Interallelic recombination in the merozoite surface protein 1 (MSP-1) gene of Plasmodium vivax from Thai isolates.

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.

Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections.

Genus- and species-specific sequences are present within the small subunit ribosomal RNA genes of the four human malaria parasites. Oligonucleotide primer pairs specific to each species were designed for specific amplification by the Polymerase Chain Reaction (PCR), to detect each malaria species. DNA equivalent to 5 microliters of blood was sufficient for the detection of each of the species. Blood samples obtained from 196 patients attending a malaria clinic in Trad province (Thailand) were analyzed. Detection and identification of the parasites, solely by electrophoretic analysis of the PCR products, has proven to be more sensitive and accurate than by routine diagnostic microscopy. A high proportion of mixed species infections were brought to light by the PCR assay. Implications for medical treatment and epidemiological studies are discussed.

Polymorphism of proteins in malaria parasites following mefloquine treatment.

Proteins in malaria parasites (Plasmodium falciparum) isolated from a patient in Thailand before treatment, and after recrudescence of infection subsequent to mefloquine treatment, were compared by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis. Nine 'pre-treatment' and six 'recrudescent' clones were studied. Variants of the enzyme glucose phosphate isomerase were also noted and mefloquine susceptibility of each clone was measured by in vitro tests. The 'pre-treatment' isolate was found to contain at least four genetically distinct clones, all sensitive to mefloquine, while the 'recrudescent' isolate contained at least two other types of clone, both showing increased tolerance to mefloquine. These two more tolerant types of clone differed from all the sensitive ones studied in regard to several different protein variants as shown by 2D-PAGE analysis. It is concluded that at least two (and probably more) genetically distinct clones of parasites with increased tolerance to mefloquine were present in the parasite population before mefloquine treatment was given, and were selected under mefloquine pressure.

Amplification of pfmdr 1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand.

Drug resistance in Plasmodium falciparum is an expanding problem in most endemic areas. Recent studies have suggested the potential involvement of genes in the MDR gene family in resistance to quinoline-containing compounds in P. falciparum. In this study a molecular analysis of pfmdr 1 in recent isolates from Thailand was done (1) to further examine the role of pfmdr 1 in drug-resistant isolates and (2) to examine the reported association of pfmdr 1 intragenic alleles and chloroquine resistance. Most of the isolates (10 of 11) were resistant to all compounds tested. Analysis of pfmdr 1 revealed an apparent association between increased gene copy number and increased level of expression of pfmdr 1 and decreased susceptibility to mefloquine and halofantrine. Sequence analysis of pfmdr 1 in these isolates revealed no association of intragenic alleles with chloroquine resistance.

Pyrimethamine resistant mutations in Plasmodium falciparum.

Three mutations in Plasmodium falciparum yielding increased resistance to pyrimethamine were obtained following treatment with chemical mutagens and selection in presence of pyrimethamine. From parasite clone TM4/8.2 a mutant, TM4/8.2/4.1, was produced which raised pyrimethamine resistance about 500 times and was found to involve an amino acid change in the DHFR-TS enzyme molecule from Ser108 to Asn108. A clone of another isolate, T9/94, yielded a mutant, T9/94/300.300, raising pyrimethamine resistance about 10 times and involving an amino acid change from Ile164 to Met164. However, another mutant from T9/94, T9/94/M1-1(b3), although it raised the pyrimethamine resistance 100 times, did not involve any changes in the coding sequence of the DHFR-TS gene, but resulted in the production of about twice as much DHFR-TS enzyme as the original clone T9/94. No amplification of the DHFR-TS gene was detected. It is concluded that changes in pyrimethamine resistance of malaria parasites may arise in at least 2 ways: (1) by structural changes in the DHFR domain of the DHFR-TS gene (as previously found by other workers); (2) by other changes, possibly affecting the expression of the DHFR-TS gene. The relative importance of these 2 mechanisms in causing resistance in wild populations of P. falciparum is discussed.

Genetic diversity of Plasmodium falciparum shows geographical variation.

Sixty Plasmodium falciparum isolates, 20 each from Thailand, Zimbabwe, and Brazil, were characterized for 20 variant genetic markers, including the enzymes glucose phosphate isomerase, adenosine deaminase and peptidase, 11 other proteins detected by 2-dimensional electrophoresis (2D-PAGE), 2 merozoite surface antigens (MSA-1 and MSA-2), one exported antigen (Exp-1), and sensitivity to the drugs chloroquine, pyrimethamine, and mefloquine. The study examines the extent of diversity between individual isolates and the differences in the frequency of certain variants of the markers between the 3 countries. The principal conclusions to be drawn from the study are that there is extensive polymorphism in many of the genetically determined characters of this parasite, multiple infections with greater than 1 genetically distinct parasite are common, and there are geographical variations in the frequencies with which variant forms of certain markers occur.

