sec; 72 °C 5 min; 4 °C hold.

5 µL of finished products from the nested PCR was used in agarose gel electrophoresis to conferm

whether the PCR was successful or not. The gel was prepared of 1 g SeaKem LE Agarose put into

100 ml of 1xTBE buffer and stored at 46 °C. Before letting the gel turn hard, about 1.5 µL of 0.625

mg/mL ethidium bromide was added to 50 mL gel.

4.5.3 DNA sequencing

After electrophoresis the PCR product was prepared to be sent for DNA sequencing. First 7.5 µL of

ExoSAP was added to 15 µL of PCR product to enzymatically clean up the PCR products of

unincorporated primers and dNTPs. Then the samples were incubated at 37 °C for 30 minutes and

at 95 °C for five minutes. 2 µL of the solution was mixed with 0.4 µL of primer (the forward primer

previously used for the internal PCR were used for sequencing) and finally distilled water was

added to each sample so that it consisted of 18 µL in total. Sequencing was done using the Sanger

method. The obtained nucleotide sequences were viewed in the program 4Peaks and compared with

database sequences of the 3D7 strain from Asia using the BLAST program. Samples that were

unreadable or varied from the 3D7 strain were again sent to DNA sequencing but this time they

were prepared with the reverse primer from the previous internal PCR.

5 Results

Out of all the patients coming to the health station between the 13th November and the 14th of

January, 38 patients proved suitable for and consented to being a part of the study. They all had an

age between 6 months and 30 years and 58 % were female. Mean parasite blood concentration at

admission was 31 670 parasites / µL. Nine patients had parasites in the day two parasite blood count

(mean concentration among these was 8570 p / µL) but they were all negative in the day three

parasite blood count, and likewise, none of the patients from which body temperature was measured

14

had fever on day three. Four of the patients did not show up for day two and three analysis.

However, since the study on K13 prevalence was done with only the day one blood samples, they

could still be part of the study.

After DNA extraction and amplification, 34 out of the 38 samples proved to contain the K13 gene

judging by the gel electrophoresis results. A new PCR round was performed with the samples that

did not contain the gene, but as they showed no different results, they were excluded from the study.

In the first DNA sequencing round 17 samples did not show any variation in their nucleotide

sequence compared to the 3D7 strain, 15 showed variation in one or more positions and finally the

last two were not readable. The once showing variation and the ones unreadable, altogether 17

samples, were prepared again for sequencing with the reverse primer. Out of these, 15 did not show

any variation, one was again not readable and one showed variation in the same nucleotide position

as in the first sequencing run.

Figure 2.

Results of the laboratory analysis.

Picture 1.

Agarose gel of the

obtained PCR

products. No 3, 6, 29 and 31 do not show

any band

representing

presence of the K13

gene.

15

The one sample showing a suspected polymorphism (origin from patient number 8) had a cytocine

instead of a guanine when comparing to the normal nucleotide sequence. This nucleotide is one of

three encoding the amino acid in position 616, and such a mutation as mentioned above would alter

the translated amino acid from a proline to a serine.

The mutation did not match any of the previously reported polymorphic codons from Asia [32].

When compared to registers of polymorphisms from Africa, no match was found either [39].

Picture 3. Illustration of the polymorphism in sample number 8 using the program 4Peaks. Polymorphisms

can be visualized by two peeks present at the same position. To the left is a picture of the forward strand

and to the right is a picture of the reverse strand.

Picture 2. Illustration from the study performed by Ariey et al. [32] showing a segment of the P. falciparum K13 gene. In

the black boxes are polymorphisms found in their study. In the red box is the suspected polymorphism found in our present study in nucleotide position number 1844 affecting the amino acid in position number 616.

16

6 Discussion

In this study, 38 patients were treated with ACT for uncomplicated P. falciparum malaria infection.

All were MDRT negative at the third day of treatment. When analyzing a segment of the K13 gene

of the infecting parasites, one out of 34 showed a possible polymorphism in its nucleotide sequence.

It is visible that ACT in this area has a good effect, curing all patients within three days. It is

however not possible to determine the prevalence of recrudescence or reinfection since the patients

were not followed up for long enough.

The choice to study the K13 propeller was made since it only required the day 1 sample to establish

its baseline prevalence before the start of ACT treatment. A longer follow-up of the patients was

planned for at the beginning of the study; however it proved complicated logistically and time-wise.

Also, the study provided by Ariey was rare in its ability to connect a molecular marker to both a

clinically and in vitro decreased ART susceptibility. Many studies only succeed in associating a

certain polymorphism to clinical results but when then trying it out in vitro, the association is lost.

