Dynamics of Plasmodium species and genotype diversity in adults with asymptomatic infection in Gabon.

OBJECTIVES: We investigated the diversity and dynamics of Plasmodium infection in serially collected samples from asymptomatic participants of a clinical trial assessing the efficacy and safety of ivermectin in Gabon. We checked whether the baseline sample reflected the P. falciparum genotype and Plasmodium species diversity seen over 7 days of follow-up. METHODS: Blood samples were collected at inclusion, every 8 hours until hour 72, daily until day 7, and on day 14. Plasmodium species was determined by qPCR and pfmsp1 length polymorphism was assessed for P. falciparum genotyping. RESULTS: In 17/48 (35%) individuals, all pfmsp1 genotypes identified during the assessed period were detected at baseline; in 31/48 (65%), new genotypes were found during follow-up. Additional sampling at hour 24 allowed the identification of all genotypes seen over 7 days in 50% of the individuals. Ivermectin did not impact the genotype dynamics. Mixed Plasmodium spp. infections were detected in 28/49 (57%) individuals at baseline, and detection of non-falciparum infections during follow-up varied. CONCLUSIONS: Our results reveal complex intra-host dynamics of P. falciparum genotypes and Plasmodium species and underscore the importance of serial sampling in clinical trials for antimalarial drugs with asymptomatically P. falciparum-infected individuals. This might allow a more accurate identification of genotypes in multiple infections, impacting the assessment of drug efficacy.

Safety and immunogenicity of the co-administered Na-APR-1 and Na-GST-1 hookworm vaccines in school-aged children in Gabon: a randomised, controlled, observer-blind, phase 1, dose-escalation trial.

BACKGROUND: A human hookworm vaccine is being developed to protect children against iron deficiency and anaemia associated with chronic infection with hookworms. Necator americanus aspartic protease-1 (Na-APR-1) and N americanus glutathione S-transferase-1 (Na-GST-1) are components of the blood digestion pathway critical to hookworm survival in the host. Recombinant Na-GST-1 and catalytically inactive Na-APR-1 (Na-APR-1[M74]) adsorbed to Alhydrogel were safe and immunogenic when delivered separately or co-administered to adults in phase 1 trials in non-endemic and endemic areas. We aimed to investigate the safety and immunogenicity of these antigens in healthy children in a hookworm-endemic area. METHODS: This was a randomised, controlled, observer-blind, phase 1, dose-escalation trial, conducted in a clinical research centre, in 60 children aged six to ten years in Lambaréné, a hookworm-endemic region of Gabon. Healthy children (determined by clinical examination and safety laboratory testing) were randomised 4:1 to receive co-administered Na-GST-1 on Alhydrogel plus Na-APR-1(M74) on Alhydrogel and glucopyranosyl lipid A in aqueous formulation (GLA-AF), or co-administered ENGERIX-B hepatitis B vaccine (HBV) and saline placebo, injected into the deltoid of each arm. Allocation to vaccine groups was observer-masked. In each vaccine group, children were randomised 1:1 to receive intramuscular injections into each deltoid on two vaccine schedules, one at months 0, 2, and 4 or at months 0, 2, and 6. 10 µg, 30 µg, and 100 µg of each antigen were administered in the first, second, and third cohorts, respectively. The intention-to-treat population was used for safety analyses; while for immunogenicity analyses, the per-protocol population was used (children who received all scheduled vaccinations). The primary outcome was to evaluate the vaccines' safety and reactogenicity in healthy children aged between six and ten years. The secondary outcome was to measure antigen-specific serum IgG antibody levels at pre-vaccination and post-vaccination timepoints by qualified ELISAs. The trial is registered with ClinicalTrials.gov, NCT02839161, and is completed. FINDINGS: Between Jan 23 and Oct 3, 2017, 137 children were screened, of whom 76 were eligible for this trial. 60 children were recruited, and allocated to either 10 µg of the co-administered antigens (n=8 for each injection schedule), 30 µg (n=8 for each schedule), 100 µg (n=8 for each schedule), or HBV and placebo (n=6 for each schedule) in three sequential cohorts. Co-administration of the vaccines was well tolerated; the most frequent solicited adverse events were mild-to-moderate injection-site pain, observed in up to 12 (75%) of 16 participants per vaccine group, and mild headache (12 [25%] of 48) and fever (11 [23%] of 48). No vaccine-related serious adverse events were observed. Significant anti-Na-APR-1(M74) and anti-Na-GST-1 IgG levels were induced in a dose-dependent manner, with peaks seen 14 days after the third vaccinations, regardless of dose (for Na-APR-1[M74], geometric mean levels [GML]=2295·97 arbitrary units [AU] and 726·89 AU, while for Na-GST-1, GMLs=331·2 AU and 21·4 AU for the month 0, 2, and 6 and month 0, 2, and 4 schedules, respectively). The month 0, 2, and 6 schedule induced significantly higher IgG responses to both antigens (p=0·01 and p=0·04 for Na-APR-1[M74] and Na-GST-1, respectively). INTERPRETATION: Co-administration of recombinant Na-APR-1(M74) and Na-GST-1 to school-aged Gabonese children was well tolerated and induced significant IgG responses. These results justify further evaluation of this antigen combination in proof-of-concept controlled-infection and efficacy studies in hookworm-endemic areas. FUNDING: European Union Seventh Framework Programme.

