Variation at position 540,437 of the dhps gene, 51,59,108 of the dhfr, 86 of the pfmdr1 and 76 of
the pfcrt in Plasmodium falciparum in Tanzania and Uganda
SunJing
Degree project in applied biotechnology, Master of Science (2 years), 2011 Examensarbete i tillämpad bioteknik 45 hp till masterexamen, 2011
Biology Education Centre and Department of medical biochemistry and microbiology, Uppsala
University
Summary
Malaria, a disease of the blood transmitted to human beings by infected mosquitoes, is one of the most severe parasitic diseases all over the world. According to WHO report, about 500,000,000 cases of malaria were recorded and the most seriously affected area is Africa.
Since the first day people found malaria, they have been trying their best to fight against it. Different drugs such as chloroquine, sulfadoxine-pyrimethamine (SP), artemisinin-based combination therapies (ACTs) were developed to treat malaria.
Although these drugs brought people hope, decreasing deaths caused by malaria, they are not so effective due to the increasing incidence of resistance.
When it comes to chloroquine resistance, it is related to mutations at different positions of two genes, mainly at position 76 of Plasmodium falciparum chloroquine resistance transporter (pfcrt) and 86 of Plasmodium falciparum multi-drug resistance gene 1 (pfmdr1). For SP resistance, mutations at position 51, 59,108 of DHFR and 437,540 of DHPS are involved.
The aim of my study was to amplify these specific genes mentioned above. In the first step, I used tris-EDTA buffer based method to extract DNA from filter paper. Then nested-PCR was used to amplify the target gene. After using the outer primers to run the first reaction, both unwanted products and the target sequence could be obtained.
Then nested primers that lie inside the outer ones were used to conduct the second reaction. Compared to basic PCR, nested-PCR is more sensitive and faithful. After that restriction enzymes were used to do the digestion and then preliminary judgments were made whether the samples were wild type, mutant or a mixture of genotypes.
I had 124 samples collected from Tanzania in total. But not all of them got clear bands since perhaps there were too little parasite in the sample. For dhps, 82/124 were successfully analyzed at position 540 and 437. At 540, 20.7% were wild type, 67.1%
were mutants and 10% were mixtures. At 437, 23.2% were wild type, 65.9% were mutants and 10.9% were mixtures. For dhfr, 42/47 were successfully analyzed at position 108, 51 and 59, mutations were dominating (97.6% at 108, 95.2% at 51, 88.1% at 59, mutant + mixture). The data above means that SP resistance in the studies area is still very high. For pfmdr1, 79/124 were successfully analyzed at position 86, wild types were in major (82.3% wild type, 2.5% mixture and 15.2%
mutant). For pfcrt, 70/124 were successfully analyzed at position 76, wild types were
also predominant (78.6% wild type, 2.8% mixture and 18.6% mutant).
Introduction
What is malaria?
Malaria, a disease of the blood that is transmitted to people by the protozoan parasites of the genus Plasmodium, still threatens human’s lives in current days.
There are five types of Plasmodium that can lead to malaria; Plasmodium malariae, Plasmodium vivax, Plasmodium falciparum, Plasmodium ovale and Plasmodium knowlesi. Among these five types, P. falciparum is the most prevalent one, responsible for nearly 90% of the malaria cases [1] and P. knowlesi is from the old world monkeys, having potential to infect human [2].
The burden of malaria
Nowadays, malaria is found in 107 countries all over the world. The world health organization (WHO) reports that 500 millions of people are suffering from malaria and more than 1,000,000 of them die yearly [3]. Sub-Saharan countries are especially severely influenced due to the presence of Anopheles gambiae, which is the vector for transmitting malaria [4]. This kind of mosquito can live for quite a long time, so it can easily transmit malaria among people. Malaria in endemic region is shown in figure 1.
Figure 1: World map of malaria transmission. Adapted from http://www.malaria.com/info/malaria-countries-map.php
Although human have been fighting against malaria since it was first discovered over
100 years ago, malaria still afflicts human all the time [5]. At the moment, the fight is
becoming harder and harder because of the appearance of drug resistance. In some
parts of Southeast Asia, P. falciparum has developed resistance to almost all the
current antimalaria drugs. The situation is almost the same in Africa, with a highly
prevalence of resistance to Chloroquine and increasing resistance to
sulphadoxine/pyrimethamine [4]. This will result in no effective treatment in Africa on a
large scale. Also, there are other factors that can increase the burden of malaria, such
as the change of climate, environmental change and increased population.
Malaria transmission
The female Anopheles mosquito acts as the vector for human malaria. Once a mosquito bites an infected person, it will carry malaria and when it bites another person to feed itself, it transmits malaria to that person. There are three types of transmission. One is through blood, as mentioned above. The other one is transmission from mother to the fetus [6]. The third one is needle stick injury, which means people who share syringes and needles to take drugs have a potential to get infected.
Life cycle of P.falciparum
The life cycle of malaria is complex, involving several stages, either in a human or a mosquito (Fig.2). A series of morphological changes are contained during the transmission [7].
Figure 2: Life cycle of malaria. Adapted from http://www.niaid.nih.gov/topics/malaria/pages/lifecycle.aspx
In the human body
When a female mosquito carrying parasites bites a human for a blood meal, it will inject parasites into the human body in the form of sporozoites with the saliva. This is the beginning of a malaria life cycle (Fig.2). The sporozoites will travel through the human body, finally arriving at the liver and invading the liver cells. Inside the liver cells, the sporozoites can multiply, forming thousands of merozoites (Fig.2). The time taken to finish this step is variable, depending on the various species of malaria.
However, not all the parasites will grow and divide, they may be dormant for a period
in the liver and will cause more severe consequence in the future [8]. Then the
merozoites burst out of the liver cells and go into the blood steam, preparing to invade
erythrocytes. In the red blood cells, the parasites continue to propagate by asexual
replication (Fig.2). These merozoites can degrade haemoglobin, living on the amino
acids, which will result in anaemia. In about 2 days, the newly formed merozoites will
exit erythrocytes, entering the bloodstream again. After this step, there will be
thousands of infected cells in the patient’s bloodstream, and it will induce fever in the patient [9]. In addition, some blood cells infected by merozoite divide into the sexual form instead of the asexual one. These cells are called gametocytes, which will also circulate in the bloodstream (Fig.2).
