Malaria is a tropical parasitic disease caused by four species of protozoon parasites of the genus Plasmodium, which are Plasmodium falciparum, P.vivax, P. ovalae and P. malariae. In 2004, the simian malaria viz. P. knowlesi has been foremost reported in the southern portion of Thailand which has been found in Malaysia since 1965 ( Jongwutiwes, et al. , 2004 ) . Of these species, P. falciparum is the most unsafe signifier of the disease which can take to intellectual malaria, coma and decease.

Malaria is still one of the universe ‘s most of import parasitic diseases of developing states. The WHO | universe malaria study 2009 has been reported that half of the universe ‘s population is at hazard of malaria, and an estimated 243 million people are infected with malaria. It is about 863,000 deceases in 2008 ; about all deceases occur in African kids under 5 old ages of age ( WHO | universe malaria study 2009: retrieved October 3rd, 2010 ) . The load of malaria is largely found in tropical and sub-tropical countries, throughout sub-Saharan Africa, Southeast Asia, the Pacific Islands, India and Central and South America ( Snow et al. , 2001 ) .

The epidemiological information of malaria state of affairs in Thailand showed a downward tendency in entire instances from about 200,000 instances in 1991 to some 100,000 instances in 1996. The one-year parasite incidence ( API ) was 2.27 per 1,000 populations in 1999. In 2006, a sum of 66,651 malaria instances ( both Thai and alien ) were reported in which Tak, Mae Hong Son and Kanchanaburi states are the high incidence of malaria instances ranking severally first ( 8,648 instances ) , 3rd ( 2,411 instances ) and 5th ( 1,250 instances ) of top 10 states with highest malaria instances reported ( Na-Bangchang ; Congpuong, 2007 ) .

The natural infections of the P. falciparum parasite frequently contain more than one parasite strain. The ground is the human and the mosquito hosts are exposed to heterogenous parasite populations. Naturally, P. falciparum infections are consist of assorted ringers, particularly in extremely endemic countries ( Zakeri et al. , 2005 ; F? rnert et al. , 2001 ) .

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The merozoite surface protein2 ( MSP2 ) cistron has been often used as a familial marker for genotyping of P. falciparum. This cistron is divided into two groups ; FC27 and 3D7 type every bit good as by length polymorphisms ( Symthe et al. , 1990 ) . The length fluctuations of this cistron are depended on the perennial sequences, and each allelomorph type can be readily distinguished after gel cataphoresis of Polymerase Chain Reaction ( PCR ) -amplified merchandises. PCR genotyping for sensing of MSP2 has been demonstrate as a great technique to qualify parasite population equally in little sum of blood samples with low parasitaemia ( F? rnert et al. , 2001 ) .

Another surface antigen cistron is the 220-kDa glutamate-rich protein ( GLURP ) found in all the developmental phases of the parasite, including the surface of freshly released merozoites ( Stricker et al. , 2000 ) . The allelomorphs are classified by size polymorphisms. This cistron besides has been used as a familial marker for genotyping of parasite population ( F? rnert et al. , 2001 ) .

The in vitro cultivation in research lab was initiated by roll uping parasitized blood samples from malaria patient in the endemic countries and so transported these samples by auto on the twenty-four hours of aggregation at ambient temperature. The P. falciparum blood samples were instantly cultured on reaching in order to avoid a loss of some parasite populations in a instance of multiple infection. The selective growing of P. falciparum subpopulation in pre-and post-culture samples was observed by Viriyakosol and co-workers ( 1994 ) utilizing three surface antigen genes- MSP1 ( barricade 2 and 4 ) , MSP2 and GLURP for parasite word picture. The consequences showed the pattern changes of molecular marker which reflected the changing in subpopulations in civilization over the clip of cultivation.

Presents, diminishing in the figure of malaria instances in Thailand has been reported. The transported sample by auto seems to be high cost consumable method for blood sample aggregation. This resulted in developing alternate method which more cost effectivity. Therefore, the malaria plan, College of Public Health Sciences, conducted the preliminary undertaking to research the possibility of utilizing EMS as sample transit. The success of in vitro civilization from EMS transported samples was acceptable. Seventy nine per centum ( 34 of 43 samples ) from Trat state, 65.8 % ( 52 of 79 samples ) from Ranong state, 80.0 % ( 28 of 35 samples ) from Tak state, 28.5 % ( 18 of 63 samples ) from Ubon Ratchathani state, 71.7 % ( 28 of 39 samples ) from Mae Hong Son state and 85.0 % ( 85 of 99 samples ) from Kanchanaburi state were successfully grown in vitro. However, the information of the changing or loss of parasite population in pre- and post-cultivation must be farther studied. Therefore, this undertaking will be used the two surface antigen cistrons ( MSP2 and GLURP ) for analyzing the changing in P. falciparum subpopulation in pre- and post-cultivation of the EMS-transported samples from three states of Thailand.