Community health workers in clinical research at the example of a phase IIIb/ IV antimalarial drug trial conducted in five African countries.

Global health, particularly in underserved settings can benefit immensely from well-trained community health workers (CHWs) supporting primary healthcare interventions. They can reduce morbidity and mortality of infectious diseases like malaria. Disease control programs can particularly benefit from a tight link between CHWs and communities and several studies have shown the benefit of the participation of non-facility-based CHWs in malaria control program activities for reducing malaria-related mortality in children. Because CHWs are often part of and trusted by served communities, they can also be an important resource to address challenges faced by their communities. Where post-marketing surveillance systems are underserved, they can relay important information about suspected safety signals and factors affecting therapeutic effectiveness in their communities. The CANTAM-Pyramax® trial was a phase IIIb/ IV cohort event monitoring study conducted at six centers in five African countries. To assess real-world effectiveness and safety of the anti-malarial pyronaridine-artesunate in 8560 malaria episodes, follow-up was not primarily conducted by medical staff but by specifically trained CHWs. This perspective paper discusses how the participation of a CHW workforce can be of benefit for effectiveness trials in limited-resource settings, using the example of the CANTAM-Pyramax trial.

Efficacy, safety, and tolerability of albendazole and ivermectin based regimens for the treatment of microfilaraemic loiasis in adult patients in Gabon: A randomized controlled assessor blinded clinical trial.

BACKGROUND: There is a lack of systematic evidence for strategies to control loiasis transmission in highly endemic regions. Here we assessed albendazole and ivermectin based treatment regimens to reduce Loa loa microfilaraemia in Gabon. METHODS: Eligible adult patients with L. loa microfilaraemia between 5,000 and 50,000 microfilariae/ml were randomized to either a control or one of three intervention groups (1:2:2:2 allocation ratio) consisting of three-week twice daily 400mg oral albendazole followed by 1) no treatment, 2) two further weeks of twice daily 400mg albendazole, or 3) a single dose of ivermectin in this open label randomized assessor blinded controlled clinical trial. The primary outcome was the proportion of participants with L. loa microfilaraemia ≤ 100 mf/ml at Day 168. RESULTS: In the efficacy-population of 42 patients 0 (0%; control group), 1 (9%; 3-week albendazole), 5 (39%; 5-weeks albendazole) and 2 (22%; 3-week albendazole plus single dose ivermectin) participants met the primary outcome of microfilaraemia below 100/ml at day 168. A 80-90% reduction of microfilaraemia was observed in the active treatment groups. CONCLUSION: The 5-week regimen of albendazole or a 3-week regimen of albendazole followed by ivermectin were most efficacious to reduce microfilaraemia. All therapeutic regimens were well tolerated and safe. TRIAL REGISTRATION: Trial registered at the Pan-African Clinical Trials Registry: PACTR201807197019027.

Pharyngeal Communities and Antimicrobial Resistance in Pangolins in Gabon.