In the mosquito
When an anopheline mosquito bites an infected person, gametocytes can be taken up into the body of the mosquito and travel to the gut. In the gut, the gametocytes can develop further into gametes, which are more mature. A zygote can be formed by the fusion of the male and female gametes, which can burrow through the gut wall and then form oocysts. Oocysts continue to multiply, forming a great amount of sporozoites, which can travel to and enter the salivary glands where they can stay, waiting for the next meal and thus they can inject the parasites into a new host. Then the cycle in the human body restarts.
Malaria symptoms
The time it takes from the initial malaria injection to the appearance of symptoms is variable according to different species of plasmodium. For Plasmodium falciparum, it takes 9 to 14 days. In the very beginning of the malaria infection, symptoms are similar to those caused by bacteria or other parasites. Such symptoms may include headache, fever, fatigue, chills and etc.
Antimalaria drugs
Chloroquine (CQ)
Chloroquine is a 4-aminoquinoline, which was first discovered in 1934 [10]. Since then, chloroquine has been used widely for its treatment due to its effectiveness and fast-acting. A document in 1948 reported that people successfully used CQ to cure P.vivax malaria [11]. Moreover, CQ is not very expensive, even the poor people can afford it. It is a quite popular drug, especially in poor areas, as, usually, cost is the major consideration, particularly to people with limited sources. Although in the past, the presence of chloroquine brought people hope, decreasing the death caused by malaria and changing the situation all over the world to some extent, nowadays it is not very effective due to the increase in chloroquine resistance [12].
Sulfadoxine-pyrimethamine (SP)
Because chloroquine can not play its role as it did in the past, Tanzania replaced it with SP on a mass scale in 2001 [13]. SP can inhibit the folate pathway which is essential for the biosynthesis reaction. The folate biosynthesis is catalyzed by enzymes dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS).
When SP is used to treat malaria, sulfadoxine can act as a competitive inhibitor of DHPS and pyrimethamine targets DHFR [38]. In addition, like CQ, SP is also inexpensive and is easy to use, and one single dose is enough for the treatment [14].
However, after using SP for a period, people also met a problem with drug resistance.
A study in 2005 investigating the efficacy of SP in Tanzania after use of SP as the first-line drug for two years showed that 49.1% patients did not get successful treatment [13]. This result indicates that after using the drug for a certain time, resistance can be developed, which will lead to treatment failure.
Artemisinin-based combination therapies (ACTs)
Artemisinin (qinghaosu) was isolated by Chinese scientists from Artemisia annua, a kind of herb. The Chinese have used it for 2000 years to treat fever as an herbal tea.
In South-East Asia, it is widely used and in Africa, it is increasingly used [15].
Compared to other antimalarial drugs, artemisinin and its derivatives can show clinical effect faster and remove parasites faster [16]. However, the half life of these drugs are short, if they are used in monotherapy, the patients need to take the drug continuously for over a week [17]. The rapid disappearance of the drugs may bring a risk with a failure in the treatment even without the presence of drug resistance. Therefore, artemisinin is used in combination with other drugs in most cases in order to increase the efficacy. This is known as artemisinin-based combination therapy. Such drugs can last longer in the patient’s body.
Drug resistance
Although there are different kinds of drugs to treat malaria, it is more and more difficult to cure it completely because of the increasing drug resistance to these drugs, especially to chloroquine [18]. Recently, ACT is the most effective one [19], with no definite report about the appearance of the resistance. However, ACT is not as effective as it was according to some occasional cases in French Guyana and Senegal [20]. In addition, resistance to ACT is found in Cambodia [21, 22]. Therefore, it can not be denied that in the near future there will be resistance to this kind of drug.
When it comes to drug resistance, several mechanisms can be involved, as listed in the following.
— Change of the drug target: mutations in the drug target which results in obstructing of drug binding and therefore the drug can not play their original role.
— More production of the target: it can be achieved by gene amplification or by increased transcription and translation which means high level of drugs is required to accomplish treatment.
— Decrease drug accumulation: fewer drugs will arrive at the target.
— Active efflux of drugs
— Drug inactivation
CQ resistance
Treatment failures of malaria by using CQ were detected in Colombia in the late
1950s [23]. In 1960s and 1970s, more and more unsuccessful treatments appeared
due to the spread of the resistance in India, Southeast Asia and South America. Africa
was not influenced until the late 1970s, with the first failure cases were found in Kenya
and Tanzania [24].
Plasmodium falciparum chloroquine resistance transporter (pfcrt)
A series of gene mutations are related to CQ resistance. Plasmodium falciparum chloroquine resistance transporter (pfcrt) is such a gene, which was recognized in 2001 [25]. PFCRT, the protein it translates, is located in the membrane of the digestive vacuole and may influence drug transportation and/or pH regulation [25]. The Pfcrt gene has 13 exons with 6-8 knew point mutations giving resistance [26]. Among those mutations, mutation in position 76 which results in a change in amino acids from lysine to threonine that plays an important role in the resistance to CQ [27]. A series of studies were followed to verify that pfcrt point mutations had relationship with CQ resistance [27, 28, 29, 30]. CQ can exist in the human body in three forms, which are CQ, CQ
+and CQ
++respectively. Only the CQ can diffuse freely and permeate the membrane into the digestive vacuole [31]. Recently, a study by Martin and his colleagues revealed that the protonated form of CQ can be transported out of the vacuole with the help of the mutation in position 76 of pfcrt gene [32]. Once a parasite becomes resistance to CQ, there will be less CQ accumulated in the digestive vacuole [33]. However, the research on the nature of the PFCRT is still ongoing, debating whether it is a channel or a carrier.
Plasmodium falciparum multi-drug resistance gene 1 (pfmdr1)
Besides pfcrt, research on drug resistance also focuses on gene pfmdr1, encoding P-glycoprotein homologue 1 (Pgh1) which located in the membrane of the parasite’s digestive vacuole. There are two homologous halves in Pgh1 with six transmembrane domains and a domain for nucleotide-binding [34]. The definite function of Pgh1 is still unclear but what is know is that it can adjust the susceptibility of parasites to certain drugs, such as chloroquine, artemisinin and mefloquine [35]. Mutations at specific points lead to related changes in amino acids (N86Y, Y184F, S1034C, N1042D and D1246Y) [36].