Certainly, the fact that many genes may interact in creating resistance may play a role there. To date,

Tanzania does not have a problem with ART resistance, nor did the patients in this study show any

signs of infection with parasites with decreased drug susceptibility since they all had their blood

cleared of parasites at day 3. The patient carrying mutated parasites did not show any clinical

anomalies i.e. prolonged clearance time or deviating body temperature. The suspected outcome of

the K13 gene sequencing would therefore be expected to show very small or no nucleotide

variability. The results in this study are coherent with that. Would the prevalence of ART resistance

in the an area be high, one would also expect more numerous polymorphisms.

The sample showing variation did not match any of the previously reported polymorphisms.

However, when comparing to Asian and even African samples one must consider that

polymorphisms may vary much in between different geographical regions. Also, when comparing to

African samples, even if the polymorphism would have matched any of the previously reported

polymorphisms, it would not necessarily have meant anything since these polymorphisms have not

yet been linked to ART resistance as they have in Cambodia via comparison to clinical therapy

outcomes. Some genomic regions are naturally prone to mutation which then might or might not be

17

linked to drug resistance. Should the polymorphism be real it is therefore hard to say what

significance it has. For the protein synthesis it has the effect that it would alter a translated amino

acid from a proline into a serine in the amino acid position 616, thus affect the final K13 protein

structure. We do not know if this could alter the function of the protein, nor yet at all what the role

of this protein is. Assuming the polymorphism is real, the conclusion one can draw is that the

mutation reported from Asia is not present in this part of Tanzania and that the method used in this

study can be used to continuously follow up the development of the prevalence of the K13 propeller

gene.

There was a big difference in the results from the first DNA round to the second one, where 15

samples were positive for possible polymorphisms at first whereas only one of them was so later on.

It is hard to say exactly what lay behind these results, but probably there was a technical problem in

the first round. First, what could have been wrong is the relationship between the availability of

primers and PCR product in the sample sent for DNA sequencing. Secondly, there could have been

something wrong with the primers used making them bind improperly to the PCR product

alternatively something wrong with the PCR product making it a bad match for the primers. The

latter is not believable here since apparently the reverse primer seemed to bind well to the DNA.

Third, it is not ideal to use the same primers for the PCR and the DNA sequencing since by-

products not visible in the agarose gel are regularly created in the PCR which can disturb the results

further along. It is hard to say how many times molecular analysis like these shall be repeated to be

able to draw reliable conclusions of the results, but in bigger studies repetition is often performed

no matter what the results look like. What one can say is that in this study when achieving the

results from the first sequencing round, the whole process from the PCR and on should have been

repeated. What made the final polymorphism likelier to be true than the previous once was that the

surrounding ‘background peaks’ were much smaller in their amplitude, and the aberrant peak was of

a high amplitude. This pattern signifies that there is more than one clone present in the sample.

If more time had been assigned to collecting data for this project, a larger number of patients would

have been followed up for a longer period of time with follow-ups scheduled for day 7, 14 and 28 as

well. Then, recrudenscent parasites as well as parasites with somewhat prolonged clearing rates

would have been particularly interesting to analyze to see if they possessed any alterations in the

K13 gene. Reinfection rates could also have been interesting to investigate if more data had been

collected, however if found common, this would rather have been an indication of a decreasing

susceptibility towards lumefanterine than towards artemisinin. Lumefanterine, being the drug that

18

stays in the body for a longer time, protects against reinfection as long as it stays in a concentration

high enough to be inhibitory for parasite growth. If more time would have been assigned for lab

analysis, it would have been interesting to do analyses of the PfMDR1 and PfCRT

mutation/wildtype prevalence to compare these with the previous studies made by Erasmus et al. An

increase in wild type prevalence would strengthen the theory that a chloroquine withdrawal and an

artemisinin-lumefanterine usage decrease the prevalence of chloroquine resistance molecule

markers in favor of return of wild type alleles.

It is important to continue the search for molecular markers for ART resistance to be able to monitor

its spread and if possible contain it. The three day resistance-definition is a subject to potential

confounding factors such as spleen function, hemoglobin abnormalities and reduced immunity,

which all can delay parasite clearance. Also the recrudescence part of the WHO definition is not

waterproof. As mentioned before it is hard to separate recrudescence from a reinfection. Even when

using PCR, one cannot be certain that a seemingly new clone of falciparum which makes a patient

fall ill again has not been present in the blood since the primary infection. A patient can be infected

by several clones at once. Blood samples for analysis are taken from the peripheral blood of a

patient and all parasites are sometimes not present in the peripheral blood at once. Therefore, it is

possible to miss a primarily infecting parasite clone. This clone could later be a source of

recrudescence and when comparing it to the original parasites with PCR and finding it different, one

would probably judge it to be a reinfection rather than a recrudescence.

