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Malaria is a tropical parasitic disease caused by four species of protozoan parasites of the genus Plasmodium, which are Plasmodium falciparum, P.vivax, P. ovalae and P. malariae. In 2004, the simian malaria namely P. knowlesi has been firstly reported in the southern part of Thailand which has been found in Malaysia since 1965 (Jongwutiwes, et al., 2004). Of these species, P. falciparum is the most dangerous form of the disease which can lead to cerebral malaria, coma and death.
Malaria is still one of the world's most important parasitic diseases of developing countries. The WHO | world malaria report 2009 has been reported that half of the world's population is at risk of malaria, and an estimated 243 million people are infected with malaria. It is nearly 863,000 deaths in 2008; almost all deaths occur in African children under 5 years of age (WHO | world malaria report 2009: retrieved October 3rd, 2010). The burden of malaria is mostly found in tropical and sub-tropical areas, throughout sub-Saharan Africa, Southeast Asia, the Pacific Islands, India and Central and South America (Snow et al., 2001).
The epidemiological data of malaria situation in Thailand showed a downward trend in total cases from approximately 200,000 cases in 1991 to some 100,000 cases in 1996. The annual parasite incidence (API) was 2.27 per 1,000 populations in 1999. In 2006, a total of 66,651 malaria cases (both Thai and foreigner) were reported in which Tak, Mae Hong Son and Kanchanaburi provinces are the high incidence of malaria cases ranking respectively first (8,648 cases), third (2,411 cases) and fifth (1,250 cases) of top ten provinces with highest malaria cases reported (Na-Bangchang ; Congpuong, 2007).
The natural infections of the P. falciparum parasite often contain more than one parasite strain. The reason is the human and the mosquito hosts are exposed to heterogeneous parasite populations. Naturally, P. falciparum infections are consist of mixed clones, especially in highly endemic areas (Zakeri et al., 2005; F?rnert et al., 2001).
The merozoite surface protein2 (MSP2) gene has been frequently used as a genetic marker for genotyping of P. falciparum. This gene is divided into two groups; FC27 and 3D7 type as well as by length polymorphisms (Symthe et al., 1990). The length variations of this gene are depended on the repeated sequences, and each allele type can be readily distinguished after gel electrophoresis of Polymerase Chain Reaction (PCR)-amplified products. PCR genotyping for detection of MSP2 has been demonstrate as a great technique to characterize parasite population evenly in small amount of blood samples with low parasitemia (F?rnert et al., 2001).
Another surface antigen gene is the 220-kDa glutamate-rich protein (GLURP) found in all the developmental stages of the parasite, including the surface of newly released merozoites (Stricker et al., 2000). The alleles are classified by size polymorphisms. This gene also has been used as a genetic marker for genotyping of parasite population (F?rnert et al., 2001).
The in vitro cultivation in laboratory was initiated by collecting parasitized blood samples from malaria patient in the endemic areas and then transported these samples by car on the day of collection at ambient temperature. The P. falciparum blood samples were immediately cultured on arrival in order to avoid a loss of some parasite populations in a case of multiple infection. The selective growth of P. falciparum subpopulation in pre-and post-culture samples was observed by Viriyakosol and colleagues (1994) using three surface antigen genes- MSP1 (block 2 and 4), MSP2 and GLURP for parasite characterization. The results showed the pattern alterations of molecular marker which reflected the changing in subpopulations in culture over the time of cultivation.
Nowadays, decreasing in the number of malaria cases in Thailand has been reported. The transported sample by car seems to be high cost consumable method for blood sample collection. This resulted in developing alternative method which more cost effectiveness. Thus, the malaria program, College of Public Health Sciences, conducted the preliminary project to explore the possibility of using EMS as sample transportation. The success of in vitro culture from EMS transported samples was acceptable. Seventy nine percent (34 of 43 samples) from Trat province, 65.8% (52 of 79 samples) from Ranong province, 80.0% (28 of 35 samples) from Tak province, 28.5% (18 of 63 samples) from Ubon Ratchathani province, 71.7% (28 of 39 samples) from Mae Hong Son province and 85.0% (85 of 99 samples) from Kanchanaburi province were successfully grown in vitro. However, the information of the changing or loss of parasite population in pre- and post-cultivation must be further studied. Therefore, this project will be used the two surface antigen genes (MSP2 and GLURP) for examining the changing in P. falciparum subpopulation in pre- and post-cultivation of the EMS-transported samples from three provinces of Thailand.
