Impact of genetic complexity on longevity and gametocytogenesis of Plasmodium falciparum during the dry and transmission-free season of eastern Sudan

Abstract

Malaria in eastern Sudan is characterised by limited seasonal transmission, with the majority of the year remaining transmission-free. Some inhabitants who contract malaria during the transmission season retain long-lasting sub-patent infections, which probably initiate transmission the following year. Here we have monitored Plasmodium falciparum infection prevalence and gametocyte production during the dry season, and examined the impact of parasite genetic multiplicity on infection longevity. A cohort of 38 individuals who were infected with P. falciparum in November 2001 was monitored monthly by microscopy and PCR until December 2002. Reverse transcriptase polymerase chain reaction of the pfg377 gene was used to detect sub-patent gametocytes. In addition, all isolates were examined for msp-2 alleles and the mean number of parasite clones per infection was estimated. We found that a large proportion (40%) of the cohort retained gametocytes throughout the dry season. The majority of patients retained asexual infection for at least 7 months. Genetic multiplicity of P. falciparum significantly influenced longevity of asexual infection and its gametocyte production. Gametocytes from mixed genotype P. falciparum infections persisted three times longer than those from single genotype infections, suggesting that genetic diversity promotes persistence. These findings are discussed in the context of the parasite biology and malaria epidemiology in the study area.

Introduction

Reproductive success is a central life history determinant of the evolutionary fitness of living organisms. For malaria parasites (Plasmodium spp.), the production of mosquito-infective gametocytes is important not only for propagation, but also for generation of new strains—e.g. those capable of evading human immune responses and anti-malarial drug pressure. An understanding of the biology of gametocytogenesis is therefore critical when considering control strategies aimed at limiting the reproductive success of the malaria parasite.

Plasmodium falciparum clones have been found to vary significantly in their capacity to produce gametocytes in culture, and gametocytogenesis has been suggested to be a stable, genetically determined characteristic (Graves et al., 1984). In vertebrates, malaria infection is initiated by mosquito-delivered sporozoites that invade the liver, then differentiate and rapidly multiply as asexual forms in the circulation. Some asexual parasites differentiate into gametocytes, forms that cannot replicate but can be transmitted to mosquitoes. It takes approximately 9–12 days for gametocytes to be produced from asexual forms. Once mature, gametocyte longevity and their infectivity to mosquitoes probably vary over time (Hawking et al., 1971, Smalley et al., 1981). Therefore, consistent gametocyte production by individual parasite clones is critical for transmission.

The low density of P. falciparum gametocytes in natural infections appears to contradict the apparently high transmission success of this parasite in nature (Taylor and Read, 1997). The recent advent of molecular diagnosis of gametocytes has in part resolved this paradox by demonstrating the occurrence of numerous sub-microscopic gametocyte reservoirs in people living in endemic areas (Menegon et al., 2000, Abdel-Wahab et al., 2002), and has also identified multiple gametocyte genotypes within a single infection (Abdel-Wahab et al., 2002).

In the present study, we have exploited these molecular techniques to examine the longevity of gametocytogenesis of P. falciparum during the transmission-free season of eastern Sudan. In this region, malaria transmission is limited to the short rainy season, and pauses over a period of 7–9 months during the dry season (Hamad et al., 2002). Previous studies have indicated that P. falciparum appearing in the transmission season gives rise to asymptomatic sub-patent asexual parasitaemia that persists throughout the dry season. Furthermore, genotypes of different clones can survive together in individual infections and fluctuate through this period (Babiker et al., 1998). Limited cross-sectional surveys have suggested that some of these long lasting infections are capable of sustaining gametocyte production during the dry season (Abdel-Wahab et al., 2002). The aim of this study was to elucidate the longevity of individual P. falciparum infections, and to examine the impact of clonal multiplicity on longevity and gametocytogenesis among clones that survive the dry season as chronic asymptomatic infections.