Gametocytes: insights gained during a decade of molecular monitoring
2008; Elsevier BV; Volume: 24; Issue: 11 Linguagem: Inglês
10.1016/j.pt.2008.08.001
ISSN1471-5007
AutoresHamza A. Babiker, Petra Schneider, Sarah E. Reece,
Tópico(s)Aquaculture disease management and microbiota
ResumoIn vertebrate hosts, malaria parasites produce specialized male and female sexual stages (gametocytes). Soon after being taken up by a mosquito, gametocytes rapidly produce gametes and, once mated, they infect their vector and can be transmitted to new hosts. Despite being the parasite stages that were first identified (over a century ago), gametocytes have remained elusive, and basic questions remain concerning their biology. However, the postgenomic era has substantiated information on the specialized molecular machinery of gametocytogenesis and expedited the development of molecular tools to detect and quantify gametocytes. The application of such highly sensitive and specific tools has opened up novel approaches and provided new insights into gametocyte biology. Here, we review the discoveries made during the past decade, highlight unanswered questions and suggest new directions. In vertebrate hosts, malaria parasites produce specialized male and female sexual stages (gametocytes). Soon after being taken up by a mosquito, gametocytes rapidly produce gametes and, once mated, they infect their vector and can be transmitted to new hosts. Despite being the parasite stages that were first identified (over a century ago), gametocytes have remained elusive, and basic questions remain concerning their biology. However, the postgenomic era has substantiated information on the specialized molecular machinery of gametocytogenesis and expedited the development of molecular tools to detect and quantify gametocytes. The application of such highly sensitive and specific tools has opened up novel approaches and provided new insights into gametocyte biology. Here, we review the discoveries made during the past decade, highlight unanswered questions and suggest new directions. Malaria is a debilitating disease that is responsible for between one and three million deaths annually, across tropical and subtropical climatic zones. The causative agents, Plasmodium parasites, replicate asexually in the blood of their vertebrate hosts, and a proportion of these asexually produced parasites differentiate into male and female sexual stages (gametocytes). Whereas the asexual stage in the life cycle of the parasite is responsible for clinical disease, gametocytes are responsible for transmission from host to vector. When taken up in the bloodmeal of a vector, male and female gametocytes immediately leave their red blood cells and produce gametes, which then mate and differentiate into stages that are infective to mosquitoes. Parasites then progress through several developmental stages in their vector, culminating in sporozoite stages that move to the salivary glands, ready to be transmitted to new hosts. Although sexual reproduction in Plasmodium parasites was discovered over a century ago, key questions about gametocyte biology remain. For example, the investment in gametocytes (the density of gametocytes relative to asexual forms, or conversion rate) and their sex ratio (the proportion of gametocytes that are male) both vary extensively across and within parasite species, populations and individual infections. Progress is being made in uncovering the genes and proximate mechanisms responsible [1Dixon M.W.A. et al.Sex in Plasmodium: a sign of commitment.Trends Parasitol. 2008; 24: 168-175Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 2Kooij T.W. Matuschewski K. Triggers and tricks of Plasmodium sexual development.Curr. Opin. Microbiol. 2007; 10: 547-553Crossref PubMed Scopus (21) Google Scholar, 3Pradel G. Proteins of the malaria parasite sexual stages: expression, function and potential for transmission blocking strategies.Parasitology. 2007; 134: 1911-1929Crossref PubMed Scopus (108) Google Scholar, 4Alano P. Plasmodium sexual stage antigens.Parasitol. Today. 1991; 7: 199-203Abstract Full Text PDF PubMed Scopus (20) Google Scholar], but the ultimate evolutionary explanations for this variation remain poorly understood [5Drakeley C. et al.The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion.Trends Parasitol. 