Lymphocyte responses to Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA) peptides in individuals with naturally acquired Plasmodium falciparum malaria.

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.

An integrated system using immunomagnetic separation, polymerase chain reaction, and colorimetric detection for diagnosis of Plasmodium falciparum.

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.

Cloning and characterization of mefloquine-resistant Plasmodium falciparum from Thailand.

Resistance to mefloquine in Plasmodium falciparum has begun to occur along the border of Thailand and Kampuchea. As a means of assessing the natural occurrence of mefloquine resistance, the admission and post-treatment parasite isolates from a mefloquine treatment failure were cloned and characterized. Clones from the admission isolate were susceptible to mefloquine in vitro (ID50 of 3.4 [2-5], G [95% CI] ng/ml) and showed a mixture of isozyme types for glucose phosphate isomerase (GPI types I and II). The post-treatment clones were resistant to mefloquine in vitro (ID50 of 17.3 [13-23] ng/ml) with only one isozyme (GPI type I) detected. These observations suggest that under mefloquine pressure a resistant parasite population was selected in the patient, indicating that the potential for mefloquine resistance already exists in the indigenous P. falciparum gene pool. In addition, the mefloquine-resistant clones showed decreased susceptibility in vitro to halofantrine suggesting possible cross-resistance to this new antimalarial drug currently under development.

Seroepidemiologic studies of humoral immune response to the Plasmodium falciparum antigens in Thailand.

We have investigated seroreactivity against Plasmodium falciparum crude parasite antigens, the P. falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), as well as against two synthetic peptides (EENV)6 and (EENVEHDA)3 that represent important epitopes of Pf155/RESA. The study population consisted of 421 children and adult Thais living in an area with moderate malaria transmission. We related these serologic findings to some important epidemiologic baseline data collected in the study area. The parasite rate in study subjects was 18.76%. Sixty-two percent were seropositive to crude P. falciparum antigens, 30.3% to the Pf155/RESA antigen, 23.05% to (EENV)6, and 20.17% to (EENVEHDA)3. Antibody responses to crude P. falciparum antigens and to Pf155/RESA were age dependent and increased with exposure. There was evidence that Pf155/RESA antibodies might play a role in protective immunity in this population. Since Pf155/RESA is a potential vaccine candidate antigen, the information obtained from these field studies will provide some seroepidemiologic baseline data for subsequent vaccine trials.

Automated flow cytometric analysis of drug susceptibility of malaria parasites.

A method is described for the fully automated reading of Plasmodium falciparum drug susceptibility tests. Cultured material was fixed and could be stored for greater than or equal to 6 months until analysis. The parasites were stained for DNA with the fluorescent dye Hoechst 33258 and analyzed by flow cytometry. The procedure was done in 96-well microtiter plates, after which the material was directed through the sensing region in the flow cytometer. The data resulting from the analysis were stored by microcomputer and processed by a program developed for this purpose. Using this method, a number of different parameters describing the growth in culture can be assessed.

Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests.

In vitro drug resistance tests of ten isolates of Plasmodium falciparum from three different collection points in Central Thailand have been carried out, and the results compared with those of similar tests with a drug-sensitive West African isolate. Judged by concentration of drug tolerated, the Thai isolates appeared to be about 10 times as resistant to chloroquine, and usually about 10(5) times as resistant to pyrimethamine, as the African isolate. A little variation amongst the Thai isolates was detected.

Variability in drug susceptibility amongst clones and isolates of Plasmodium falciparum.

Heterogeneity within isolates of Plasmodium falciparum in regard to drug susceptibility is described from studies with three Thai isolates and some clones derived from them. One isolate (T9), which before cloning was resistant in vitro to chloroquine and pyrimethamine, contained a diverse assortment of clones, some of which were sensitive to one or other, or both, of these drugs. Another isolate (CH150) was initially sensitive to mefloquine in vitro, but, on recrudescence of infection in the patient, developed a number of clones all of which had diminished susceptibility to mefloquine. Drug pressure in a laboratory culture of CH150 resulted in a similar change in susceptibility. Hence resistant clones are thought to have been present as a minority component of the original isolate CH150. On testing uncloned isolates at different times after growth in culture, drug susceptibility showed considerable variability, but clones remained stable. Reaction in vitro of these isolates to some other drugs (amodiaquine, Fansidar, quinine) is also described, and the results are discussed in relation to changes in drug resistance of malaria parasites which may occur in laboratory cultures and under field conditions.