Wildlife can be a reservoir and source of zoonotic pathogens for humans. For instance, pangolins were considered one of the potential animal reservoirs of SARS-CoV-2. The aim of this study was to assess the prevalence of antimicrobial-resistant species (e.g., extended-spectrum ß-lactamase [ESBL]-producing Enterobacterales) and Staphylococcus aureus-related complex and to describe the bacterial community in wild Gabonese pangolins. The pharyngeal colonization of pangolins sold in Gabon (n = 89, 2021 to 2022) was analyzed using culture media selective for ESBL-producing Enterobacterales, S. aureus-related complex, Gram-positive bacteria and nonfermenters. Phylogenetic analyses of ESBL-producing Enterobacterales was done using core-genome multilocus sequence typing (cgMLST) and compared with publicly available genomes. Patterns of cooccurring species were detected by network analysis. Of the 439 bacterial isolates, the majority of species belonged to the genus Pseudomonas (n = 170), followed by Stenotrophomonas (n = 113) and Achromobacter (n = 37). Three Klebsiella pneumoniae isolates and one Escherichia coli isolate were ESBL-producers, which clustered with human isolates from Nigeria (MLST sequence type 1788 [ST1788]) and Gabon (ST38), respectively. Network analysis revealed a frequent cooccurrence of Stenotrophomonas maltophilia with Pseudomonas putida and Pseudomonas aeruginosa. In conclusion, pangolins can be colonized with human-related ESBL-producing K. pneumoniae and E. coli. Unlike in other African wildlife, S. aureus-related complex was not detected in pangolins. IMPORTANCE There is an ongoing debate if pangolins are a relevant reservoir for viruses such as SARS-CoV-2. Here, we wanted to know if African pangolins are colonized with bacteria that are relevant for human health. A wildlife reservoir of antimicrobial resistance would be of medical relevance in regions were consumption of so-called bushmeat is common. In 89 pangolins, we found three ESBL-producing Klebsiella pneumoniae strains and one ESBL-producing Escherichia coli strains, which were closely related to isolates from humans in Africa. This points toward either a transmission between pangolins and humans or a common source from which both humans and pangolins became colonized.

Plasmodium malariae contributes to high levels of malaria transmission in a forest-savannah transition area in Cameroon.

BACKGROUND: Malaria control efforts are highly skewed towards Plasmodium falciparum while overlooking other Plasmodium species such as P. malariae. A better understanding of the role of Plasmodium species other than P. falciparum is needed to strengthen malaria elimination initiatives. The aim of the present study was to elucidate the contribution of P. malariae to malaria transmission in Cameroon. METHODS: The study was conducted in the Ngatti Health District, a forest-savannah transition area in the Adamawa Region, Cameroon. A total of 497 individuals aged from 1 to 85 years were diagnosed with malaria in November 2020 using a rapid diagnostic test (RDT) and microscopy. Adult mosquitoes were collected between September 2019 and March 2020 by indoor aspiration and identified morphologically and molecularly. The infection status of Plasmodium spp. was also determined by quantitative PCR, and dried blood spots were collected from 156 participants with the aim to detect different Plasmodium species by nested PCR. RESULTS: The overall Plasmodium prevalence was 50.3%, 51.8% and 64.7%, as detected by microscopy, the RDT and PCR, respectively. Based on the PCR results, P. falciparum was the most prevalent species (43%); followed by co-infections P. falciparum/P. malariae (17%), P. falciparum/P. ovale (1.3%), P. falciparum/P. ovale/P. malariae (1.3%); and then by P. malariae mono-infection (2.5%). The same trend was observed using microscopy, with 35% of participants infected with P. falciparum, 11% co-infected with P. falciparum/P. malariae and 4% infected with P. malariae. The prevalence and parasite density of malaria infection varied significantly with age group (P < 0.05), with the highest prevalence rate observed in children aged 6-10 years (P = 0.0001) while the density of Plasmodium infection increased significantly in children aged < 5 years compared to the other age groups (P = 10-3). Among the 757 Anopheles mosquitoes collected, 737 (97.35%) were An. funestus sensu stricto, 15 (1.9%) were An. gambiae and 5 (0.6%) were An. hancocki. The Plasmodium species recorded at the head/thorax level were P. falciparum and P. malariae, with a sporozoite infection rate of 8.4%; the highest sporozoite infection rate was recorded at Mibellon village (13.6%). CONCLUSION: The results of this study reveal the significant contribution of P. malariae, in addition to P. falciparum, to the high malaria transmission rate in this region. These findings highlight the need to deploy initiatives to also tackle this Plasmodium species to eliminate malaria in the region.

