Elucidating barriers to malaria elimination: An analysis of the role of low-density and imported Plasmodium falciparum infections to onward malaria transmission

Despite yearly investments of more than US $4.1 billion dollars and extensive efforts, malaria continues to be one of the greatest contributors to morbidity and mortality in endemic areas. To continue to reduce the malaria burden, new control strategies, therapeutics, and vaccines are needed. While the malaria burden has declined since the last decade, the rate of change has plateaued in recent years, calling for new and innovative strategies to continue to drive malaria elimination. To prioritize resource allocation, accurately assess the effectiveness of candidate vaccines and therapeutics, and develop effective control strategies, it is essential to better understand the drivers of Plasmodium transmission, identify malaria hotspots, and develop new methodologies that allow for a more in-depth understanding of the true burden of disease.

There are several threats to malaria elimination efforts. Among these include a large reservoir of low-density Plasmodium infections in endemic areas and importation of cases. Low-density infections are highly prevalent in endemic areas and can contribute to onward transmission. However, since at low densities, they do not cause immediate clinical illness and are below the limit of detection (LoD) of standard field diagnostics, there is a paucity of data on their natural history of infection, which limits our understanding of the necessity of targeting these types of infections. Imported Plasmodium infections, which can be further transmitted, and introduce new strains or lines of resistance, are of rising concern, especially with increasing human mobility. This dissertation utilizes advanced epidemiologic methods and novel laboratory techniques to study low-density and imported Plasmodium infections.

In the first analysis, household clustering of subpatent infections around rapid diagnostic test (RDT) positive infections on Bioko Island, Equatorial Guinea was analyzed. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA was used to identify Plasmodium falciparum (Pf) parasite infections that were not detected by RDT (referred to as subpatent infections) on a subset of household samples from the 2015 Malaria Indicator Survey (MIS). The association between living in a household with an RDT(+) individual and having a subpatent infection was evaluated using multivariate hierarchical logistic regression models with inverse probability weights for selection. To evaluate possible modification of the association by potential importation of the RDT(+) case, the analysis was repeated among strata of matched sets based on the reported eight-week travel history of the RDT(+) individual(s). The adjusted prevalence odds of subpatent infection were 2.59-fold greater (95% CI: 1.31, 5.09) for those in a household with an RDT(+) individual compared to individuals in a household without RDT(+) individuals. When stratifying by travel history of the RDT(+) individual, the association between subpatent infections and RDT(+) infections was stronger in the strata in which the RDT(+) individual(s) had not recently travelled (adjusted Prevalence Odds Ratio (aPOR) 2.95; 95% CI:1.17, 7.41), and attenuated in the strata in which recent travel was reported (aPOR 1.76; 95% CI: 0.54, 5.67).

In the second analysis, data collected before and after a travel moratorium imposed in response to COVID-19 was leveraged to estimate the impact of imported Plasmodium infections on prevalence in areas of historically high travel prevalence on Bioko Island. A difference in differences approach was used to estimate the change in odds of Pf infection for individuals living in historically high travel areas following the travel moratorium relative to those living in historically low travel areas. A survey generalized linear model with robust standard errors and a logit link function was fit with an interaction term between travel area and year to estimate the impact of the travel moratorium on risk of Plasmodium infection. Comparing the change from 2019 to 2020 in high travel areas to low travel areas, the adjusted odds of infection were 39% lower in high travel areas (aOR: 0.61; 95% CI: 0.43, 0.88) than would have been expected in the absence of a travel moratorium.

Finally, in a third study, the feasibility of using daily at-home dried blood spot (DBS) collection as a method to study the natural history of asymptomatic, low-density infections was assessed in a small pilot study in Katakwi district, Uganda. One hundred and two adults and 29 children who were RDT-negative and asymptomatic for malaria at screening were invited to selfcollect DBS daily for 28 days. Venous blood samples and clinic-collected DBS were taken at enrollment and at four weekly clinic visits as well. Participant opinions about the DBS collection process were solicited through daily Diary Cards and a 5-point Likert scale survey on acceptability administered at the final clinic visit. The DBS and venous blood were analyzed by qRT-PCR. The number of participants completing the study, the total number of DBS collected, and the opinions of the process and any reported pain were analyzed to determine compliance and acceptability of the study procedure. The human internal control mRNA and Plasmodium 18S rRNA were evaluated for the at-home versus clinic-collected DBS and venous blood to assess quality of the at-home collected samples and evaluate the accuracy of DBS as a parasite detection tool. At-home DBS collection was found to be a feasible approach to studying low-density infections. Almost all participants (92%) completed the study, and only three individuals withdrew due to pain or inconvenience of the study procedure. Overall, 97% of participants collected ≥ 16 of 24 home DBS, and 87% of all spots had ≥ 40 µL of blood. The procedure was well tolerated and viewed favorably by participants. At-home collected DBS were acceptable for qRT-PCR and showed only slightly lower concentrations of human control mRNA compared to clinic-collected DBS (human internal control TBP mRNA cycle threshold 0.8 cycles earlier for clinic-collected DBS; 95%CI: 0.65, 0.98), though this latter difference was not clinically impactful. Correlation between Pf 18S rRNA from paired whole blood and DBS samples was high (R=0.93)

Conclusions: The results of these studies provide new evidence on the contribution and characteristics of low-density and imported Plasmodium carriage to malaria transmission. In addition, a new method for studying Plasmodium infections over time that is particularly well suited for the study of low-density infections has been validated. At-home DBS collection should be widely implemented for epidemiological surveillance, clinical trial baseline surveys and parasite monitoring, as well as for in-depth analyses of parasite dynamics, immune responses, and genetics. The increased understanding of low-density infections that will come from the utilization of this evaluation strategy will improve control strategies and development of new therapeutics and vaccines to combat malaria.