2.1 Geographic distribution and causative agents

Malaria remains the most of import of the tropical diseases, widespread throughout the semitropicss and Torrid Zones, but besides happening in many temperate zones. The disease exacts a heavy toll of unwellness and decease, particularly amongst kids in Africa. It besides poses a hazard to concern travellers, tourers and immigrants, and imported instances of malaria are progressively seen in non-endemic countries such as Europe and North America. The transmittal is frequent in rural countries particularly in economic developing state. Treatment and control have become more hard with the spread of P. falciparum resistant to antimalarial drugs, and insecticide immune strains of the mosquito vectors ( WHO, 1990 ) .

The causative agents of human malaria are four species of one-celled protozoon parasites of the genus Plasmodium ; P. falciparum, P. vivax, P. ovale, and P. malariae. The geographic distribution of malaria chiefly follows the distribution of Anopheles, disease vector. Anopheles can last and reproduce good in tropical climes. 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- chiefly in tropical West Africa

P. malariae-worldwide but really patchy distribution

In Thailand, the prevalence of the disease remains reported from along the boundary lines between Thailand and Myanmar, Cambodia and Malaysia ( Konchom et. Al, 2003 ) .

The parasite life rhythm involves two hosts. The first 1 is a craniate host ( human ) and the 2nd 1 is an invertebrate host ( mosquito ) . During a blood repast, a malaria-infected female Anopheles mosquito with a malaria infection inoculates the sporozoites into the human host. Sporozoites penetrate into the blood stream so infect liver cells. The sporozoites develop and mature into schizonts ( exo-erythrocytic phase ) , which rupture and release merozoites into blood circulation. This merozoites invade the ruddy cells and develop to maturate schizonts in the ruddy cell ( erythrocytic phase ) . The multiple infections usually found in P. falciparum infection. The ring, trophozoites mature into schizonts, which rupture and let go ofing merozoites once more. Some parasites, get downing at pealing phase differentiate into male and female gametocytes ( sexual erythrocytic phase ) . The blood phase parasites are responsible for the clinical manifestations of the disease.

The sexual rhythm get downing with, male ( microgametocytes ) and female ( macrogametocytes ) , are ingested by female Anopheles mosquito during a blood repast. The gametocytes develop and multiply in the mosquito ‘s tummy beginning of the sporogonic rhythm. The fertilisation is started with the microgametes penetrate into the macrogametes to bring forth fertilized ovums. The zygotes become motile and elongated in form called ookinetes which this form can easy occupy 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 secretory organs and inoculate into a new human host for get downing with new life rhythm and malaria transmittal go oning.

2.3 Familial diverseness of malaria parasites

P. falciparum population in endemic countries is often assorted strain or ringer, normally incorporating 1-4 or more clonal populations. The parasite genotypes are indistinguishable for each strain or clonal. The mechanisms of familial recombination, cistron transition, and duplicate usually bring forth new strain in P. falciparum populations. Different parasite strains with different genotype ensuing in different phenotypes. This ground consequence on the diverseness of P. falciparum population in endemic countries. Analysis of the familial diverseness could bespeak the construction of parasite population ( Snounou G. and Beck H-P. , 1998 ) .

P. falciparum shows extended familial fluctuation in the merozoite surface antigens, largely establishing in merozoite phase of its life rhythm. The pronounced diverseness has been found in cistrons encoding the merozoite surface protein 1 ( MSP1 ) , merozoite surface protein2 ( MSP2 ) and the glutamine rich protein ( GLURP ) . These extremely polymorphous cistrons have been often used as familial markers for genotyping of P. falciparum ( Orht et al. , 1997 ) .

2.3.1 The merozoite surface protein1 ( MSP1 cistron )

MSP1 is a major protein on the surface of the blood phases of the parasite. It is composed of a 190-kDa membrane-anchored protein composite, which undergoes proteolytic cleaved into four fragments that remain on the merozoite surface. Before the merozoite ( erythrocyte phase ) invasion, the full MSP1 composite is shed, except for the C-terminal 19kDa ( MSP119 ) , which remains on the surface as the merozoite enters the red blood cell ( Blackman et al. , 1990 ) .