2006; 22: 424-430Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 6Paul R.E.L. et al.The evolutionary ecology of Plasmodium.Ecol. Lett. 2003; 6: 866-880Crossref Scopus (58) Google Scholar, 7Talman A.M. et al.Gametocytogenesis: the puberty of Plasmodium falciparum.Malaria J. 2004; 3: 14pCrossref PubMed Scopus (139) Google Scholar, 8West S.A. et al.Evolution of gametocyte sex ratios in malaria and related apicomplexan (protozoan) parasites.Trends Parasitol. 2001; 17: 525-531Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar]. Given the essential role that gametocytes have in transmission and the drive to develop clinical interventions that disrupt sexual reproduction, why are there still fundamental unanswered questions about their biology? This is, at least in part, because gametocytes occur at much lower densities than asexual parasites, which makes them more difficult to detect. However, with the development of a gametocyte-specific PCR assay for P. falciparum in 1999 [9Babiker H.A. et al.Detection of low level Plasmodium falciparum gametocytes using reverse transcriptase polymerase chain reaction.Mol. Biochem. Parasitol. 1999; 99: 143-148Crossref PubMed Scopus (66) Google Scholar], the elusive gametocytes became detectable with greater sensitivity. Over the past decade [10Babiker H.A. Schneider P. Application of molecular methods for monitoring transmission stages of malaria parasites.Biomed. Mat. 2008; 3: 34007Crossref Scopus (17) Google Scholar], the continued development of molecular techniques has provided assays that are sensitive enough to: (i) detect and quantify gametocytes at low densities [9Babiker H.A. et al.Detection of low level Plasmodium falciparum gametocytes using reverse transcriptase polymerase chain reaction.Mol. Biochem. Parasitol. 1999; 99: 143-148Crossref PubMed Scopus (66) Google Scholar, 11Paganotti G.M. et al.Genetic complexity and gametocyte production of Plasmodium falciparum in Fulani and Mossi communities in Burkina Faso.Parasitology. 2006; 132: 607-614Crossref PubMed Scopus (18) Google Scholar, 12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar, 13Wargo A.R. et al.Plasmodium chabaudi: reverse transcription PCR for the detection and quantification of transmission stage malaria parasites.Exp. Parasitol. 2006; 112: 13-20Crossref PubMed Scopus (21) Google Scholar]; (ii) differentiate gametocytes at early and late stages of development [14Schneider P. et al.Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification.Mol. Biochem. Parasitol. 2004; 137: 35-41Crossref PubMed Scopus (119) Google Scholar]; (iii) quantify gametocytes produced by different parasite genotypes in multi-genotype infections [15Menegon M. et al.Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction.Mol. Biochem. Parasitol. 2000; 111: 153-161Crossref PubMed Scopus (41) Google Scholar, 16Wargo A.R. et al.Competitive release and facilitation of drug-resistance parasites after therapeutic chemotherapy in a rodent malaria model.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19914-19919Crossref PubMed Scopus (121) Google Scholar]; (iv) determine the expression pattern of sexual-stage-specific genes [17Niederwieser I. et al.Plasmodium falciparum: expression of gametocyte-specific genes in monolayer cultures and malaria-positive blood samples.Exp. Parasitol. 2000; 95: 163-169Crossref PubMed Scopus (28) Google Scholar]; and (v) distinguish between male and female gametocytes, including those produced by different genotypes in multi-genotype infections [18Drew D.R. Reece S.E. Development of reverse-transcription PCR techniques to analyse the density and sex ratio of gametocytes in genetically diverse Plasmodium chabaudi infections.Mol. Biochem. Parasitol. 2007; 156: 199-209Crossref PubMed Scopus (35) Google Scholar]. These assays have stimulated major lines of research, from epidemiological and population-level surveys of gametocyte carriers and their transmission potential to the performance and behaviour of individual parasite genotypes during infections and the assessment of transmission outcomes after anti-malarial therapy. Here, we provide an overview of the insights into Plasmodium gametocyte and transmission biology gained using molecular tools over the past decade (Box 1). We suggest approaches to tackle some of the remaining questions and discuss the implications for control measures.Box 1Developments and discoveries made during a decade of molecular gametocyte detection1999 Qualitative detection of P. falciparum gametocytes by reverse transcriptase (RT)-PCR confirms the presence of submicroscopic gametocytes [9Babiker H.A. et al.Detection of low level Plasmodium falciparum gametocytes using reverse transcriptase polymerase chain reaction.Mol. Biochem. Parasitol. 