Abstract.Equatorial Guinea (EG) has implemented a successful malaria control program on Bioko Island. A highly effective vaccine would be an ideal complement to this effort and could lead to halting transmission and eliminating malaria. Sanaria® PfSPZ Vaccine (Plasmodium falciparum sporozoite Vaccine) is being developed for this purpose. To begin the process of establishing the efficacy of and implementing a PfSPZ Vaccine mass vaccination program in EG, we decided to conduct a series of clinical trials of PfSPZ Vaccine on Bioko Island. Because no clinical trial had ever been conducted in EG, we first successfully established the ethical, regulatory, quality, and clinical foundation for conducting trials. We now report the safety, tolerability, and immunogenicity results of the first clinical trial in the history of the country. Thirty adult males were randomized in the ratio 2:1 to receive three doses of 2.7 × 105 PfSPZ of PfSPZ Vaccine (N = 20) or normal saline placebo (N = 10) by direct venous inoculation at 8-week intervals. The vaccine was safe and well tolerated. Seventy percent, 65%, and 45% of vaccinees developed antibodies to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) by enzyme-linked immunosorbent assay, PfSPZ by automated immunofluorescence assay, and PfSPZ by inhibition of sporozoite invasion assay, respectively. Antibody responses were significantly lower than responses in U.S. adults who received the same dosage regimen, but not significantly different than responses in young adult Malians. Based on these results, a clinical trial enrolling 135 subjects aged 6 months to 65 years has been initiated in EG; it includes PfSPZ Vaccine and first assessment in Africa of PfSPZ-CVac. ClinicalTrials.gov identifier: NCT02418962.

Abstract. From 1992-1999, we have assessed the therapeutic efficacy of three malaria treatment regimens (chloroquine 25 mg/kg over three days, pyrimethamine/sulfadoxine 1.25/25 mg/kg in one dose, and quinine 25-30 mg/kg daily in three oral doses over a four-, five-, or seven-day period) in 1,189 children under age 10 at Malabo Regional Hospital in Equatorial Guinea. Of those children, 958 were followed up clinically and parasitologically for 14 days. With chloroquine, the failure rate varied from 55% in 1996 to 40% in 1999; the early treatment failure rate increased progressively over the years, from 6% in 1992 to 30% in 1999. With pyrimethamine/sulfadoxine, the failure rate varied from 0% in 1996 to 16% in 1995. The short quinine treatment regimens used in 1992 and 1993 (4 and 5 days, respectively) resulted in significantly higher failure rates (19% and 22%, respectively) than the 7d regimen (3-5.5%). We conclude that: a) failure rates for chloroquine are in the change period (>25%), and urgent action is needed; b) pyrimethamine/ sulfadoxine failure rates are in the alert period (6-15%), and surveillance must be continued; and c) quinine failure rates are in the grace period (<6%), so quinine can be recommended.

Background Progress towards malaria elimination has stagnated, partly because infections persisting at low parasite densities comprise a large reservoir contributing to ongoing malaria transmission and are difficult to detect. This study compared the performance of an ultrasensitive rapid diagnostic test (uRDT) designed to detect low density infections to a conventional RDT (cRDT), expert microscopy using Giemsa-stained thick blood smears (TBS), and quantitative polymerase chain reaction (qPCR) during a controlled human malaria infection (CHMI) study conducted in malaria exposed adults (NCT03590340). Methods Blood samples were collected from healthy Equatoguineans aged 18–35 years beginning on day 8 after CHMI with 3.2 × 103 cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ Challenge, strain NF54) administered by direct venous inoculation. qPCR (18s ribosomal DNA), uRDT (Alere™ Malaria Ag P.f.), cRDT [Carestart Malaria Pf/PAN (PfHRP2/pLDH)], and TBS were performed daily until the volunteer became TBS positive and treatment was administered. qPCR was the reference for the presence of Plasmodium falciparum parasites. Results 279 samples were collected from 24 participants; 123 were positive by qPCR. TBS detected 24/123 (19.5% sensitivity [95% CI 13.1–27.8%]), uRDT 21/123 (17.1% sensitivity [95% CI 11.1–25.1%]), cRDT 10/123 (8.1% sensitivity [95% CI 4.2–14.8%]); all were 100% specific and did not detect any positive samples not detected by qPCR. TBS and uRDT were more sensitive than cRDT (TBS vs. cRDT p = 0.015; uRDT vs. cRDT p = 0.053), detecting parasitaemias as low as 3.7 parasites/µL (p/µL) (TBS and uRDT) compared to 5.6 p/µL (cRDT) based on TBS density measurements. TBS, uRDT and cRDT did not detect any of the 70/123 samples positive by qPCR below 5.86 p/µL, the qPCR density corresponding to 3.7 p/µL by TBS. The median prepatent periods in days (ranges) were 14.5 (10–20), 18.0 (15–28), 18.0 (15–20) and 18.0 (16–24) for qPCR, TBS, uRDT and cRDT, respectively; qPCR detected parasitaemia significantly earlier (3.5 days) than the other tests. Conclusions TBS and uRDT had similar sensitivities, both were more sensitive than cRDT, and neither matched qPCR for detecting low density parasitaemia. uRDT could be considered an alternative to TBS in selected applications, such as CHMI or field diagnosis, where qualitative, dichotomous results for malaria infection might be sufficient.