The WHO definition of ART resistance will certainly adapt over time when established molecular

markers or better in vitro methods are available. Finally, understanding the gene, or probably the

several genes, underlying artemisinin resistance would help us understand not only the mechanism

of this resistance but also the mechanism of artemisinin itself.

19

References

1. World Health Organization & Global Ma-

laria Programme. World malaria report

2012. (World Health Organization, 2012)

2. Miller, L. H. & Su, X. Artemisinin: Dis-

covery from the Chinese Herbal Garden.

Cell 146, 855–858 (2011).

3. Ding, X. C., Beck, H.-P. & Raso, G.

Plasmodium sensitivity to artemisinins:

magic bullets hit elusive targets. Trends

in Parasitology 27, 73–81 (2011).

4. White, N. Antimalarial drug resistance

and combination chemotherapy. Philo-

sophical Transactions of the Royal Socie-

ty B: Biological Sciences 354, 739–749

(1999).

5. Stepniewska, K. et al. In Vivo Parasito-

logical Measures of Artemisinin Suscep-

tibility. The Journal of Infectious Diseas-

es 201, 570–579 (2010).

6. Noedl H, Se Y, Schaecher K, Smith BL,

Socheat D, Fukuda MM. Evidence of ar-

temisinin-resistant malaria in Western

Cambodia. N Engl J Med 2008;

359:2619-2620.

7. Dondorp, A. M. et al. Artemisinin re-

sistance in Plasmodium falciparum ma-

laria. New England Journal of Medicine

361, 455–467 (2009).

8. Phyo, A. P. et al. Emergence of artemis-inin-resistant malaria on the western

border of Thailand: a longitudinal study.

The Lancet 379, 1960–1966 (2012).

9. Carrara, V. I. et al. Changes in the Treat-

ment Responses to Artesunate-

Mefloquine on the Northwestern Border

of Thailand during 13 Years of Continu-

ous Deployment. PLoS ONE 4, e4551

(2009).

10. Kyaw, M. P. et al. Reduced Susceptibility

of Plasmodium falciparum to Artesunate

in Southern Myanmar. PLoS ONE 8,

e57689 (2013).

11. T Mita, K Tanabe. Evolution of Plasmo-

dium falciparum drug resistance: implica-

tions for the development and contain-

ment of artemisinin resistance. Japanese

Journal of Infectious Diseases 65, 465-

475 (2012)

12. Roper, C. et al. Intercontinental spread of

pyrimethamine-resistant malaria. Science

305, 1124–1124 (2004).

13. World Health Organization & Global Ma-

laria Programme. Update on artemisinin

resistance – April 2012. (World Health

Organization, 2012).

14. White, N. J. The parasite clearance curve.

Malaria journal 10, 278 (2011).

15. Anderson, T. J. C. et al. High Heritability

of Malaria Parasite Clearance Rate Indi-cates a Genetic Basis for Artemisinin Re-

sistance in Western Cambodia. The

Journal of Infectious Diseases 201, 1326–

1330 (2010).

16. Sidhu, A. B. S. Chloroquine Resistance in

Plasmodium falciparum Malaria Parasites

Conferred by pfcrt Mutations. Science

298, 210–213 (2002).

17. Cooper, R. A. et al. Alternative mutations

at position 76 of the vacuolar transmem-

brane protein PfCRT are associated with

chloroquine resistance and unique stereo-specific quinine and quinidine responses

inPlasmodium falciparum. Molecular

pharmacology 61, 35–42 (2002).

18. Valderramos, S. G. et al. Identification of

a Mutant PfCRT-Mediated Chloroquine

Tolerance Phenotype in Plasmodium fal-

ciparum. PLoS Pathogens 6, e1000887

(2010).

19. Reed, M. B., Saliba, K. J., Caruana, S. R.,

Kirk, K. & Cowman, A. F. Pgh1 modu-

lates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum.

Nature 403, 906–909 (2000).

20. Wellems, T. E. & Plowe, C. V. Chloro-

quine-resistant malaria. Journal of Infec-

tious Diseases 184, 770–776 (2001).

