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1.
Exp Parasitol ; 98(2): 59-70, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11465989

ABSTRACT

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22-46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed.


Subject(s)
Antimalarials/pharmacology , Plasmodium falciparum/genetics , Pyrimethamine/pharmacology , Tetrahydrofolate Dehydrogenase/genetics , Amino Acids/chemistry , Amino Acids/genetics , Animals , Blotting, Southern , Culture Media , DNA, Protozoan/chemistry , Drug Resistance/genetics , Electrophoresis, Gel, Pulsed-Field , Humans , Immunoblotting , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Point Mutation , Polymerase Chain Reaction , Tetrahydrofolate Dehydrogenase/chemistry
2.
Asian Pac J Allergy Immunol ; 17(1): 23-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10403006

ABSTRACT

Two types of antimalaria antibodies in the serum of 54 villagers living in a malaria endemic area of Thailand were determined by indirect immunofluorescence assay in order to define the status of malaria immunity within the group. Antibodies to parasite-derived antigens in the membrane of ring stage-infected erythrocytes were very high (> or = 1:1,250) in 44%, moderate to low (< or = 1:250) in 37% of the sera, and the rest did not have the antibody. However, all the sera had antibodies to antigens of the intraerythrocytic mature parasites, showing a very high level in 65% and moderate to low levels in 37% of the sera. Sera with high antibody titers to either type of antigen significantly inhibited cytoadherence of P. falciparum-infected erythrocytes. All the sera variably inhibited rosette formation of the parasites but showed no association with the antibody titers. These results suggest that the antibodies to cytoadherence and rosette formation can be elicited and sustained in the malaria experienced host while living in the endemic area. This may be a natural preventive mechanism against the severity of P. falciparum infection in the infected host. How long the antiparasite adherence activity will last remains to be investigated.


Subject(s)
Erythrocytes/parasitology , Malaria, Falciparum/blood , Plasmodium falciparum/immunology , Adolescent , Adult , Animals , Antibodies, Protozoan/blood , Cell Adhesion , Endemic Diseases , Erythrocytes/cytology , Erythrocytes/immunology , Humans , Incidence , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Middle Aged , Parasitemia/blood , Parasitemia/epidemiology , Rural Population , Thailand/epidemiology , Tumor Cells, Cultured
3.
Trans R Soc Trop Med Hyg ; 93(4): 369-74, 1999.
Article in English | MEDLINE | ID: mdl-10674079

ABSTRACT

Plasmodium falciparum isolates were obtained from Thai patients attending a malaria clinic on the Thai-Kampuchean border over 4 cross-sectional surveys carried out at 3-monthly intervals. The genetic structure of the parasite populations was determined by nested polymerase chain reaction (PCR) amplification of polymorphic regions of 3 P. falciparum antigen genes: msp1, msp2 and glurp. Although a high degree of diversity characterized these isolates, the overall population structure of the parasites associated with patent malaria infections was observed to remain relatively stable over time. The highest degree of polymorphism was observed with msp2, and the mean number of lines per infection (multiplicity of infection) calculated with this marker was higher than that obtained using msp1 or glurp alone, or combined. Infections with > or = 2 parasite lines were seen in 76% of the samples, and were proportionally more numerous at the start and end of the rainy season. Two interesting exceptions to the random distribution were observed and involved 2 allelic variants which in one case were found dissociated (msp1 MAD20-family) and in the other were associated (msp2 FC27-family). The epidemiological significance of these types of data is discussed.


Subject(s)
Antigens, Protozoan/genetics , Malaria, Falciparum/genetics , Merozoite Surface Protein 1/genetics , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Animals , Bias , Cross-Sectional Studies , Gene Frequency , Humans , Malaria, Falciparum/epidemiology , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Thailand/epidemiology
4.
Article in Chinese | MEDLINE | ID: mdl-12078268

ABSTRACT

AIM: To develop a genotyping method based on amplifying glutamate-rich protein (GLURP) gene for the diagnosis and identification of Plasmodium falciparum. METHODS: Two pairs of primers specific for GLURP gene of P. falciparum were designed and synthesized. R2 polymorphic domain of GLURP gene was amplified by nested PCR, which was applied to genotyping of P. falciparum isolates obtained from patients attending the malaria clinic at the village of Borai, Thailand. RESULTS: Conspicuous polymorphism of GLURP alleles in natural populations of P. falciparum was found. 290 GLURP alleles were detected in 154 P. falciparum infections. Among the above-mentioned alleles, 12 different GLURP genotypes were distinguished according to different DNA sizes. Of them, the most frequently found allele was a variant of 770 bp, the least allele was that of 1,100 bp. More than 43% of the patients were found to be infected with mixed alleles. No apparent change for frequencies of the 12 different alleles was found in the 9-month longitudinal study. CONCLUSION: A genotyping method is developed for the research of strain taxonomy and pathogenesis of malaria parasites.