1999; 99: 143-148Crossref PubMed Scopus (66) Google Scholar].2000 Qualitative genotype-specific RT-PCR to detect P. falciparum gametocytes reveals that multiple co-infecting genotypes within an infection can simultaneously produce gametocytes [15Menegon M. et al.Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction.Mol. Biochem. Parasitol. 2000; 111: 153-161Crossref PubMed Scopus (41) Google Scholar].2002 RT-PCR used to show that gametocytes at submicroscopic densities are present in infections during the transmission-free season in Sudan [19Abdel-Wahab A. et al.Dynamics of gametocytes among Plasmodium falciparum clones in natural infections in an area of highly seasonal transmission.J. Infect. Dis. 2002; 185: 1838-1842Crossref PubMed Scopus (73) Google Scholar].2004 Quantitative nucleic acid sequence-based amplification (NASBA) developed to distinguish P. falciparum gametocytes at early and late stages of development reveals that gametocytes are produced soon after parasites are released from the liver [14Schneider P. et al.Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification.Mol. Biochem. Parasitol. 2004; 137: 35-41Crossref PubMed Scopus (119) Google Scholar].2005 Genotype-specific RT-PCR reveals that multi-genotype infections of P. falciparum persist and produce gametocytes for longer during transmission-free season than single-genotype infections [20Nassir E. et al.Impact of genetic complexity on longevity and gametocytogenesis of Plasmodium falciparum during the dry and transmission-free season of eastern Sudan.Int. J. Parasitol. 2005; 35: 49-55Crossref PubMed Scopus (92) Google Scholar].2006 Real-time quantitative assays developed to quantify gametocyte densities for P. falciparum[12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar] and P. chabaudi[13Wargo A.R. et al.Plasmodium chabaudi: reverse transcription PCR for the detection and quantification of transmission stage malaria parasites.Exp. Parasitol. 2006; 112: 13-20Crossref PubMed Scopus (21) Google Scholar]. In P. falciparum, gametocytes at submicroscopic densities are common post-treatment and these infections contribute considerably to transmission [12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar, 21Bousema J.T. et al.Moderate effect of artemisinin-based combination therapy on transmission of Plasmodium falciparum.J. Infect. Dis. 2006; 193: 1151-1159Crossref PubMed Scopus (161) Google Scholar]. Previous beliefs, based on microscopic data, are that treatment can result in increased gametocyte prevalence. Molecular assays show that this post-treatment peak in gametocyte prevalence reflects an increase in gametocyte density to levels that are more likely detected by microscopy [12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar, 53Ali E. et al.Increased density but not prevalence of gametocytes following drug treatment of Plasmodium falciparum.Trans. R. Soc. Trop. Med. Hyg. 2006; 100: 176-183Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. Furthermore, artemisinin-based combination therapy (ACT) reduces gametocyte density and the proportion of mosquitoes infected but does not affect the proportion of patients with infectious gametocytes (infectious reservoir) [12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar, 21Bousema J.T. et al.Moderate effect of artemisinin-based combination therapy on transmission of Plasmodium falciparum.J. Infect. Dis. 2006; 193: 1151-1159Crossref PubMed Scopus (161) Google Scholar].2007 Real-time RT-PCR reveals that P. chabaudi genotypes do not alter their gametocyte conversion rate in response to competition with a co-infecting genotype [65Wargo A.R. et al.Transmission stage investment of malaria parasites in response to in-host competition.Proc. R. Soc. London Ser. B Biol. Sci. 2007; 274: 2629-2638Crossref PubMed Scopus (58) Google Scholar]. However, when competition occurs between a drug-resistant and a drug-sensitive genotype and infections are treated to clear the sensitive genotype, the resistant genotype achieves higher gametocyte densities than when in single-genotype infections [16Wargo A.R. et al.Competitive release and facilitation of drug-resistance parasites after therapeutic chemotherapy in a rodent malaria model.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19914-19919Crossref PubMed Scopus (121) Google Scholar]. Quantitative genotype-specific and sex-specific RT-PCR developed to quantify P. chabaudi gametocyte densities and sex ratios of individual genotypes in multi-genotype infections [18Drew D.R. Reece S.E. Development of reverse-transcription PCR techniques to analyse the density and sex ratio of gametocytes in genetically diverse Plasmodium chabaudi infections.Mol. Biochem. Parasitol. 