Research Article: Demographical, hematological and serological risk factors for Plasmodium falciparum gametocyte carriage in a high stable transmission zone in Cameroon

Date Published: April 25, 2019

Publisher: Public Library of Science

Author(s): Estelle Essangui, Carole Else Eboumbou Moukoko, Niels Nguedia, Michele Tchokwansi, Umaru Banlanjo, Franklin Maloba, Balotin Fogang, Christiane Donkeu, Marie Biabi, Glwadys Cheteug, Sylvie Kemleu, Emmanuel Elanga-Ndille, Léopold Lehman, Lawrence Ayong, Luzia Helena Carvalho.

http://doi.org/10.1371/journal.pone.0216133

Abstract

Presence of mature gametocyte forms of malaria parasites in peripheral blood is a key requirement for malaria transmission. Yet, studies conducted in most malaria transmission zones report the absence of gametocyte in the majority of patients. We therefore sought to determine the risk factors of both all-stage and mature gametocyte carriage in an area with high stable transmission of Plasmodium falciparum in Cameroon. Gametocyte positivity was determined using three complementary methods: thick blood smear microscopy, RT-PCR and RT-LAMP, whereas exposure to the infection was assessed by enzyme-linked immunosorbent assay. Of 361 malaria endemic residents randomly included in the study (mean age: 28±23 years, age range: 2–100 years, male/female sex ratio: 1.1), 87.8% were diagnosed with P. falciparum infection, of whom 45.7% presented with fever (axillary body temperature ≥37.5°C). Mature gametocyte positivity was 1.9% by thick blood smear microscopy and 8.9% by RT-PCR targeting the mature gametocyte transcript, Pfs25. The gametocyte positivity rate was 24.1% and 36.3% by RT-PCR or RT-LAMP, respectively, when targeting the sexual stage marker, Pfs16. Multivariate analyses revealed anemia as a common independent risk factor for both mature and all-stage gametocyte carriage, whereas fever and low anti-gametocyte antibody levels were independently associated with all-stage gametocyte carriage only. Taken together, the data suggest important differences in risk factors of gametocyte carriage depending on stage analyzed, with anemia, fever and low antiplasmodial plasma antibody levels representing the major contributing risk factors.