SP resistance
In Gabon in 2005, treatment with SP was found to be associated with 12%-14%
failures among children under the age of 10 years [37]. The failure is related to mutations in the gene dhfr and dhps which encode for DHFR and DHPS. Therefore, drug binding is reduced.
Dihydrofolate reductase (dhfr)
DHFR can catalyze folate to H
2-folate and further to H
4-folate, which a key step in
folate pathway [39]. In the late 1980s, sequences of DHFR were depicted [40] and it is
well known that pyrimethamine resistance is caused by the accumulation of the
mutations in the dhfr. Resistance is at a low level with a single mutation S108N/T and
is increased with double mutations N51I and S108N/T or C59R and S108N/T. When
triple mutations N51I, C59R and S108N/T are formed, the level of resistance is even
higher. In addition, mutations in position 16 and 164 also contribute to the resistance
[41].
Dihydropteroate synthase (dhps)
Specific mutations in dhps are related for Plasmodium falciparum resistance to sulfadoxine [42]. It is reported that mutations in codons 436, 437, 581 and 613 contribute to the resistance to sulfadoxine. Later, variation in codon 540 also plays a vital role in the resistance [43, 44]. These mutations can decrease drug affinity. By using sulfadoxine and pyrimethamine together, a synergistic effect can be achieved and it can take more time to develop related drug resistance [45]. Research on the mechanism of the resistance to certain drugs is essential, people can find new ways to cure malaria effectively and monitor drug activity.
Aim of the study
My studies focused on the gene dhfr, dhps, pfmdr1 and pfcrt. In the first step, I used
nested PCR to amplify certain genes and then the enzyme digestions were followed
by using the successful PCR products as samples. Finally, I made preliminary
judgments that whether the sample is wild type, mutant or mixture.
Results
Dhps amplification and digestion PCR results
Figure 3: PCR amplification of Dhps. Lane 1: 100bp ladder. Lane 2-18: samples from Uganda.
Lane19-20: 3d7 and dd2 as positive control. Lane 21: negative control.
Digestion results
At position 540, if it is wild type, it can not be digested with FokⅠ, but if it is mutant, it can be digested in two fragments. The sizes of the two parts are 94 bp and 634 bp respectively. At position 437, the situation is the same with that at position 540. After digestion with AvaⅡ, two fragments with the size of 402 bp and 326 bp can be detected by using UV transillumination.
Figure 4: Restriction digestion of dhps PCR products. Lane 1-8: Ava digestion. Ⅱ Lane 10-17:
FokⅠdigestion. Ladder: 100 bp.
Samples from Uganda
Totally 68 samples were amplified, however, only 14 of them worked successfully (Fig.3). These products were used to do enzyme digestion to make a preliminary judgment that whether the sample is wild type (wt), mutant (mut) or mixture (mix) (contains both wild type and mutant, a mixed infection) (Fig.4). The results are shown in the table 1. 7 (50%) of the samples in position 437 show a wild type genotype, while the remaining ones are mutant. In position 540, 7 (50%) of the samples also show a wild type genotype.
Table 1: Amplification and digestion of dhps gene at position 540 and 437. Samples collected in Uganda.
No. AvaⅡ(437) FokⅠ(540) No. AvaⅡ(437) FokⅠ(540)
39 Mut Mut 224 Mut Mut
46 Wt Wt 247 Mut Mut
183 Wt Wt 502 Wt Wt
195 Mut Mut 505 Wt Wt
210 Wt Wt 508 Wt Wt
216 Mut Mut 571 Wt Wt
223 Mut Mut 625 Mut Mut
Samples from Tanzania
124 samples were analyzed. Among these samples, 85 were successfully amplified and 82 were digested (Table.2). At position 540, 17 (20.7%) of the samples were wild type, 55 (67.1%) expose a mutant genotype, and the remaining 10 (12.2%) gave a mixture genotype. At position 437, 19 (23.2%) were wild type, 54 (65.9%) showed a mutant genotype and 9 (10.9%) were mixture.
Table 2: Amplification of dhps gene at position 540 and 437. Samples are from Tanzania.
No. AvaⅡ (437)
FokⅠ
(540) No. AvaⅡ (437)
FokⅠ
(540) No. AvaⅡ (437)
FokⅠ
(540) No. AvaⅡ (437)
FokⅠ (540) 08
Day 0 --- --- 17
Day14 --- --- 34
Day 0 --- --- 61
Day14 Wt Wt 08
Day 3 --- --- 18
Day 0 Mut Mut 35
Day 0 Mut Mut 61
Day28 --- --- 08
Day14 Wt Wt 18
Day28 Mut Mut 36
Day 0 --- --- 62
Day 0 Mut Mut 08
Day28 Wt Wt 19
Day 0 Mut Mut 37
Day 0 Mut Mut 63
