Anti-Gametocyte Antibody Levels and Submicroscopic Gametocytaemia in an Area of Seasonal Malaria Transmission

Abstract

Background: Malaria is one of the leading vector-borne infectious diseases in the world with alarming death rates, which is of public health concern. Only 0.1-1 gametocyte per μL blood is sufficient to spread malaria; however microscopy which is the gold standard for detection has a detection limit of 8 gametocytes per μL. Therefore, some individuals who may be diagnosed by microscopy as negative for gametocyte may be possible reservoirs of malaria transmission. Main Objective: This study determined sub-microscopic gametocyte prevalence and antigametocyte antibody levels to gamete surface antigens Pfs230, Pfs48/45 (transmission blocking vaccine candidates) and crude gametocyte antigen (G. Extract) from collected blood samples in recruited volunteers from the Bongo District in the Upper East Region of Ghana, which is an area of seasonal malaria transmission. Also, a determination of the correlation between the prevalence of submicroscopic gametocyte levels and anti-gametocyte antibody levels was conducted. Design: Indirect enzyme immunosorbent assay (ELISA) was used to quantify the levels of antigens (Pfs230, Pfs48/45, crude gametocyte antigen, glutamate rich protein (GLURP) and merozoite surface protein 3 (MSP3)) specific antibodies in archived serum samples. Messenger ribonucleic Acid (mRNA) was extracted from archived blood samples preserved in RNA later (an RNA preservative) at the Noguchi Memorial Institute for Medical Research (NMIMR). Complementary DNA (cDNA) was synthesized from the mRNA using a reverse transcriptase. Nested PCR was carried out to amplify the Pfs25 gene from the cDNA products and visualized on a 2% agarose gel. Results: It was established that prevalence of gametocyte carriage at submicroscopic level (2.76 %) was two times higher than gametocyte carriage estimated by microscopy (1.26 %).Antibodies to all the antigens tested were lowest among the under-five (5) years and seroprevalence (%) frequently increased with age. Linear regression analysis revealed that, gametocyte-specific antibody response was influenced by level of gametocytaemia and other confounding factors. A moderate but significant correlation was found to exist between Pfs230 antibody titres and antibody titres against the crude gametocyte antigen during both the wet and dry seasons (Wet season: Spearman r = 0.414, p < 0.0001; Dry season: Spearman r = 0.390, p < 0.0001). Conclusion: There were antibody levels to gametocyte antigens Pfs48/45, Pfs230 and crude gametocyte antigens and generally antibody levels decreased in the dry season. In addition, antibody seroprevalence increased with age for both wet and dry seasons. This is a confirmation that these antigens are potential candidates for transmission blocking vaccines. A submicroscopic level of gametocytes was over two-fold higher compared to microscopic estimates of gametocytes. Also, there was weak but significant correlation between Pfs230 and G. Extract. This means that Pfs230 can be used in place of crude gametocyte antigen for the estimation of gametocyte exposure. Finally, from this study it was established that the gametocytespecific antibody response was influenced by level of gametocytaemia and other confounding factors.