2.1 Geographic distribution and causative agents
Malaria remains the most important of the tropical diseases, widespread throughout the subtropics and tropics, but also occurring in many temperate zones. The disease exacts a heavy toll of illness and death, especially amongst children in Africa. It also poses a risk to business travelers, tourists and immigrants, and imported cases of malaria are increasingly seen in non-endemic areas such as Europe and North America. The transmission is frequent in rural areas especially in economic developing country. Treatment and control have become more difficult with the spread of P. falciparum resistant to antimalarial drugs, and insecticide resistant strains of the mosquito vectors (WHO, 1990).
The causative agents of human malaria are four species of single-celled protozoan parasites of the genus Plasmodium; P. falciparum, P. vivax, P. ovale, and P. malariae. The geographic distribution of malaria mainly follows the distribution of Anopheles, disease vector. Anopheles can survive and reproduce well in tropical climates. The distribution for each species is showed below:
P. falciparum- throughout tropical Africa, Asia and Latin America
P. vivax- worldwide in tropical and some temperate zones
P. ovale- mainly in tropical West Africa
P. malariae-worldwide but very patchy distribution
In Thailand, the prevalence of the disease remains reported from along the borders between Thailand and Myanmar, Cambodia and Malaysia (Konchom et. al, 2003).
The parasite life cycle involves two hosts. The first one is a vertebrate host (human) and the second one is an invertebrate host (mosquito). During a blood meal, a malaria-infected female Anopheles mosquito with a malaria infection inoculates the sporozoites into the human host. Sporozoites penetrate into the bloodstream then infect liver cells. The sporozoites develop and mature into schizonts (exo-erythrocytic stage), which rupture and release merozoites into blood circulation. This merozoites invade the red cells and develop to mature schizonts in the red cell (erythrocytic stage). The multiple infections normally found in P. falciparum infection. The ring, trophozoites mature into schizonts, which rupture and releasing merozoites again. Some parasites, starting at ring stage differentiate into male and female gametocytes (sexual erythrocytic stage). The blood stage parasites are responsible for the clinical manifestations of the disease.
The sexual cycle starting with, male (microgametocytes) and female (macrogametocytes), are ingested by female Anopheles mosquito during a blood meal. The gametocytes develop and multiply in the mosquito's stomach beginning of the sporogonic cycle. The fertilization is started with the microgametes penetrate into the macrogametes to generate zygotes. The zygotes become motile and elongated in shape called ookinetes which this shape can easily invade the midgut wall of the mosquito where they develop into oocysts. The mature oocysts rupture and release sporozoites which migrate to the mosquito's salivary glands and inoculate into a new human host for starting with new life cycle and malaria transmission happening.
2.3 Genetic diversity of malaria parasites
P. falciparum population in endemic areas is frequently mixed strain or clone, usually containing 1-4 or more clonal populations. The parasite genotypes are identical for each strain or clonal. The mechanisms of genetic recombination, gene conversion, and duplication normally generate new strain in P. falciparum populations. Different parasite strains with different genotype resulting in different phenotypes. This reason effect on the diversity of P. falciparum population in endemic areas. Analysis of the genetic diversity could indicate the structure of parasite population (Snounou G. and Beck H-P., 1998).
P. falciparum displays extensive genetic variation in the merozoite surface antigens, mostly founding in merozoite stage of its life cycle. The marked diversity has been found in genes encoding the merozoite surface protein 1 (MSP1), merozoite surface protein2 (MSP2) and the glutamine rich protein (GLURP). These highly polymorphic genes have been frequently used as genetic markers for genotyping of P. falciparum (Orht et al., 1997).
2.3.1 The merozoite surface protein1 (MSP1 gene)
MSP1 is a major protein on the surface of the blood stages of the parasite. It is composed of a 190-kDa membrane-anchored protein complex, which undergoes proteolytic cleaved into four fragments that remain on the merozoite surface. Before the merozoite (erythrocyte stage) invasion, the entire MSP1 complex is shed, except for the C-terminal 19kDa (MSP119), which remains on the surface as the merozoite enters the erythrocyte (Blackman et al., 1990).