The objectives of this study were: 1) to evaluate the safety and efficacy of combination artesunate (AS)/amodiaquine (AQ) therapy, and 2) to determine the difference between recrudescence and resistance. An in vivo efficacy study was conducted in Equatorial Guinea. A total of 122 children 6–59 months of age from two regional hospitals were randomized and subjected to a 28-day clinical and parasitological follow-up. A blood sample on Whatman paper was taken on Days 0, 7, 14, 21, and 28 or on any day in cases of treatment failure, with the parasite DNA then being extracted for molecular analysis purposes. A total of 4 children were excluded, and 9 cases were lost to follow-up. There were 17 cases of late parasitological failure, 3 cases of late clinical failure, and 89 cases of adequate clinical and parasitological response. The parasitological failure rate was 18.3% (20 of 109) and the success rate 81.70% (95% confidence interval [72.5–87.9%]). After molecular correction, real treatment efficacy stood at 97.3%. Our study showed the good efficacy of combination AS/AQ therapy. This finding enabled this treatment to be recommended to Equatorial Guinea's National Malaria Control Program to change the official treatment policy as of March 2008.

Objectives. The objectives of the study were (i) to evaluate the efficacy of combination drugs, such as artesunate + sulphadoxine-pyrimethamine (AS + SP) and amodiaquine + sulphadoxine-pyripethamine (AQ + SP) in treatment of uncomplicated falciparum malaria (ii) to differentiate recrudescence from reinfection by analysing msp-1 and msp-2 genes of Plasmodium falciparum in treatment failure cases. Methods. We carried out an in vivo study in the year 2005 in 206 children between 6 to 59 months age groups. Of the 206, 120 received AQ + SP, and 86 received AS + SP. A clinical and parasitological followup during 14 days was undertaken. Finger-prick blood sample from each patient was taken on Whatman filter paper (no. 3) on days 0, 7, 14 and also the day when the parasite and symptoms reappeared for PCR analysis. Results. Late treatment failure was observed in 3.5% (4/114) with AQ + SP, and 2.5% (2/79) with AS + SP. The success rate was 96.5% with AQ + SP and 97.5% with AS + SP. No deaths and severe reactions were recorded. Out of the 6 treatment failure cases, one was reinfection as observed by PCR analysis of msp-1 and msp-2 genes on day 14. Discussion. Both the combinations found to be efficacious and safe and could be used as a first-line treatment for uncomplicated falciparum malaria in Equatorial Guinea.

Conventional malaria diagnosis based on microscopy raises serious difficulties in weak health systems. Cost-effective and sensitive rapid diagnostic tests have been recently proposed as alternatives to microscopy. In Equatorial Guinea, a study was conducted to assess the reliability of a rapid diagnostic test compared to microscopy. The study was designed in accordance with the directives of the Standards for Reporting Diagnostic Accuracy Initiative (STARD). Peripheral thick and thin films for the microscopy diagnosis and a rapid immunochromatographic test (ICT Malaria Combo Cassette Test) were performed on under five-year-old children with malaria suspicion. The ICT test detected Plasmodium spp. infection with a sensitivity of 81.5% and a specificity of 81.9% while P. falciparum diagnosis occurred with a sensitivity of 69.7% and a specificity of 73.7%. The sensitivity of the ICT test increased with higher parasitemias. The general results showed little concordance between the ICT test and microscopy (kappa = 0.28, se: 0.04). In Equatorial Guinea, the ICT Malaria Combo Cassette Test has proven to be an acceptable test to detect high P. falciparum parasitemias. However, the decrease of sensitivity at medium and low parasitemias hampers that ICT can replace properly performed microscopy at present in the diagnosis of malaria in children.

