Surveillance of Plasmodium malariae infection among inhabitants of rural areas in Ouidah-Kpomasse-Tori Bossito health district, Benin.

Among the Plasmodium species that infect humans, P. falciparum has been largely studied in malaria endemic areas. However, P. malariae infection is less documented among the human population. This study aimed to monitor the prevalence and distribution of P. malariae in Southern Benin. A cross-sectional survey was conducted in rural localities in the Ouidah-Kpomasse-Tori Bossito (OKT) health district in Southern Benin from June to October 2019. Socio-demographic data were collected using a questionnaire, while malaria infection data were obtained on the one hand by microscopy diagnosis and, on the other, by nested polymerase chain reaction (PCR). Based on microscopy, the prevalence of P. malariae mono-infection and coinfection of P. falciparum, P. malariae was respectively 2.3% and 1.2% in the OKT health district. This prevalence was higher (P < 0.01) than that reported by Damien et al. (2010) 10 years ago in the same study area with 0.7% and 0.3% of P. malariae and P. falciparum/P. malariae, respectively. Based on PCR analysis, P. malariae prevalence was 14.1%, including 5.2% of mono-infection and 8.9% of mixed infection with P. falciparum. Sub-microscopic Plasmodium infections were high (30.6%) and more pronounced in older participants (>20 years). The present study revealed that P. malariae increased in the OKT health district with a high prevalence of submicroscopic infection. Since our results provide valuable evidence of increasing P. malariae infection, the National Malaria Control Programs (NMCPs) must consider P. malariae when designing future measures for effective control and malaria treatment.

Experimental Transmission of Plasmodium malariae to Anopheles gambiae.

Our current knowledge of the clinical burden, biology, and transmission of Plasmodium malariae is extremely scarce. To start addressing some of those questions, we experimentally infected Anopheles gambiae mosquitoes with fresh P. malariae isolates obtained from asymptomatic individuals in Lambaréné, Gabon. The proportion of mosquitoes infected via direct membrane feeding assay with either P. malariae monoinfections (16% [19 of 121]) or coinfections (28% [31 of 112]) was higher after serum replacement than in parallel groups without serum replacement (4% [4 of 102] and 4% [2 of 45], respectively; P < .01). Our results show that isolates from asymptomatic carriers can be used for experimental studies of P. malariae transmission.

First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria.

BACKGROUND: Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual "blood-stage" parasites in the placenta, the major virulence mechanism. METHODS: The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 µg (n = 9) or 50 µg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization. RESULTS: All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing P. falciparum-infected erythrocytes to CSA in a standardized functional assay. CONCLUSIONS: PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area. CLINICAL TRIALS REGISTRATION: EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489.

Persistent Plasmodium falciparum Infection in Women With an Intent to Become Pregnant as a Risk Factor for Pregnancy-associated Malaria.

Background: Pregnant women are more susceptible to Plasmodium falciparum than before pregnancy, and infection has consequences for both mother and offspring. The World Health Organization recommends that pregnant woman in areas of transmission receive intermittent preventive treatment (IPTp) starting in the second trimester. Consequently, women are not protected during the first trimester, although P. falciparum infections are both frequent and harmful. Methods: A cohort of nulligravid women was followed up during subsequent pregnancy. Malaria was diagnosed by means of microscopy and polymerase chain reaction. Parasites were genotyped at polymorphic loci. Results: Among 275 nulligravidae enrolled, 68 women became pregnant and were followed up during pregnancy. Before pregnancy, P. falciparum prevalence rates were 15% by microscopy and 66% by polymerase chain reaction. Microscopic infection rates increased to 29% until IPTp administration, and their density increased by 20-fold. Conversely, submicroscopic infection rates decreased. After IPTp administration, all types of infections decreased, but they increased again late in pregnancy. The risk of infection during pregnancy was higher in women with a microscopic (odds ratio, 6.5; P = .047) or submicroscopic (3.06; P = .05) infection before pregnancy and was not related to the season of occurrence. Most infections during pregnancy were persistent infections acquired before pregnancy. Conclusions: Microscopic and submicroscopic malaria infections were frequent in nulligravid women from south Benin. During the first trimester of pregnancy, microscopic infections were more frequent, with a higher parasite density, and mainly derived from parasites infecting the woman before conception. Preventive strategies targeting nonpregnant women with a desire for conception need to be designed.