2007; 156: 199-209Crossref PubMed Scopus (35) Google Scholar]. In P. falciparum, more gametocyte carriers are detected using QT-NASBA than microscopy in Burkina Faso [22Ouédraogo A.L. et al.Age-dependent distribution of Plasmodium falciparum gametocytes quantified by pfs25 real-time QT-NASBA in a cross-sectional study in Burkina Faso.Am. J. Trop. Med. Hyg. 2007; 76: 626-630PubMed Google Scholar] and Tanzania [23Shekalaghe S. et al.Primaquine clears submicroscopic Plasmodium falciparum gametocytes that persist after treatment with sulphadoxine-pyrimethamine and artesunate.PLoS One. 2007; 2: e1023Crossref PubMed Scopus (108) Google Scholar, 24Shekalaghe S.A. et al.Submicroscopic Plasmodium falciparum gametocyte carriage is common in an area of low and seasonal transmission in Tanzania.Trop. Med. Int. Health. 2007; 12: 547-553Crossref PubMed Scopus (96) Google Scholar], and submicroscopic gametocytes contribute to transmission [26Schneider P. et al.Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection.Am. J. Trop. Med. Hyg. 2007; 76: 470-474Crossref PubMed Scopus (232) Google Scholar]. Primaquine treatment can clear all gametocytes remaining after treatment with ACT and sulphadoxine–pyrimethamine [23Shekalaghe S. et al.Primaquine clears submicroscopic Plasmodium falciparum gametocytes that persist after treatment with sulphadoxine-pyrimethamine and artesunate.PLoS One. 2007; 2: e1023Crossref PubMed Scopus (108) Google Scholar].2008 Quantitative genotype-specific and sex-specific RT-PCR for P. chabaudi reveals: genetic variation for patterns of sex-ratio variation observed throughout infections; sex-ratio patterns correlate with host anaemia, and total parasite and gametocyte density; and genotypes increase their investment in male, relative to female, gametocytes in response to the presence of co-infecting genotypes. Genotype-specific RT-PCR for P. falciparum reveals that minority genotypes in multiple-clone infections are able to transmit to mosquitoes [25Nwakanma D. et al.High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia.Int. J. Parasitol. 2008; 38: 219-227Crossref PubMed Scopus (51) Google Scholar]. In mixed-species infections with Plasmodium malariae, P. falciparum gametocyte densities are higher than in single-species infections [79Bousema J.T. et al.Increased Plasmodium falciparum gametocyte production in mixed infections with P. malariae.Am. J. Trop. Med. Hyg. 2008; 78: 442-448PubMed Google Scholar]. 1999 Qualitative detection of P. falciparum gametocytes by reverse transcriptase (RT)-PCR confirms the presence of submicroscopic gametocytes [9Babiker H.A. et al.Detection of low level Plasmodium falciparum gametocytes using reverse transcriptase polymerase chain reaction.Mol. Biochem. Parasitol. 1999; 99: 143-148Crossref PubMed Scopus (66) Google Scholar]. 2000 Qualitative genotype-specific RT-PCR to detect P. falciparum gametocytes reveals that multiple co-infecting genotypes within an infection can simultaneously produce gametocytes [15Menegon M. et al.Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction.Mol. Biochem. Parasitol. 2000; 111: 153-161Crossref PubMed Scopus (41) Google Scholar]. 2002 RT-PCR used to show that gametocytes at submicroscopic densities are present in infections during the transmission-free season in Sudan [19Abdel-Wahab A. et al.Dynamics of gametocytes among Plasmodium falciparum clones in natural infections in an area of highly seasonal transmission.J. Infect. Dis. 2002; 185: 1838-1842Crossref PubMed Scopus (73) Google Scholar]. 2004 Quantitative nucleic acid sequence-based amplification (NASBA) developed to distinguish P. falciparum gametocytes at early and late stages of development reveals that gametocytes are produced soon after parasites are released from the liver [14Schneider P. et al.Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification.Mol. Biochem. Parasitol. 2004; 137: 35-41Crossref PubMed Scopus (119) Google Scholar]. 2005 Genotype-specific RT-PCR reveals that multi-genotype infections of P. falciparum persist and produce gametocytes for longer during transmission-free season than single-genotype infections [20Nassir E. et al.Impact of genetic complexity on longevity and gametocytogenesis of Plasmodium falciparum during the dry and transmission-free season of eastern Sudan.Int. J. Parasitol. 2005; 35: 49-55Crossref PubMed Scopus (92) Google Scholar]. 