Identifying individual, household and environmental risk factors for malaria infection on Bioko Island to inform interventions.

BACKGROUND: Since 2004, malaria transmission on Bioko Island has declined significantly as a result of the scaling-up of control interventions. The aim of eliminating malaria from the Island remains elusive, however, underscoring the need to adapt control to the local context. Understanding the factors driving the risk of malaria infection is critical to inform optimal suits of interventions in this adaptive approach. METHODS: This study used individual and household-level data from the 2015 and 2018 annual malaria indicator surveys on Bioko Island, as well as remotely-sensed environmental data in multilevel logistic regression models to quantify the odds of malaria infection. The analyses were stratified by urban and rural settings and by survey year. RESULTS: Malaria prevalence was higher in 10-14-year-old children and similar between female and male individuals. After adjusting for demographic factors and other covariates, many of the variables investigated showed no significant association with malaria infection. The factor most strongly associated was history of travel to mainland Equatorial Guinea (mEG), which increased the odds significantly both in urban and rural settings (people who travelled had 4 times the odds of infection). Sleeping under a long-lasting insecticidal net decreased significantly the odds of malaria across urban and rural settings and survey years (net users had around 30% less odds of infection), highlighting their contribution to malaria control on the Island. Improved housing conditions indicated some protection, though this was not consistent across settings and survey year. CONCLUSIONS: Malaria risk on Bioko Island is heterogeneous and determined by a combination of factors interacting with local mosquito ecology. These interactions grant further investigation in order to better adapt control according to need. The single most important risk factor identified was travel to mEG, in line with previous investigations, and represents a great challenge for the success of malaria control on the Island.

Feasibility of community at-home dried blood spot collection combined with pooled reverse transcription PCR as a viable and convenient method for malaria epidemiology studies.

BACKGROUND: Many Plasmodium infections in endemic regions exist at densities below the limit of detection of standard diagnostic tools. These infections threaten control efforts and may impact vaccine and therapeutic drug studies. Simple, cost-effective methods are needed to study the natural history of asymptomatic submicroscopic parasitaemia. Self-collected dried blood spots (DBS) analysed using pooled and individual quantitative reverse transcription polymerase chain reaction (qRT-PCR) provide such a solution. Here, the feasibility and acceptability of daily at-home DBS collections for qRT-PCR was studied to better understand low-density infections. METHODS: Rapid diagnostic test (RDT)-negative individuals in Katakwi District, northeastern Uganda, were recruited between April and May 2021. Venous blood samples and clinic-collected DBS were taken at enrollment and at four weekly clinic visits. Participants were trained in DBS collection and asked to collect six DBS weekly between clinic visits. Opinions about the collection process were solicited using daily Diary Cards and a Likert scale survey at the final study visit. Venous blood and DBS were analysed by Plasmodium 18S rRNA qRT-PCR. The number of participants completing the study, total DBS collected, and opinions of the process were analysed to determine compliance and acceptability. The human internal control mRNA and Plasmodium 18S rRNA were evaluated for at-home vs. clinic-collected DBS and venous blood to assess quality and accuracy of at-home collected samples. RESULTS: One-hundred two adults and 29 children were enrolled, and 95 and 26 completed the study, respectively. Three individuals withdrew due to pain or inconvenience of procedures. Overall, 96% of participants collected ≥ 16 of 24 at-home DBS, and 87% of DBS contained ≥ 40 µL of blood. The procedure was well tolerated and viewed favourably by participants. At-home collected DBS were acceptable for qRT-PCR and showed less than a one qRT-PCR cycle threshold shift in the human control mRNA compared to clinic-collected DBS. Correlation between Plasmodium falciparum 18S rRNA from paired whole blood and DBS was high (R = 0.93). CONCLUSIONS: At-home DBS collection is a feasible, acceptable, and robust method to obtain blood to evaluate the natural history of low-density Plasmodium infections by qRT-PCR.

Clustering of subpatent infections in households with asymptomatic rapid diagnostic test-positive cases in Bioko Island, Equatorial Guinea independent of travel to regions of higher malaria endemicity: a cross-sectional study.

BACKGROUND: Prevalence of falciparum malaria on Bioko Island remains high despite sustained, intensive control. Progress may be hindered by high proportions of subpatent infections that are not detected by rapid diagnostic tests (RDT) but contribute to onward transmission, and by imported infections. Better understanding of the relationship between subpatent infections and RDT-detected infections, and whether this relationship is different from imported versus locally acquired infections, is imperative to better understand the sources of infection and mechanisms of transmission to tailor more effective interventions. METHODS: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on a sub-set of samples from the 2015 Malaria Indicator Survey to identify subpatent infections. Households with RDT(+) individuals were matched 1:4 with households with no RDT(+) individuals. 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). RESULTS: There were 142 subpatent infections detected in 1,400 individuals (10.0%). The prevalence of subpatent infections was higher in households with versus without an RDT(+) individual (15.0 vs 9.1%). 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). CONCLUSIONS: There is clustering of subpatent infections around RDT(+) individual(s) when both imported and local infection are suspected. Future control strategies that aim to treat whole households in which an RDT(+) individual is found may target a substantial portion of infections that would otherwise not be detected.