The sequence of MSP1gene can be divided into 17 blocks based on sequence variableness. Most of the sequence in MSP1 groups into two distinguishable allele households ( Tanabe et al. , 1987 ) , with the exclusion of block 2, which is insistent part that consists of four allele households ( Takala et al. , 2002 ) . Block 17 contains MSP119, which has been the focal point of malaria vaccinum development because of its extremely conserved sequence and hypothesized critical map. ( Takala S.L, 2009 ) .

2.3.2 The Merozoite surface protein2 ( MSP2 ) cistron

Merozoite surface protein2 ( MSP2 ) cistron is a 2nd household of merozoite surface antigens, usually found in the erythrocytic phases. 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 ) mediety ( Gerold et al. , 1966 ) . MSP2 is extremely polymorphous with conserved N- and C-terminal spheres flanking a cardinal variable part, which contains tandemly arrayed insistent sequences. All MSP2 allelomorphs have been categorized into two groups typified by 3D7 and FC27 allelomorphs, severally, because of differences in the repetitions and flanking variable sequences. The MSP2 of P. falciparum shows extreme size and allelomorphic polymorphism.

The MSP2 is divided into five blocks of whole cistron sequence. The N- terminus and C-terminal translated sequence, block 1 and barricade 5, severally ( Figure 3 ) . The block 3 of MSP2 is a polymorphous cardinal part flanked by two non-repetitive parts ( barricade 2 and barricade 4 ) . The extended polymorphism of MSP2 allelomorphs is usually used for pronounced diverseness for analysis of P. falciparum population.

2.2.3 The glutamine rich protein ( GLURP ) cistron

The glutamine rich protein is a 220-kDa an exoantigen found in the parasitophorous vacuole of P. fafciparum. The individual full-length GLURP sequence ( F32 strain ) shows two amino acid repeat parts viz. R1 and R2 parts ( Figure 4 ) . Diverseness in GLURP has been indicated by difference sized PCR merchandises the R2 part of assorted laboratory-adapted and field isolates. The sequence of this cistron showed extremely conserved among geographically dispersed isolates, and a merger protein with this cistron sequence was able to excite B- and T-cells ( Borre et al. and Stricker et al. ) .

2.4 Genotyping of P. falciparum

The most often used techniques with interesting potency for placing familial fluctuations of P. falciparum are based on the PCR ( Mullis et. Al, 1986 ) PCR is a major discovery in molecular research because its sensitiveness allows the analysis of cistrons from little sum of parasite stuffs.

Nested PCR uses two sets of elaboration primers ( Van et al. , 2002 ) . The mark DNA sequence of one set of primers ( termed ‘inner ‘ primers ) is located within the mark sequence of the 2nd set of primers ( termed ‘outer ‘ primers ) .

In pattern, a standard PCR reaction is first tally with the patient sample utilizing the outer primers. Then a 2nd PCR reaction is run with the inner primers utilizing the merchandise of the first reaction as the elaboration mark. This process increases the sensitiveness of the check by re-amplifying the merchandise of the first reaction in a 2nd reaction. The specificity of the check is increased because of the interior primers amplify merely if the first PCR reaction yielded a specific merchandise. However, nested PCR could non place different DNA sequences with the same size.

Long and co-workers ( 1995 ) were used a standard PCR protocol to magnify P. falciparum DNA which was extracted by methanol extraction from dried blood topographic point on filter paper. All specimens were collected from participants in a Plasmodium falciparum vaccinum test and from samples collected during a hospital-based survey in Thailand. The samples were shipped by mail to the research lab in Maryland where the check was carried out. The PCR was performed by elaboration of the P. falciparum circumsporozoite protein cistron based on a antecedently published sequence. Sensitivity was 100 % when compared with thick blood movie consequences in the vaccinum test ( range = 4-60 parasites/ ? cubic decimeter, average = 8 parasites/ ? cubic decimeter ) and 94.6 % ( range = 3-133,988 parasites/ ? cubic decimeter, average = 616 parasites ) in the infirmary survey.

