Treatment efficacy of artesunate-amodiaquine and prevalence of Plasmodium falciparum drug resistance markers in Zanzibar, 2002-2017 May 2019 Aung Paing Soe Master in Global Heath, 30 credits (Spring 2019) International Maternal and Child Health (IMCH) Department of Women’s and Children’s Health Supervisors: Dr. Soorej Jose Puthoopparambil and Prof. Mats Målqvist IMCH, Uppsala External Supervisors: Dr. Ulrika Morris and Prof. Anders Björkman Molecular, Cell and Tumor Department, Biomedicum, Karolinska Institutet, Stockholm (9739 words)
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Treatment efficacy of artesunate-amodiaquine and
prevalence of Plasmodium falciparum drug
resistance markers in Zanzibar, 2002-2017
May 2019
Aung Paing Soe Master in Global Heath, 30 credits (Spring 2019)
International Maternal and Child Health (IMCH)
Department of Women’s and Children’s Health
Supervisors: Dr. Soorej Jose Puthoopparambil and Prof. Mats Målqvist
IMCH, Uppsala
External Supervisors: Dr. Ulrika Morris and Prof. Anders Björkman
Molecular, Cell and Tumor Department, Biomedicum, Karolinska Institutet,
Stockholm
(9739 words)
UPPSALA UNIVERSITET Resistance Markers to Artesunate and Amodiaquine
2.1 Study Design .................................................................................................................................... 11
All 7/144 (4.9%) 3/144 (2.1%) 97/144 (67.4%) 2/144 (1.4%) N/A
(1) All mixed infections (i.e. mutant + wildtype) were also counted as resistance alleles.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
20
02
-3 (
199
)
20
05
(1
74)
20
10
(1
05)
20
13
(9
4)
20
17
(1
42)
20
02
-3 (
196
)
20
05
(1
71)
20
10
(1
10)
20
13
(9
8)
20
17
(1
42)
20
02
-3 (
198
)
20
05
(1
73)
20
10
(1
12)
20
13
(9
4)
20
17
(1
42)
20
02
-3 (
200
)
20
05
(1
75)
20
10
(1
05)
20
13
(9
4)
20
17
(1
44)
pfcrt K76T pfmdr1 N86Y pfmdr1 Y184F pfmdr1 D1246Y
Fre
qu
ency
%
76T; 86Y; Y184; 1246Y Mixed K76; N86; 184F; D1246
UPPSALA UNIVERSITET Resistance Markers to Artesunate and Amodiaquine
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A total of nine samples positive by qPCR on Day 28 were also processed for nested PCR to
examine the status of SNPs. However, these samples have shown very low (<1 p/µl) parasite
density by quantitative PCR, and they were all consistently negative in the nested PCR used for
genotyping SNPs. Therefore, the Day 28 samples were not successfully genotyped.
The pfmdr1 YYY and YYD are the most frequent haplotypes in 2002-3, in contrast, the NYD and
NFD haplotypes were most frequent in 2017. There was zero YYY and only three YYD in 2017
study. There was significant decline of YYY (p < 0.001) and YYD (p <0.001) between 2002-3
and 2017 (Figure 6).
Figure 6. Haplotypes shift from YYY to FDN between 2002 & 2017, representing the
frequencies of pfmdr1 haplotypes. (N86Y, Y184F, and D1246Y). Fisher exact test was
performed to compare the frequencies between all years. Asterisk (*) and (**) indicate p value
below 0.05 and 0.001 respectively.
A PfK13 pilot study conducted in 2013 identified no SNPs in the PfK13 propeller region in
Zanzibar, yet the finding still needs validation due to small sample size (N=34) (unpublished).
However, regarding genotyping of PfK13 gene, 98% (139 out of 142) samples were successfully
sequenced in 2017. Among them, 5 (3.6%) samples had the previously reported synonymous SNP
cysteine to cysteine at amino acid position 469 (CYS 469 CYS) which was previously reported
from Fukayosai, Tanzania in 2012 [37]. One additional synonymous SNP serine to serine at amino
acid position 477 (SER 477 SER) was also identified in a single (0.7%) sample Table 5. In 2017,
only 1 out of 6 isolates reported history of travelling to mainland, Tanzania in the past one month.
Moreover, the association between PfK13 and presence of travel history cannot be proven due to
small sample size. Nevertheless, the SNPs identified in 2017 study are not associated with
artemisinin resistance [37].