21. Sisowath, C. et al. The role of pfmdr1 in

Plasmodium falciparum tolerance to ar-

temether-lumefantrine in Africa: pfmdr1

and artemether-lumefantrine. Tropical

Medicine & International Health 12,

736–742 (2007).

22. Sisowath, C. et al. In vivo selection of Plasmodium falciparum pfmdr1 86N cod-

ing alleles by artemether-lumefantrine

(Coartem). Journal of Infectious Diseases

191, 1014–1017 (2005).

23. Sisowath, C. et al. In Vivo Selection of

Plasmodium falciparum Parasites Carry-

ingthe Chloroquine‐Susceptible pfcrt K76

Allele after Treatment with Artemether‐Lumefantrine in Africa. The Journal of

Infectious Diseases 199, 750–757 (2009). 24. Kublin, J. G. et al. Reemergence of chlo-

roquine-sensitive Plasmodium falciparum

malaria after cessation of chloroquine use

1

in Malawi. Journal of Infectious Diseases

187, 1870–1875 (2003).

25. Laufer, M. K. et al. Return of Chloro-

quine‐Susceptible Falciparum Malaria in

Malawi Was a Reexpansion of Diverse

Susceptible Parasites. The Journal of In-

fectious Diseases 202, 801–808 (2010).

26. Tukwasibwe, S. et al. Differential preva-lence of transporter polymorphisms in

symptomatic and asymptomatic falcipa-

rum malaria infections in Uganda. Jour-

nal of Infectious Diseases 2014 Jan 19.

27. Mvumbi, D. M. et al. Assessment of pfcrt

72-76 haplotypes eight years after chloro-

quine withdrawal in Kinshasa, Democrat-

ic Republic of Congo. Malaria journal

12, 459 (2013).

28. Mang’era, C. M., Mbai, F. N., Omedo, I.

A., Mireji, P. O. & Omar, S. A. Changes

in genotypes of Plasmodium falciparum human malaria parasite following with-

drawal of chloroquine in Tiwi, Kenya.

Acta Tropica 123, 202–207 (2012).

29. Eyase, F. L. et al. The Role of Pfmdr1

and Pfcrt in Changing Chloroquine,

Amodiaquine, Mefloquine and Lumefan-

trine Susceptibility in Western-Kenya P.

falciparum Samples during 2008–2011.

PLoS ONE 8, e64299 (2013).

30. Takala-Harrison, S. et al. Genetic loci as-

sociated with delayed clearance of Plas-modium falciparum following artemisinin

treatment in Southeast Asia. Proceedings

of the National Academy of Sciences 110,

240–245 (2012).

31. Cheeseman, I. H. et al. A Major Genome

Region Underlying Artemisinin Re-

sistance in Malaria. Science 336, 79–82

(2012).

32. Ariey, F. et al. A molecular marker of ar-

temisinin-resistant Plasmodium falcipa-

rum malaria. Nature 505, 50–55 (2013).

33. Mugittu, K. et al. Therapeutic efficacy of

sulfadoxine-pyrimethamine and preva-

lence of resistance markers in Tanzania

prior to revision of malaria treatment pol-

icy: Plasmodium falciparum dihydro-

folate reductase and. American Journal of

Tropical Medicine and Hygiene 71, 696–

702 (2004).

34. Ministry of Health and Social Welfare. Tanzania Malaria Programme Review

2010.

35. Agarwal, A. et al. A randomized trial of

artemether-lumefantrine and dihydroar-

temisinin-piperaquine in the treatment of

uncomplicated malaria among children in

western Kenya. Malar J 12, 254 (2013).

36. Schramm, B. et al. RESEARCH Open

Access. (2013). at

http://www.biomedcentral.com/content/p

df/1475-2875-12-251.pdf

37. Malmberg, M. et al. Temporal trends of molecular markers associated with arte-

mether-lumefantrine tolerance/resistance

in Bagamoyo district, Tanzania. Malar J

12, 103 (2013).

38. Kamugisha, E. et al. Efficacy of arteme-

ther-lumefantrine in treatment of malaria

among under-fives and prevalence of

drug resistance markers in Igombe-

Mwanza, north-western Tanzania. Malar

J 11, 58 (2012).