Subject(s)
DNA, Protozoan/genetics , Genes, Protozoan , Plasmodium falciparum/classification , Protozoan Proteins/genetics , Alleles , Amino Acid Sequence , Animals , Base Sequence , Genotype , Humans , Longitudinal Studies , Molecular Sequence Data , Plasmodium falciparum/chemistry , Plasmodium falciparum/genetics , Polymorphism, Genetic , Protozoan Proteins/isolation & purification
5.
Am J Trop Med Hyg ; 56(3): 322-8, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9129537

ABSTRACT

An integrated system for sample preparation and DNA detection of the malaria parasite using immunomagnetic separation in combination with the polymerase chain reaction (PCR) and colorimetric analysis is described. A cocktail of three monoclonal antibodies towards merozoite surface antigen-1 was used for magnetic capture of parasites from microliter amounts of whole blood. A sensitivity down to a parasitemia of 10(-6)% was achieved using cultured parasites as a model. The integrated approach was evaluated in a field study. A total of 410 blood samples from patients attending malaria clinics in Trat province and Kanchanaburi province in Thailand were analyzed. The samples were processed by immunomagnetic separation and transferred to central laboratory for PCR-based detection. Microscopic examinations on blood smears were done in parallel; 53% were positive using the DNA-based assay, while only 32% were positive using conventional microscopic analysis. This field study suggests a possible model for large-scale testing of malaria with an increased sensitivity compared with conventional methods.


Subject(s)
DNA, Protozoan/analysis , Malaria, Falciparum/diagnosis , Parasitemia/diagnosis , Plasmodium falciparum/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Antigens, Protozoan/immunology , Antigens, Surface/immunology , Colorimetry , Humans , Immunomagnetic Separation , Malaria, Falciparum/parasitology , Merozoite Surface Protein 1 , Parasitemia/parasitology , Plasmodium falciparum/genetics , Polymerase Chain Reaction , Protein Precursors/immunology , Protozoan Proteins/immunology , Sensitivity and Specificity
6.
Mol Biochem Parasitol ; 84(1): 49-56, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9041520

ABSTRACT

The merozoite of Plasmodium vivax possesses a high molecular mass surface protein called Pv-merozoite surface protein 1, PvMSP-1, which exhibits antigenic diversity among isolates. In this study, the extent of sequence variation in the polymorphic region and the flanking interspecies conserved blocks (ICBs) 5 and 6 of the PvMSP-1 gene was analyzed using the polymerase chain reaction to amplify the DNA fragment encompassing these regions, followed by sequencing. Twenty different alleles were obtained from 15 Thai isolates. Results revealed five distinct sequence types of the polymorphic region, two of which were newly identified in this study: one probably generated by intragenic recombination at a site different from that previously reported and the other by duplication of a 30 nucleotide (nt) sequence at the 3' end of the region. On the other hand, almost all nucleotide substitutions in the flanking regions, ICB5 and ICB6, were dimorphic, creating microheterogeneity in the region. Furthermore, stretches of nucleotide substitutions were found to be linked in ICB6, suggesting the potential recombination sites between these stretches. It is also noted that extensive sequence variation in the PvMSP-1 gene and coinfection with different PvMSP-1 alleles occurred among the P. vivax population in the endemic areas of Thailand.


Subject(s)
Genes, Protozoan/genetics , Plasmodium vivax/genetics , Protein Precursors/genetics , Protozoan Proteins/genetics , Animals , Base Sequence , Cloning, Molecular , Merozoite Surface Protein 1 , Molecular Sequence Data , Recombination, Genetic , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid
7.
Cytometry ; 27(1): 84-91, 1997 Jan 01.
Article in English | MEDLINE | ID: mdl-9000589