The sequence of MSP1gene can be divided into 17 blocks based on sequence variability. Most of the sequence in MSP1 groups into two distinct allele families (Tanabe et al., 1987), with the exception of block 2, which is repetitive region that consists of four allele families (Takala et al., 2002). Block 17 contains MSP119, which has been the focus of malaria vaccine development because of its highly conserved sequence and hypothesized critical function. (Takala S.L, 2009).
2.3.2 The Merozoite surface protein2 (MSP2) gene
Merozoite surface protein2 (MSP2) gene is a second family of merozoite surface antigens, normally found in the erythrocytic stages. It is a 43-50-kDa glycoprotein and has been localized on the plasma membrane of intracellular and free merozoites. This molecule is anchored on the plasma membrane of the merozoite by a C-terminal glycosylphosphatidylinositol (GPI) moiety (Gerold et al., 1966). MSP2 is highly polymorphic with conserved N- and C-terminal domains flanking a central variable region, which contains tandemly arrayed repetitive sequences. All MSP2 alleles have been categorized into two groups typified by 3D7 and FC27 alleles, respectively, because of differences in the repeats and flanking variable sequences. The MSP2 of P. falciparum displays extreme size and allelic polymorphism.
The MSP2 is divided into five blocks of whole gene sequence. The N- terminal and C-terminal translated sequence, block 1 and block 5, respectively (Figure 3). The block 3 of MSP2 is a polymorphic central region flanked by two non-repetitive regions (block 2 and block 4). The extensive polymorphism of MSP2 alleles is normally used for marked diversity for analysis of P. falciparum population.
2.2.3 The glutamine rich protein (GLURP) gene
The glutamine rich protein is a 220-kDa an exoantigen found in the parasitophorous vacuole of P. fafciparum. The single full-length GLURP sequence (F32 strain) shows two amino acid repeat regions namely R1 and R2 regions (Figure 4). Diversity in GLURP has been indicated by difference sized PCR products the R2 region of various laboratory-adapted and field isolates. The sequence of this gene showed highly conserved among geographically dispersed isolates, and a fusion protein with this gene sequence was able to stimulate B- and T-cells (Borre et al. and Stricker et al.).
2.4 Genotyping of P. falciparum
The most frequently used techniques with interesting potential for identifying genetic variations of P. falciparum are based on the PCR (Mullis et. al, 1986) PCR is a major breakthrough in molecular research because its sensitivity allows the analysis of genes from small amount of parasite materials.
Nested PCR uses two sets of amplification primers (Van et al., 2002). The target DNA sequence of one set of primers (termed 'inner' primers) is located within the target sequence of the second set of primers (termed 'outer' primers).
In practice, a standard PCR reaction is first run with the patient sample using the outer primers. Then a second PCR reaction is run with the inner primers using the product of the first reaction as the amplification target. This procedure increases the sensitivity of the assay by re-amplifying the product of the first reaction in a second reaction. The specificity of the assay is increased because of the inner primers amplify only if the first PCR reaction yielded a specific product. However, nested PCR could not identify different DNA sequences with the same size.
Long and colleagues (1995) were used a standard PCR protocol to amplify P. falciparum DNA which was extracted by methanol extraction from dried blood spot on filter paper. All specimens were collected from participants in a Plasmodium falciparum vaccine trial and from samples collected during a hospital-based study in Thailand. The samples were shipped by mail to the laboratory in Maryland where the assay was carried out. The PCR was performed by amplification of the P. falciparum circumsporozoite protein gene based on a previously published sequence. Sensitivity was 100% when compared with thick blood film results in the vaccine trial (range = 4-60 parasites/?l, median = 8 parasites/ ?l) and 94.6% (range = 3-133,988 parasites/?l, median = 616 parasites) in the hospital study.
Bereczky and colleagues (2005) described a new Tris-EDTA (TE) buffer-based method for extraction of DNA from blood dried on filter paper. The P. falciparum blood samples was blotted onto two types of filter paper; 3MM? filter paper and 903? Schleicher & Schuell filter paper and store for 1-2 year. The DNA extraction was extracted by the three methods; TE buffer-based method, methanol extraction and Chelex? method respectively. The PCR was performed by nested amplification of merozoite surface protein 2 (msp2) which is widely used in molecular epidemiological studies, drug trails to determine the number or types of parasite genotypes of P. falciparum infections. The sensitivity and reproducibility were higher in the TE buffer method than in the Chelex? and methanol method when performed on the two filter paper types stored for 15 and 29 months, respectively. For 903? Schleicher & Schuell filter paper blood spots were lower in sensitivity and reproducibility due to a long storage of these samples. The DNA extraction using TE buffer protocol was done in a few hours prior to the PCR amplification. The effect on long-term storage on the quality of DNA extracted by the TE buffer method still needs to be evaluated.
