Comprehensive study of proteasome inhibitors against Plasmodium falciparum laboratory strains and field isolates from Gabon

<p>Abstract</p> <p>Background</p> <p>The emergence and spread of <it>Plasmodium falciparum </it>resistance to almost all available antimalarial drugs necessitates the search for new chemotherapeutic compounds. The ubiquitin/proteasome system plays a major role in overall protein turnover, especially in fast dividing eukaryotic cells including plasmodia. Previous studies show that the 20S proteasome is expressed and catalytically active in plasmodia and treatment with proteasome inhibitors arrests parasite growth. This is the first comprehensive screening of proteasome inhibitors with different chemical modes of action against laboratory strains of <it>P. falciparum</it>. Subsequently, a selection of inhibitors was tested in field isolates from Lambaréné, Gabon.</p> <p>Methods</p> <p>Epoxomicin, YU101, YU102, MG132, MG115, Z-L₃-VS, Ada-Ahx₃-L₃-VS, lactacystin, bortezomib (Velcade^®), gliotoxin, PR11 and PR39 were tested and compared to chloroquine- and artesunate-activities in a standardized <it>in vitro </it>drug susceptibility assay against <it>P. falciparum </it>laboratory strains 3D7, D10 and Dd2. Freshly obtained field isolates from Lambaréné, Gabon, were used to measure the activity of chloroquine, artesunate, epoxomicin, MG132, lactacystin and bortezomib. Parasite growth was detected through histidine-rich protein 2 (HRP2) production. Raw data were fitted by a four-parameter logistic model and individual inhibitory concentrations (50%, 90%, and 99%) were calculated.</p> <p>Results</p> <p>Amongst all proteasome inhibitors tested, epoxomicin showed the highest activity in chloroquine-susceptible (IC50: 6.8 nM [3D7], 1.7 nM [D10]) and in chloroquine-resistant laboratory strains (IC50: 10.4 nM [Dd2]) as well as in field isolates (IC50: 8.5 nM). The comparator drug artesunate was even more active (IC50: 1.0 nM), whereas all strains were chloroquine-resistant (IC50: 113 nM).</p> <p>Conclusion</p> <p>The peptide α',β'-epoxyketone epoxomicin is highly active against <it>P. falciparum </it>regardless the grade of the parasite's chloroquine susceptibility. Therefore, inhibition of the proteasome is a highly promising strategy to develop new antimalarials. Epoxomicin can serve as a standard to compare new inhibitors with species-specific activity.</p

A rapid malaria appraisal in the Venezuelan Amazon

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Assessment of LED fluorescence microscopy for the diagnosis of Plasmodium falciparum infections in Gabon

<p>Abstract</p> <p>Background</p> <p>Rapid and accurate diagnosis of malaria is central to clinical management and the prevention of drug-overuse, which may lead to resistance development, toxicity and economic losses. So far, light microscopy (LM) of Giemsa-stained thick blood smears is the gold standard. Under optimal conditions the procedure is fast and reliable; nevertheless a gain in speed would be a great advantage. Rapid diagnosis tests are an alternative, although they cost more and give qualitative instead of quantitative results. Light-emitting diode (LED) fluorescence microscopy (ledFM 400 ×, 1000 ×) may offer a reliable and cheap alternative, which can be used at the point of care.</p> <p>Methods</p> <p>LedFM and conventional fluorescence microscopy (uvFM) were compared to LM in 210 samples from patients with history of fever in the last 24 hours admitted to the Albert Schweitzer Hospital in Lambaréné, Gabon.</p> <p>Results</p> <p>Sensitivities were 99.1% for ledFM and 97.0% for uvFM, specificities 90.7% for ledFM 400 × and 92.6% for ledFM 1000 × and uvFM. High agreement was found in Bland-Altman-plot and Kappa coefficient (ledFM 1000 ×: 0.914, ledFM 400 × and uvFM: 0.895). The time to diagnosis for both FM methods was shorter compared to LM (LM: 43 min, uvFM: 16 min, ledFM 1000 ×: 14 min, ledFM 400 ×: 10 min).</p> <p>Conclusion</p> <p>ledFM is a reliable, accurate, fast and inexpensive tool for daily routine malaria diagnosis and may be used as a point of care diagnostic tool.</p

Prospective evaluation of artemether-lumefantrine for the treatment of non-falciparum and mixed-species malaria in Gabon