A randomised, double-blind clinical phase II trial of the efficacy, safety, tolerability and pharmacokinetics of a single dose combination treatment with artefenomel and piperaquine in adults and children with uncomplicated Plasmodium falciparum malaria.

BACKGROUND: The clinical development of a single encounter treatment for uncomplicated malaria has the potential to significantly improve the effectiveness of antimalarials. Exploratory data suggested that the combination of artefenomel and piperaquine phosphate (PQP) has the potential to achieve satisfactory cure rates as a single dose therapy. The primary objective of the study was to determine whether a single dose of artefenomel (800 mg) plus PQP in ascending doses is an efficacious treatment for uncomplicated Plasmodium falciparum malaria in the 'target' population of children ≤ 5 years of age in Africa as well as Asian patients of all ages. METHODS: Patients in six African countries and in Vietnam were randomised to treatment with follow-up for 42-63 days. Efficacy, tolerability, safety and pharmacokinetics were assessed. Additional key objectives were to characterise the exposure-response relationship for polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response at day 28 post-dose (ACPR28) and to further investigate Kelch13 mutations. Patients in Africa (n = 355) and Vietnam (n = 82) were included, with 85% of the total population being children < 5 years of age. RESULTS: ACPR28 in the per protocol population (95% confidence interval) was 70.8% (61.13-79.19), 68.4% (59.13-76.66) and 78.6% (70.09-85.67) for doses of 800 mg artefenomel with 640 mg, 960 mg and 1440 mg of PQP respectively. ACPR28 was lower in Vietnamese than in African patients (66.2%; 54.55-76.62 and 74.5%; 68.81-79.68) respectively. Within the African population, efficacy was lowest in the youngest age group of ≥ 0.5 to ≤ 2 years, 52.7% (38.80-66.35). Initial parasite clearance was twice as long in Vietnam than in Africa. Within Vietnam, the frequency of the Kelch13 mutation was 70.1% and was clearly associated with parasite clearance half-life (PCt1/2). The most significant tolerability finding was vomiting (28.8%). CONCLUSIONS: In this first clinical trial evaluating a single encounter antimalarial therapy, none of the treatment arms reached the target efficacy of > 95% PCR-adjusted ACPR at day 28. Achieving very high efficacy following single dose treatment is challenging, since > 95% of the population must have sufficient concentrations to achieve cure across a range of parasite sensitivities and baseline parasitaemia levels. While challenging, the development of tools suitable for deployment as single encounter curative treatments for adults and children in Africa and to support elimination strategies remains a key development goal. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02083380 . Registered on 7 March 2014.

Recognition of Plasmodium falciparum mature gametocyte-infected erythrocytes by antibodies of semi-immune adults and malaria-exposed children from Gabon.

BACKGROUND: Transmission of malaria from man to mosquito depends on the presence of gametocytes, the sexual stage of Plasmodium parasites in the infected host. Naturally acquired antibodies against gametocytes exist and may play a role in controlling transmission by limiting the gametocyte development in the circulation or by interrupting gamete development and fertilization in the mosquito following ingestion. So far, most studies on antibody responses to sexual stage antigens have focused on a subset of gametocyte-surface antigens, even though inhibitory Ab responses to other gametocyte antigens might also play a role in controlling gametocyte density and fertility. Limited information is available on natural antibody response to the surfaces of gametocyte-infected erythrocytes. METHODS: Ab responses to surface antigens of erythrocytes infected by in vitro differentiated Plasmodium falciparum mature gametocytes were investigated in sera of semi-immune adults and malaria-exposed children. In addition, the effect of immunization with GMZ2, a blood stage malaria vaccine candidate, and the effect of intestinal helminth infection on the development of immunity to gametocytes of P. falciparum was evaluated in malaria-exposed children and adults from Gabon. Serum samples from two Phase I clinical trials conducted in Gabon were analysed by microscopic and flow-cytometric immunofluorescence assay. RESULTS: Adults had a higher Ab response compared to children. Ab reactivity was significantly higher after fixation and permeabilization of parasitized erythrocytes. Following vaccination with the malaria vaccine candidate GMZ2, anti-gametocyte Ab concentration decreased in adults compared to baseline. Ab response to whole asexual stage antigens had a significant but weak positive correlation to anti-gametocyte Ab responses in adults, but not in children. Children infected with Ascaris lumbricoides had a significantly higher anti-gametocyte Ab response compared to non-infected children. CONCLUSION: The current data suggest that antigens exposed on the gametocyte-infected red blood cells are recognized by serum antibodies from malaria-exposed children and semi-immune adults. This anti-gametocyte immune response may be influenced by natural exposure and vaccination. Modulation of the natural immune response to gametocytes by co-infecting parasites should be investigated further and may have an important impact on malaria control strategies.