Mapping and enumerating houses and households to support malaria control interventions on Bioko Island.

BACKGROUND: Housing mapping and household enumeration are essential for the planning, implementation, targeting, and monitoring of malaria control interventions. In many malaria endemic countries, control efforts are hindered by incomplete or non-existent housing cartography and household enumeration. This paper describes the development of a comprehensive mapping and enumeration system to support the Bioko Island Malaria Control Project (BIMCP). RESULTS: A highly detailed database was developed to include every housing unit on Bioko Island and uniquely enumerate the associated households residing in these houses. First, the island was divided into a virtual, geo-dereferenced grid of 1 × 1 km sequentially numbered map-areas, each of which was in turn subdivided into one hundred, 100 × 100 m sequentially numbered map-sectors. Second, high-resolution satellite imagery was used to sequentially and uniquely identify all housing units within each map-sector. Third, where satellite imagery was not available, global positioning systems (GPS) were used as the basis for uniquely identifying and mapping housing units in a sequential manner. A total of 97,048 housing units were mapped by 2018, 56% of which were concentrated in just 5.2% of Bioko Island's total mapped area. Of these housing units, 70.7% were occupied, thus representing uniquely identified households. CONCLUSIONS: The housing unit mapping and household enumeration system developed for Bioko Island enabled the BIMCP to more effectively plan, implement, target, and monitor malaria control interventions. Since 2014, the BIMCP has used the unique household identifiers to monitor all household-level interventions, including indoor residual spraying, long-lasting insecticide-treated nets distribution, and annual malaria indicator surveys. The coding system used to create the unique housing unit and household identifiers is highly intuitive and allows quick location of any house within the grid without a GPS. Its flexibility has permitted the BIMCP to easily take into account the rapid and substantial changes in housing infrastructure. Importantly, by utilizing this coding system, an unprecedented quantity and diversity of detailed, geo-referenced demographic and health data have been assembled that have proved highly relevant for informing decision-making both for malaria control and potentially for the wider public health agenda on Bioko Island.

Assessing the daily natural history of asymptomatic Plasmodium infections in adults and older children in Katakwi, Uganda: a longitudinal cohort study.

BACKGROUND: Low-density asymptomatic Plasmodium infections are prevalent in endemic areas, but little is known about their natural history. The trajectories of these infections and their propensity to fluctuate to undetectable densities can affect detection in clinical trials and field studies. We aimed to classify the natural history of these infections in a high transmission area over 29 days. METHODS: In this longitudinal cohort study, we enrolled healthy, malaria-asymptomatic, afebrile, adults (age 18-59 years) and older children (age 8-17 years) in Katakwi District, Uganda, who were negative for Plasmodium infection on rapid diagnostic tests. Participants were instructed to self-collect one dried blood spot (DBS) per day for a maximum of 29 days. We excluded people if they were pregnant or taking antimalarials. During weekly clinic visits, staff collected a DBS and a 4 mL sample of venous blood. We analysed DBSs by Plasmodium 18S rRNA quantitative RT-PCR (qRT-PCR). We classified DBS by infection type as negative, P falciparum, non-P falciparum, or mixed. We plotted infection type over time for each participant and categorised trajectories as negative, new, cleared, chronic, or indeterminate infections. To estimate the effect of single timepoint sampling, we calculated the daily prevalence for each study day and estimated the number of infections that would have been detected in our population if sampling frequency was reduced. FINDINGS: Between April 9 and May 20, 2021, 3577 DBSs were collected by 128 (40 male adults, 60 female adults, 12 male children, and 16 female children) study participants. 2287 (64%) DBSs were categorised as negative, 751 (21%) as positive for P falciparum, 507 (14%) as positive for non-P falciparum, and 32 (1%) as mixed infections. Daily Plasmodium prevalence in the population ranged from 45·3% (95% CI 36·6-54·1) at baseline to 30·3% (21·9-38·6) on day 24. 37 (95%) of 39 P falciparum and 35 (85%) of 41 non-P falciparum infections would have been detected with every other day sampling, whereas, with weekly sampling, 35 (90%) P falciparum infections and 31 (76%) non-P falciparum infections would have been detected. INTERPRETATION: Parasite dynamics and species are highly variable among low-density asymptomatic Plasmodium infections. Sampling every other day or every 3 days detected a similar proportion of infections as daily sampling, whereas testing once per week or even less frequently could misclassify up to a third of the infections. Even using highly sensitive diagnostics, single timepoint testing might misclassify the true infection status of an individual. FUNDING: US National Institutes of Health and Bill and Melinda Gates Foundation.