Susceptibility of Plasmodium falciparum to five drugs: an in vitro study of isolates mainly from Thailand.

This paper describes the results of testing the susceptibility of 60 isolates of the human malaria parasite Plasmodium falciparum from Thailand, and single isolates from five other countries, to five drugs: chloroquine, pyrimethamine, quinine, mefloquine and amodiaquine. The Thai isolates were obtained from patients in three different regions of the country (Chantaburi, Songkhla and Mae Sod), and were first grown in culture by the Trager-Jensen candle-jar technique. Samples were then exposed to a range of concentrations of the five drugs, in Falcon microtest culture wells, for 72 hours, with daily changes of medium (with or without added drug solutions). Presence or absence of parasites was then determined by microscope observations on thin-film Giemsa-stained preparations. Most Thai isolates showed a minimum inhibitory concentration (MIC) for chloroquine of 10(-6) M or higher, and were classified as highly resistant, though one cloned isolate was as sensitive to this drug as a chloroquine-sensitive isolate from West Africa. Similarly most Thai isolates showed a very high resistance to pyrimethamine (MIC 10(-4) M to 10(-6) M), but a few clones were sensitive (MIC 10(-9)) to it. Susceptibility to quinine showed some variation (MIC varied between 10(-6) M and 10(-8) M), and some isolates were thought to be incapable of responding to a therapeutically permissible dose of this drug. Little variation was found in the reaction of any of the isolates to mefloquine or amodiaquine, and by the in vitro technique used in this study, it was found that chloroquine-resistant and chloroquine-sensitive isolates were equally susceptible to amodiaquine. In general the survey showed the existence of a marked correlation between development of drug resistance of Plasmodium falciparum and the extent to which a given drug had been used in Thailand.

Enzyme typing of some isolates of Plasmodium falciparum from Thailand.

One hundred and eighty nine isolates of Plasmodium falciparum collected in Thailand, and eleven originating from Cambodia, have been typed by starch-gel electrophoresis of six enzymes (GPI, LDH, GDH, PGD, ADA, PEPE). Substantial polymorphism was found only with GPI. Occasional variants occurred with ADA, while the other four enzymes appeared to be invariant by the tests used. The results are compared with those of similar studies on African isolates, and lead to the provisional conclusion that P. falciparum isolates from different different endemic areas constitute a single, world wide species, containing potentially interbreeding individual organisms.

Evidence of increased chloroquine sensitivity in Thai isolates of Plasmodium falciparum.

Sensitivity of Thai isolates of Plasmodium falciparum to chloroquine collected over the years 1978-1986 was measured by two methods: (i) by growth of previously cultured isolates for 72 h in presence of drug, and (ii) by the WHO standard in vitro microtest. During this period there were signs of a gradual increase in drug sensitivity, coinciding with the withdrawal of chloroquine for treatment of falciparum malaria in Thailand.

Differentiation of Plasmodium falciparum clones by means of a repetitive DNA probe.

A recombinant DNA probe, pBRK1-14, was constructed by inserting a repetitive DNA fragment of 753 base pairs obtained from Plasmodium falciparum, isolate K1, into the EcoRI/PstI cloning sites of pBR322. This probe could discriminate between different parasite clones derived from a single isolate by hybridization with Southern genomic blots.

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.

Clonal diversity in a single isolate of the malaria parasite Plasmodium falciparum.

Clones of an isolate of Plasmodium falciparum from Mae Sod (Thailand) were prepared by a dilution procedure. Some of the parasite cultures thus obtained have been typed for the following characters: (i) electrophoretic variants of three enzymes; (ii) susceptibility to chloroquine and pyrimethamine; (iii) antigen diversities recognized by ten strain-specific monoclonal antibodies; (iv) presence or absence of knobs on infected erythrocytes and (v) two-dimensional PAGE variants of seven proteins. Amongst the clones there was variation involving each of these five characters. At least seven different types of clones were found in ten cultures produced by dilution. The amount of phenotypic variation within a single isolate has thus been shown to be surprisingly great. Variations in drug susceptibility and antigens are considered to be particularly important in view of their relevance to anti-malarial treatments.

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.