Background Extensive malaria control measures have been implemented on Bioko Island, Equatorial Guinea over the past 16 years, reducing parasite prevalence and malaria-related morbidity and mortality, but without achieving elimination. Malaria vaccines offer hope for reducing the burden to zero. Three phase 1/2 studies have been conducted successfully on Bioko Island to evaluate the safety and efficacy of whole Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccines. A large, pivotal trial of the safety and efficacy of the radiation-attenuated Sanaria® PfSPZ Vaccine against P. falciparum is planned for 2022. This study assessed the incidence of malaria at the phase 3 study site and characterized the influence of socio-demographic factors on the burden of malaria to guide trial design. Methods A cohort of 240 randomly selected individuals aged 6 months to 45 years from selected areas of North Bioko Province, Bioko Island, was followed for 24 weeks after clearance of parasitaemia. Assessment of clinical presentation consistent with malaria and thick blood smears were performed every 2 weeks. Incidence of first and multiple malaria infections per person-time of follow-up was estimated, compared between age groups, and examined for associated socio-demographic risk factors. Results There were 58 malaria infection episodes observed during the follow up period, including 47 first and 11 repeat infections. The incidence of malaria was 0.25 [95% CI (0.19, 0.32)] and of first malaria was 0.23 [95% CI (0.17, 0.30)] per person per 24 weeks (0.22 in 6–59-month-olds, 0.26 in 5–17-year-olds, 0.20 in 18–45-year-olds). Incidence of first malaria with symptoms was 0.13 [95% CI (0.09, 0.19)] per person per 24 weeks (0.16 in 6–59-month-olds, 0.10 in 5–17-year-olds, 0.11 in 18–45-year-olds). Multivariate assessment showed that study area, gender, malaria positivity at screening, and household socioeconomic status independently predicted the observed incidence of malaria. Conclusion Despite intensive malaria control efforts on Bioko Island, local transmission remains and is spread evenly throughout age groups. These incidence rates indicate moderate malaria transmission which may be sufficient to support future larger trials of PfSPZ Vaccine. The long-term goal is to conduct mass vaccination programmes to halt transmission and eliminate P. falciparum malaria.

Background: Extensive malaria control measures have been implemented on Bioko Island, Equatorial Guinea over the past 16 years, reducing parasite prevalence and malaria-related morbidity and mortality but without achieving elimination. Malaria vaccines offer hope for reducing the burden to zero. Three Phase 1/2 studies have been conducted successfully on Bioko Island to evaluate the safety and efficacy of whole Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccines. A large, pivotal trial of the safety and efficacy of the radiation-attenuated Sanaria® PfSPZ Vaccine against Pf is planned for 2022. This study assessed the incidence of malaria at the Phase 3 study site and characterized the influence of socio-demographic factors on the burden of malaria to guide trial design. Methods: A cohort of 240 randomly selected individuals aged 6 months to 45 years from North Bioko Province, Bioko Island, was followed for 24 weeks after clearance of parasitemia. Assessment of clinical presentation consistent with malaria and thick blood smears were performed every two weeks. Incidence of first and multiple malaria infections per person-time of follow-up was estimated, compared between age groups, and examined for associated socio-demographic risk factors. Results: 58 malaria infection episodes (malaria) were observed during the follow up period, including 47 first and 11 repeat infections. The incidence of malaria was 0.25 [95% CI (0.19, 0.32)] and of first malaria was 0.23 [95% CI (0.17, 0.30)] per person per 24 weeks (0.22 in 6-59-month-olds, 0.26 in 5-17-year-olds, 0.20 in 18-45-year-olds). Incidence of first malaria with symptoms was 0.13 [95% CI (0.09, 0.19)] per person per 24 weeks (0.16 in 6-59-month-olds, 0.10 in 5-17-year-olds, 0.11 in 18-45-year-olds). Multivariate assessment showed that study area, gender, malaria positivity at screening, and household socioeconomic status independently predicted the observed incidence of malaria. Conclusion: Despite intensive malaria control efforts on Bioko Island, local transmission remains and is spread fairly evenly throughout age groups. These incidence rates are sufficient to support the planned future trial of PfSPZ Vaccine. The long-term goal is to conduct mass vaccination programs to halt transmission and eliminate Pf malaria.