Impact of six-month COVID-19 travel moratorium on Plasmodium falciparum prevalence on Bioko Island, Equatorial Guinea.

Importation of malaria infections is a suspected driver of sustained malaria prevalence on areas of Bioko Island, Equatorial Guinea. Quantifying the impact of imported infections is difficult because of the dynamic nature of the disease and complexity of designing a randomized trial. We leverage a six-month travel moratorium in and out of Bioko Island during the initial COVID-19 pandemic response to evaluate the contribution of imported infections to malaria prevalence on Bioko Island. Using a difference in differences design and data from island wide household surveys conducted before (2019) and after (2020) the travel moratorium, we compare the change in prevalence between areas of low historical travel to those with high historical travel. Here, we report that in the absence of a travel moratorium, the prevalence of infection in high travel areas was expected to be 9% higher than observed, highlighting the importance of control measures that target imported infections.

Plasmodium 18S Ribosomal RNA Biomarker Clearance After Food and Drug Administration-Approved Antimalarial Treatment in Controlled Human Malaria Infection Trials.

Background: Sensitive molecular assays, such as quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of Plasmodium 18S ribosomal RNA (rRNA), are increasingly the primary method of detecting infections in controlled human malaria infection (CHMI) trials. However, thick blood smears (TBSs) remain the main method for confirming clearance of parasites after curative treatment, in part owing to uncertainty regarding biomarker clearance rates. Methods: For this analysis, 18S rRNA qRT-PCR data were compiled from 127 Plasmodium falciparum-infected participants treated with chloroquine or atovaquone-proguanil in 6 CHMI studies conducted in Seattle, Washington, over the past decade. A survival analysis approach was used to compare biomarker and TBS clearance times among studies. The effect of the parasite density at which treatment was initiated on clearance time was estimated using linear regression. Results: The median time to biomarker clearance was 3 days (interquartile range, 3-5 days), while the median time to TBS clearance was 1 day (1-2 days). Time to biomarker clearance increased with the parasite density at which treatment was initiated. Parasite density did not have a significant effect on TBS clearance. Conclusions: The Plasmodium 18S rRNA biomarker clears quickly and can be relied on to confirm the adequacy of Food and Drug Administration-approved treatments in CHMI studies at nonendemic sites.

Safety and Immunogenicity of Radiation-Attenuated PfSPZ Vaccine in Equatoguinean Infants, Children, and Adults.

The radiation-attenuated Plasmodium falciparum sporozoites (PfSPZ) Vaccine has demonstrated safety and immunogenicity in 5-month-old to 50-year-old Africans in multiple trials. Except for one, each trial has restricted enrollment to either infants and children or adults < 50 years old. This trial was conducted in Equatorial Guinea and assessed the safety, tolerability, and immunogenicity of three direct venous inoculations of 1.8 × 106 or 2.7 × 106 PfSPZ, of PfSPZ Vaccine, or normal saline administered at 8-week intervals in a randomized, double-blind, placebo-controlled trial stratified by age (6-11 months and 1-5, 6-10, 11-17, 18-35, and 36-61 years). All doses were successfully administered. In all, 192/207 injections (93%) in those aged 6-61 years were rated as causing no or mild pain. There were no significant differences in solicited adverse events (AEs) between vaccinees and controls in any age group (P ≥ 0.17). There were no significant differences between vaccinees and controls with respect to the rates or severity of unsolicited AEs or laboratory abnormalities. Development of antibodies to P. falciparum circumsporozoite protein occurred in 67/69 vaccinees (97%) and 0/15 controls. Median antibody levels were highest in infants and 1-5-year-olds and declined progressively with age. Antibody responses in children were greater than in adults protected against controlled human malaria infection. Robust immunogenicity, combined with a benign AE profile, indicates children are an ideal target for immunization with PfSPZ Vaccine.

Rethinking detection of pre-existing and intervening Plasmodium infections in malaria clinical trials.

Pre-existing and intervening low-density Plasmodium infections complicate the conduct of malaria clinical trials. These infections confound infection detection endpoints, and their immunological effects may detract from intended vaccine-induced immune responses. Historically, these infections were often unrecognized since infrequent and often analytically insensitive parasitological testing was performed before and during trials. Molecular diagnostics now permits their detection, but investigators must weigh the cost, complexity, and personnel demands on the study and the laboratory when scheduling such tests. This paper discusses the effect of pre-existing and intervening, low-density Plasmodium infections on malaria vaccine trial endpoints and the current methods employed for their infection detection. We review detection techniques, that until recently, provided a dearth of cost-effective strategies for detecting low density infections. A recently deployed, field-tested, simple, and cost-effective molecular diagnostic strategy for detecting pre-existing and intervening Plasmodium infections from dried blood spots (DBS) in malaria-endemic settings is discussed to inform new clinical trial designs. Strategies that combine sensitive molecular diagnostic techniques with convenient DBS collections and cost-effective pooling strategies may enable more thorough and informative infection monitoring in upcoming malaria clinical trials and epidemiological studies.