The research of F?rnert and colleagues (1997) was focused on the observation of the daily dynamics of P. falciparum subpopulations in asymptomatic children in rural Tanzania by using a nested PCR on msp1, msp2 and glurp genes. The P. falciparum subpopulations were observed in the eight children harboring P. falciparum throughout the study period. The parasite populations were found to be highly complex with daily changes in both parasite density and genotyping pattern. These findings implicated that the use of the parasite samples from peripheral blood may be only partly reflect the whole parasite population in an infected individual.
3.1.1 Study site
Blood samples were collected from willing patients who attended for malaria diagnosis at clinics in three provinces (Mae Hong Son, Tak and Kanchanaburi provinces) in Thailand during the year 2007 to 2011.
3.1.2 Sample size
A sample size was calculated as following equation:
Sample size (n) = Z2? PQ/(P- lower limit)2
When, Z? = Confidence level at 95% (Standard value of 1.96)
P = Estimated prevalence of multiple infection in the study area
According to the result of Pumpaibool et al., 2009 found that the prevalence of multiple infected samples from six provinces in Thailand were 44% in Mae Hong Son, 31% in Tak, 27% in Kanchanaburi, 22% in Ubon Ratchathani, 36% in Trat and 32% in Ranong. The selected provinces in this study are Mae Hong Son, Tak and Kanchanaburi provinces with multiple infection from 44%, 31% and 27% respectively. The mean value is equal 34%, so the P value is equal 0.34
Q = 1-p = 1-0.34 = 0.66
The expected multiple infection in this study area is estimated at 0.3 and the lower limit is
estimated at 0.2 then the maximum permissible error (d) is equal 0.1
Sample size (n) = (1.96)2(0.34x0.66)/(0.1)2
= 86 cases
So the sample will be collected at least 86 cases from three provinces in Thailand during the year 2007 to 2011. The total number of samples both before and after culture are finally equal at least 172 samples for the experiment.
- The patient is not less than 5 years old and willing to participate in this research project.
- The patient who has a new infection with P. falciparum diagnosed by a malaria officer.
- The patient is present of one or more of the general danger signs or any sign of severe or complicated malaria.
3.1.4 Ethical considerations
The research proposal will be submitted to Ethical Review Committee for Research Involving Human Research Subjects, Health Science Group, Chulalongkorn University (ECCU) for the ethical clearance of this study project. Informed consent will be obtained from each participant by signature or thumbprint in the presence of a witness. For foreigner participants who do not understand in Thai language both speaking and listening; informed consent will be asked by a malaria official with communicable language.
3.1.5 Parasite collection
The P. falciparum infected patient identified by a malaria official will be taken a small amount of peripheral blood from the selected finger by a malaria official. Before piercing, patient's finger will be cleaned with cotton wool lightly soaked in 70% ethanol for at least two times, allow to dry, puncture the ball of the finger with a sterile lancet. The first drop of blood (about 20-50 ?l) is sucked by using a sterile pipette tip, then put in a micro-centrifuge tube containing 500 ?l of transport medium with 5 units of Heparin, mix by inverting the tube. Another 20-50 of blood is kept in the second tube. The samples are kept in refrigerator (4-10oC). The second drop of blood is absorbed on filter paper, air-dried and put in Zip-locked plastic bag after completely dry. The last drop of blood is used for making the thin and thick film on the same slide. The code number is written on the slide with a soft lead pencil. All specimens are put in a small mail box on the morning of Wednesday and sent by EMS.
The partial samples from the research project under the Malaria program, College of Public Health Sciences were used in this study.