0
10
20
30
40
50
60
70
80
90
100
YYY YYD YFD NYD NFY NFD
freq
uec
y %
of
Pfm
dr1
Ha
plo
typ
es
Figure 4. Haplotypes shift from YYY to FDN between
2002 & 2017
2002 2005 2010 2013 2017
** **
** **
**
*
* *
*
*
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Table 5. Representing PfK13 single nucleotide polymorphisms identified in 2017 according to
travel history to mainland Tanzania
Study
Site
Age (years
=yr or
month=mth)
Sex
(M/F)
Travel
(Y/N) PfK13 SNP1
Previous
Reported
SNP (Y/N)
Bububu 22yr F N Synonymous SNP: CYS 469 CYS Y
Bububu 6 yr M N Synonymous SNP: CYS 469 CYS Y
Uzini 7 yr M Y Synonymous SNP: CYS 469 CYS Y
Bububu 21 mth F N Synonymous SNP: CYS 469 CYS Y
Bububu 15 yr M N Synonymous SNP: CYS 469 CYS Y
Bububu 41 yr F N Synonymous SNP: SER 477 SER N
1Synonymous mutation within PfK13 alleles C469C was previously reported in Fukayosi,
Tanzania in 2012 [37].
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4. Discussion
Zanzibar is one of the countries in the Africa Region with the most remarkable decrease in both
malaria morbidity and mortality since 2005 [6,50]. To achieve malaria elimination, maintaining
the drug efficacy and prevention of development of resistance is critical. The aim of this study is
to assess the drug efficacy of ASAQ and track the development of molecular markers that are
known to be associated with amodiaquine and artesunate resistance. The following sections
discussed the evidence provided as the result of this study in relation to the drug efficacy of ASAQ
and polymorphisms associated with resistance to amodiaquine and artesunate.
4.1 Efficacy of Artesunate-Amodiaquine
Efficacy studies conducted in Zanzibar (2002-3, 2005 and 2017) consistently showed that the first
line ACT (ASAQ) treatment for uncomplicated P. falciparum malaria remains effective. Malaria
diagnosis by microscopy is the conventional tool to monitor drug resistance in therapeutic efficacy
studies, and revisions of national malaria treatment guideline can be considered if day 3
parasitemia and day 28 cure rates are more than 10% and less than 90%, respectively [71].
Microscopic examination of malaria parasites showed that parasitemia on day 3 were as low as
0.5%, 0% and 1%, and cure rates on day 28 remains as high as 94%, 96% and 100% in 2002-3,
2005 and 2017 respectively. In addition, the proportion of recurrent infection on day 28 (after PCR
correction) decreased during the period from 2002-3 to 2017. Findings regarding treatment
efficacy could provide as an evidence to maintain ASAQ as a first line treatment in Zanzibar.
4.2 Single Nucleotide Polymorphisms
Polymorphisms in pfcrt and pfmdr1gene have been associated with reduced sensitivity to ASAQ
and AL, yet the characteristic of these alleles are not fully understood [72]. The selection of pfcrt
76T, pfmdr1 86Y, 184Y and 1246Y alleles have been associated with reduce sensitivity to ASAQ,
whilst in reverse, selection of wild types alleles are related to reduced sensitivity in AL [29–
31,33,35,73]. Hence, pfmdr1 86N, 184F and 1246D alleles were found to be associated with high
proportion of treatment failure in Tanzania where AL was the first line drug to treat falciparum
malaria [36]. Similarly, a study conducted in Burkina Faso reported that there was significant
increase in pfcrt 76K and decreased in pfcrt 76T after AL treatment whereas increased in pfcrt
76T and decreased in pfcrt 76K after ASAQ treatment [73]. Regarding pfmdr1 N86Y alleles, 86Y
has been associated with increased with ASAQ treatment failure, but the evidence is less
convincing for pfmdr1 Y184F [73].
On the one hand, the mechanism of higher selection of wild types in this study SNPs - pfcrt 76K,
pfmdr1 86N, 184F and 1246D alleles - are not fully understood. On the other hand, continuous
wide-scale use of ASAQ is more likely to select mutant type due to presence of sustain drug
UPPSALA UNIVERSITET Resistance Markers to Artesunate and Amodiaquine
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pressure at population level [27,29,30,35,36]. However, in 2012, Froberg et al. reported the first
observation of decreased prevalence of SNPs that were associated with ASAQ resistance in
Zanzibar and suggested three possible underlying causes of selecting pfcrt 76K, pfmdr1 86N and
1246D alleles were genetic dilution caused by imported cases form mainland Tanzania, selection
by artesunate per se, and fitness cost of the parasite itself. [74]. Parasite fitness seems to have
advantage over selection of circulating resistant parasites [75]. The prevalence of mutants pfcrt
76T and pfmdr1 86Y found to be decreased in low transmission areas when drug pressure is
diminishing [65,75,76]. However, information regarding mediating factors of resistance
polymorphisms on parasite fitness is not known sufficiently [76]. In 2015, Morris et al. also
reported that significant further decline in pfcrt 76T and pfmdr1 86Y alleles in Zanzibar [65].
Current study (2017) has shown further reductions in markers associated with tolerance and
resistance to ASAQ since the last time report in 2015. The molecular findings of 2017 study are
consistent with two previous studies, hence, continuous monitoring of genetic changes in SNPs
have strong implication for shaping malaria treatment policy in Zanzibar.