Immunogenicity and Protective Efficacy of Radiation-Attenuated and Chemo-Attenuated PfSPZ Vaccines in Equatoguinean Adults.

Plasmodium falciparum sporozoite (PfSPZ) Vaccine (radiation-attenuated, aseptic, purified, cryopreserved PfSPZ) and PfSPZ-CVac (infectious, aseptic, purified, cryopreserved PfSPZ administered to subjects taking weekly chloroquine chemoprophylaxis) have shown vaccine efficacies (VEs) of 100% against homologous controlled human malaria infection (CHMI) in nonimmune adults. Plasmodium falciparum sporozoite-CVac has never been assessed against CHMI in African vaccinees. We assessed the safety, immunogenicity, and VE against homologous CHMI of three doses of 2.7 × 106 PfSPZ of PfSPZ Vaccine at 8-week intervals and three doses of 1.0 × 105 PfSPZ of PfSPZ-CVac at 4-week intervals with each arm randomized, double-blind, placebo-controlled, and conducted in parallel. There were no differences in solicited adverse events between vaccinees and normal saline controls, or between PfSPZ Vaccine and PfSPZ-CVac recipients during the 6 days after administration of investigational product. However, from days 7-13, PfSPZ-CVac recipients had significantly more AEs, probably because of Pf parasitemia. Antibody responses were 2.9 times higher in PfSPZ Vaccine recipients than PfSPZ-CVac recipients at time of CHMI. Vaccine efficacy at a median of 14 weeks after last PfSPZ-CVac dose was 55% (8 of 13, P = 0.051) and at a median of 15 weeks after last PfSPZ Vaccine dose was 27% (5 of 15, P = 0.32). The higher VE in PfSPZ-CVac recipients of 55% with a 27-fold lower dose was likely a result of later stage parasite maturation in the liver, leading to induction of cellular immunity against a greater quantity and broader array of antigens.

The Equatoguinean Malaria Vaccine Initiative: From the Launching of a Clinical Research Platform to Malaria Elimination Planning in Central West Africa.

Fifteen years of investment in malaria control on Bioko Island, Equatorial Guinea (EG), dramatically reduced malaria-associated morbidity and mortality, but the impact has plateaued. To progress toward elimination, EG is investing in the development of a malaria vaccine. We assessed the unique public-private partnership that has had such a significant impact on malaria on Bioko Island and now added a major effort on malaria vaccine development. As part of a $79M commitment, the EG government (75%) and three American energy companies (25%) have invested since 2012 greater than $55M in the Equatoguinean Malaria Vaccine Initiative (EGMVI) to support clinical development of Sanaria® PfSPZ vaccines (Sanaria Inc., Rockville, MD). In turn, the vaccine development program is building human capital and physical capacity. The EGMVI established regulatory and ethical oversight to ensure compliance with the International Conference on Harmonization and Good Clinical Practices for the first importation of investigational product, ethical approval, and conduct of a clinical trial in Equatoguinean history. The EGMVI has completed three vaccine trials in EG, two vaccine trials in Tanzania, and a malaria incidence study, and initiated preparations for a 2,100-volunteer clinical trial. Personnel are training for advanced degrees abroad and have been trained in Good Clinical Practices and protocol-specific methods. A new facility has established the foundation for a national research institute. Biomedical research and development within this visionary, ambitious public-private partnership is fostering major improvements in EG. The EGMVI plans to use a PfSPZ Vaccine alongside standard malaria control interventions to eliminate Pf malaria from Bioko, becoming a potential model for elimination campaigns elsewhere.

Human mobility patterns and malaria importation on Bioko Island.

Malaria burden on Bioko Island has decreased significantly over the past 15 years. The impact of interventions on malaria prevalence, however, has recently stalled. Here, we use data from island-wide, annual malaria indicator surveys to investigate human movement patterns and their relationship to Plasmodium falciparum prevalence. Using geostatistical and mathematical modelling, we find that off-island travel is more prevalent in and around the capital, Malabo. The odds of malaria infection among off-island travelers are significantly higher than the rest of the population. We estimate that malaria importation rates are high enough to explain malaria prevalence in much of Malabo and its surroundings, and that local transmission is highest along the West Coast of the island. Despite uncertainty, these estimates of residual transmission and importation serve as a basis for evaluating progress towards elimination and for efficiently allocating resources as Bioko makes the transition from control to elimination.