Clinical features, treatment outcomes and mortality risk of tuberculosis sepsis in HIV-negative patients: a systematic review and meta-analysis of case reports.

PURPOSE: Tuberculosis sepsis (TBS) is sepsis due to the Mycobacterium species causing tuberculosis (TB). It seems to be rare in HIV-negative patients and mainly individual case reports have been reported. This systematic review summarizes the epidemiology, clinical features, and treatment outcomes of TBS in HIV-negative patients. METHODS: An electronic search of PubMed, Embase, Web of Science, and Google Scholar was performed to identify published case reports of TBS between January 1991 and September 2022. RESULTS: Twenty-five articles reported 28 cases of TBS in HIV-negative patients, among which 54% (15/28) were women; with 50% (14/28) of patients not having reported predisposing factors. A total of 64% (18/28) of patients died, and the diagnosis was obtained for many of them only post-mortem. Two of the reports mentioned the BCG vaccination status. A higher proportion of deaths occurred in patients with delayed diagnosis of sepsis. The probability of survival of patients diagnosed with tuberculosis sepsis was 68% on day 10; 41% on day 20; and 33% on day 30 after admission. CONCLUSIONS: Our review showed TBS occurred in HIV-negative patients and some of them have no known immunocompromised underlying co-morbidity. TBS might not be rare as clinicians thought but might be prone to be missed. In endemic settings, M. tuberculosis etiology of sepsis should be accounted for early, irrespective of HIV infection status.

Rapid and Specific Action of Methylene Blue against Plasmodium Transmission Stages.

Methylene blue (MB) is the oldest synthetic anti-infective. Its high potency against asexual and sexual stages of malaria parasites is well documented. This study aimed to investigate possible additional activities of MB in interfering with parasite transmission and determine target stages in Anopheles vectors and humans. MB's transmission-blocking activity was first evaluated by an ex vivo direct membrane feeding assay (DMFA) using Plasmodium falciparum field isolates. To investigate anti-mosquito stage activity, Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal on mice that had been treated with methylene blue, 3, 6- and 15-days after the initial infectious blood meal. Anti-sporozoite and liver stage activities were evaluated in vitro and in vivo via sporozoite invasion and liver stage development assays, respectively. MB exhibited a robust inhibition of P. falciparum transmission in An. gambiae, even when added shortly before the DMFA but only a moderate effect against P. berghei oocyst development. Exposure of mature P. berghei and P. falciparum sporozoites to MB blocked hepatocyte invasion, yet P. berghei liver stage development was unaffected by MB. Our results indicate previously underappreciated rapid specific activities of methylene blue against Plasmodium transmission stages, preventing the establishment of both mosquito midgut and liver infections as the first essential steps in both hosts.

Urinary Metabolic Profiling in Volunteers Undergoing Malaria Challenge in Gabon.

The interaction of malaria parasites with their human host is extensively studied, yet only few studies reported how P. falciparum infection affects urinary metabolite profiles and how this is associated with immunity. We present a longitudinal study of the urinary metabolic profiles of twenty healthy Africans with lifelong exposure to malaria and five malaria-naïve Europeans, who were all challenged with direct venous inoculation of live P. falciparum sporozoïtes (PfSPZ) and followed up until they developed symptoms or became thick blood smear positive (TBS). Urine samples were collected before and at 2, 5, 9 and 11 days post challenge and were analysed. Upon infection, all Europeans became TBS positive, while Africans showed either a delay in time to parasitaemia or controlled infection. Our metabolic data showed that Europeans and Africans had distinct alterations in metabolite patterns, with changes mostly seen on days 5 and 9 post PfSPZ infection, and more prominently in Europeans. Within the African group, the levels of formate, urea, trimethylamine, threonine, choline, myo-inositol and acetate were significantly higher in TBS positive whereas the levels of pyruvate, 3-methylhistidine and dimethylglycine were significantly lower in individuals who remained TBS negative. Notably, before inoculation with PfSPZ, a group of metabolites including phenylacetylglutamine can potentially be used to predict parasitaemia control among Africans. Taken together, this study highlights the difference in urinary metabolic changes in response to malaria infection as a consequence of lifelong exposure to malaria and that change detectable before challenge might predict the control of parasitaemia in malaria-endemic areas.