Intramuscular Artesunate for Severe Malaria in African Children: A Multicenter Randomized Controlled Trial.

BACKGROUND: Current artesunate (ARS) regimens for severe malaria are complex. Once daily intramuscular (i.m.) injection for 3 d would be simpler and more appropriate for remote health facilities than the current WHO-recommended regimen of five intravenous (i.v.) or i.m. injections over 4 d. We compared both a three-dose i.m. and a three-dose i.v. parenteral ARS regimen with the standard five-dose regimen using a non-inferiority design (with non-inferiority margins of 10%). METHODS AND FINDINGS: This randomized controlled trial included children (0.5-10 y) with severe malaria at seven sites in five African countries to assess whether the efficacy of simplified three-dose regimens is non-inferior to a five-dose regimen. We randomly allocated 1,047 children to receive a total dose of 12 mg/kg ARS as either a control regimen of five i.m. injections of 2.4 mg/kg (at 0, 12, 24, 48, and 72 h) (n = 348) or three injections of 4 mg/kg (at 0, 24, and 48 h) either i.m. (n = 348) or i.v. (n = 351), both of which were the intervention arms. The primary endpoint was the proportion of children with ≥ 99% reduction in parasitemia at 24 h from admission values, measured by microscopists who were blinded to the group allocations. Primary analysis was performed on the per-protocol population, which was 96% of the intention-to-treat population. Secondary analyses included an analysis of host and parasite genotypes as risks for prolongation of parasite clearance kinetics, measured every 6 h, and a Kaplan-Meier analysis to compare parasite clearance kinetics between treatment groups. A post hoc analysis was performed for delayed anemia, defined as hemoglobin ≤ 7 g/dl 7 d or more after admission. The per-protocol population was 1,002 children (five-dose i.m.: n = 331; three-dose i.m.: n = 338; three-dose i.v.: n = 333); 139 participants were lost to follow-up. In the three-dose i.m. arm, 265/338 (78%) children had a ≥ 99% reduction in parasitemia at 24 h compared to 263/331 (79%) receiving the five-dose i.m. regimen, showing non-inferiority of the simplified three-dose regimen to the conventional five-dose regimen (95% CI -7, 5; p = 0.02). In the three-dose i.v. arm, 246/333 (74%) children had ≥ 99% reduction in parasitemia at 24 h; hence, non-inferiority of this regimen to the five-dose control regimen was not shown (95% CI -12, 1; p = 0.24). Delayed parasite clearance was associated with the N86YPfmdr1 genotype. In a post hoc analysis, 192/885 (22%) children developed delayed anemia, an adverse event associated with increased leukocyte counts. There was no observed difference in delayed anemia between treatment arms. A potential limitation of the study is its open-label design, although the primary outcome measures were assessed in a blinded manner. CONCLUSIONS: A simplified three-dose i.m. regimen for severe malaria in African children is non-inferior to the more complex WHO-recommended regimen. Parenteral ARS is associated with a risk of delayed anemia in African children. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201102000277177.

A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants.