Partial Text

Despite considerable global efforts against malaria, the disease remains a major public health problem globally. In 2017, approximately 219 million malaria cases and 435,000 related deaths were recorded worldwide; the majority (92%) of which occurred in sub-Saharan Africa. In the same period, 390,130 of the cases were reported in Cameroon, where malaria remains highly endemic [1]. Malaria is caused by Plasmodium parasites, and 6 species (Plasmodium falciparum, Plasmodium malaria, Plasmodium ovale, Plasmodium vivax, Plasmodium knowlesi and Plasmodium cynomolgi) are responsible for malaria in humans [2,3]. Plasmodium parasites are transmitted through the bite of infected female Anopheles mosquitoes. Only carriers of the sexual parasite stages known as mature gametocytes are infectious to mosquitoes, and gametocyte carriage is dependent on host and parasite factors that may vary between individuals or geo-epidemiological transmission zones[4–6]. Gametocyte production in the human host is thought to initiate immediately after asexual division, resulting in the production and release of sexually committed rings that then develop into transmissible mature gametocyte also known as stage V gametocytes [7,8]. Indeed, only a small proportion (<5%) of the asexually multiplying parasite population often commit to sexual development, and only a small portion of the sexually committed parasites may develop into transmissible mature gametocyte forms [6]. The host and parasite factors that favor sexual commitment and maturation in malaria parasites are not fully understood but are believed to involve both parasite genetics and host immunological and/or stress-related responses [9]. Risk factors for mature gametocyte carriage in P. falciparum infected patients include patient age [10–12], gender [13], asexual parasite densities, [13–15], blood hemoglobin levels [15–17], infection duration [13], presence of a fever [5,11,14,16,18,19], as well as patient’s blood group [11,15]. Blood levels of Plasmodium gametocytes may also depend on the gametocytogenic potential of the infecting clones and the ability of the host environment to either promote or block gametocyte production in vivo [6,20]. Indeed, under certain stress conditions including antimalarial medication [21–24], anemia [15], and host immune activity [9], a strong gametocyte surge can be observed [25,26]. However, it is not known if all or some of the above factors constitute risk factors for sexual commitment and circulation of early gametocyte forms in infected persons. Understanding gametocyte production and dynamics as well as the associated risk factors from population-based studies is essential to effectively combating the disease in all transmission zones. Unfortunately, epidemiological studies on gametocyte carriage have been limited by lack of diagnostic tools capable of detecting all circulating gametocyte forms. With the advent of modern genomics technology, several molecular techniques including loop-mediated isothermal amplification and polymerase chain reaction based methods now exist for effective measurement of gametocyte carriage at endemic country levels. Amongst these, methods based on the early gametocytogenesis marker Pfs16 and the mature gametocyte stage marker Pfs25 are the most widely used [27–31]. This study aimed to determine the risk factors of gametocyte carriage in a high stable malaria transmission zone in Cameroon. We employed a recently developed RT-LAMP method [33] to include submicroscopically P. falciparum infected subjects in our analysis. Furthermore, we used a combination of three gametocyte detection techniques (RT-LAMP, RT-PCR, light microscopy) to identify active carriers of P. falciparum gametocytes in the study population. Exposure to gametocyte carriage was also investigated using an ELISA-based method. Together, our data show an extremely low prevalence (1.9%) of mature gametocyte carriage in the study area when using light microscopy. This finding is similar to those reported in other regions in Cameroon by Songue et al (0%), Van der Kolk et al (4.4%), and Sandeu et al (8.9%) using light microscopy as diagnostic method [36–38]. The mature gametocyte prevalence in our study population was only slightly increased to 8.9% when using RT-PCR as diagnostic method. We therefore sought to understand if such low prevalence rates also applied to total gametocytaemia that include both mature gametocytes and newly committed sexual stage parasites. Interestingly, the RT-PCR targeting the early gametocyte marker gene, Pfs16, revealed a gametocyte prevalence three-fold (24.1%) higher than the Pfs25 RT-PCR. Considering that the Pfs16 transcript is expressed at a two-fold higher level in mature gametocytes when compared to the Pfs25 transcript [39], it is arguable that the observed difference in gametocyte prevalence may be due to differences in assay sensitivities. However, considering the generally low densities of mature gametocytes in the peripheral blood, it was conceivable that the increased sensitivity of the Pfs16 target was due to the presence of early stage gametocytes (sexually committed rings) in the majority of infected blood samples. Indeed, Plasmodium gametocytogenesis is known to initiate in mid-trophozoite stage parasites leading to the release of sexually committed merozoites that infect and develop within circulating red blood cells as sexually committed rings before maturing through older gametocyte stages (stages I-V) [40]. However, only the sexually committed rings and mature stage V gametocytes appear in the circulation, as the remaining stages sequester in deep tissues, notably in the spleen and bone marrow [41,42]. Approximately 6% of the gametocyte positives (Pfs16-based RT-LAMP and Pfs25-based RT-PCR) were negative for P. falciparum infection as determined by light microscopy and PfExp1-based RT-LAMP. We interpreted such observations as corresponding to individuals who have recently cleared the infection either naturally or following antimalarial drug administration. This is consistent with several previous reports indicating that gametocyte positivity can proceed for several weeks after the clearance of asexual parasites, depending on the treatment or host immunity [43–45] In conclusion, gametocyte rates in the study population were comparatively high, and depended on test sensitivity and the gametocyte stage being targeted. On the basis of gametocyte positivity in the study population, we identified anemia as a common risk factor for both all stage and mature stage gametocyte carriage. Importantly, we identified low anti-Plasmodial antibody responses as major contributing factors to mature gametocyte carriage whereas low antibody responses to sexual stage antigens may associate strongly with gametocytogenesis and the initial release of early stage gametocytes into the peripheral blood. The observed negative association between the anti-gametocyte plasma antibody levels strongly support ongoing efforts to develop novel malaria transmission-blocking vaccines targeting Plasmodium gametocytogenesis.   Source: http://doi.org/10.1371/journal.pone.0216133

 

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