3.2 Microscopic examination and confirmation
Thin and thick blood smears are stained with 10% Giemsa solution for 10-15 minutes. In thin blood film, parasites are counted against 10,000 red blood cells using a magnification of 1,000 X. it can be reported as parasites per 10,000 red blood cells and parasitaemia can be calculated by multiplication of parasite count with red blood cells, then divided by 10,000 (number of red blood cells counted)
Example: Parasite count is 500 per 10,000 red blood cells
Standard red blood cell counted is 4,500,000 cells/?l of blood
Parasitaemia = (500 x 4,500,000)/10,000
= 225,000 parasites/?l of blood
In thick blood films, parasite density can be defined by counting asexual parasites (rings, trophozoites and schizonts) against 200 white blood cells and reported as parasites per 200 white blood cells. The number of parasites relative to leucocytes counted can be calculated and expressed as parasites/?l of blood, from the formula:
(Parasites counted/No. of leukocytes) x 8,000 = parasites/?l of blood
(Karbwang and Harinasuta, 1992)
3.3 In vitro culture of P. falciparum
This study will be used the combined techniques of Trager and Jensen (1976) and Thaithong and colleagues (1994) for culturing of the P. falciparum finger-prick blood samples. The blood sample in transport medium is spun down at 1500 rpm for 2-3 min, the upper layer of transport medium is pipetted off, and the packed cells are transferred to 3 wells of 96-well microtitre plate containing 100 ?l of complete RPMI medium plus 10% pooled serum. The contents of the wells are thoroughly mixed. Then the plates are covered with lids, placed in a candle-jar (Trager and Jensen, 1976), and incubate at 37oC. When the number of parasites has reached 3-5%, the cultures are transferred to Petri dishes for expanding the number and the quantity of parasites.
3.4 DNA extraction protocol
In this study, two DNA extraction protocol will be used: the first one is Chelex? extraction protocol for extraction of genomic DNA of P. falciparum from preserved blood on filter paper as described below:
3.4.1 Chelex? extraction protocol (Bereczky et al., 2005)
One blood spot on filter paper is cut and placed in eppendorf tube and incubated overnight at 4oC in 1 ml of 0.5% saponin in phosphate-buffered saline (PBS). The punches are washed for 30 minutes in PBS at 4oC, then transfered into new tubes containing 25 ?l of stock solution (20% Chelex-100) and 75 ?l of distilled water, and vortexed for 30 seconds. The tubes are heated at 99oC for 15 minutes to elute the DNA, vortexed and centrifuged at 10,000xg for 2 minutes. The supernatant (65 ?l) are transferred into new tubes.
The second one is Phenol-Chloroform extraction protocol will be used for extraction of fresh or frozen P. falciparum blood samples in case of a reference or control case for all PCR reactions. The protocol is described below:
3.4.2 Phenol extraction protocol (Snounou et al., 1993)
Parasite samples are washed twice by 200 ?l ice-cold PBS. 500?l of 0.5% saponin in PBS is mixed to each sample. The mixture is placed at room temperature for 5 minutes or until lysis is observed. The samples are centrifuged at 10,000 rpm for 5 minutes and the supernatant is removed. Pellet is washed 2-3 times with ice-cold PBS and 100 ?l of lysis buffer (Lysis buffer: 40mM Tris-HCl, pH 8.0; 80mM EDTA, pH 8.0; 2% SDS) with 2 mg/ml proteinase K is added. To dilute the mixture, 50 ?l of sterile double distilled water (ddH2O) is added and gently mix. The mixture is incubated overnight at 37oC, at the end of the incubation period; the mixture is adjusted to 30 ?l by adding sterile ddH2O and mix. DNA is extracted with an equal volume of saturated phenol: chloroform (1:1). To precipitate the DNA 1:10 volume of 3 M sodium acetate, pH 5.2 and 2 volume of cold absolute ethanol is added to the extracted solution. After incubation for overnight at -20oC, DNA is pelleted by centrifugation at 13,000 rpm for 10 minutes at 4oC and the supernatant is carefully removed. The pellet is washed with 1 ml cold 70% absolute ethanol and dissolved in 20 ?l of TE buffer (TE buffer: 100mM Tris-HCL, pH 8.0; 10mM EDTA, pH 8.0). One microliter of dissolved DNA is used for PCR amplification.