BACKGROUND: The candidate malaria vaccine RTS,S/AS01 reduced episodes of both clinical and severe malaria in children 5 to 17 months of age by approximately 50% in an ongoing phase 3 trial. We studied infants 6 to 12 weeks of age recruited for the same trial. METHODS: We administered RTS,S/AS01 or a comparator vaccine to 6537 infants who were 6 to 12 weeks of age at the time of the first vaccination in conjunction with Expanded Program on Immunization (EPI) vaccines in a three-dose monthly schedule. Vaccine efficacy against the first or only episode of clinical malaria during the 12 months after vaccination, a coprimary end point, was analyzed with the use of Cox regression. Vaccine efficacy against all malaria episodes, vaccine efficacy against severe malaria, safety, and immunogenicity were also assessed. RESULTS: The incidence of the first or only episode of clinical malaria in the intention-to-treat population during the 14 months after the first dose of vaccine was 0.31 per person-year in the RTS,S/AS01 group and 0.40 per person-year in the control group, for a vaccine efficacy of 30.1% (95% confidence interval [CI], 23.6 to 36.1). Vaccine efficacy in the per-protocol population was 31.3% (97.5% CI, 23.6 to 38.3). Vaccine efficacy against severe malaria was 26.0% (95% CI, -7.4 to 48.6) in the intention-to-treat population and 36.6% (95% CI, 4.6 to 57.7) in the per-protocol population. Serious adverse events occurred with a similar frequency in the two study groups. One month after administration of the third dose of RTS,S/AS01, 99.7% of children were positive for anti-circumsporozoite antibodies, with a geometric mean titer of 209 EU per milliliter (95% CI, 197 to 222). CONCLUSIONS: The RTS,S/AS01 vaccine coadministered with EPI vaccines provided modest protection against both clinical and severe malaria in young infants. (Funded by GlaxoSmithKline Biologicals and the PATH Malaria Vaccine Initiative; RTS,S ClinicalTrials.gov number, NCT00866619.).

Severe malaria in children leads to a significant impairment of transitory otoacoustic emissions--a prospective multicenter cohort study.

BACKGROUND: Severe malaria may influence inner ear function, although this possibility has not been examined prospectively. In a retrospective analysis, hearing impairment was found in 9 of 23 patients with cerebral malaria. An objective method to quickly evaluate the function of the inner ear are the otoacoustic emissions. Negative transient otoacoustic emissions are associated with a threshold shift of 20 dB and above. METHODS: This prospective multicenter study analyses otoacoustic emissions in patients with severe malaria up to the age of 10 years. In three study sites (Ghana, Gabon, Kenya) 144 patients with severe malaria and 108 control children were included. All malaria patients were treated with parental artesunate. RESULTS: In the control group, 92.6 % (n = 108, 95 % confidence interval 86.19-6.2 %) passed otoacoustic emission screening. In malaria patients, 58.5 % (n = 94, malaria vs controls p < 0.001, 95 % confidence interval 48.4-67.9 %) passed otoacoustic emission screening at the baseline measurement. The value increased to 65.2 % (n = 66, p < 0.001, 95 % confidence interval 53.1-75.5 %) at follow up 14-28 days after diagnosis of malaria. The study population was divided into severe non-cerebral malaria and severe malaria with neurological symptoms (cerebral malaria). Whereas otoacoustic emissions in severe malaria improved to a passing percentage of 72.9 % (n = 48, 95 % confidence interval 59-83.4 %) at follow-up, the patients with cerebral malaria showed a drop in the passing percentage to 33 % (n = 18) 3-7 days after diagnosis. This shows a significant impairment in the cerebral malaria group (p = 0.012 at days 3-7, 95 % confidence interval 16.3-56.3 %; p = 0.031 at day 14-28, 95 % confidence interval 24.5-66.3 %). CONCLUSION: The presented data show that 40 % of children have involvement of the inner ear early in severe malaria. In children, audiological screening after severe malaria infection is not currently recommended, but is worth investigating in larger studies.

Delayed hemolysis after treatment with parenteral artesunate in African children with severe malaria--a double-center prospective study.