Day 0 Mix Mix 09
Day 0 Wt Wt 20
Day 0 Mut Mut 38
Day 0 Mut Mut 63
Day14 --- --- 09
Day 3 Wt Wt 20
Day14 --- --- 39
Day 0 Mut Mut 63
Day28 --- --- 09
Day14 Wt Wt 20
Day28 --- --- 40
Day 0 Mut Mut 64
Day 0 Mut Mut 09
Day28 --- --- 21
Day 0 --- --- 41
Day 0 Mut Mut 64
Day28 --- --- 10
Day 0 mix mix 22
Day 0 Mut Mut 42
Day 0 Mut Mut 65
Day 0 Mut Mut 10
Day 3 mix mix 22
Day14 Mut Mut 43
Day 0 Mut Mut 66
Day 0 Mut Mut 10
Day14 Wt Wt 22
Day28 --- --- 44
Day 0 Mut Mut 66
Day14 Wt Wt 10
Day28 Wt Wt 23
Day 0 Mut Mut 45
Day 0 Wt Wt 66
Day28 --- --- 11
Day 0 Mut Mut 23
Day14 --- --- 46
Day 0 Mix Mix 67
Day 0 --- --- 11
Day14 Wt Wt 23
Day28 --- --- 47
Day 0 Mix Mix 68
Day 0 Wt Mix 11
Day28 Wt Wt 24
Day 0 Mut Mut 48
Day 0 Mut Mut 70
Day 0 Mut Mut
12
Day 0 Mut Mut 24
Day14 --- --- 49
Day 0 Mut Mut 70
Day14 Mut Mut 12
Day14 --- --- 24
Day28 --- --- 50
Day 0 Mut Mut 71
Day 0 Mix Mix 12
Day28 --- --- 25
Day 0 Mut Mut 51
Day 0 Wt Wt 71
Day14 --- --- 13
Day 0 Mut Mut 25
Day14 --- --- 52
Day 0 Mut Mut 72
Day 0 Mut Mut 13
Day14 --- --- 26
Day 0 Mut Mut 53
Day 0 Mut Mut 74
Day 0 Mut Mut 13
Day28 --- --- 26
Day14 Mut Mut 54
Day 0 Mut Mut 75
Day 0 Mut Mut 14
Day 0 Wt Mix 26
Day28 --- --- 54
Day14 --- --- 75
Day14 --- --- 14
Day14 --- --- 27
Day 0 --- --- 54
Day28 --- --- 76
Day 0 Mix Mix 14
Day28 --- --- 27
Day14 Mix Mix 55
Day 0 Mut Mut 77
Day 0 Mut Mut 15
Day 0 Mut Mut 27
Day28 --- --- 56
Day 0 Mix Mix 78
Day 0 Mut Mut 15
Day14 Mut Mut 28
Day 0 --- --- 57
Day 0 Mut Mut 79
Day 0 --- --- 15
Day28 --- --- 29
Day 0 --- --- 58
Day 0 Mut Mut 80
Day 0 Mut Mut 16
Day0 Mut Mut 30
Day 0 --- --- 58
Day14 --- --- 81
Day 0 Mut Mut 16
Day14 Wt Wt 31
Day 0 Wt Wt 59
Day 0 Mut Mut 82
day 0 Wt Wt 16
Day28 --- --- 32
Day 0 Mut Mut 60
Day 0 Wt Wt 83
Day 0 Mut Mut 17
Day 0 Mut Mut 33
Day 0 --- --- 61
Day 0 Mut Mut 84
Day 0 Mut Mut
Dhfr amplification and digestion
PCR results
The sizes of PCR products were 523 bp (primer M3 and F/) and 215 bp (primer M3
and pfdhfr6). The PCR products generated by using primer (pfdhfr3+pfdhfr4) and
(pfdhfr5+pfdhfr6) were 290 bp and 176 bp respectively.
Figure 5: dhfr PCR results. Left: nested-PCR reaction whose primers were M3 and F/. Middle: amplified by using primers pfdhfr3 and pfdhfr4. Right: primers used for nested-PCR were pfdhfr5 and pfdhfr6.
Ladder:100 bp.
Digestion results
At position 108, if it is wild type, it can be digested into two fragments, but if it is mutant, it can not be digested. The size of the digestion product is the same as that of the PCR product. At position 51, the situation is the same with that at position 108. And at position 59, the size of the wild type’s band is 30bp larger than that of the mutant’s band.
Figure 6: Enzyme digestion. Upper: Samples were from Uganda. Bottom: samples were from Tanzania.
Left: Alu digestion. Middle: Ⅰ Tsp5091 digestion. Right: Taq digestion. Ladder: 100bp. Ⅰ
Samples from Uganda
Totally 68 samples were amplified, only 18 of them were successful (Fig.5). Among there 18 products, at position 108, 5 (27.8%) were mutant, 3 (16.7%) were wild type and the remaining 10 (55.5%) were mixture (Fig.6). At position 51, 4 (22.2%) showed mutant, 3 (16.7%) were wild type and 11 (61.1%) gave a mixture genotype. At position 59, only 1 (5.6%) was mutant, 8 (44.4%) were wild type and 9 (50%) were mixture.
Table 3: Amplification of dhfr gene at position 108, 51 and 59. Samples are from Uganda.
No.
AluⅠ (108)
Tsp5091 (51)
TaqⅠ
(59) No.
AluⅠ (108)
Tsp5091 (51)
TaqⅠ (59)
39 Mut Mut Mut 501 Wt Wt Wt
46 Mix Mix Wt 502 Mut Mix Mix
183 Mix Mix Wt 505 Wt Wt Wt
195 Mix Mix Mix 508 Mut Mut Mix
210 Mix Mix Wt 543 Mix Mix Mix
216 Mix Mix Mix 577 Mix Mix Mix
223 Mut Mut Mix 579 Mix Mix Wt
224 Mut Mut Mix 609 Mix Mix Wt
247 Mix Mix Mix 625 Wt Wt Wt
Samples from Tanzania
The number of the total samples was 47 and 42 of them were successfully amplified and digested. At position 108, 39 (92.9%) were mutant, 1 (2.4%) was wild type and 2 (4.7%) were mixtures. At position 51, 26 (61.9%) showed a mutant genotype, 2 (4.8%) showed a wild type genotype and the remaining 14 (33.3%) showed a mixed genotype. At position 59, 23 (54.8%) gave a mutant genotype, 5 (11.9%) gave a wild type genotype and 14 (33.3%) gave a mixed genotype.
Table 4: Amplification of dhfr gene at position 108, 51 and 59. Samples are from Tanzania.