Long-term validation of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 from March 2020 to October 2021 in Central Africa, Gabon.

BACKGROUND: Despite the development of several methods for diagnosing COVID-19, long-term validation of such methods remains limited. In the early phase of the COVID-19 pandemic, we developed a rapid and sensitive diagnostic method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology, which is suitable for point-of-care application or for use in resource-limited settings to detect SARS-CoV-2. To assess the applicability of the RT-LAMP assay technique to resource-limited regions, such as rural areas in Africa, and to verify the usability of the method against various SARS-CoV-2 variants, the method was validated using clinical samples collected longitudinally during the pandemic. METHODOLOGY/PRINCIPAL FINDINGS: First, the sensitivity of the RT-LAMP assay for detecting 10 SARS-CoV-2 variants was evaluated using viral RNA samples extracted from cell culture with a portable battery-supported device, resulting in the successful detection of 20-50 copies of the viral genome within 15 min, regardless of the variant. COVID-19 positive samples collected in Gabon between March 2020 and October 2021 were used to evaluate the sensitivity of the assay and to calculate the copy number of the SARS-CoV-2 genome. More than 292 copies of the viral genome were detected with 100% probability within 15 min in almost all tests. CONCLUSIONS: This long-term validation study clearly demonstrated the applicability of the RT-LAMP assay for the clinical diagnosis of COVID-19 in resource-limited settings of Africa, such as rural areas in Gabon. The results show the potential of the assay as a promising COVID-19 diagnostic method, especially in rural and remote regions located far from the official diagnosis facilities in urban or semi-urban areas.

Effectiveness of antimalarial drug combinations in treating concomitant urogenital schistosomiasis in malaria patients in Lambaréné, Gabon: A non-randomised event-monitoring study.

BACKGROUND: Urogenital schistosomiasis is prevalent in many malaria endemic regions of sub-Saharan Africa and can lead to long-term health consequences if untreated. Antimalarial drugs used to treat uncomplicated malaria have shown to exert some activity against Schistosoma haematobium. Here, we explore the efficacy on concomitant urogenital schistosomiasis of first-line recommended artemisinin-based combination therapies (ACTs) and investigational second-generation ACTs when administered for the treatment of uncomplicated malaria in Gabon. METHODS: Microscopic determination of urogenital schistosomiasis was performed from urine samples collected from patients with confirmed uncomplicated malaria. Egg excretion reduction rate and cure rate were determined at 4-weeks and 6-weeks post-treatment with either artesunate-pyronaridine, artemether-lumefantrine, artesunate-amodiaquine or artefenomel-ferroquine. RESULTS: Fifty-two (16%) out of 322 malaria patients were co-infected with urogenital schistosomiasis and were treated with antimalarial drug combinations. Schistosoma haematobium egg excretion rates showed a median reduction of 100% (interquartile range (IQR), 17% to 100%) and 65% (IQR, -133% to 100%) at 4-weeks and 6-weeks post-treatment, respectively, in the artesunate-pyronaridine group (n = 20) compared to 35% (IQR, -250% to 70%) and 65% (IQR, -65% to 79%) in the artemether-lumefantrine group (n = 18). Artesunate-amodiaquine (n = 2) and artefenomel-ferroquine combination (n = 3) were not able to reduce the rate of eggs excreted in this limited number of patients. In addition, cure rates were 56% and 37% at 4- and 6-weeks post-treatment, respectively, with artesunate-pyronaridine and no cases of cure were observed for the other antimalarial combinations. CONCLUSIONS: Antimalarial treatments with artesunate-pyronaridine and artemether-lumefantrine reduced the excretion of S. haematobium eggs, comforting the hypothesis that antimalarial drugs could play a role in the control of schistosomiasis. TRIAL REGISTRATION: This trial is registered with clinicaltrials.gov, under the Identifier NCT04264130.

Investigation of urinary metabolomics in a phase I hookworm vaccine trial in Gabon.