BACKGROUND: Parenteral artesunate is recommended as first-line therapy for severe malaria. While its efficacy is firmly established, data on safety are still incomplete. Delayed hemolysis has been described in hyperparasitemic nonimmune travelers, but it is unknown if African children are equally at risk. METHODS: Children aged 6 to 120 months with severe malaria were followed up after treatment with parenteral artesunate in Lambaréné, Gabon, and Kumasi, Ghana. The primary outcome was incidence of delayed hemolysis on day 14. RESULTS: In total, 72 children contributed complete data sets necessary for primary outcome assessment. Delayed hemolysis was detected in 5 children (7%), with 1 child reaching a nadir in hemoglobin of 2.8 g/dL. Patients with delayed hemolysis had higher parasite counts on admission (geometric mean parasite densities (GMPD) 306 968/µL vs 92 642/µL, P = .028) and were younger (median age: 24 months vs 43 months, P = .046) than the rest of the cohort. No correlation with sickle cell trait or glucose-6-phosphate-dehydrogenase deficiency was observed. CONCLUSIONS: Delayed hemolysis is a frequent and relevant complication in hyperparasitemic African children treated with parenteral artesunate for severe malaria. Physicians should be aware of this complication and consider prolonged follow-up. CLINICAL TRIALS REGISTRATION: Pan-African Clinical Trials Registry: PACTR201102000277177 (www.pactr.org).

Randomized, controlled, assessor-blind clinical trial to assess the efficacy of single- versus repeated-dose albendazole to treat ascaris lumbricoides, trichuris trichiura, and hookworm infection.

In many regions where soil-transmitted helminth infections are endemic, single-dose albendazole is used in mass drug administration programs to control infections. There are little data on the efficacy of the standard single-dose administration compared to that of alternative regimens. We conducted a randomized, controlled, assessor-blinded clinical trial to determine the efficacies of standard and extended albendazole treatment against soil-transmitted helminth infection in Gabon. A total of 175 children were included. Adequate cure rates and egg reduction rates above 85% were found with a single dose of albendazole for Ascaris infection, 85% (95% confidence interval [CI], 73, 96) and 93.8% (CI, 87.6, 100), respectively, while two doses were necessary for hookworm infestation (92% [CI, 78, 100] and 92% [CI, 78, 100], respectively). However, while a 3-day regimen was not sufficient to cure Trichuris (cure rate, 83% [CI, 73, 93]), this regimen reduced the number of eggs up to 90.6% (CI, 83.1, 100). The rate ratios of two- and three-dose regimens compared to a single-dose treatment were 1.7 (CI, 1.1, 2.5) and 2.1 (CI, 1.5, 2.9) for Trichuris and 1.7 (CI, 1.0, 2.9) and 1.7 (CI, 1.0, 2.9) for hookworm. Albendazole was safe and well tolerated in all regimens. A single-dose albendazole treatment considerably reduces Ascaris infection but has only a moderate effect on hookworm and Trichuris infections. The single-dose option may still be the preferred regimen because it balances efficacy, safety, and compliance during mass drug administration, keeping in mind that asymptomatic low-level helminth carriage may also have beneficial effects. (This study has been registered at ClinicalTrials.gov under registration number NCT01192802.).

First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children.