Bereczky and co-workers ( 2005 ) described a new Tris-EDTA ( TE ) buffer-based method for extraction of Deoxyribonucleic acid 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 & A ; Schuell filter paper and shop for 1-2 twelvemonth. The DNA extraction was extracted by the three methods ; TE buffer-based method, methanol extraction and Chelex? method severally. The PCR was performed by nested elaboration of merozoite surface protein 2 ( msp2 ) which is widely used in molecular epidemiological surveies, drug trails to find the figure or types of parasite genotypes of P. falciparum infections. The sensitiveness and duplicability 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, severally. For 903? Schleicher & A ; Schuell filter paper blood musca volitanss were lower in sensitiveness and duplicability due to a long storage of these samples. The Deoxyribonucleic acid extraction utilizing TE buffer protocol was done in a few hours prior to the PCR elaboration. The consequence on long-run storage on the quality of DNA extracted by the TE buffer method still needs to be evaluated.

The research of F? rnert and co-workers ( 1997 ) was focused on the observation of the day-to-day kineticss of P. falciparum subpopulations in symptomless kids in rural Tanzania by utilizing a nested PCR on msp1, msp2 and glurp cistrons. The P. falciparum subpopulations were observed in the eight kids harbouring P. falciparum throughout the survey period. The parasite populations were found to be extremely complex with day-to-day alterations in both parasite denseness and genotyping form. These findings implicated that the usage of the parasite samples from peripheral blood may be merely partially reflect the whole parasite population in an septic person.

3.1 Sampling

3.1.1 Study site

Blood samples were collected from willing patients who attended for malaria diagnosing at clinics in three states ( Mae Hong Son, Tak and Kanchanaburi states ) in Thailand during the twelvemonth 2007 to 2011.

3.1.2 Sample size

A sample size was calculated as following equation:

Sample size ( n ) = Z2? PQ/ ( P- lower bound ) 2

When, Z? = Confidence degree at 95 % ( Standard value of 1.96 )

P = Estimated prevalence of multiple infection in the survey country

Harmonizing to the consequence of Pumpaibool et al. , 2009 found that the prevalence of multiple septic samples from six states 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 states in this survey are Mae Hong Son, Tak and Kanchanaburi states with multiple infection from 44 % , 31 % and 27 % severally. The average 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 survey country is estimated at 0.3 and the lower bound is

estimated at 0.2 so the maximal allowable mistake ( vitamin D ) is equal 0.1

Sample size ( n ) = ( 1.96 ) 2 ( 0.34×0.66 ) / ( 0.1 ) 2

= 3.8416×0.2244/0.01

= 86 instances

So the sample will be collected at least 86 instances from three states in Thailand during the twelvemonth 2007 to 2011. The entire figure of samples both before and after civilization are eventually equal at least 172 samples for the experiment.

3.1.3 Standards

Inclusion standards

– The patient is non less than 5 old ages old and willing to take part in this research undertaking.

– The patient who has a new infection with P. falciparum diagnosed by a malaria officer.

Exclusion standards

– The patient is present of one or more of the general danger marks or any mark of terrible 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 survey undertaking. Informed consent will be obtained from each participant by signature or thumbprint in the presence of a informant. For alien participants who do non understand in Thai linguistic communication both speech production and hearing ; informed consent will be asked by a malaria functionary with catching linguistic communication.

3.1.5 Parasite aggregation

The P. falciparum infected patient identified by a malaria functionary will be taken a little sum of peripheral blood from the selected finger by a malaria functionary. Before piercing, patient ‘s finger will be cleaned with cotton wool lightly soaked in 70 % ethyl alcohol for at least two times, let to dry, puncture the ball of the finger with a unfertile lancet. The first bead of blood ( about 20-50? cubic decimeter ) is sucked by utilizing a unfertile pipette tip, so put in a micro-centrifuge tubing incorporating 500? cubic decimeter of conveyance medium with 5 units of Heparin, mix by inverting the tubing. Another 20-50 of blood is kept in the 2nd tubing. The samples are kept in icebox ( 4-10oC ) . The 2nd bead of blood is absorbed on filter paper, air-dried and put in Zip-locked plastic bag after wholly dry. The last bead of blood is used for doing the thin and thick movie on the same slide. The codification figure is written on the slide with a soft lead pencil. All specimens are put in a little mail box on the forenoon of Wednesday and sent by EMS.

The partial samples from the research undertaking under the Malaria plan, College of Public Health Sciences were used in this survey.