2006 Real-time quantitative assays developed to quantify gametocyte densities for P. falciparum[12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar] and P. chabaudi[13Wargo A.R. et al.Plasmodium chabaudi: reverse transcription PCR for the detection and quantification of transmission stage malaria parasites.Exp. Parasitol. 2006; 112: 13-20Crossref PubMed Scopus (21) Google Scholar]. In P. falciparum, gametocytes at submicroscopic densities are common post-treatment and these infections contribute considerably to transmission [12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar, 21Bousema J.T. et al.Moderate effect of artemisinin-based combination therapy on transmission of Plasmodium falciparum.J. Infect. Dis. 2006; 193: 1151-1159Crossref PubMed Scopus (161) Google Scholar]. Previous beliefs, based on microscopic data, are that treatment can result in increased gametocyte prevalence. Molecular assays show that this post-treatment peak in gametocyte prevalence reflects an increase in gametocyte density to levels that are more likely detected by microscopy [12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar, 53Ali E. et al.Increased density but not prevalence of gametocytes following drug treatment of Plasmodium falciparum.Trans. R. Soc. Trop. Med. Hyg. 2006; 100: 176-183Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. Furthermore, artemisinin-based combination therapy (ACT) reduces gametocyte density and the proportion of mosquitoes infected but does not affect the proportion of patients with infectious gametocytes (infectious reservoir) [12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar, 21Bousema J.T. et al.Moderate effect of artemisinin-based combination therapy on transmission of Plasmodium falciparum.J. Infect. Dis. 2006; 193: 1151-1159Crossref PubMed Scopus (161) Google Scholar]. 2007 Real-time RT-PCR reveals that P. chabaudi genotypes do not alter their gametocyte conversion rate in response to competition with a co-infecting genotype [65Wargo A.R. et al.Transmission stage investment of malaria parasites in response to in-host competition.Proc. R. Soc. London Ser. B Biol. Sci. 2007; 274: 2629-2638Crossref PubMed Scopus (58) Google Scholar]. However, when competition occurs between a drug-resistant and a drug-sensitive genotype and infections are treated to clear the sensitive genotype, the resistant genotype achieves higher gametocyte densities than when in single-genotype infections [16Wargo A.R. et al.Competitive release and facilitation of drug-resistance parasites after therapeutic chemotherapy in a rodent malaria model.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19914-19919Crossref PubMed Scopus (121) Google Scholar]. Quantitative genotype-specific and sex-specific RT-PCR developed to quantify P. chabaudi gametocyte densities and sex ratios of individual genotypes in multi-genotype infections [18Drew D.R. Reece S.E. Development of reverse-transcription PCR techniques to analyse the density and sex ratio of gametocytes in genetically diverse Plasmodium chabaudi infections.Mol. Biochem. Parasitol. 2007; 156: 199-209Crossref PubMed Scopus (35) Google Scholar]. In P. falciparum, more gametocyte carriers are detected using QT-NASBA than microscopy in Burkina Faso [22Ouédraogo A.L. et al.Age-dependent distribution of Plasmodium falciparum gametocytes quantified by pfs25 real-time QT-NASBA in a cross-sectional study in Burkina Faso.Am. J. Trop. Med. Hyg. 2007; 76: 626-630PubMed Google Scholar] and Tanzania [23Shekalaghe S. et al.Primaquine clears submicroscopic Plasmodium falciparum gametocytes that persist after treatment with sulphadoxine-pyrimethamine and artesunate.PLoS One. 2007; 2: e1023Crossref PubMed Scopus (108) Google Scholar, 24Shekalaghe S.A. et al.Submicroscopic Plasmodium falciparum gametocyte carriage is common in an area of low and seasonal transmission in Tanzania.Trop. Med. Int. Health. 2007; 12: 547-553Crossref PubMed Scopus (96) Google Scholar], and submicroscopic gametocytes contribute to transmission [26Schneider P. et al.Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection.Am. J. Trop. Med. Hyg. 2007; 76: 470-474Crossref PubMed Scopus (232) Google Scholar]. Primaquine treatment can clear all gametocytes remaining after treatment with ACT and sulphadoxine–pyrimethamine [23Shekalaghe S. et al.Primaquine clears submicroscopic Plasmodium falciparum gametocytes that persist after treatment with sulphadoxine-pyrimethamine and artesunate.PLoS One. 2007; 2: e1023Crossref PubMed Scopus (108) Google Scholar]. 