BACKGROUND: An ongoing phase 3 study of the efficacy, safety, and immunogenicity of candidate malaria vaccine RTS,S/AS01 is being conducted in seven African countries. METHODS: From March 2009 through January 2011, we enrolled 15,460 children in two age categories--6 to 12 weeks of age and 5 to 17 months of age--for vaccination with either RTS,S/AS01 or a non-malaria comparator vaccine. The primary end point of the analysis was vaccine efficacy against clinical malaria during the 12 months after vaccination in the first 6000 children 5 to 17 months of age at enrollment who received all three doses of vaccine according to protocol. After 250 children had an episode of severe malaria, we evaluated vaccine efficacy against severe malaria in both age categories. RESULTS: In the 14 months after the first dose of vaccine, the incidence of first episodes of clinical malaria in the first 6000 children in the older age category was 0.32 episodes per person-year in the RTS,S/AS01 group and 0.55 episodes per person-year in the control group, for an efficacy of 50.4% (95% confidence interval [CI], 45.8 to 54.6) in the intention-to-treat population and 55.8% (97.5% CI, 50.6 to 60.4) in the per-protocol population. Vaccine efficacy against severe malaria was 45.1% (95% CI, 23.8 to 60.5) in the intention-to-treat population and 47.3% (95% CI, 22.4 to 64.2) in the per-protocol population. Vaccine efficacy against severe malaria in the combined age categories was 34.8% (95% CI, 16.2 to 49.2) in the per-protocol population during an average follow-up of 11 months. Serious adverse events occurred with a similar frequency in the two study groups. Among children in the older age category, the rate of generalized convulsive seizures after RTS,S/AS01 vaccination was 1.04 per 1000 doses (95% CI, 0.62 to 1.64). CONCLUSIONS: The RTS,S/AS01 vaccine provided protection against both clinical and severe malaria in African children. (Funded by GlaxoSmithKline Biologicals and the PATH Malaria Vaccine Initiative; RTS,S ClinicalTrials.gov number, NCT00866619 .).

The risk to import ESBL-producing Enterobacteriaceae and Staphylococcus aureus through chicken meat trade in Gabon.

BACKGROUND: A main export market for chicken meat from industrialized countries is sub-Saharan Africa. We hypothesized that antibiotic resistant bacteria could be exported to developing countries through chicken meat trade. The objective was to investigate the occurrence and molecular types of ESBL-producing Enterobacteriaceae and Staphylococcus aureus in chicken meat in Gabon and to assess their dissemination among humans. RESULTS: Frozen chicken meat samples imported from industrialized countries to Gabon (n = 151) were screened for ESBL-producing Enterobacteriaceae and S. aureus. Genotypes and resistance genes (SHV, TEM, CTX-M, CMY-2) of isolates from meat were compared with isolates derived from humans. The contamination rate per chicken part (i. e. leg, wing) with ESBL-producing Escherichia coli (ESBL E. coli, no other ESBL-producing Enterobacteriaceae were found) and S. aureus was 23% and 3%, respectively. The beta-lactamase CTX-M 1 was predominant in ESBL E. coli from meat samples but was not found in isolates from cases of human colonization or infection. S. aureus belonging to spa type t002 (multilocus sequence type ST5) were found both in chicken meat and humans. CONCLUSION: There is a risk to import ESBL E. coli to Gabon but molecular differences between isolates from humans and chicken meat argue against a further dissemination. No MRSA isolate was detected in imported chicken meat.

The malaria vaccine candidate GMZ2 elicits functional antibodies in individuals from malaria endemic and non-endemic areas.

BACKGROUND: GMZ2 is a hybrid protein consisting of the N-terminal region of the glutamate-rich protein fused in frame to the C-terminal region of merozoite surface protein 3 (MSP3). GMZ2 formulated in Al(OH)3 has been tested in 3 published phase 1 clinical trials. The GMZ2/alum formulation showed good safety, tolerability, and immunogenicity, but whether antibodies elicited by vaccination are functional is not known. METHODS: Serum samples prior to vaccination and 4 weeks after the last vaccination from the 3 clinical trials were used to perform a comparative assessment of biological activity against Plasmodium falciparum. RESULTS: We showed that the maximum level of immunoglobulin G (IgG) antibodies obtained by GMZ2 vaccination is independent of ethnicity, time under malaria-exposure, and vaccine dose and that GMZ2 elicits high levels of functionally active IgG antibodies. Both, malaria-naive adults and malaria-exposed preschool children elicit vaccine-specific antibodies with broad inhibitory activity against geographically diverse P. falciparum isolates. Peptide-mapping studies of IgG subclass responses identified IgG3 against a peptide derived from MSP3 as the strongest predictor of antibody-dependent cellular inhibition. CONCLUSIONS: These findings suggest that GMZ2 adjuvanted in Al(OH)3 elicits high levels of specific and functional antibodies with the capacity to control parasite multiplication.

Retrospective analysis of antimicrobial resistance and bacterial spectrum of infection in Gabon, Central Africa.