Metabolomics provides a powerful tool to study physiological changes in response to various perturbations such as vaccination. We explored whether metabolomic changes could be seen after vaccination in a phase I trial where Gabonese adults living either in rural or semi-urban areas received the subunit hookworm vaccine candidates (Na-GST-1 and Na-APR-1 (M74) adjuvanted with Alhydrogel plus GLA-AF (n = 24) or the hepatitis B vaccine (n = 8) as control. Urine samples were collected and assayed using targeted 1H NMR spectroscopy. At baseline, a set of metabolites significantly distinguished rural from semi-urban individuals. The pre- and post-vaccination comparisons indicated significant changes in few metabolites but only one day after the first vaccination. There was no relationship with immunogenicity. In conclusion, in a small phase 1 trial, urinary metabolomics could distinguish volunteers with different environmental exposures and reflected the safety of the vaccines but did not show a relationship to immunogenicity.

Codiversification of gut microbiota with humans.

The gut microbiomes of human populations worldwide have many core microbial species in common. However, within a species, some strains can show remarkable population specificity. The question is whether such specificity arises from a shared evolutionary history (codiversification) between humans and their microbes. To test for codiversification of host and microbiota, we analyzed paired gut metagenomes and human genomes for 1225 individuals in Europe, Asia, and Africa, including mothers and their children. Between and within countries, a parallel evolutionary history was evident for humans and their gut microbes. Moreover, species displaying the strongest codiversification independently evolved traits characteristic of host dependency, including reduced genomes and oxygen and temperature sensitivity. These findings all point to the importance of understanding the potential role of population-specific microbial strains in microbiome-mediated disease phenotypes.

Performance of Field's Stain Compared with Conventional Giemsa Stain for the Rapid Detection of Blood Microfilariae in Gabon.

Filarial infections caused by Loa loa and Mansonella perstans are a considerable public health burden in rural regions of Central Africa. Rapid diagnostic tools for the detection of microfilariae in the blood are needed. Field's stain is a rapid staining technique for microscopic slides originally established for malaria diagnostics. It requires less than 1 minute of staining compared with conventional staining protocols requiring at least 15 to 20 minutes for staining and could thus significantly accelerate diagnostics for human filariasis. Here we evaluated Field's stain as a rapid staining technique in comparison to Giemsa stain for the detection of microfilariae in peripheral blood. Blood smears were collected from 175 participants residing in the region of Lambaréné and Fougamou, Gabon. Each participant's samples were stained in parallel with Field's stain and conventional Giemsa stain. Slides were then microscopically assessed and compared for qualitative and quantitative results by a blinded assessor for the two endemic filarial blood pathogens M. perstans and L. loa. Field's stain shows excellent diagnostic performance characteristics for L. loa microfilariae compared with Giemsa staining. Concordance was favorable for M. perstans although lower than for L. loa. Field's stain offers a rapid alternative to Giemsa stain for detection of L. loa microfilariae in thick blood smears. This could help accelerate diagnostics of blood filarial pathogens in mass screening programs or resource constrained health care institutions with high patient load.

Assessment of malaria transmission intensity and insecticide resistance mechanisms in three rural areas of the Moyen Ogooué Province of Gabon.