3.2 Microscopic scrutiny and verification

Thin and thick blood vilifications are stained with 10 % Giemsa solution for 10-15 proceedingss. In thin blood movie, parasites are counted against 10,000 ruddy blood cells utilizing a magnification of 1,000 X. it can be reported as parasites per 10,000 ruddy blood cells and parasitemia can be calculated by generation of parasite count with ruddy blood cells, so divided by 10,000 ( figure of ruddy blood cells counted )

Example: Parasite count is 500 per 10,000 ruddy blood cells

Standard red blood cell counted is 4,500,000 cells/ ? cubic decimeter of blood

Parasitaemia = ( 500 x 4,500,000 ) /10,000

= 225,000 parasites/ ? cubic decimeter of blood

In thick blood movies, parasite denseness can be defined by numbering nonsexual parasites ( rings, trophozoites and schizonts ) against 200 white blood cells and reported as parasites per 200 white blood cells. The figure of parasites relative to leukocytes counted can be calculated and expressed as parasites/ ? cubic decimeter of blood, from the expression:

( Parasites counted/No. of leucocytes ) x 8,000 = parasites/ ? cubic decimeter of blood

( Karbwang and Harinasuta, 1992 )

3.3 In vitro civilization of P. falciparum

This survey will be used the combined techniques of Trager and Jensen ( 1976 ) and Thaithong and co-workers ( 1994 ) for culturing of the P. falciparum finger-prick blood samples. The blood sample in conveyance medium is spun down at 1500 revolutions per minute for 2-3 min, the upper bed of conveyance medium is pipetted away, and the jammed cells are transferred to 3 Wellss of 96-well microtitre home base incorporating 100? cubic decimeter of complete RPMI medium plus 10 % pooled serum. The contents of the Wellss are exhaustively assorted. Then the home bases are covered with palpebras, placed in a candle-jar ( Trager and Jensen, 1976 ) , and incubate at 37oC. When the figure of parasites has reached 3-5 % , the civilizations are transferred to Petri dishes for spread outing the figure and the measure of parasites.

3.4 DNA extraction protocol

In this survey, two DNA extraction protocol will be used: the first 1 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 topographic point on filter paper is cut and placed in eppendorf tubing and incubated overnight at 4oC in 1 milliliter of 0.5 % saponin in phosphate-buffered saline ( PBS ) . The clouts are washed for 30 proceedingss in PBS at 4oC, so transfered into new tubings incorporating 25? cubic decimeter of stock solution ( 20 % Chelex-100 ) and 75? cubic decimeter of distilled H2O, and vortexed for 30 seconds. The tubings are heated at 99oC for 15 proceedingss to elute the Deoxyribonucleic acid, vortexed and centrifuged at 10,000xg for 2 proceedingss. The supernatant ( 65? cubic decimeter ) are transferred into new tubings.

The 2nd one is Phenol-Chloroform extraction protocol will be used for extraction of fresh or frozen P. falciparum blood samples in instance of a mention or control instance 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? fifty ice-cold PBS. 500? cubic decimeter of 0.5 % saponin in PBS is assorted to each sample. The mixture is placed at room temperature for 5 proceedingss or until lysis is observed. The samples are centrifuged at 10,000 revolutions per minute for 5 proceedingss and the supernatant is removed. Pellet is washed 2-3 times with ice-cold PBS and 100? cubic decimeter 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 thin the mixture, 50? cubic decimeter of unfertile dual distilled H2O ( ddH2O ) is added and gently blend. The mixture is incubated overnight at 37oC, at the terminal of the incubation period ; the mixture is adjusted to 30? cubic decimeter by adding unfertile ddH2O and mix. Deoxyribonucleic acid is extracted with an equal volume of concentrated phenol: trichloromethane ( 1:1 ) . To precipitate the Deoxyribonucleic acid 1:10 volume of 3 M Na ethanoate, pH 5.2 and 2 volume of cold absolute ethyl alcohol is added to the extracted solution. After incubation for nightlong at -20oC, DNA is pelleted by centrifugation at 13,000 revolutions per minute for 10 proceedingss at 4oC and the supernatant is carefully removed. The pellet is washed with 1 milliliters cold 70 % absolute ethyl alcohol and dissolved in 20? cubic decimeter 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 elaboration.

