Determination of the prevalence and density of true plasmodium falciparum infections and gametocyte carriage during two malaria outbreaks in Zambezi region, Namibia
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Date
2023
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University of Namibia
Abstract
Malaria is a disease caused by parasites from the genus Plasmodium, which are transmitted to humans by the bite of an infected female Anopheles mosquito. Malaria remains as one of the leading causes of mortality globally. Namibia has successfully reduced the malaria burden by over 90% between 2004 and 2014 and aimed to eliminate the disease by 2020, however, this progress has stalled and it has experienced outbreaks. New approaches are needed to understand and address the reasons for persistent transmission and outbreaks. Accurate diagnosis is a challenge with the routinely used Rapid Diagnostic Tests (RDTs) at low transmission settings as they have been reported to give both false-positive and false-negative results, both outcomes compromise the detection and treatment of all malaria cases. Furthermore, RDTs do not give much information about epidemiological parameters that affect malaria transmission and can be used to assess and mitigate against onward transmission of the disease which includes transmission intensity and risk of transmission. Molecular tools have been reported as being able to identify true malaria infections. This includes parasite species and strains as well as being able to give a more accurate and quantitative reflection of the malaria burden and potential risk of transmission, additional information that RDTs cannot provide. This study was conducted to confirm and identify the Plasmodium species confirmed by RDTs; quantify parasite densities and gametocytes during the 2017 and 2021 outbreak seasons to determine the importance of these parameters in assessing transmission intensity and risk. A total of 589 malaria RDT positives, (251 from the 2017 malaria outbreak and 338 from the 2021 outbreak) from the Zambezi region with corresponding dried blood spots (DBS) were analysed using DNA extracted using the Chelex method for conventional PCR. Only 223/589 (38%) RDT positive samples were confirmed as being true malaria positive: (71/251, 28% for 2017 and 152/338, 45% for 2021). All confirmed samples were identified as Plasmodium falciparum only. The QIAamp DNA kit was used to extract DNA from the 223 confirmed malaria positive samples for quantitative PCR to calculate parasite density for each malaria outbreak season (71 for 2017 and 152 for the 2021 season, a total of 223). The parasitaemia of the samples from each season was calculated and classified as follows: for 2017, low - 13%, moderate - 24%, high - 51%, hyperparasitaemia - 13%; for 2021 low - 24%, moderate - 29%, high - 40%, hyperparasitaemia - 7%. The mean parasite density was higher in 2017 (46088 p/μL) as compared to 2021 (35211 p/μL) (Mann-Whitney, p=0.008). Gametocyte carriage was determined by RTqPCR to detect and quantify the presence of Pfs25 transcripts for female gametocytes. The study showed that as parasitaemia increased, there was a higher chance of detecting gametocytes. Gametocyte detection was found in 59/71 (83%) for 2017 and 98/152 (64%) for 2021 using a Ct value of ≤35 as a cut-off point. There was a significant difference in gametocyte mean for 2017 (116 g/μL) and 2021 (892 g/μL) (Mann-Whitney, p=0.003). There was a moderate correlation between parasitaemia and gametocyte carriage during the 2017 outbreak (rho = 0.6) and a strong correlation for the 2021 outbreak (rho = 1) using Spearman’s Correlation. The Median test showed that during the 2021 outbreak there was no significant difference (p=0.910) between gametocyte carriage in low parasitaemia infections and moderate parasitaemia infections. This showed that low density infections are actually a risk factor for onward transmission of malaria. This study confirmed that P. falciparum is the dominant parasite causing malaria in the Zambezi region during the 2017 and 2021 outbreaks. It also showed that the use of RDTs for Reactive Case Detection (RACD) does not give an accurate picture of true malaria cases during outbreaks, only 38% of cases were confirmed by PCR. Both outbreaks showed moderate parasite density on average although both had high parasitaemia and hyper parasitaemia cases, signals indicative of an outbreak. Parasitaemia also correlated with gametocyte carriage showing that the parasite density was linked with higher transmission risk through gametocyte carriage. Thus, parasite density and gametocyte carriage as determined through the use of molecular tools can be used to assess the risk of transmission. However, further studies should be conducted in dry seasons to have a better understanding of residual transmission, parasite reservoir, and malaria epidemiology as a comparison to the outbreak seasons. The use of additional molecular analysis such as the Multiplicity of Infection (MOI), heterozygosity index and parasite transmission networks can also enrich the data collected during molecular surveillance and its usefulness in heterogeneous malaria transmission settings. It is recommended that molecular surveillance be used during passive surveillance at clinics; used during reactive case detection and Mass Screen and Treat and when targeting interventions to high-risk groups to understand the drivers of sustained transmission. This will contribute to the tailoring of cost-effective interventions and not a one-size fits all blanket intervention.
Description
A thesis submitted in fulfilment of the requirements for the Degree of Master of Science (Molecular Biology)
Keywords
Quantification, Plasmodium, Rapid Diagnostic Tests