3.5 PCR amplification for the block 2 of MSP1, block 3 of MSP2 and GLURP sequence
The primers will be used in this research from Snounou et al., 1999 which were designed from published sequences as listed in the UNDP/World Bang/WHO-TDR Malaria Database complied by Ross Coppel. The sequence of the primers and other details in the respective genes are presented in Table 1
The nested PCR amplifications are performed with one microliter of the genomic DNA of P. falciparum, 0.5 units of i-Taq DNA polymerase (Invitrogen), 125 ?M of each dNTPs, 1x PCR buffer, 2 mM MgCl2 and 125 nM of each primer in a total volume of 20 ?l per reaction, in the first reactions. The second amplification is performed in the same condition as in the first amplification, except 1?l of the products are used for the amplification. The reaction mixture is incubated in a Thermocycler (GeneAmp 9700 and Veriti ). The PCR parameter is composed of an initial denaturation period of 5 min at 95oC , annealing for 30 sec at 58oC (the first and nested reactions for GLURP) or 61oC (all nested reactions for msp1 & 2), extension for 30 sec at 72oC, denaturation for 30 sec at 95oC, the last extension for 7 min. The PCR cycles are performed for 20-25 cycles for the outer amplification and 30 cycles for nested amplification. Five microliters of PCR products and 1 ?l of 5x gel loading buffer are analyzed by agarose gel electrophoresis in 1x TBE buffer.
3.6 Visualization of PCR products (Snounou et al., 1993)
Five microliters of 1kb ladder marker and five microlitres of amplification products are mixed with 1 ?l of 5x loading buffer and loaded into each well of 1.5-2% agarose or NuSieve agarose gel. The gel is run at 80 volts for 25-30 minutes. The agarose gel is stained with 0.5 ?g/ml ethibromide for 10-20 min and destained with distilled water for 5-10 min. The stained agarose gel is visualized and photographed with AutoChemi?System by using Labwork 4.0?software (UVP Biomaging Systems) and Cannon Digital camera.
3.7 Data analysis
The estimation for the P. falciparum population in before and after culture of EMS-transported blood samples by the use of PCR technique is generally observed from the multiple bands in the PCR product of PCR amplification which reflect the presence of many different strains in an isolate. The P. falciparum population in all samples will be estimated from the number of bands observed in an agarose gel. For MSP2, the number of FC27 and IC1/3D7 bands is counted.
4.1 Polymorphisms in MSP2 of P. falciparum
The polymorphisms of P. falciparum in each sample were assessed by determining the number and the size variation (alleles) of MSP2 gene. The amplification products form nested PCR of MSP2 as showed by the gel image and table.
Fifteen samples partially used in this study were successfully amplified for MSP2. The number of allele types in each sample analyzed by nested PCR was shown in the table 2. From these observations, it appears that the numbers of allele types of MSP2 ranged from 1 to 2 but it was mostly 1 in the sample after in vitro culture Table 2: The number of allele types analyzed by nested PCR of MSP2 in before and after culture of P. falciparum isolates
Two alleles were detected in four isolates before culture (MH62, MH65, MH67, and T222) but remained one allele type after culture. For MH62, MH65, MH67 isolates were found two bands of FC27 type but mixed FC27 and IC1 type was found in T222 isolate only.
In conclusion, 4/15 (26.66%) samples were changed after culture by disappeared of the band, 1/15 (6.66%) samples was different in size but same allele types.
At the MSP2 loci, 12/15 (80%) samples contained FC27 type and 3/15 (20%) samples contain IC1 type. The predominant allele of MSP2 was FC27 as 12 out of 15 samples (80.0%) harboured FC27 type, 3 out of 15 samples (33.33%) were IC1/3D7 type.
The data presented in this study demonstrate that nested PCR can distinguish FC27 and IC1/3D7 alleles of MSP2 gene. The result showed the changing of allele pattern comparing between before and after culture in MH62, MH65, MH67 and T222 isolates. In T222 isolate disappeared of IC1/3D7 allele in after culture remaining only FC27 allele with smaller size.
This result indicated that the P. falciparum isolates from the endemic areas, transported by EMS to laboratory in Bangkok following in vitro culture were changed in parasite population when using only one MSP2 marker gene. The alterations of P. falciparum population could effect in characteristic of sample using for research experiment.
Viriyakosol and colleagues (1994) used a PCR technique for parasite genotyping with the three surface antigen genes: msp1 block 2 and 4, the whole gene of msp2 and glurp before and after in vitro culture in 101 of a total 269 samples. The changes were detected in one or more of the three markers in the remaining 37 cases. In this research article, the malaria blood samples were collected from all the patients who attended at malarial clinic in the day of collection. The blood samples were taken from venous blood by a doctor and transported to the laboratory in Bangkok on a day of collection and in vitro culture was done as soon as possible on arrival.