Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction

doi:10.1016/S0166-6851(00)00314-5

Molecular and Biochemical Parasitology

Volume 111, Issue 1, November 2000, Pages 153-161

https://doi.org/10.1016/S0166-6851(00)00314-5 Get rights and content

Abstract

A molecular assay has been developed for the specific detection and genetic characterisation of Plasmodium falciparum gametocytes in the blood of malaria infected individuals. The assay is based on the reverse transcription and polymerase chain reaction (RT-PCR) amplification of the messenger RNA of gene pfg377, a sexual-stage specific transcript abundantly produced in maturing gametocytes. The gene contains four regions of repetitive sequences, of which region 3 was shown to be the most polymorphic in laboratory clones and field isolates of the parasite. Analysis of samples of malaria infected blood by RT-PCR specific for region 3 has enabled identification of multiple gametocyte-producing clones within single infections. The assay is able to detect gametocytes below the threshold of microscopic detection, and is highly specific for its gametocyte targets also in the presence of a vast excess of asexual forms.

Introduction

Studies on the genetic structure and epidemiology of natural malaria parasite populations, especially of Plasmodium falciparum, have been greatly facilitated by polymerase chain reaction (PCR) methodology. Amplification of genomic DNA, especially repetitive sequences of variable length, has allowed the detection of multiple alleles of polymorphic parasite genes from the small samples of infected blood. Such an approach has revealed the frequent presence of multiple clones of P. falciparum within infected individuals, and has generated much data on the diversity and the distribution of allelic variants of genes within and between parasite populations [1].

An unanswered question is whether all or only some clones in a mixed infection are producing gametocytes at the time of examination. In situ PCR using allele-specific primers of polymorphic genes [2] can potentially be used to determine the genetic constitution of gametocytes in blood smears. However, this method is not applicable to sub-patent gametocytes in infected patients, which have been shown to be capable of producing infections in mosquitoes [3]. This question is important in the population biology of P. falciparum, since gametocytes are the only mosquito-transmissible stages, and the frequency of crossing between genetically different gametocytes in mosquitoes determines the likelihood of the generation of new genotypes by recombination [4], [5].

A method based on reverse transcriptase-PCR (RT-PCR) to detect expression of the pfs25 gene, expressed only in sexual stages, has recently been developed [6], which is proving useful for detecting low or sub-patent levels of these forms in natural infections. Here we report on an RT-PCR specific for the pfg377 gene which is also expressed exclusively in gametocytes [7], and has the added advantage for such studies of being highly polymorphic, as shown in the present work. This method can thus potentially detect which clones in a mixed infection are producing gametocytes.

Section snippets

Parasites and patients

The following gametocyte-producing P. falciparum cloned lines were used; clones 3D7 [8], HB3 [8], FCR3A2 [9], K1 [10] and 1776S8 [11]. Clone F12, unable to produce gametocytes and derived from 3D7 [11], was used as a negative control. Venous blood from patients who had imported malaria into Italy from other malarious countries was collected in acid citrate dextrose (ACD) at the ‘Lazzaro Spallanzani’ Hospital, Rome. Field samples were collected in epidemiological surveys carried out in Asar

The gametocyte-specific gene pfg377 contains repetitive regions exhibiting length polymorphism

Oligonucleotide primers were designed from the sequence of pfg377 to amplify specifically regions 1–4, which contain repeated amino acid motifs, from five isolates and clones of P. falciparum of different geographic origin. The size of region 1 was similar in all five isolates, while the other three regions showed some degree of size polymorphism, region 3 being the most polymorphic (Fig. 1, panel A).

In order to ascertain the molecular basis of the observed polymorphism, PCR fragments of region

Discussion

We have described here an RT-PCR assay for detection and typing of P. falciparum gametocytes based on the sexual stage-specific gene pfg377 among laboratory clones and field isolates of the parasite. The technique has proved to be able to identify multiple gametocyte-producing clones in a single infection. At present the method is inadequate for determining the precise ratios of each type of clone. Results obtained with artificial mixtures of HB3 and 3D7 gametocytes indicated that the relative

Acknowledgements

This work was supported by the Wellcome Trust (054597/98/Z), the Medical Research Council (UK), the Contract INCO-DC ERBIC 18CT97 0217 of the European Commission, and a Grant of Istituto Superiore di Sanità. We are indebted to Professor G. Girelli, Centro Trasfusionale, Dipartimento di Biopatologia Umana, Università di Roma ‘La Sapienza’, for the gift of red blood cells for parasite cultivation, and to Dr Marco Gentile, IRCCS ‘Lazzaro Spallanzani’, Rome, for providing the samples from imported