ABSTRACT

The resurgence of drug-resistant malaria makes urgent the evaluation of new antimalarial agents. This study describes a flow cytometric method (FCM) for testing in vitro drug susceptibility of Plasmodium falciparum malaria to several orally active hydroxypyridinone (CP) iron chelators and to the parenteral iron chelator desferrioxamine (DF). After exposure of parasites to various concentrations of iron chelating agents, aliquots of cultures were fixed with glutaraldehyde. The fixed samples were washed and stained for parasite DNA with propidium iodide and analyzed by flow cytometry. The remaining cells were pulsed with 3H-hypoxanthine, using the microdilution radioisotope method. Both CP and DF showed dose-dependent inhibition of parasite growth. Of the compounds studied, DF exerted a stronger inhibitory effect. Fifty percent of inhibitory concentrations (IC50) of CP and DF determined by DNA fluorescence profiles in the flow cytometer were consistent with those obtained from the radioisotope method and by microscopic examination. Moreover, the minimum inhibitory concentrations (MIC) of drug required to inhibit parasite growth, as detected by the decreasing DNA fluorescence intensity of the schizont, correlated with observed abnormal microscopic morphology. The validity of the MIC, as indicated by decreased fluorescence intensity, was confirmed by subsequent parasite culture. Our FCM study demonstrated the sensitivity of both chloroquine- and pyrimethamine-resistant malaria parasites to iron chelators. Addition of equimolar concentrations of ferric ion completely abolished the inhibitory effect of iron chelators, indicating the importance of iron for parasite growth and the primary effect of the compounds as iron (III) chelating agents. These data demonstrate that FCM provides a simple and reliable means for antimalarial drug susceptibility testing, and suggest that iron chelators have potential for the treatment of drug-resistant malaria.


Subject(s)
Antimalarials/pharmacology , Flow Cytometry/methods , Iron Chelating Agents/pharmacology , Plasmodium falciparum/drug effects , Pyridones/pharmacology , Animals , Coloring Agents , DNA, Protozoan/analysis , Deferoxamine/pharmacology , Drug Resistance , Erythrocytes/parasitology , Iron/pharmacology , Microbial Sensitivity Tests , Plasmodium falciparum/growth & development , Propidium
8.
J Med Assoc Thai ; 79 Suppl 1: S40-8, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9071066

ABSTRACT

Pf155/RESA, an antigen found on the surface of Plasmodium falciparum red blood cell membrane was once a proposed malarial vaccine candidate. The complete sequence of Pf155/RESA gene from one strain and partial sequence from two other isolates revealed that the gene is well conserved. But polymorphism of other antigenic encoded regions occurs with high frequency among isolates especially those collected from the field. Using solid phase sequencing technique, the nested PCR products of upstream 3' repeated region of exon 2 RESA gene were studied in 150 P. falciparum isolates. Of which 117 isolates were directly collected from the field and sequenced. Other samples studied include clones and cryopreserved of previously cultured isolates. The resulting sequences are compared with previously existing data of F32 (Tanzania) and FC27 (Papua New Guinea) designated as allelic type I and II respectively. Sequence analysis of the 150 P. falciparum showed that the amplified region of RESA gene was highly variable with substitution ranging from one to six bases and these allelic variables can be divided into 10 types. The frequency of type I(F32) occurrence is 70.86 percent, type III 13.38 per cent and 0.78 percent to 5.51 per cent for others. As a result of allelic polymorphism, the amino acid sequence is highly variable and this may cause Pf155/RESA to be an inefficient antigen.


Subject(s)
Antigenic Variation , Antigens, Protozoan/genetics , DNA, Protozoan/genetics , Genes, Protozoan/genetics , Plasmodium falciparum/genetics , Alleles , Animals , DNA Primers , Polymorphism, Genetic , Thailand
9.
Antimicrob Agents Chemother ; 40(8): 1846-54, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8843292

ABSTRACT

We have synthesized several 4-aminoquinolines with shortened side chains that retain activity against chloroquine-resistant isolates of Plasmodium falciparum malaria (W. Hofheinz, C. Jaquet, and S. Jolidon, European patent 94116281.0, June 1995). We report here an assessment of the activities of four selected compounds containing ethyl, propyl, and isopropyl side chains. Reasonable in vitro activity (50% inhibitory concentration, < 100 nM) against chloroquine-resistant P. falciparum strains was consistently observed, and the compounds performed well in a variety of plasmodium berghei animal models. However, some potential drawbacks of these compounds became evident upon in-depth testing. In vitro analysis of more than 70 isolates of P. falciparum and studies with a mouse in vivo model suggested a degree of cross-resistance with chloroquine. In addition, pharmacokinetic analysis demonstrated the formation of N-dealkylated metabolites of these compounds. These metabolites are similarly active against chloroquine-susceptible strains but are much less active against chloroquine-resistant strains. Thus, the clinical dosing required for these compounds would probably be greater for chloroquine-resistant strains than for chloroquine-susceptible strains. The clinical potential of these compounds is discussed within the context of chloroquine's low therapeutic ratio and toxicity.