3.5 PCR elaboration 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 inside informations in the several cistrons are presented in Table 1

The nested PCR elaborations are performed with one microliter of the genomic Deoxyribonucleic acid of P. falciparum, 0.5 units of i-Taq DNA polymerase ( Invitrogen ) , 125? M of each dNTPs, 1x PCR buffer, 2 millimeter MgCl2 and 125 nanometer of each primer in a entire volume of 20? cubic decimeter per reaction, in the first reactions. The 2nd elaboration is performed in the same status as in the first elaboration, except 1? cubic decimeter of the merchandises are used for the elaboration. The reaction mixture is incubated in a Thermocycler ( GeneAmp 9700 and Veriti ) . The PCR parametric quantity is composed of an initial denaturation period of 5 min at 95oC, tempering for 30 sec at 58oC ( the first and nested reactions for GLURP ) or 61oC ( all nested reactions for msp1 & A ; 2 ) , extension for 30 sec at 72oC, denaturation for 30 sec at 95oC, the last extension for 7 min. The PCR rhythms are performed for 20-25 rhythms for the outer elaboration and 30 rhythms for nested elaboration. Five microliters of PCR merchandises and 1? cubic decimeter of 5x gel lading buffer are analyzed by agarose gel cataphoresis in 1x TBE buffer.

3.6 Visualization of PCR merchandises ( Snounou et al. , 1993 )

Five microliters of 1kb ladder marker and five microlitres of elaboration merchandises are assorted with 1? cubic decimeter of 5x lading buffer and loaded into each well of 1.5-2 % agarose or NuSieve agarose gel. The gel is run at 80 Vs for 25-30 proceedingss. The agarose gel is stained with 0.5? g/ml ethibromide for 10-20 min and destained with distilled H2O for 5-10 min. The stained agarose gel is visualized and photographed with AutoChemi? System by utilizing Labwork 4.0? package ( UVP Biomaging Systems ) and Cannon Digital camera.

3.7 Data analysis

The appraisal for the P. falciparum population in before and after civilization of EMS-transported blood samples by the usage of PCR technique is by and large observed from the multiple sets in the PCR merchandise of PCR elaboration which reflect the presence of many different strains in an isolate. The P. falciparum population in all samples will be estimated from the figure of sets observed in an agarose gel. For MSP2, the figure of FC27 and IC1/3D7 sets is counted.

4.1 Polymorphisms in MSP2 of P. falciparum

The polymorphisms of P. falciparum in each sample were assessed by finding the figure and the size fluctuation ( allelomorphs ) of MSP2 cistron. The elaboration merchandises form nested PCR of MSP2 as showed by the gel image and tabular array.

Fifteen samples partly used in this survey were successfully amplified for MSP2. The figure of allele types in each sample analyzed by nested PCR was shown in the tabular array 2. From these observations, it appears that the Numberss of allele types of MSP2 ranged from 1 to 2 but it was largely 1 in the sample after in vitro civilization Table 2: The figure of allele types analyzed by nested PCR of MSP2 in earlier and after civilization of P. falciparum isolates

Two allelomorphs were detected in four isolates before civilization ( MH62, MH65, MH67, and T222 ) but remained one allelomorph type after civilization. For MH62, MH65, MH67 isolates were found two sets of FC27 type but assorted FC27 and IC1 type was found in T222 isolate merely.

In decision, 4/15 ( 26.66 % ) samples were changed after civilization by disappeared of the set, 1/15 ( 6.66 % ) samples was different in size but same allele types.

At the MSP2 venue, 12/15 ( 80 % ) samples contained FC27 type and 3/15 ( 20 % ) samples contain IC1 type. The prevailing allelomorph 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 information presented in this survey demonstrate that nested PCR can separate FC27 and IC1/3D7 allelomorphs of MSP2 cistron. The consequence showed the changing of allele form comparing between before and after civilization in MH62, MH65, MH67 and T222 isolates. In T222 isolate disappeared of IC1/3D7 allelomorph in after civilization staying merely FC27 allelomorph with smaller size.

This consequence indicated that the P. falciparum isolates from the endemic countries, transported by EMS to research lab in Bangkok following in vitro civilization were changed in parasite population when utilizing merely one MSP2 marker cistron. The changes of P. falciparum population could consequence in feature of sample utilizing for research experiment.

Viriyakosol and co-workers ( 1994 ) used a PCR technique for parasite genotyping with the three surface antigen cistrons: msp1 block 2 and 4, the whole cistron of msp2 and glurp before and after in vitro civilization in 101 of a entire 269 samples. The alterations were detected in one or more of the three markers in the staying 37 instances. In this research article, the malaria blood samples were collected from all the patients who attended at malarial clinic in the twenty-four hours of aggregation. The blood samples were taken from venous blood by a physician and transported to the research lab in Bangkok on a twenty-four hours of aggregation and in vitro civilization was done every bit shortly as possible on reaching.




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