BACKGROUND: Physicians depend on reliable information on the local epidemiology of infection and antibiotic resistance rates to guide empiric treatment in critically ill patients. As these data are scarce for Central Africa, we performed a retrospective analysis of microbiological findings from a secondary care hospital in Gabon. METHODS: Microbiological reports from 2009 to 2012 were used to assess the non-susceptibility rates of the three most common isolates from six major types of infections (bloodstream, ear-eye-nose-throat, surgical site, skin and soft tissue, urinary tract and wound infection). RESULTS: A high diversity of pathogens was found, but Staphylococcus aureus was predominant in the majority of infections. Overall, the three most prevalent pathogens in children were S. aureus (33.7%), Streptococcus pyogenes (8.1%) and Escherichia coli (4.5%) and in adults S. aureus (23.5%), E. coli (15.1%) and Klebsiella pneumoniae (7.4%). In total, 5.8% (n = 19) of all S. aureus isolates were methicillin resistant. The proportion of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae was 15.4% (n = 78), 49.4% of all K. pneumoniae were ESBL-producer (n = 42). CONCLUSION: The high diversity of potential pathogens and high resistance rates in Gram-negative bacteria challenge a rational empiric use of antibiotics. Countrywide continuous sentinel surveillance is therefore urgently needed.

A simplified intravenous artesunate regimen for severe malaria.

BACKGROUND: We compared a conventional empirically derived regimen with a simplified regimen for parenteral artesunate in severe malaria. METHODS: This was a randomized, double-blind, placebo-controlled comparison to assess the noninferiority of a simplified 3-dose regimen (given at 0, 24, and 48 hours) compared with the conventional 5-dose regimen of intravenous artesunate (given at 0, 12, 24, 48, and 72 hours) in African children with Plasmodium falciparum malaria with a prespecified delta of 0.2. The total dose of artesunate in each group was 12 mg/kg. The primary end point was the proportion of children clearing ≥ 99% of their admission parasitemia at 24 hours. Safety data, secondary efficacy end points, and pharmacokinetics were also analyzed. RESULTS: In 171 children (per protocol), 78% of the recipients (95% confidence interval [CI], 69%-87%) in the 3-dose group achieved ≥ 99% parasite clearance 24 hours after the start of treatment, compared with 85% (95% CI, 77%-93%) of those receiving the conventional regimen (treatment difference, -7.2%; 95% CI, -18.9% to 4.4%). Dihydroartemisinin was cleared slightly more slowly in those children receiving the higher 3-dose regimen (7.4 vs 8.8 L/h for a 13-kg child; P 5 .008). CONCLUSIONS: Pharmacodynamic analysis suggests that 3 doses of artesunate were not inferior to 5 doses for the treatment of severe malaria in children. CLINICAL TRIALS REGISTRATION: NCT00522132.

Pharmacokinetics of ferroquine, a novel 4-aminoquinoline, in asymptomatic carriers of Plasmodium falciparum infections.

Ferroquine (SSR97193), a ferrocene-quinoline conjugate, is a promising novel antimalarial currently undergoing clinical evaluation. This study characterizes its pharmacokinetic properties. Young male African volunteers with asymptomatic Plasmodium falciparum infection were administered a single oral dose (n = 40) or a repeated oral dose (n = 26) given over 3 days of ferroquine in two dose-escalation, double-blind, randomized, placebo-controlled clinical trials. In addition, a food interaction study was performed in a subsample of participants (n = 16). The studies were carried out in Lambaréné, Gabon. After single-dose administration of ferroquine, dose linearity was demonstrated in a dose range of 400 to 1,200 mg for maximum mean blood concentrations ([C(max)] 82 to 270 ng/ml) and in a dose range of 400 to 1,600 mg for overall exposure to ferroquine (area under the concentration-time curve [AUC], 13,100 to 49,200 ng · h/ml). Overall mean estimate for blood apparent terminal half-life of ferroquine was 16 days and 31 days for its active and major metabolite desmethylferroquine (SSR97213). In the 3-day repeated-dose study, C(max) and overall cumulated exposure to ferroquine (AUC(cum)) increased in proportion with the dose from day 1 to day 3 between 400 and 800 mg. No major food effect on ferroquine pharmacokinetics was observed after single administration of 100 mg of ferroquine except for a slight delay of time to maximum blood concentration (t(max)) by approximately 3 h. The pharmacokinetics of ferroquine and its active main metabolite are characterized by sustained levels in blood, and the properties of ferroquine as a partner drug in antimalarial combination therapy should be evaluated.