Subject(s)
Aminoquinolines/pharmacology , Antimalarials/pharmacology , Chloroquine/pharmacology , Plasmodium falciparum/drug effects , Aminoquinolines/metabolism , Aminoquinolines/pharmacokinetics , Aminoquinolines/toxicity , Animals , Antimalarials/metabolism , Antimalarials/pharmacokinetics , Antimalarials/toxicity , Chloroquine/analogs & derivatives , Chloroquine/pharmacokinetics , Drug Evaluation, Preclinical , Drug Resistance , Malaria/drug therapy , Malaria/parasitology , Male , Mice , Plasmodium berghei/drug effects , Plasmodium berghei/growth & development , Plasmodium falciparum/growth & development , Plasmodium yoelii/drug effects , Rats , Specific Pathogen-Free Organisms
10.
Article in Spanish | PAHO | ID: pah-22224

ABSTRACT

La epidemiología del paludismo es el resultado de la interacción que tienen entre sí y con el medio circundante tres acervos genéticos: el del parásito, el del ser humano y el del mosquito vector. Actualmente se están elaborando métodos para caracterizar la genética de las poblaciones humanas en riesgo y de los posibles vectores, y a fin de llegar a conocer más a fondo la epidemiología, mecanismos patógenos y biología de este parásito también sería enormemente útil caracterizar las poblaciones naturales de Plasmodium y su distribución en huéspedes humanos y en insectos de zonas de estudio determinadas, especialmente si este enfoque se combina con estudios simultáneos en seres humanos y con los vectores. En este trabajo se describe un ensayo basado en la reacción en cadena de la polimerasa (RCP), que proporciona un método sensible, práctico y reproducible para caracterizar distintas poblaciones de parásitos de una misma especie. Con el fin de ilustrar la idoneidad de este tipo de ensayo, se escogieron y amplificaron con la RCP cuatro dominios polimórficos de los genes de tres proteínas de P. falciparum (los bloques 2 y 4 de la proteína de superficie del merozoito tipo 1 (PSM1), la tipo 2 (PSM2) y la proteína rica en glutamato (PRGLU) y una región casi enteramente conservada (el bloque 17 de la PSM1). Sirvieron de molde para amplificar con la RCP los ADN derivados de 15 líneas de P. falciparum cultivadas in vitro (siete de las cuales fueron clonadas) y de muestras de sangre de pacientes infectados procedentes de Tailandia. Los productos de la amplificación se analizaron por electroforesis en gel para detectar polimorfismos de longitud. Se detectaron siete variantes alélicas de la PRGLU, cinco del bloque 2 de la PSM1, tres del bloque 4 de la PSM1 y nueve de la PSM2. Este alto grado de polimorfismo se puede usar para caracterizar la composición genética de cualquier población de parásitos en un momento dado. En este trabajo se examina la aplicabilidad de esta forma de identificar genotipos en el campo de la epidemiología y se recomienda la adopción de patrones internacionales para su empleo, de tal modo que se puedan comparar los datos obtenidos en distintos lugares y momentos (AU)


Subject(s)
Malaria, Falciparum/epidemiology , Polymerase Chain Reaction , Plasmodium falciparum/genetics
11.
Article | PAHO-IRIS | ID: phr-15500

