Plasmodium falciparum genotype and gametocyte prevalence in children with uncomplicated malaria in coastal Ghana

Ayanful‑Torgby et al. Malar J (2016) 15:592 DOI 10.1186/s12936-016-1640-8 Malaria Journal Open Access RESEARCH Plasmodium falciparum genotype and gametocyte prevalence in children with uncomplicated malaria in coastal Ghana Ruth Ayanful‑Torgby1, Akua Oppong1, Joana Abankwa1, Festus Acquah1, Kimberly C. Williamson2 and Linda Eva Amoah1* Abstract Background: Plasmodium falciparum gametocytes are vital to sustaining malaria transmission. Parasite densities, multiplicity of infection as well as asexual genotype are features that have been found to influence gametocyte pro‑ duction. Measurements of the prevalence of Plasmodium sp. gametocytes may serve as a tool to monitor the success of malaria eradication efforts. Methods: Whole blood was collected from 112 children aged between 6 months and 13 years with uncomplicated P. falciparum malaria attending three health facilities in southern Ghana from June to August, 2014 before (day 0) and 4 days after completion of anti-malaria drug treatment (day 7). Malaria parasites were observed by microscopy and polymerase chain reaction (PCR); submicroscopic gametocyte carriage was measured by a Pfs25 (PF3D7_1031000) mRNA real time reverse transcriptase polymerase chain reaction (RT-PCR). Parasite genotyping was performed on gDNA extracted from dried filter paper blood blots by amplification of the polymorphic regions of msp1 (PF3D7_0930300) and msp2 (PF3D7_0206800) using PCR. Results: Microscopy estimated 3.1% (3/96) of the total population to carry gametocytes on day 0, which decreased to 2.1% (2/96) on day 7. In contrast, reverse transcriptase-real time PCR (RT-PCR) analysis of a subset of 35 samples estimated submicroscopic gametocyte carriage to be as high as 77% (27/35) using primers specific for Pfs25 (CT < 35) on day 0 and by day 7 this only declined to 60% (21/35). Genotyping the msp2 gene identified higher levels of MOI than the msp1 gene. Conclusions: Although below detection by microscopy, gametocyte prevalence at submicroscopic levels are high in this region and emphasize the need for more effective elimination approaches like the development of transmissionblocking vaccines and safer gametocytocidal drugs. Keywords: Gametocytes, Genetic diversity, Multiplicity of infection Background In Ghana, malaria is still one of the leading causes of outpatient attendance and mortality in children under the age of 5 years [1], despite enhanced control efforts. Plasmodium falciparum, the most lethal of the five species that cause human malaria, is responsible for about *Correspondence: 1 Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana Full list of author information is available at the end of the article 90% of all malaria cases in Ghana [2]. Malaria transmission requires the production of sexual stage parasites that are stimulated to fertilize after being taken up during a blood meal by a mosquito [3]. The zygote continues development in the mosquito producing an oocyst containing sporozoites that can initiate an infection in humans during a subsequent blood meal. Sexual reproduction coupled with high genetic diversity in the local parasite population and concurrent infections with polymorphic parasite lines provides genetic flexibility that allow adaptation to immune and drug pressure [4] and © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ayanful‑Torgby et al. Malar J (2016) 15:592 also influences malaria transmission success [5]. For example, an increase in the rate of sexual recombination has been found to give rise to parasites with different drug resistant profiles [6–9]. Low haematocrit and history of prolonged illness have been associated with gametocyte prevalence detected using microscopy [10]. Genetic factors are also likely to play a role since gametocyte production and mosquito infectivity have been shown to vary between parasite lines [11–14]. Together the dynamics of parasite diversity and gametocyte production have important implications for the acquisition of immunity by the host and the spread of drug resistant parasites. However, monitoring gametocyte production in the human host is complicated by low production levels and sequestration of immature gametocytes during the 10–12 days required for the development of stage V P. falciparum gametocytes. Only mature stage V gametocytes circulate and can be detected in peripheral blood. Previous work in East Africa and Asia demonstrated that gametocytes are resistant to artemisinin-based combination therapy (ACT) and, consequently, patients remain infectious for over a week after asexual parasite clearance [15, 16]. The role of the immune response in controlling gametocyte levels in the human host has not been well established [17]. However, Pfs230 and Pfs48/45 are expressed on the gametocyte surface during development in the RBC in the human host [18–20] and anti-Pfs230 and Pfs48/45 antibodies are generated during a natural infection [19–24] and thus can serve as a marker for recent gametocyte exposure. This study assessed the prevalence of submicroscopic gametocytes levels and asexual parasite diversity in patients aged between 6 months and 13 years with uncomplicated P. falciparum infections. Understanding these patterns is critical to the development of intervention strategies in high transmission areas. The persistence of gametocytes in children with uncomplicated malaria 4 days after a 3-day anti-malarial drug course (day 7) was also analysed. Methods Ethical considerations The study was approved by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research (NMIMR) and Ghana Health Services. Before recruitment each parent/guardian was informed of the objectives, methods, anticipated benefits and potential hazards of the study. The parents/guardians were encouraged to ask questions about any aspect of the study that was unclear to them and informed about their liberty to withdraw their children at any time without penalty. Children were enrolled only after written parental consent had been obtained. All patient information is treated as confidential. Page 2 of 10 Study site and population The study was conducted in three health facilities, the Ghana Atomic Energy Commission (GAEC) Clinic in Accra, Ewim Health Centre and Elmina Health Centre, both in Cape Coast. Cape Coast (05°05′ N, 01°15′ W), an urban setting, has an estimated population of 227,269 an (...truncated)