No. AluⅠ (108)
Tsp5091 (51)
TaqⅠ (59)
No. AluⅠ (108)
Tsp5091 (51)
TaqⅠ (59) 49
Day 0
Mut Mut Mut 65
Day 0
Mut Mut Wt
50 Day 0
Mut Mix Mut 66
Day 0
Mut Wt Mut
51 Day 0
Mut Mix Mix 66
Day14
Mut Mut Mut
52 Day 0
Mut Mix Mut 66
Day28
Mut Mut Mix
53 Day 0
Mut Mix Wt 67
Day 0
Mut Mut Mix
54 Day 0
Mut Mix Mut 68
Day 0
Mut Mut Mut
54 Day14
--- --- --- 70
Day 0
Mut Mut Mut
54 Day28
--- --- --- 70
Day14
Mut Mut Mix
55 Day 0
Mut Mix Mut 71
Day 0
Mut Mut Mut
56 Day 0
Mut Mut Mix 71
Day14
Mut Mut Mix
57 Day 0
Mut Mix Mut 72
Day 0
Mut Mut Mut
58 Day 0
Mut Mix Mut 74
Day 0
Mut Mut Mut
58 Day14
--- --- --- 75
Day 0
Mut Mut Mut
59 Day 0
Mut Mut Wt 75
Day14
Mut Mut Mix
60 Day 0
Mut Mix Wt 76
Day 0
Mut Mut Mix
61 Day 0
Mut Mix Mix 77
Day 0
Mut Mut Mut
61 Day14
Mix Mix Mix 78
Day 0
Mut Mut Mut
61 Day28
Wt Wt Wt 79
Day 0
Mut Mut Mix
62 Day 0
Mut Mix Mut 80
Day 0
Mut Mut Mut
63 Day 0
Mut Mix Mut 81
Day 0
Mut Mut Mut
63 Day14
--- --- --- 82
Day 0
Mut Mut Mut
63 Day28
--- --- --- 83
Day 0
Mut Mut Mix
64 Day 0
Mut Mut Mut 84
Day 0
Mut Mut Mix
64 Day28
Mix Mix Mix
Pfmdr1 amplification and digestion
PCR results
The sizes of PCR products were 560 bp (Fig.7).
Figure 7: pfmdr1 PCR results. Ladder: 100 bp.
Digestion results
At position 86, if it is wild type, it can be digested into three parts whose sizes are 230
bp, 245 bp, 74 bp respectively. But only two fragments can be detected by using UV
transillumination as the difference between the former two bands is quite small. If it is
a mutant genotype, it can be digested into two parts. The sizes of these two are 480
bp and 74 bp (Fig.8).
Figure 8: Apo digestion. Ladder: 100Ⅰ bp.
There are 124 samples in total. 81 of them were successfully amplified and 79 of them got clear digestion results. At position 86, 12 (15.2%) showed a mutant genotype, 65 (82.3%) showed a wild type genotype and 2 (2.5%) showed a mixture genotype.
Table 5: Amplification of pfmdr1 gene at position 86. Samples are from Tanzania.
No. ApoⅠ No. ApoⅠ No. ApoⅠ No. ApoⅠ No. ApoⅠ No. ApoⅠ 08
Day 0 --- 14
Day 0 Wt 23
Day 0 Wt 35
Day 0 Mut 54
Day28 --- 67
Day 0 --- 08
Day 3 --- 14
Day14 --- 23
Day14 --- 36
Day 0 --- 55
Day 0 Wt 68
Day 0 Mix 08
Day14 Mut 14
Day28 --- 23
Day28 --- 37
Day 0 Wt 56
Day 0 Wt 70
Day 0 Wt 08
Day28 Mut 15
Day 0 Wt 24
Day 0 Wt 38
Day 0 Wt 57
Day 0 Wt 70
Day14 --- 09
Day 0 --- 15
Day14 Wt 24
Day14 --- 39
Day 0 Wt 58
Day 0 Wt 71
Day 0 Mix 09
Day 3 --- 15
Day28 --- 24
Day28 --- 40
Day 0 Wt 58
Day14 --- 71
Day14 --- 09
Day14 Mut 16
Day0 Wt 25
Day 0 Mut 41
Day 0 Wt 59
Day 0 Wt 72
Day 0 Wt 09
Day28 Mut 16
Day14 --- 25
Day14 --- 42
Day 0 Wt 60
Day 0 Wt 74
Day 0 Wt 10
Day 0 Wt 16
Day28 --- 26
Day 0 Mut 43
Day 0 Wt 61
Day 0 Wt 75
Day 0 Wt 10
Day 3 Wt 17
Day 0 Wt 26
Day14 --- 44
Day 0 Mut 61
Day14 Wt 75
Day14 --- 10
Day14 mut 17
Day14 Wt 26
Day28 --- 45
Day 0 Wt 61
Day28 --- 76
Day 0 Wt 10
Day28 Mut 18
Day 0 Wt 27
Day 0 --- 46
Day 0 Wt 62
Day 0 Wt 77
Day 0 Wt 11
Day 0 Wt 18
Day28 Wt 27
Day14 Wt 47
Day 0 Wt 63
Day 0 Wt 78
Day 0 Wt 11
Day14 mut 19
Day 0 Wt 27
Day28 --- 48
Day 0 Wt 63
Day14 --- 79
Day 0 --- 11
Day28 Mut 20
Day 0 Wt 28
Day 0 --- 49
Day 0 Wt 63
Day28 --- 80
Day 0 Wt 12
Day 0 Wt 20
Day14 Wt 29
Day 0 --- 50
Day 0 Wt 64
Day 0 Wt 81
Day 0 Wt 12
Day14 --- 20
Day28 --- 30
Day 0 Wt 51
Day 0 Wt 64
Day28 --- 82
day 0 Wt 12
Day28 --- 21
Day 0 --- 31
Day 0 Wt 52
Day 0 Wt 65
Day 0 Wt 83
Day 0 Wt 13
Day 0 Wt 22
Day 0 Wt 32
Day 0 --- 53
Day 0 Wt 66
Day 0 Wt 84
Day 0 Wt
13
Day14 --- 22
Day14 Wt 33
Day 0 --- 54
Day 0 Wt 66
Day14 ---
13
Day28 --- 22
Day28 --- 34
Day 0 --- 54
Day14 --- 66
Day28 ---
Pfcrt amplification and digestion
PCR results
The sizes of PCR products were 145 bp (Fig.9).
Figure 9: pfcrt PCR results. Ladder: 100 bp.
Digestion results
At position 76, if it is wild type, it can be digested into two parts whose sizes are 95 bp and 50 bp respectively. If it is a mutant genotype, it can not be digested (Fig.10).
Figure 10: Apo digestion. Ladder: 100Ⅰ bp.
For pfcrt, there were 70 samples successfully amplified and digested under a total number 124. At position 76, 13 (18.6%) were mutant, 55 (78.6%) were wild type and 2 (2.8%) were mixture.
Table 6: Amplification of pfcrt gene at position 76. Samples are from Tanzania.