BACKGROUND: Vector control is considered to be the most successful component of malaria prevention programs and a major contributor to the reduction of malaria incidence over the last two decades. However, the success of this strategy is threatened by the development of resistance to insecticides and behavioural adaptations of vectors. The aim of this study was to monitor malaria transmission and the distribution of insecticide resistance genes in Anopheles populations from three rural areas of the Moyen Ogooué Province of Gabon. METHODS: Anopheles spp. were collected using human landing catches in Bindo, Nombakélé and Zilé, three villages located in the surroundings of Lambaréné, during both the rainy and dry seasons. Mosquitoes were identified morphologically, and DNA was extracted from heads and thoraces. Members of the Anopheles gambiae complex were identified by molecular methods using the PCR SINE200 protocol and by sequencing of the internal transcribed spacer 2 region. Taqman assays were used to determine Plasmodium infection and the presence of resistance alleles. RESULTS: Anopheles gambiae sensu lato (97.7%), An. moucheti (1.7%) and An. coustani (0.6%) were the three groups of species collected. Anopheles gambiae sensu stricto (98.5%) and An. coluzzii (1.5%) were the only species of the An. gambiae complex present in the collection. Of the 1235 Anopheles collected, 1193 were collected during the rainy season; these exhibited an exophagic behaviour, and consistently more mosquitoes were collected outdoor than indoor in the three study areas. Of the 1166 Anopheles screened, 26 (2.2%) were infected with Plasmodium species, specifically Plasmodium falciparum (66.7%), P. malariae (15.4%), P. ovale curtisi (11.5%) and P. ovale wallikeri (3.8%). Malaria transmission intensity was high in Zilé, with an average annual entomological inoculation rate (aEIR) of 243 infective bites per year, while aEIRs in Bindo and Nombakélé were 80.2 and 17 infective bites per year, respectively. Both the L1014F and L1014S mutations were present at frequencies > 95% but no Ace1G119S mutation was found. CONCLUSION: Our results demonstrate that malaria transmission intensity is heterogeneous in these three rural areas of Moyen Ogooué Province, with areas of high transmission, such as Zilé. The exophagic behaviour of the mosquitoes as well as the high frequency of resistance mutations are serious challenges that need to be addressed by the deployment of control measures adapted to the local setting.

Cellular and antibody response in GMZ2-vaccinated Gabonese volunteers in a controlled human malaria infection trial.

BACKGROUND: Antibody and cellular memory responses following vaccination are important measures of immunogenicity. These immune markers were quantified in the framework of a vaccine trial investigating the malaria vaccine candidate GMZ2. METHODS: Fifty Gabonese adults were vaccinated with two formulations (aluminum Alhydrogel and CAF01) of GMZ2 or a control vaccine (Verorab). Vaccine efficacy was assessed using controlled human malaria infection (CHMI) by direct venous inoculation of 3200 live Plasmodium falciparum sporozoites (PfSPZ Challenge). GMZ2-stimulated T and specific B-cell responses were estimated by flow cytometry before and after vaccination. Additionally, the antibody response against 212 P. falciparum antigens was estimated before CHMI by protein microarray. RESULTS: Frequencies of pro- and anti-inflammatory CD4+ T cells stimulated with the vaccine antigen GMZ2 as well as B cell profiles did not change after vaccination. IL-10-producing CD4+ T cells and CD20+ IgG+ B cells were increased post-vaccination regardless of the intervention, thus could not be specifically attributed to any malaria vaccine regimen. In contrast, GMZ2-specific antibody response increased after the vaccination, but was not correlated to protection. Antibody responses to several P. falciparum blood and liver stage antigens (MSP1, MSP4, MSP8, PfEMP1, STARP) as well as the breadth of the malaria-specific antibody response were significantly higher in protected study participants. CONCLUSIONS: In lifelong malaria exposed adults, the main marker of protection against CHMI is a broad antibody pattern recognizing multiple stages of the plasmodial life cycle. Despite vaccination with GMZ2 using a novel formulation, expansion of the GMZ2-stimulated T cells or the GMZ2-specific B cell response was limited, and the vaccine response could not be identified as a marker of protection against malaria. Trial registration PACTR; PACTR201503001038304; Registered 17 February 2015; https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1038.

P. falciparum msp1 and msp2 genetic diversity in P. falciparum single and mixed infection with P. malariae among the asymptomatic population in Southern Benin.

Plasmodium falciparum and Plasmodium malariae infections are prevalent in malaria-endemic countries. However, very little is known about their interactions especially the effect of P. malariae on P. falciparum genetic diversity. This study aimed to assess P. falciparum genetic diversity in P. falciparum and mixed infection P. falciparum/P. malariae isolates among the asymptomatic populations in Southern Benin. Two hundred and fifty blood samples (125 of P. falciparum and 125 P. falciparum/P. malariae isolates) were analysed by a nested PCR amplification of msp1 and msp2 genes. The R033 allelic family was the most represented for the msp1 gene in mono and mixed infection isolates (99.2% vs 86.4%), while the K1 family had the lowest frequency (38.3% vs 20.4%). However, with the msp2 gene, the two allelic families displayed similar frequencies in P. falciparum isolates while the 3D7 allelic family was more represented in P. falciparum/P. malariae isolates (88.7%). Polyclonal infections were also lower (62.9%) in P. falciparum/P. malariae isolates (p < 0.05). Overall, 96 individual alleles were identified (47 for msp1 and 49 for msp2) in P. falciparum isolates while a total of 50 individual alleles were identified (23 for msp1 and 27 for msp2) in P. falciparum/P. malariae isolates. The Multiplicity of Infection (MOI) was lower in P. falciparum/P. malariae isolates (p < 0.05). This study revealed a lower genetic diversity of P. falciparum in P. falciparum/P. malariae isolates using msp1 and msp2 genes among the asymptomatic population in Southern Benin.