Background: The recommendation of artemisinin combination therapy (ACT) as first-line treatment for uncomplicated falciparum malaria is supported by a plethora of high quality clinical trials. However, their recommendation for the treatment of mixed-species malaria and the large-scale use for the treatment of non-falciparum malaria in endemic regions is based on anecdotal rather than systematic clinical evidence. Methods: This study prospectively observed the efficacy of artemether-lumefantrine for the treatment of uncomplicated non-falciparum or mixed-species malaria in two routine district hospitals in the Central African country of Gabon. Results: Forty patients suffering from uncomplicated Plasmodium malariae, Plasmodium ovale or mixed-species malaria (including Plasmodium falciparum) presenting at the hospital received artemether-lumefantrine treatment and were followed up. All evaluable patients (n = 38) showed an adequate clinical and parasitological response on Day 28 after oral treatment with artemether-lumefantrine (95% confidence interval: 0.91,1). All adverse events were of mild to moderate intensity and completely resolved by the end of study. Conclusions: This first systematic assessment of artemether-lumefantrine treatment for P. malariae, P. ovale and mixed-species malaria demonstrated a high cure rate of 100% and a favourable tolerability profile, and thus lends support to the practice of treating non-falciparum or mixed-species malaria, or all cases of malaria without definite species differentiation, with artemether-lumefantrine in Gabon. Trial Registration: ClinicalTrials.gov Identifier: NCT0072577

Natural infection of Plasmodium brasilianum in humans: Man and monkey share quartan malaria parasites in the Venezuelan Amazon

AbstractBackgroundThe quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now.MethodsWe investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing.FindingsBased on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys.InterpretationThis study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts

Genetic Diversity of Enteric Viruses in Children under Five Years Old in Gabon

Enteric viruses are the leading cause of diarrhea in children globally. Identifying viral agents and understanding their genetic diversity could help to develop effective preventive measures. This study aimed to determine the detection rate and genetic diversity of four enteric viruses in Gabonese children aged below five years. Stool samples from children <5 years with (n = 177) and without (n = 67) diarrhea were collected from April 2018 to November 2019. Norovirus, astrovirus, sapovirus, and aichivirus A were identified using PCR techniques followed by sequencing and phylogenetic analyses. At least one viral agent was identified in 23.2% and 14.9% of the symptomatic and asymptomatic participants, respectively. Norovirus (14.7%) and astrovirus (7.3%) were the most prevalent in children with diarrhea, whereas in the healthy group norovirus (9%) followed by the first reported aichivirus A in Gabon (6%) were predominant. The predominant norovirus genogroup was GII, consisting mostly of genotype GII.P31-GII.4 Sydney. Phylogenetic analysis of the 3CD region of the aichivirus A genome revealed the presence of two genotypes (A and C) in the study cohort. Astrovirus and sapovirus showed a high diversity, with five different astrovirus genotypes and four sapovirus genotypes, respectively. Our findings give new insights into the circulation and genetic diversity of enteric viruses in Gabonese children.Peer Reviewe

Naturally acquired immune responses to malaria vaccine candidate antigens MSP3 and GLURP in Guahibo and Piaroa indigenous communities of the Venezuelan Amazon

BACKGROUND: Malaria transmission in most of Latin America can be considered as controlled. In such a scenario, parameters of baseline immunity to malaria antigens are of specific interest with respect to future malaria eradication efforts. METHODS: A cross-sectional study was carried out in two indigenous population groups in Amazonas/Venezuela. Data from the regional malaria documentation system were extracted and participants from the ethnic groups of the Guahibo (n = 180) and Piaroa (n = 295) were investigated for the presence of Plasmodium parasites and naturally acquired antibodies to Plasmodium falciparum antigens in serum. The GMZ2 vaccine candidate proteins MSP3 and GLURP were chosen as serological markers. RESULTS: The incidence of P. falciparum in both communities was found to be less than 2%, and none of the participants harboured P. falciparum at the time of the cross-sectional. Nearly a quarter of the participants (111/475; 23,4%) had positive antibody titres to at least one of the antigens. 53/475 participants (11.2%) were positive for MSP3, and 93/475 participants (19.6%) were positive for GLURP. High positive responses were detected in 36/475 participants (7.6%) and 61/475 participants (12.8%) for MSP3 and GLURP, respectively. Guahibo participants had significantly higher antibody titres than Piaroa participants. CONCLUSIONS: Considering the low incidence of P. falciparum, submicroscopical infections may explain the comparatively high anti-P. falciparum antibody concentrations

A phase 2b randomized, controlled trial of the efficacy of the GMZ2 malaria vaccine in African children.

BACKGROUND: GMZ2 is a recombinant protein malaria vaccine, comprising two blood-stage antigens of Plasmodium falciparum, glutamate-rich protein and merozoite surface protein 3. We assessed efficacy of GMZ2 in children in Burkina Faso, Gabon, Ghana and Uganda. METHODS: Children 12-60months old were randomized to receive three injections of either 100μg GMZ2 adjuvanted with aluminum hydroxide or a control vaccine (rabies) four weeks apart and were followed up for six months to measure the incidence of malaria defined as fever or history of fever and a parasite density ⩾5000/μL. RESULTS: A cohort of 1849 children were randomized, 1735 received three doses of vaccine (868 GMZ2, 867 control-vaccine). There were 641 malaria episodes in the GMZ2/Alum group and 720 in the control group. In the ATP analysis, vaccine efficacy (VE), adjusted for age and site was 14% (95% confidence interval [CI]: 3.6%, 23%, p-value=0.009). In the ITT analysis, age-adjusted VE was 11.3% (95% CI 2.5%, 19%, p-value=0.013). VE was higher in older children. In GMZ2-vaccinated children, the incidence of malaria decreased with increasing vaccine-induced anti-GMZ2 IgG concentration. There were 32 cases of severe malaria (18 in the rabies vaccine group and 14 in the GMZ2 group), VE 27% (95% CI -44%, 63%). CONCLUSIONS: GMZ2 is the first blood-stage malaria vaccine to be evaluated in a large multicenter trial. GMZ2 was well tolerated and immunogenic, and reduced the incidence of malaria, but efficacy would need to be substantially improved, using a more immunogenic formulation, for the vaccine to have a public health role