ABSTRACT

La epidemiología del paludismo es el resultado de la interacción que tienen entre sí y con el medio circundante tres acervos genéticos: el del parásito, el del ser humano y el del mosquito vector. Actualmente se están elaborando métodos para caracterizar la genética de las poblaciones humanas en riesgo y de los posibles vectores, y a fin de llegar a conocer más a fondo la epidemiología, mecanismos patógenos y biología de este parásito también sería enormemente útil caracterizar las poblaciones naturales de Plasmodium y su distribución en huéspedes humanos y en insectos de zonas de estudio determinadas, especialmente si este enfoque se combina con estudios simultáneos en seres humanos y con los vectores. En este trabajo se describe un ensayo basado en la reacción en cadena de la polimerasa (RCP), que proporciona un método sensible, práctico y reproducible para caracterizar distintas poblaciones de parásitos de una misma especie. Con el fin de ilustrar la idoneidad de este tipo de ensayo, se escogieron y amplificaron con la RCP cuatro dominios polimórficos de los genes de tres proteínas de P. falciparum (los bloques 2 y 4 de la proteína de superficie del merozoito tipo 1 (PSM1), la tipo 2 (PSM2) y la proteína rica en glutamato (PRGLU) y una región casi enteramente conservada (el bloque 17 de la PSM1). Sirvieron de molde para amplificar con la RCP los ADN derivados de 15 líneas de P. falciparum cultivadas in vitro (siete de las cuales fueron clonadas) y de muestras de sangre de pacientes infectados procedentes de Tailandia. Los productos de la amplificación se analizaron por electroforesis en gel para detectar polimorfismos de longitud. Se detectaron siete variantes alélicas de la PRGLU, cinco del bloque 2 de la PSM1, tres del bloque 4 de la PSM1 y nueve de la PSM2. Este alto grado de polimorfismo se puede usar para caracterizar la composición genética de cualquier población de parásitos en un momento dado. En este trabajo se examina la aplicabilidad de esta forma de identificar genotipos en el campo de la epidemiología y se recomienda la adopción de patrones internacionales para su empleo, de tal modo que se puedan comparar los datos obtenidos en distintos lugares y momentos (AU)


Se publica también en inglés en el Bull. WHO. Vol. 73(1), 1995


Subject(s)
Malaria, Falciparum , Plasmodium falciparum , Polymerase Chain Reaction
12.
Article in English | MEDLINE | ID: mdl-8629047

ABSTRACT

An in vivo study of the response of P. falciparum to the combination drug, MSP, was conducted among gem miners who contracted malaria from Cambodia in 1991-1992. High level resistance (RII, RIII responses) was observed in 22.5% of the 40 cases attending Mae Sot malaria clinic, west Thailand border, and in 28.1% of the 96 cases attending Bo Rai malaria clinic, east Thailand border. The observations on in vitro studies conducted prior to the MSP treatment and after recrudescence, together with the findings on adequate mefloquine blood levels strongly indicated the serious deterioration of mefloquine efficacy. The first line treatment for the malaria control program needs to be revised and the use of qinghaosu derivatives considered. Intensive measures to combat spreading of the highly resistant strains to other parts of the country should be taken into account.


Subject(s)
Antimalarials/therapeutic use , Malaria, Falciparum/drug therapy , Mefloquine/analogs & derivatives , Pyrimethamine/therapeutic use , Sulfadoxine/therapeutic use , Transients and Migrants , Adult , Animals , Antimalarials/pharmacology , Cambodia , Chi-Square Distribution , Drug Combinations , Drug Resistance , Female , Humans , Malaria, Falciparum/blood , Male , Mefloquine/pharmacology , Mefloquine/therapeutic use , Mining , Plasmodium falciparum/drug effects , Pyrimethamine/pharmacology , Sulfadoxine/pharmacology , Thailand
13.
Bull World Health Organ ; 73(1): 85-95, 1995.
Article in English | MEDLINE | ID: mdl-7704931

ABSTRACT

The epidemiology of malaria results from the interactions of three gene pools--parasite, human, and mosquito vector--with one another and with their environment. Methods are being developed for characterizing the genetics of human populations at risk and of potential vectors. The characterization of natural populations of Plasmodium and knowledge of their distribution within the human and insect hosts in any given area under study would also greatly enhance understanding of the epidemiology, pathology and biology of this parasite, particularly when combined with simultaneous human and vector studies. This paper describes a polymerase chain reaction (PCR)-based assay which provides a sensitive, reproducible and practical method by which parasite populations within species can be characterized. In order to illustrate the suitability of the PCR assay, four polymorphic domains on the genes of three P. falciparum proteins (MSP1 blocks 2 and 4, MSP2, and GLURP) and one largely conserved region (MSP1 block 17) were chosen for amplification by PCR. DNA derived from 15 in-vitro cultured lines of P. falciparum (7 of which were cloned) and from blood samples obtained from infected patients in Thailand were used as templates for PCR amplification. The amplification products were analysed by gel electrophoresis for length polymorphisms. Seven allelic variants of GLURP, five of MSP1 block 2, three of MSP1 block 4, and nine of MSP2 were detected. This high degree of polymorphism can be used to characterize the genetic composition of any parasite population, at a given time. The paper discusses the applicability of this type of genotyping to epidemiology and urges the adoption of international standards for its use so that data from different areas and different times can be compared.