No. ApoⅠ No. ApoⅠ No. ApoⅠ No. ApoⅠ No. ApoⅠ No. ApoⅠ 08
Day 0 Wt 14
Day 0 --- 23
Day 0 Wt 35
Day 0 Mut 54
Day28 --- 67
Day 0 Wt 08
Day 3 --- 14
Day14 --- 23
Day14 --- 36
Day 0 Mut 55
Day 0 Wt 68
Day 0 Wt 08
Day14 --- 14
Day28 --- 23
Day28 --- 37
Day 0 Mut 56
Day 0 Wt 70
Day 0 Wt 08
Day28 --- 15
Day 0 Wt 24
Day 0 Wt 38
Day 0 Wt 57
Day 0 Wt 70
Day14 --- 09
Day 0 --- 15
Day14 Mut 24
Day14 --- 39
Day 0 Wt 58
Day 0 Wt 71
Day 0 Wt 09
Day 3 --- 15
Day28 --- 24
Day28 --- 40
Day 0 Wt 58
Day14 --- 71
Day14 --- 09
Day14 --- 16
Day0 wt 25
Day 0 wt 41
Day 0 Wt 59
Day 0 Wt 72
Day 0 wt 09 --- 16 --- 25 --- 42 Wt 60 Wt 74 Wt
Day28 Day14 Day14 Day 0 Day 0 Day 0 10
Day 0 --- 16
Day28 --- 26
Day 0 Mut 43
Day 0 Mut 61
Day 0 Wt 75
Day 0 Wt 10
Day 3 Wt 17
Day 0 Wt 26
Day14 Wt 44
Day 0 Wt 61
Day14 Wt 75
Day14 --- 10
Day14 --- 17
Day14 --- 26
Day28 --- 45
Day 0 Wt 61
Day28 --- 76
Day 0 Wt 10
Day28 --- 18
Day 0 Wt 27
Day 0 --- 46
Day 0 Wt 62
Day 0 Mut 77
Day 0 Wt 11
Day 0 --- 18
Day28 Mut 27
Day14 Mix 47
Day 0 Wt 63
Day 0 Mix 78
Day 0 Wt 11
Day14 --- 19
Day 0 Wt 27
Day28 --- 48
Day 0 Wt 63
Day14 --- 79
Day 0 Wt 11
Day28 --- 20
Day 0 Wt 28
Day 0 --- 49
Day 0 Wt 63
Day28 --- 80
Day 0 Mut 12
Day 0 --- 20
Day14 Mut 29
Day 0 --- 50
Day 0 Wt 64
Day 0 Wt 81
Day 0 Wt 12
Day14 --- 20
Day28 --- 30
Day 0 --- 51
Day 0 Mut 64
Day28 --- 82
day 0 Wt 12
Day28 --- 21
Day 0 --- 31
Day 0 Wt 52
Day 0 Wt 65
Day 0 Wt 83
Day 0 Wt 13
Day 0 --- 22
Day 0 Wt 32
Day 0 Mut 53
Day 0 Wt 66
Day 0 Wt 84
Day 0 Wt 13
Day14 --- 22
Day14 Mut 33
Day 0 --- 54
Day 0 Wt 66
Day14 ---
13
Day28 --- 22
Day28 --- 34
Day 0 --- 54
Day14 --- 66
Day28 ---
Table 7: Summary of all the results.
Gene dhps dhfr pfmdr1 pfcrt
position 540 437 51 59 108 86 76
Wild type 20.7% 23.2% 4.8% 11.9% 2.4% 82.3% 78.6%
Mutant 67.1% 65.9% 61.9% 54.8% 92.9% 15.2% 18.6%
Mixture 12.2% 10.9% 33.3% 33.3% 4.7% 2.5% 2.8%
Discussion
Samples from Uganda
There were only 14 of 68 samples successfully amplified. The number was not enough to illustrate the trend in this area.
Samples from Tanzania
From the PCR results, most Day 14 and Day 28 samples were not successful no matter which gene was amplified, which meant there was too little parasite in the samples.
For dhps, at position 540 and 437, mutations were in major (79.3% at 540 and 76.8%
at 437, mutant+ mixture). Samples no.10, 11, 16, 61 and 66 got successful PCR
results on both Day 14 and Day28. From these results, on Day 0, the type of the parasite was mutant, but after treated for 3 to 28 days, it changed to wild type. This might mean that before treatment with the current used drugs, parasites in the human body were mutant and resistance to the former drugs, but after using this new drug for a while, the parasites became wild type and finally disappeared. For dhfr, at position 108, 51 and 59, mutations were also in major (97.6% at 108, 95.2% at 51, 88.1% at 59, mutant + mixture). The data above means that SP resistance in the study area is still very high.
For pfmdr1, at position 86, wild types were in major (84.8%, wild type+ mixture). For pfcrt, at position 76, wild types were also predominant (81.4%, wild type+ mixture).
Samples no.15, 18, 20, 22 got successful PCR results on Day 14. And the results showed that the type of the parasite changed from wild type to a mutant genotype, which might mean that after treated for a while, the parasites in the human body became resistance to the drugs.
Materials and methods
Study areas
Samples were collected both from Uganda and Tanzania. In Uganda, Lganga district was studied. And in Tanzania, samples came from Lgombe.
Sample collection
Blood samples from the patients were collected on filter paper (Whatman 3mm) and dried in a clean container for three hours. The dry filter paper could be used to extract DNA if it was required, otherwise they were stored in room temperature. The samples I used in this study had two parts. One part was older, collected from October to November 2008 in Uganda. The other one was new, obtained in November 2010 in Tanzania. Samples from patients who did not receive treatment were marked as Day 0. Samples taken from patients who were treated by the drug for three days were labeled Day 3. 14 days after treatment were named Day 14 and Day 28 meant the patients had already been treated for 28 days.
Extraction of parasite DNA
Tris-EDTA buffer based extraction was used to obtain parasite DNA.
Material
Tris (10mM, pH 8.0) EDTA (0.1mM) Distilled water
Use a scissor to cut the blood filter paper into a piece and put it in an Eppendorf tube.
Add 65μl Tris-EDTA buffer to each tube and keep them in room temperature for 1 hour.
Then incubate the tubes at 50 for 15 minutes using a heat block. After that, use ℃
pipette tips to push the filter papers to the bottom of the tube gently several times.
Then incubate again at 97 for another 15minutes in order to elute the DNA. Finally, ℃ centrifuge the tubes for a few seconds and transfer the solution to new tubes. Store at -20℃.