39. PathogenSeq - GGV: PlasmoView. at <http://pathogenseq.lshtm.ac.uk/plasmovi

ew>

40. EFFICACY, O. A. D. Assessment and

monitoring of antimalarial drug efficacy

for the treatment of uncomplicated falci-

parum malaria. (2003). at

<http://whqlibdoc.who.int/Hq/2003/WH

O_HTM_RBM_2003.50.pdf>

1

Appendix

Exclusion criteria for this study were:

presence of general danger signs according to the definitions of WHO [40];

mixed or mono-infection with other Plasmodium species detected by microscopy;

presence of severe malnutrition (defined as a child whose growth standard is below -3 z-

score, has symmetrical edema involving at least the feet or has a mid-upper arm circumference

<110 mm);

presence of febrile conditions due to other than malaria (e.g. Measles, acute respiratory tract

infection, severe diarrhea with dehydration) or other known underlying chronic or severe diseases

(e.g. cardiologic, renal and hepatic diseases, HIV/AIDS);

antimalarial treatment within previous 28 days;

regular medication, which may interfere with antimalarial pharmacokinetics including

traditional medicines;

history of hypersensitivity reactions or contraindications to any of the medicine(s) being

tested or used.

2

P

atie

nt d

ata

Patie

nt no.

Age in

Sex

Day

1 M

RD

TD

ay

1 P

ara

site

count

Day

2 M

RD

TD

ay

2 P

ara

site

count

Day

3 M

RD

T

years

(y)

& m

onth

s (

m) fem

ale

(f)

/ m

ale

(m

) p

os / n

eg

p/u

l p

os / n

eg

p/u

l p

os / n

eg

16 m

fpos

48000

pos

8800

neg

228 y

fpos

520

neg

neg

32 y

5 m

fpos

128

neg

neg

428 y

mpos

1040

neg

neg

57 m

fpos

2400

neg

neg

617 y

fpos

368

neg

neg

713 y

mpos

16000

Patie

nt did

not show

up

Patie

nt did

not show

up

87 y

mpos

480

neg

neg

92 y

mpos

128000

pos

64000

neg

10

4 y

fpos

132000

pos

2400

neg

11

1 y

10 m

fpos

40000

pos

400

neg

12

8 m

mpos

2800

neg

neg

13

11 y

fpos

16000

pos

48

neg

14

22 y

fpos

8800

neg

neg

15

5 y

mpos

120000

neg

neg

16

6 y

mpos

20000

neg

neg

17

1 y

10 m

fpos

400

neg

neg

18

19 y

fpos

24000

neg

neg

19

18 y

fpos

20000

neg

neg

20

1 y

3 m

mpos

600

neg

neg

21

5 y

fpos

80000

neg

neg

22

2 y

2 m

fpos

49000

Patie

nt did

not show

up

Patie

nt did

not show

up

23

7 m

mpos

40000

Patie

nt did

not show

up

Patie

nt did

not show

up

24

14 y

fpos

16000

Patie

nt did

not show

up

Patie

nt did

not show

up

25

2 y

fpos

4000

pos

320

neg

26

1 y

mpos

160000

neg

neg

27

7 y

fpos

12000

neg

neg

28

4 y

mpos

16000

pos

600

neg

29

12 y

fpos

400

neg

neg

30

3 y

fpos

120000

neg

neg

31

16 y

mpos

800

neg

neg

32

7 y

fpos

16000

400

neg

33

2 y

8 m

mpos

60000

160

neg

34

8 m

fpos

20000

neg

neg

35

3 y

mpos

40000

neg

neg

36

7 y

mpos

16000

neg

neg

37

3 y

mpos

1200

neg

neg

38

30 y

fpos

20000

neg

neg

3

Body temperature in case meas-

ured (°C) Patient no. Day 1 Day 2 Day 3

1 39,3 35,1

2 36,8 36,0

3 39,3 35,8 36,2

4 36,4 35,5

5

6 39,6

7 40,3

8 37,4 36,4 36,5

9 39,0 37,9 35,8

10 38,7 36,8 35,6

11 38,7 36,5 36,7

12 36,9 36,5 36,5

13 36,3 34,8 34,0

14 34,3 36,5 35,7

15 39,5 38,9 36,4

16 39,3 35,6 36,2

17 36,3 36,2 36,4

18 35,8

19 35,9 36,9 36,7

20 39,6 36,5

21 40,1 36,6

22 39,5

23 36,1

24 37,2

25 38,4

26 39,4 39,3 35,3

27 38,4 36,4

28 37,8 35,8 36,7

29 35,1 36,0 36,5

30 36,2 36,1 36,2

31 36,7 36,3 35,6

32 38,2 35,6 34,2

33 35,3 35,5 35,5

34 36,7 35,6 35,0

35 37,7 36,6

36 39,4 37,1 35,6

37 40,5 35,4

38 37,1