Subject(s)
Genotype , Plasmodium falciparum/genetics , Polymerase Chain Reaction , Amino Acid Sequence , Animals , Epidemiologic Methods , Genetics, Population , Molecular Sequence Data , Polymorphism, Genetic , Reproducibility of Results , Sensitivity and Specificity
16.
Article in English | MEDLINE | ID: mdl-7777902

ABSTRACT

Malaria is still a serious health problem in Thailand. Present attempts at controlling the disease by drug treatment and other means remain unsatisfactory. Thus, development of vaccination against malaria is a major research goal of malaria immunology. The objective of this study was to acquire epidemiological base line data for subsequent vaccine trials. A cross-sectional descriptive survey was conducted among 451 local inhabitants during the beginning of the transmission season in June 1989 at Pong Nam Ron District, Chanthaburi Province, Eastern Thailand where malaria transmission was likely to be high. Following the cross-sectional survey weekly morbidity surveillance was started to detect new cases of malaria by using active and passive case detection at the district hospital, local health centers and at neighboring malaria clinics. Entomological observations were made monthly to determine inoculation rates. Forty-six percent of the population were male and 54% female; one third were under the age of 15 and 14% under the age of 5 years. Eighty percent of the adults were married. Sixty percent of the subjects interviewed gave a history of malarial illness in the past. Malaria, malnutrition, abnormal hemoglobin diseases and parasitic infestation were the main health problems in the study area. The annual parasite incidence of malaria was 149.6/1,000 population and two-thirds of them were asymptomatic indicating a semi-immune condition among these subjects. It was difficult to interpret the results of entomological studies due to low density of the malaria vector.


Subject(s)
Malaria/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Infant , Male , Middle Aged , Morbidity , Thailand/epidemiology
18.
Am J Trop Med Hyg ; 50(4): 465-71, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8166354

ABSTRACT

Antibody titers and lymphocyte responses to synthetic peptides corresponding to repeated amino acid sequences of the 3' and 5' regions of Pf155/ring-infected erythrocyte surface antigen (RESA) were studied in two groups of Thai subjects, soldiers (Rangers), and villagers who differed in their history of malaria exposure. The frequency of Pf155/RESA seropositivity was similar in the two groups while the frequency of high titer antibody was significantly greater in villagers than in Rangers. Lymphocyte responsiveness in vitro to all Pf155/RESA peptides was infrequent for both groups although half of the subjects studied responded to crude Plasmodium falciparum asexual blood stage malaria antigen (MA). Among responders, Pf155/RESA peptides elicited lymphocyte responses in which proliferation and interferon-gamma (IFN-gamma) production were not associated, whereas with MA, the two responses were associated. The MA-stimulated lymphocyte proliferation and IFN-gamma production for both groups of volunteers appeared to be independent of antibody titer. In this study, antibody, but not lymphocyte, responses to Pf155/RESA peptides were shown to reflect differences in prior exposure and levels of acquired immunity to falciparum malaria.


Subject(s)
Antigens, Protozoan/immunology , Antigens, Surface/immunology , Lymphocytes/immunology , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Adult , Animals , Antibodies, Protozoan/biosynthesis , Humans , Interferon-gamma/biosynthesis , Lymphocyte Activation , Middle Aged , Military Personnel , Thailand
20.
J Clin Microbiol ; 31(10): 2715-9, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8253971

ABSTRACT

The detection of pathogens by polymerase chain reaction (PCR) in clinical samples, such as blood, urine, or feces, requires initial sample preparation to remove polymerase inhibitors and to concentrate the target DNA. Here we show for the first time that immunomagnetic separation can be used to recover pathogens from whole blood and then used for PCR analysis. With antibodies to the merozoite surface protein (MSP1), the malaria-causing parasite Plasmodium falciparum was purified and concentrated from clinical samples. The recovered parasites were used directly for in vitro DNA amplification. The PCR product was subsequently analyzed by a colorimetric 96-well microtiter plate assay. The results from examining 117 patients attending a clinic in the Borai district, Thailand, demonstrate that the combined method with immunomagnetic separation followed by PCR increases the group of positively diagnosed patients compared with microscopic examination of stained blood films. Analysis of 1 microliter of whole blood resulted in a 12% (14 of 117) increase in positively diagnosed patients while a 10-microliters sample volume increased the positives diagnosed to 20.5% (24 of 117).


Subject(s)
DNA, Protozoan/analysis , Immunomagnetic Separation , Malaria, Falciparum/diagnosis , Plasmodium falciparum/genetics , Animals , Base Sequence , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Rabbits
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