Amplification of certain gene by PCR
PCR system
Table 8: PCR system used for amplification
PCR-outer PCR-nested
Green Buffer 2 μl Green Buffer 2 μl
dNTP 2 μl dNTP 2 μl
Primer forward 1 μl Primer forward 1 μl
Primer reverse 1 μl Primer reverse 1 μl
Dream Taq 0.5 μl Dream Taq 0.5 μl
DNA(sample) 2 μl DNA(sample from PCR-outer reaction) 1 μl
H2O 11.5 μl H2O 12.5 μl
total 20 μl total 20 μl
Dhps amplification
Primer used for outer and nested PCR
Table 9: Primers used for dhps amplification. The size of the nested reaction products was 728 bp.
Name Sequence N185(outer forward) 5’-TGA TAC CCG AAT ATA AGC ATA ATG-3’
N218(outer reverse) 5’-ATA ATA GCT GTA GGA AGC AAT TG-3’
Rc(nested forward) 5’-GGT ATT TTT GTT GAA CCT AAA CG-3’
Rd(nested reverse) 5’-ATC CAA TTG TGT GAT TTG TGG AC-3’
Program for amplification
Table 10: Program used for dhps amplification
Name PCR program outer 94 for 3min; 25cycles:94 1min, 51 2min, 72 1min; 74 10min; 4 hold℃ ℃ ℃ ℃ ℃ ℃
Nested 94 for 3min; 25cycles:94 1min, 54 2min, 72 1min; 72 10min; 4 hold℃ ℃ ℃ ℃ ℃ ℃
Dhfr amplification
Samples from Uganda
Primer used for outer and nested PCR
Table 11: Primers used for dhfr amplification. After the outer reaction, two nested reactions were proceeded, the primers used for nested reaction were M3 and F/, M3 and pfdhfr6 respectively. The size of product amplified by M3 and F/ was 523 bp. And the size of the product amplified by M3 and pfdhfr6 was 215 bp.
Name Sequence M1(outer forward) 5’-TTT ATG ATG GAA CAA GTC TGC-3’
M5(outer reverse) 5’-AGT ATA TAC ATC GCT AAC AGA-3’
M3(nested forward) 5’-TTT ATG ATG GAA CAA GTC TGC GAC GTT-3’
F/(nested reverse) 5’-AAA TTC TTG ATA AAC AAC GGA ACC TTT TA-3’
Pfdhfr6(nested reverse) 5’-CAT ATT TTG ATT CAT TCA CAT ATG TTG TAA CTA CTC-3’
Program used for dhfr amplification
Table 12: Program used for dhfr amplification.
Name PCR program Outer 94 2min; 45 cycles:94 30sec, 50 45sec, 72℃ ℃ ℃ ℃ 1min; 72 5min; 4 hold℃ ℃
Nested
(M3+F/) 94 3min; 40 cycles:94 1min, 45 1min, 72 1min; 72 10min; 4 hold℃ ℃ ℃ ℃ ℃ ℃ Nested
(M3+pfdhfr6) 94 1min; 10 cycles: 94 20sec, 60 30sec, 72 30sec; 35 cycles: 94 20sec, ℃ ℃ ℃ ℃ ℃ 58 30sec, 72 30sec; 4 hold℃ ℃ ℃
Samples from Tanzania
Primer used for outer and nested PCR
Table 13: Primers used for dhfr amplification. After the outer reaction, two nested reactions were proceeded, the primers used for nested reaction were pfdhfr3 and pfdhfr4, pfdhfr5 and pfdhfr6 respectively. The size of product amplified by pfdhfr3 and pfdhfr4 was 290 bp. And the size of the product amplified by pfdhfr5 and pfdhfr6 was 176 bp.
Name Sequence Pfdhfr1(outer forward) 5’-ATG ATG GAA CAA GTC TGC GAC-3’
Pfdhfr2(outer reverse) 5’-C TTG ATA AAC AAC GGA ACC TCC-3’
Pfdhfr3(nested forward) 5’-ACT ACA CAT TTA GAG GTC TAG G-3’
Pfdhfr4(nested reverse) 5’-GG TTC TAG ACA ATA TAA CAT TTA TCC-3’
Pfdhfr5(nested forward) 5’-GCC ATA TGT GCA TGT TGT AAG GTT GAA AG-3’
Pfdhfr6(nested reverse) 5’-CAT ATT TTG ATT CAT TCA CAT ATG TTG TAA CTG CTC-3’
Program used for dhfr amplification
Table 14: Program used for dhfr amplification.
Name PCR program Outer 94 3min; 5 cycles:94 30sec, 56 30sec, 72 45sec; 8 cycles: 92 30sec, ℃ ℃ ℃ ℃ ℃
55 30sec, 72 45sec; 12 cycles: 92 30sec, 53 30sec, 72 45sec; 18 ℃ ℃ ℃ ℃ ℃ cycles: 92 30sec, 50 30sec, 72 45sec; 16 hold℃ ℃ ℃ ℃
Nested
(pfdhfr3+pfdhfr4) 94 3min; 30 cycles:94 1min, 50 1min℃ ℃ ℃ , 72 1min; 72 10min; 4 hold℃ ℃ ℃ Nested
(pfdhfr5+pfdhfr6) 94 3min; 30 cycles: 94 1min, 55 1min, 72 1min; 72 10min; 4 hold℃ ℃ ℃ ℃ ℃ ℃
Pfmdr1 amplification
Primer used for outer and nested PCR
Table 15: Primer used for pfmdr1 amplification. The size of the nested product was 560 bp.
Name Sequence
O1(outer forward) 5’-TGT TGA AAG ATG GGT AAA GAG CAG AAA GAG-3’
O2(outer reverse) 5’-TAC TTT CTT ATT ACA TAT GAC ACC ACA AAC-3’
N1(nested reverse) 5’-GTC AAA CGT GCA TTT TTT ATT AAT GAC CAA ATA-3’
N2(nested forward) 5’-AAA GAT GGT AAC CTC AGT ATC AAA GAA GAG-3’
Program for amplification
Table 16: Program used for pfmdr1 amplification
Name PCR program outer 94 for 3min; 35cycles:94 1min, 45 1min, 72 1min; 72 10min; 4 hold℃ ℃ ℃ ℃ ℃ ℃
Nested 94 for 3min; 30cycles:94 1min, 45 2min, 72 1min; 72 10min; 4 hold℃ ℃ ℃ ℃ ℃ ℃
Pfcrt amplification
Primer used for outer and nested PCR
Table 17: Primers used for pfcrt amplification. The size of the nested product was 145 bp.