2008 Quantitative genotype-specific and sex-specific RT-PCR for P. chabaudi reveals: genetic variation for patterns of sex-ratio variation observed throughout infections; sex-ratio patterns correlate with host anaemia, and total parasite and gametocyte density; and genotypes increase their investment in male, relative to female, gametocytes in response to the presence of co-infecting genotypes. Genotype-specific RT-PCR for P. falciparum reveals that minority genotypes in multiple-clone infections are able to transmit to mosquitoes [25Nwakanma D. et al.High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia.Int. J. Parasitol. 2008; 38: 219-227Crossref PubMed Scopus (51) Google Scholar]. In mixed-species infections with Plasmodium malariae, P. falciparum gametocyte densities are higher than in single-species infections [79Bousema J.T. et al.Increased Plasmodium falciparum gametocyte production in mixed infections with P. malariae.Am. J. Trop. Med. Hyg. 2008; 78: 442-448PubMed Google Scholar]. Surveys based on the examination of blood smears by microscopy have consistently shown that gametocytes are observed only in a subset of infected patients [7Talman A.M. et al.Gametocytogenesis: the puberty of Plasmodium falciparum.Malaria J. 2004; 3: 14pCrossref PubMed Scopus (139) Google Scholar]. However, malaria parasites can transmit from these supposedly 'non-infectious' hosts, and the presence of gametocytes at extremely low densities has long been suspected [5Drakeley C. et al.The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion.Trends Parasitol. 2006; 22: 424-430Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar]. Molecular-amplification-based techniques are sensitive enough to detect and quantify gametocytes at very low densities (e.g. 0.02–10 gametocytes per microlitre [12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar]) and have confirmed the presence of gametocytes at densities too low to be detected by standard microscopy methods. We refer to gametocyte densities that are unlikely to be detected by microscopy but can be detected by molecular methods as 'submicroscopic'. Submicroscopic gametocyte carriers exist at a higher frequency than expected and at all levels of endemicity [12Schneider P. et al.(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate.Int. J. Parasitol. 2006; 36: 403-408Crossref PubMed Scopus (76) Google Scholar, 19Abdel-Wahab A. et al.Dynamics of gametocytes among Plasmodium falciparum clones in natural infections in an area of highly seasonal transmission.J. Infect. Dis. 2002; 185: 1838-1842Crossref PubMed Scopus (73) Google Scholar, 20Nassir E. et al.Impact of genetic complexity on longevity and gametocytogenesis of Plasmodium falciparum during the dry and transmission-free season of eastern Sudan.Int. J. Parasitol. 2005; 35: 49-55Crossref PubMed Scopus (92) Google Scholar, 21Bousema J.T. et al.Moderate effect of artemisinin-based combination therapy on transmission of Plasmodium falciparum.J. Infect. Dis. 2006; 193: 1151-1159Crossref PubMed Scopus (161) Google Scholar, 22Ouédraogo A.L. et al.Age-dependent distribution of Plasmodium falciparum gametocytes quantified by pfs25 real-time QT-NASBA in a cross-sectional study in Burkina Faso.Am. J. Trop. Med. Hyg. 2007; 76: 626-630PubMed Google Scholar, 23Shekalaghe S. et al.Primaquine clears submicroscopic Plasmodium falciparum gametocytes that persist after treatment with sulphadoxine-pyrimethamine and artesunate.PLoS One. 2007; 2: e1023Crossref PubMed Scopus (108) Google Scholar, 24Shekalaghe S.A. et al.Submicroscopic Plasmodium falciparum gametocyte carriage is common in an area of low and seasonal transmission in Tanzania.Trop. Med. Int. Health. 2007; 12: 547-553Crossref PubMed Scopus (96) Google Scholar, 25Nwakanma D. et al.High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia.Int. J. Parasitol. 2008; 38: 219-227Crossref PubMed Scopus (51) Google Scholar], and, most importantly, make considerable contributions to transmission [26Schneider P. et al.Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection.Am. J. Trop. Med. Hyg. 2007; 76: 470-474Crossref PubMed Scopus (232) Google Scholar]. The successful infection of vectors is generally positively correlated to gametocyte density [26Schneider P. et al.Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection.Am. J. Trop. Med. Hyg. 2007; 76: 470-474Crossref PubMed Scopus (232) Google Scholar, 27Jeffery G.M. Eyles D.E. Infectivity to mosquitoes of Plasmodium falciparum as related to gametocyte de
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