4.2.1 Haplotypes
In the study, pfmdr1 (86Y, 184Y, 1246Y) haplotypes YYY and YYD are the most frequent in
2002-3, but interestingly, NYD and NFD haplotypes were most frequent in 2017. NFD haplotypes
were associated with reduced sensitivity to AL [77]. There was only three YYD and zero YYY
haplotypes that are otherwise known to be the risk factor for treatment failure in patients treated
with ASAQ in African settings [35,77,78]. There was a significant decline of YYY (p < 0.001)
and YYD (p <0.001) between 2002-3 and 2017. A study conducted in Western Kenya reported
that when the drug pressure of AQ is decreased, there was significant increase in selection of
pfmdr1 and pfcrt wild type alleles within three year period (between 2008 – 2010) of replacing
AQ with ACT as a first line treatment [79]. This provides additional evidence that ASAQ remains
an effective first line treatment in Zanzibar.
4.3 Generalizability
This study was a follow-up study to monitor efficacy of local first line ACT in Zanzibar with
adaptation of WHO TES guideline to surveillance of artemisinin resistance. Findings of this paper
may contribute to reaching the elimination target in Zanzibar by ensuring the effectiveness of
ASAQ plus single low dose PQ. The aim is to eliminate local malaria transmission by 2023,
according to the Zanzibar Malaria Elimination Programme. Moreover, this study can contribute to
pooled analysis of resistance data initiated by such as “Worldwide Antimalarial Resistance
Network” (WWARN) and WHO world artemisinin status reports. Although the findings in this
study are specific to the Zanzibar setting, and are not generalizable to other settings with varied
UPPSALA UNIVERSITET Resistance Markers to Artesunate and Amodiaquine
Page 28 of 42
malaria endemicities, and applicable to other ACTs, the study protocol involving satellite sites and
recruitment centers in order to achieve a large enough sample size to power a clinical trial can be
applicable to other low-transmission settings.
4.4 Study Limitations
4.4.1 Limitation of Study Designs
The original studies involved in this analysis are three clinical trials and two surveys. Two surveys
conducted in 2010 and 2013 were health facility based, providing a snap-shot measurement of the
prevalence of drug resistance markers, and did not assess treatment efficacy. Regarding three
clinical trials, follow-up period in 2017 study was 28 days, whilst it was 42 days for the two
previous studies (2002-3 and 2005). Although WHO recommends 28 days follow-up period for
antimalarials including amodiaquine - which have elimination half-lives of less than 7 days, longer
duration of follow-up up to 42 days in clinical trials is beneficial to evaluate the true cure rate [24].
The study conducted in Zanzibar (2002-3) reported that high proportion of recrudescence after
day-28 follow-up [68]. Additionally, laboratory analysis to differentiate re-infection or
recrudescence was not done for day-28 in 2017 study since there was no parasite positivity by
microscopy on day-28.
4.4.2 Participants Characteristics
Despite the patient’s demographic and clinical information such as age, sex, history of fever,
hemoglobin level, treatment history, parasite and gametocyte carriage and parasite density are
available for clinical trials, there was no comprehensive data for two surveys conducted in 2010
and 2013. Therefore, no complete data was available for comparison of fever clearance,
parasitemia and hemoglobin level assessments for all studies.
4.4.3 Molecular Methods
Overall, the limitation of highly sensitive PCR is risk of contamination which can lead to false
positive results. However, even highly sensitivity PCR can still give negative results if the parasite
density approaches to the detection limit. For such very low density sub-microscopic infection can
also give inconsistent results owing to the lower reproducibility [80]. Moreover, the sensitivity of
RFLP assay is low to detect mixed infection. Regarding running of gel-electrophoresis, it is
moderately time consuming and requires minor adjustments to get a better visualization.
Repetition is unavoidable until the DNA bands are clear enough for identification of interest.
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5. Conclusion
Despite the wide-scale use of ASAQ as first-line drug to treat uncomplicated P. falciparum
malaria, efficacy remains high after fourteen years of first deployment in 2003. The molecular
markers associated with ASAQ resistance - SNPs pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y alleles
and pfmdr1 (86Y, 184Y and 1246Y) YYY haplotypes were significantly decreased between 2002-
3 and 2017. Although two synonymous SNPS in the PfK13 gene (five CYS469CYS and one
SER477SER isolates) were identified, these SNPs have not been associated with artemisinin
resistance. Continuous monitoring of drug efficacy and resistance markers is warranted to track
the development and characteristics of artemisinin resistance in Zanzibar.
UPPSALA UNIVERSITET Resistance Markers to Artesunate and Amodiaquine
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Annex
Appendix 1. Showing the remarkable decreasing community prevalences of asexual P.
falciparum parasitemia by microscopy or RDT; all age groups in May/June in two study