A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals.

BACKGROUND: Antibodies play a central role in naturally acquired immunity against Plasmodium falciparum. Current assays to detect anti-plasmodial antibodies against native antigens within their cellular context are prone to bias and cannot be automated, although they provide important information about natural exposure and vaccine immunogenicity. A novel, cytometry-based workflow for quantitative detection of anti-plasmodial antibodies in human serum is presented. METHODS: Fixed red blood cells (RBCs), infected with late stages of P. falciparum were utilized to detect malaria-specific antibodies by flow cytometry with subsequent automated data analysis. Available methods for data-driven analysis of cytometry data were assessed and a new overlap subtraction algorithm (OSA) based on open source software was developed. The complete workflow was evaluated using sera from two GMZ2 malaria vaccine trials in semi-immune adults and pre-school children residing in a malaria endemic area. RESULTS: Fixation, permeabilization, and staining of infected RBCs were adapted for best operation in flow cytometry. As asexual blood-stage vaccine candidates are designed to induce antibody patterns similar to those in semi-immune adults, serial dilutions of sera from heavily exposed individuals were compared to naïve controls to determine optimal antibody dilutions. To eliminate investigator effects introduced by manual gating, a non-biased algorithm (OSA) for data-driven gating was developed. OSA-derived results correlated well with those obtained by manual gating (r between 0.79 and 0.99) and outperformed other model-driven gating methods. Bland-Altman plots confirmed the agreement of manual gating and OSA-derived results. A 1.33-fold increase (p=0.003) in the number of positive cells after vaccination in a subgroup of pre-school children vaccinated with 100 µg GMZ2 was present and in vaccinated adults from the same region we measured a baseline-corrected 1.23-fold, vaccine-induced increase in mean fluorescence intensity of positive cells (p=0.03). CONCLUSIONS: The current workflow advances detection and quantification of anti-plasmodial antibodies through improvement of a bias-prone, low-throughput to an unbiased, semi-automated, scalable method. In conclusion, this work presents a novel method for immunofluorescence assays in malaria research.

Phase I randomized dose-ascending placebo-controlled trials of ferroquine--a candidate anti-malarial drug--in adults with asymptomatic Plasmodium falciparum infection.

BACKGROUND: The development and spread of drug resistant Plasmodium falciparum strains is a major concern and novel anti-malarial drugs are, therefore, needed. Ferroquine is a ferrocenic derivative of chloroquine with proven anti-malarial activity against chloroquine-resistant and -sensitive P. falciparum laboratory strains. METHODS: Adult young male aged 18 to 45 years, asymptomatic carriers of P. falciparum, were included in two-dose escalation, double-blind, randomized, placebo-controlled Phase I trials, a single dose study and a multiple dose study aiming to evaluate oral doses of ferroquine from 400 to 1,600 mg. RESULTS: Overall, 54/66 patients (40 and 26 treated in the single and multiple dose studies, respectively) experienced at least one adverse event, 15 were under placebo. Adverse events were mainly gastrointestinal symptoms such as abdominal pain (16), diarrhoea (5), nausea (13), and vomiting (9), but also headache (11), and dizziness (5). A few patients had slightly elevated liver parameters (10/66) including two patients under placebo. Moderate changes in QTc and morphological changes in T waves were observed in the course of the study. However, no adverse cardiac effects with clinical relevance were observed. CONCLUSIONS: These phase I trials showed that clinically, ferroquine was generally well-tolerated up to 1,600 mg as single dose and up to 800 mg as repeated dose in asymptomatic young male with P. falciparum infection. Further clinical development of ferroquine, either alone or in combination with another anti-malarial, is highly warranted and currently underway.