A Randomized Controlled Phase Ib Trial of the Malaria Vaccine Candidate GMZ2 in African Children

BACKGROUND: GMZ2 is a fusion protein of Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP) that mediates an immune response against the blood stage of the parasite. Two previous phase I clinical trials, one in naïve European adults and one in malaria-exposed Gabonese adults showed that GMZ2 was well tolerated and immunogenic. Here, we present data on safety and immunogenicity of GMZ2 in one to five year old Gabonese children, a target population for future malaria vaccine efficacy trials. METHODOLOGY/PRINCIPAL FINDINGS: Thirty children one to five years of age were randomized to receive three doses of either 30 µg or 100 µg of GMZ2, or rabies vaccine. GMZ2, adjuvanted in aluminum hydroxide, was administered on Days 0, 28 and 56. All participants received a full course of their respective vaccination and were followed up for one year. Both 30 µg and 100 µg GMZ2 vaccine doses were well tolerated and induced antibodies and memory B-cells against GMZ2 as well as its antigenic constituents MSP3 and GLURP. After three doses of vaccine, the geometric mean concentration of antibodies to GMZ2 was 19-fold (95%CI: 11,34) higher in the 30 µg GMZ2 group than in the rabies vaccine controls, and 16-fold (7,36) higher in the 100 µg GMZ2 group than the rabies group. Geometric mean concentration of antibodies to MSP3 was 2.7-fold (1.6,4.6) higher in the 30 µg group than in the rabies group and 3.8-fold (1.5,9.6) higher in the 100 µg group. Memory B-cells against GMZ2 developed in both GMZ2 vaccinated groups. CONCLUSIONS/SIGNIFICANCE: Both 30 µg as well as 100 µg intramuscular GMZ2 are immunogenic, well tolerated, and safe in young, malaria-exposed Gabonese children. This result confirms previous findings in naïve and malaria-exposed adults and supports further clinical development of GMZ2. TRIAL REGISTRATION: ClinicalTrials.gov NCT00703066

Hemolysis Is Associated with Low Reticulocyte Production Index and Predicts Blood Transfusion in Severe Malarial Anemia

Background: Falciparum Malaria, an infectious disease caused by the apicomplexan parasite Plasmodium falciparum, is among the leading causes of death and morbidity attributable to infectious diseases worldwide. In Gabon, Central Africa, one out of four inpatients have severe malarial anemia (SMA), a life-threatening complication if left untreated. Emerging drug resistant parasites might aggravate the situation. This case control study investigates biomarkers of enhanced hemolysis in hospitalized children with either SMA or mild malaria (MM). Methods and Findings: Ninety-one children were included, thereof 39 SMA patients. Strict inclusion criteria were chosen to exclude other causes of anemia. At diagnosis, erythrophagocytosis (a direct marker for extravascular hemolysis, EVH) was enhanced in SMA compared to MM patients (5.0 arbitrary units (AU) (interquartile range (IR): 2.2–9.6) vs. 2.1 AU (IR: 1.3–3.9), p<0.01). Furthermore, indirect markers for EVH, (i.e. serum neopterin levels, spleen size enlargement and monocyte pigment) were significantly increased in SMA patients. Markers for erythrocyte ageing, such as CD35 (complement receptor 1), CD55 (decay acceleration factor) and phosphatidylserine exposure (annexin-V-binding) were investigated by flow cytometry. In SMA patients, levels of CD35 and CD55 on the red blood cell surface were decreased and erythrocyte removal markers were increased when compared to MM or reconvalescent patients. Additionally, intravascular hemolysis (IVH) was quantified using several indirect markers (LDH, alpha-HBDH, haptoglobin and hemopexin), which all showed elevated IVH in SMA. The presence of both IVH and EVH predicted the need for blood transfusion during antimalarial treatment (odds ratio 61.5, 95% confidence interval (CI): 8.9–427). Interestingly, this subpopulation is characterized by a significantly lowered reticulocyte production index (RPI, p<0.05). Conclusions: Our results show the multifactorial pathophysiology of SMA, whereby EVH and IVH play a particularly important role. We propose a model where removal of infected and non-infected erythrocytes of all ages (including reticulocytes) by EVH and IVH is a main mechanism of SMA. Further studies are underway to investigate the mechanism and extent of reticulocyte removal to identify possible interventions to reduce the risk of SMA development