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A major challenge to the success of malaria control program in Saudi Arabia is the high influx of expatriates and holy visitors from malaria endemic countries. In the present study we examined whether drug resistant parasite genotypes reported in Jazan region, southwest of Saudi Arabia are imported or developed locally. We examined 178 Plasmodium falciparum isolates for alleles of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), associated with Sulfadoxine-Pyrimethamine (SP) resistance, and three microsatellites flanking each gene. In addition, we examined a neutral polymorphic gene (Pfg377). We compared the dhfr and dhps haplotypes in Jazan, using network analysis, to an existing similar data set of 94 P. falciparum isolates from eastern Sudan. In Jazan, double mutant dhfr allele (51I, 108N) occurred with a prevalence of 33%. The vast majority (99%) of dhps were wild-type alleles. The mean expected heterozygosity (H_e) of microsatellites around mutant dhfr alleles (H_e = 0.312; n = 60) was lower (P ⩽ 0.05) than that around the wild-type allele (H_e = 0.834; n = 116). Also, the mutant dhfr isolates showed high H_e for dhps (H_e = 0.80) and the non-drug resistance locus Pfg377 (H_e = 0.63) indicative of selection for mutant dhfr only. The predominant double mutant dhfr haplotype in Jazan (73%), was prevalent among P. falciparum in east Africa. Network analysis suggests the mutant haplotype of dhfr gene was possibly introduced into Jazan from East Africa. The absence of mutations in dhps as well as triple mutant dhfr haplotype associated with SP failure support the current use of SP as a partner with artesunate as a first line therapy in Saudi Arabia. However, the close relationship between the major mutant dhfr haplotype in Sudan and Saudi Arabia, favour the hypothesis of recent migration as a source of the major resistant dhfr lineage. Thus, regular monitoring of the dhfr and dhps haplotypes is of high priority to guard possible importation of high level SP resistant lineages.
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Citation Excerpt :
These parasites were subsequently adapted to in vitro culture, and gametocyte production was assessed by the same technique. Pfg377 is a gametocyte-specific P. falciparum gene, in which polymorphisms in the sequence length of region 3 allow the typing of alleles by PCR (Menegon et al., 2000). We were able to compare the predominant asexual parasite pfg377 alleles with those of the gametocytes from the same infections, in order to determine which clones were producing gametocytes.
Understanding why some malaria-infected individuals are infective to mosquitoes while others are not, is of great importance when considering interventions to stop malaria transmission. Whether gametocytes are produced in every individual infected with Plasmodium falciparum remains unclear. Using a highly sensitive reverse transcription (RT)-PCR assay, we attempted to detect gametocyte-specific mRNA transcripts in isolates from Thai patients which newly adapted to continuous in vitro culture. We then compared the allelic types of the pfg377 gene between patient blood and culture-adapted parasites in order to determine whether the same parasite lines were producing gametocytes in vivo and in vitro. Transcripts of pfg377 were detected in all parasite isolates and in the corresponding cultured isolates, revealing that all patients had gametocytes circulating in their blood at the time of sampling. For isolates in continuous in vitro culture, there was a match between pfg377 allelic types detected by PCR from genomic DNA (and thus indicative of the dominant allelic type of asexual parasites) and those detected by RT-PCR of mRNA (gametocyte-specific), whereas in freshly isolated patient blood there were some differences between the asexual parasite allelic type and that of the gametocytes in the same infection. Seven isolates contained asexual stage parasites harbouring pfg377 alleles that were not detectable in gametocytes from the same infections, suggesting that some clones were not producing gametocytes at the time of sampling, or that they were below the level of detection.
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In addition to their ability to detect gametocytes at low density, molecular assays also enable the discrimination and quantification of gametocytes produced by different genotypes within the same infection. So far, genotype-specific assays have been developed for P. falciparum and P. chabaudi gametocytes [15,16]. The application of these assays has revealed that co-infecting genotypes can simultaneously produce gametocytes and transmit, throughout both clinical and asymptomatic P. falciparum infections [25].
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Molecular and Biochemical Parasitology

Abstract

Introduction

Section snippets

Parasites and patients

The gametocyte-specific gene pfg377 contains repetitive regions exhibiting length polymorphism

Discussion

Acknowledgements

References (21)

Current views on the population structure of Plasmodium falciparum: implications for control

Parasitol. Today

Intragenic recombination of Plasmodium falciparum identified by in situ polymerase chain reaction

Mol. Biochem. Parasitol.

Detection of low level Plasmodium falciparum gametocytes using reverse transcriptase polymerase chain reaction

Mol. Biochem. Parasitol.

Cos cell expression cloning of Pfg377, a Plasmodium falciparum gametocyte antigen associated with osmiophilic bodies

Mol. Biochem. Parasitol.

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

Trans. R. Soc. Trop. Med. Hyg.

Plasmodium falciparum: parasites defective in early stages of gametocytogenesis

Exp. Parasitol.

PCR and strain identification in Plasmodium falciparum

Parasitol. Today

The production of the osmiophilic body protein Pfg377 is associated with stage of maturation and sex in Plasmodium falciparum

Mol. Biochem. Parasitol.

Pfcrk1, a developmentally regulated cdc2-related protein kinase of Plasmodium falciparum

Mol. Biochem. Parasitol.

Factors determining the true reservoir of infection of Plasmodium falciparum and Wuchereria bancrofti in a west African village

Trans. R. Soc. Trop. Med. Hyg.

Cited by (43)

Identification of a long noncoding RNA required for temperature induced expression of stage-specific rRNA in malaria parasites

Plasmodium Gametocytes in Field Studies: Do We Measure Commitment to Transmission or Detectability?

Source of drug resistant Plasmodium falciparum in a potential malaria elimination site in Saudi Arabia

In vivo and in vitro gametocyte production of Plasmodium falciparum isolates from Northern Thailand

Anopheles dirus co-infection with human and monkey malaria parasites in Vietnam

Gametocytes: insights gained during a decade of molecular monitoring