Challenges in the clinical development pathway for triple and multiple drug combinations in the treatment of uncomplicated falciparum malaria.

The addition of a third anti-malarial drug matching the pharmacokinetic characteristics of the slowly eliminated partner drug in artemisinin-based combination therapy (ACT) has been proposed as new therapeutic paradigm for the treatment of uncomplicated falciparum malaria. These triple artemisinin-based combination therapy (TACT) should in theory more effectively prevent the development and spread of multidrug resistance than current ACT. Several clinical trials evaluating TACT-or other multidrug anti-malarial combination therapy (MDACT)-have been reported and more are underway. From a regulatory perspective, these clinical development programmes face a strategic dilemma: pivotal clinical trials evaluating TACT are designed to test for non-inferiority of efficacy compared to standard ACT as primary endpoint. While meeting the endpoint of non-inferior efficacy, TACT are consistently associated with a slightly higher frequency of adverse drug reactions than currently used ACT. Moreover, the prevention of the selection of specific drug resistance-one of the main reasons for TACT development-is beyond the scope of even large-scale clinical trials. This raises important questions: if equal efficacy is combined with poorer tolerability, how can then the actual benefit of these drug combinations be demonstrated? How should clinical development plans be conceived to provide objective evidence for or against an improved management of patients and effective prevention of anti-malarial drug resistance by TACT? What are the objective criteria to ultimately convince regulators to approve these new products? In this Opinion paper, the authors discuss the challenges for the clinical development of triple and multidrug anti-malarial combination therapies and the hard choices that need to be taken in the further clinical evaluation and future implementation of this new treatment paradigm.

Creatine kinase-(MB) and hepcidin as candidate biomarkers for early diagnosis of pulmonary tuberculosis: a proof-of-concept study in Lambaréné, Gabon.

BACKGROUND: The present study aimed to evaluate the diagnostic utility of creatine kinase-MB (CK-MB), hepcidin (HEPC), phospholipase A2 group IIA (PLa2G2A), and myosin-binding protein C (MYBPC1) for tuberculosis (TB). These four biomarkers are differentially regulated between quiescent Mycobacterium tuberculosis (Mtb) infected individuals (non-progressors to TB disease) and Mtb-infected TB disease progressors 6 months before the onset of symptoms. METHODS: We enrolled samples from patients experiencing moderate-to-severe pulmonary infections diseases including 23 TB cases confirmed by smear microscopy and culture, and 34 TB-negative cases. For each participant, the serum levels of the four biomarkers were measured using ELISA. RESULTS: The levels of CK-MB and HEPC were significantly reduced in patients with active TB disease. CK-MB median level was 2045 pg/ml (1455-4000 pg/ml) in active TB cases and 3245 pg/ml (1645-4000 pg/ml) in non-TB pulmonary diseases. Using the receiver operating characteristic curve (ROC) analysis, HEPC and CK-MB had the Area Under the Curve (AUC) of 79% (95% CI 67-91%) and 81% (95% CI 69-93%), respectively. Both markers correlated with TB diagnosis as a single marker. PLa2G2A and MYBPC1 with AUCs of 48% (95% CI 36-65%) and 62% (95% CI 48-76%) did not performed well as single biomarkers. The three markers'model (CK-MB-HEPC-PLa2G2A) had the highest diagnostic accuracy at 82% (95% CI 56-82%) after cross-validation. CONCLUSION: CK-MB and HEPC levels were statistically different between confirmed TB cases and non-TB cases. This study yields promising results for the rapid diagnosis of TB disease using a single marker or three biomarkers model.