Name Sequence Crt P1(outer forward) 5’-CCG TTA ATA ATA AAT ACA CGC AG-3’
Crt P2(outer reverse) 5’-CGG ATG TTA CAA AAC TAT AGT TAC C-3’
Crt D1(nested forward) 5’-TGT GCT CAT GTG TTT AAA CTT-3’
Crt D2(nested reverse) 5’-CAA AAC TAT AGT TAC CAA TTT TG-3’
Program for amplification
Table 18: Program used for pfcrt amplification.
Name PCR program outer 94 for 3min; 35cycles:94 30sec, 56 30sec, 72 1min; 72 5min; 4 hold℃ ℃ ℃ ℃ ℃ ℃
Nested 94 for 3min; 25cycles:94 30sec, 56 30sec, 72 1min; 72 5min; 4 hold℃ ℃ ℃ ℃ ℃ ℃
Enzyme digestion
Digestion system
Table 19: System used for digestion.
buffer 2 μl
DNA(PCR products) 5 μl
Enzyme 0.5 μl
H2O 12.5 μl
Total 20 μl
Dhps digestion
Use Ava Ⅱ and Fok Ⅰ to digest the PCR products at 37 for 2 hours respectively. ℃
Dhfr digestion
The PCR products amplified by primer (M3+F/) and (pfdhfr3+pfdhfr4) were digested by Alu at 37 Ⅰ ℃. While products amplified by (M3+pfdhfr6) and (pfdhfr5+pfdhfr6) were digested by Tsp5091 and Taq at 65 for 2 hours separately. Ⅰ ℃
Pfmdr1 digestion
Apo Ⅰ was used to digest the PCR products at 37 for 2 hours. ℃
Pfcrt digestion
The PCR products were also digested by Apo . Ⅰ
Acknowledgement
I want to appreciate to Göte Swedberg’s group in department of medical biochemistry and microbiology. Thanks to Göte, the group leader, also my best supervisor ever, his great knowledge and hard working impress me a lot. He gave me detailed guidance during my whole work, trusted me and left me space to develop my experiment skills.
Thanks for the help he offered and patience and tolerance he had with everything,
especially with my mistakes. Also thanks to other members in the group. Thanks for
the encouragement and help they provided. Because of these members, the corridor
is very lovely. Thank you.
Reference
[1] WHO 2008b. WHO Global Malaria Programme. World Malaria Report: 2008. Geneva: World Health Organization, 2008.
[2] Anderios, F. NoorRain, A. Vythilingam, I, 2010. In vivo study of human Plasmodium knowlesi in Macaca fascicularis. Experimental Parasitology, 124, 181-189.
[3] Budi Setiawan. Current Malaria Management: Guideline 2009. 2010. Clinical practice. October 2010 Vol 42, Number 4.
[4] Greenwood, Mutabingwa.B.T., 2003. Malaria in 2002. Nature, 415,670-672.
[5] Kublin,J.G., F.K.Dzinjalamala, D.D.Kamwendo,etc. 2002. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis, 185, 380-388.
[6] Gutman.J., Slutsker.L.. Malaria control in pregnancy: still a long way to go. Online/Articles DOI:10.1016/S1473-3099(10)70295-4
[7] Hanssen.E., McMillan.P.J, Tilley.L. 2010. Cellular architecture of Plasmodium falciparum-infected erythrocytes. International joural for parasitology, 40, 1127-1135
[8] Bannister,L.H., Mitchell, G.H.. 2009. The malaria merozoite, forty years on. Parasitology, 136, 1435-1444.
[9] Miller,L.H., Baruch, D.I., Marsh, K., Doumbo, O.K.. 2002. The pathogenic basis of malaria. Nature, 415, 673-679.
[10] Thompson PE, Werbel LM. 1972. Antimalarial agents: chemistry and pharmacology. In: DeStevens G, ed. Medicinal chemistry. Vol. 12. New York: Academic Press.
[11] Pullman TN, Craige Jr B, Alving AS, Whorton CM, Jones Jr R, Eichelberger. L. 1948. Comparison of chloroquine, quinacrine (atabrine) and quinine in the treatment of acute attacks of sporozoite-induced vivax malaria, Chesson strain. J Clin Invest, 27(3 Pt1), 46–50.
[12] Savarino.A, Boelaert.J.R, Cassone.A, Majori.G, and Cauda.R.. 2003. Effects of chloroquine on viral infections: an old drug against today’s diseases? Lancet Infect Dis, 3, 722–27
[13] Mugittu.K, Abdulla.S, Falk.N, Masanja.H, Felger.I, Mshinda.H, Beck.H.P., Genton.B. 2005. Efficacy of sulfadoxine-pyrimethamine in Tanzania after two years as first-line drug for uncomplicated malaria:
assessment protocol and implication for treatment policy strategies. Malaria journal, 4,55
[14] Hien.T.T, Arnold.K, Hung.N.T, Loc.P.P, Dung.N.T, Cuong.B.M, Toan.L.M, Phung.M.Q, Anh.L.H.V, Mai.P.P. 1994. Single dose artemisinin-mefloquine treatment for acute uncomplicated falciparum malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene. 88(6), 688-691.
[15] Agtmael.M.A.V, Eggelte T.A, Boxtel.C.J.V.. 1999. Artemisinin drugs in the treatment of malaria:
from medicinal herb to registered medication. Review, 20, 199-205.
[16] Adjuik M, Babiker A, Garner P, Olliaro P, Taylor W, White N, International Artemisinin Study Group.
2004. Artesunate combinations for treatment of malaria: meta-analysis. The Lancet,363(9402),9–17.
[17] Meshnick SR, Taylor TE, Kamchonwongpaisan S. 1996. Artemisinin and the antimalarial endoperoxides: from herbal remedy to targeted chemotherapy. Microbiological Reviews, 60(2), 301–15.
[18] Bloland.P.B.. 2001. Drug resistance in malaria. In: WHO/CDS/CSR/DRS/2001.4, WHO, Geneva,
