Plasmodium falciparum: gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine.

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.

Antiparasite adherence activity in Thai individuals living in a P. falciparum endemic area.

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.

Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand.

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.

Plasmodium falciparum: selection of parasite subpopulations with decreased sensitivity for antibody-mediated growth inhibition in vitro.

The chronic, recrudescent nature of malaria has been linked to antigenic diversity of the parasite in which protective immunity against Plasmodium falciparum may be obtained after repeated exposure to infection during a long time. In this study we show that parasite populations with decreased sensitivity to antibody-mediated growth inhibition are readily generated in vitro. A laboratory strain, F32, was cultured for long periods (10-12 weeks) in the presence of suboptimal inhibitory antibody concentrations. The antibodies used were the human monoclonal antibody 33G2 reacting with a linear epitope of the P. falciparum blood-stage antigen 332 and rabbit antibodies to repeat sequences of the blood-stage antigen Pf155/RESA. Our data indicate that the P. falciparum parasites adapt to antibody pressure as reflected by their specifically decreased sensitivity to growth inhibition. A relative resistance of the parasite to growth inhibition mediated by the antibodies used in the culture developed successively, while the parasite remained sensitive to growth inhibition by other antibodies. When the antibody pressure was removed a successive return of sensitivity to growth inhibition developed. Immunoflourescence did not reveal any significant difference in antigen expression between the parasite populations. However, PCR analysis showed that a new population appeared in the parasites grown in the presence of mAb 33G2, while no such change was detected in those grown in the presence of the rabbit antibodies. Our data suggest that the specific decrease in sensitivity to growth inhibition may either be due to down-regulation of antigen synthesis or expression by antibody pressure or, that antibody pressure selects for parasites with low expression of a specific antigen from a heterogeneous parasite population.

Identificación de genotipos de cepas aisladas de Plasmodium falciparum mediante la reacción en cadena de la polimerasa y sus posibles usos en estudios epidemiológicos / Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies

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)

Identificación de genotipos de cepas aisladas de Plasmodium falciparum mediante la reacción en cadena de la polimerasa y sus posibles usos en estudios epidemiológicos / Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies

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

Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies.

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.

Plasmodium falciparum: selective growth of subpopulations from field samples following in vitro culture, as detected by the polymerase chain reaction.

Analysis of the Plasmodium falciparum parasites circulating in the blood of infected persons frequently reveals the presence of two or more genetically distinct parasite populations. P. falciparum parasites cultured in vitro, from blood specimens collected in the field, are often used for biological, immunological, and drug-resistance investigations relating to the epidemiology in the area concerned or on the assumption that the parasites which grow in vitro are in general representative of all P. falciparum parasites. By using the polymerase chain reaction to detect and characterize a number of parasite polymorphic genes with great sensitivity, the composition of P. falciparum populations from 51 isolates were compared on the day of collection and following 2 months of in vitro culture. It was found that substantial changes in the parasite population profile could be detected in ca. 70% of the samples analyzed. The implications of this observation for studies using parasite isolates cultured in vitro are discussed.

Electrophoretic variants of enzymes in isolates of Plasmodium falciparum, P. malariae and P. vivax from Thailand.

A new electrophoretic variant of glucose phosphate isomerase (GPI), which we now denote GPI-3, has been found in isolates of Plasmodium falciparum from 6 patients, all of whom acquired the infection in the same region (in or near Prachinburi province) of Thailand. In other regions, from which 453 isolates have been tested, only GPI-1 and/or GPI-2 have been found. Two isolates of P. malariae from patients at Kanchanaburi showed a band of GPI activity on cellulose acetate gels at a cathodal position quite distinct from that of any previously known GPI variants in other human malaria parasites. Thirty-nine isolates of P. vivax from 3 regions of Thailand have been examined for variants of GPI and lactate dehydrogenase (LDH). Three forms of GPI were found, corresponding approximately in band positions to GPI-1, 2 and 3 of P. falciparum. The position of the band of LDH activity in P. vivax was the same in all the isolates examined, and different from that of LDH-1 in P. falciparum.