Impact of piperaquine resistance in Plasmodium falciparum on malaria treatment effectiveness in The Guianas: a descriptive epidemiological study.

BACKGROUND: Plasmodium falciparum is an apicomplexan parasite responsible for lethal cases of malaria. According to WHO recommendations, P falciparum cases are treated with artemisinin-based combination therapy including dihydroartemisinin-piperaquine. However, the emergence of resistant parasites against dihydroartemisinin-piperaquine was reported in southeast Asia in 2008 and, a few years later, suspected in South America. METHODS: To characterise resistance emergence, a treatment efficacy study was performed on the reported patients infected with P falciparum and treated with dihydroartemisinin-piperaquine in French Guiana (n=6, 2016-18). Contemporary isolates collected in French Guiana were genotyped for P falciparum chloroquine resistance transporter (pfCRT; n=845) and pfpm2 and pfpm3 copy number (n=231), phenotyped using the in vitro piperaquine survival assay (n=86), and analysed through genomic studies (n=50). Additional samples from five Amazonian countries and one outside the region were genotyped (n=1440). FINDINGS: In field isolates, 40 (47%) of 86 (95% CI 35·9-57·1) were resistant to piperaquine in vitro; these phenotypes were more associated with pfCRTC350R (ie, Cys350Arg) and pfpm2 and pfpm3 amplifications (Dunn test, p<0·001). Those markers were also associated with dihydroartemisinin-piperaquine treatment failure (n=3 [50%] of 6). A high prevalence of piperaquine resistance markers was observed in Suriname in 19 (83%) of 35 isolates and in Guyana in 579 (73%) of 791 isolates. The pfCRTC350R mutation emerged before pfpm2 and pfpm3 amplification in a temporal sequence different from southeast Asia, and in the absence of artemisinin partial resistance, suggesting a geographically distinctive epistatic relationship between these genetic markers. INTERPRETATION: The high prevalence of piperaquine resistance markers in parasite populations of the Guianas, and the risk of associated therapeutic failures calls for caution on dihydroartemisinin-piperaquine use in the region. Furthermore, greater attention should be given to potential differences in genotype to phenotype mapping across genetically distinct parasite populations from different continents. FUNDING: Pan American Health Organization and WHO, French Ministry for Research, European Commission, Santé publique France, Agence Nationale de la Recherche, Fundação de Amparo à Pesquisa do Estado do Amazonas, Ministry of Health of Brazil, Oswaldo Cruz Foundation, and National Institutes of Health. TRANSLATIONS: For the French and Portuguese translations of the abstract see Supplementary Materials section.

Seroepidemiology of the Seasonal Human Coronaviruses NL63, 229E, OC43 and HKU1 in France.

Background: The seasonal human coronaviruses (HCoV) NL63, 229E, OC43, and HKU1 are globally endemic, yet the majority of HCoV infections remain undiagnosed. Methods: In a cross-sectional study, 2389 serum samples were collected from children and adults in France in 2020. In a longitudinal cohort study, 2520 samples were collected from 898 French individuals followed up between 2020 and 2021. Antibodies to HCoVs were measured using a bead-based multiplex assay. Results: The rate of waning of anti-HCoV spike immunoglobulin G antibodies was estimated as 0.22-0.47 year-1 for children, and 0.13-0.27 year-1 for adults. Seroreversion was estimated as 0.31-1.37 year-1 in children and 0.19-0.72 year-1 in adults. The estimated seroconversion rate in children was consistent with 20%-39% of children being infected every year with each HCoV. Conclusions: The high force of infection in children indicates that HCoVs may be responsible for a substantial proportion of fever episodes experienced by children.

Molecular detection of human Plasmodium species using a multiplex real time PCR.

Molecular detection methods have revealed higher sensitivity and specificity than conventional microscopy or rapid diagnostic tests for malaria diagnosis. In this study, we implemented, evaluated and validated according to the ISO 15,189 requirements, a multiplex real-time PCR assay to detect and identify the five human malaria parasites. DNA samples were extracted from whole blood or dried blood spots drawn from patients. Based on the External Quality Assessment (whole blood), this method shows 100% sensitivity and specificity. This PCR detected P. vivax up to 0.25 p/µl, P. falciparum and P. knowlesi up to 0.5 p/µl, P. ovale up to 1 p/µl and P. malariae up to 5 p/µl of blood. From blood spots (extraction from four punches), it detected P. vivax at 5 p/µl, P. falciparum, P. ovale and P. knowlesi at 20 p/µl and P. malariae at 125 p/µl. In conclusion, this quantitative PCR shows excellent performance, is easy to use and DNA saver. It is especially useful to actively screen large population groups and identify the five human malaria parasites in a context of low malaria transmission.

Estimated protection against COVID-19 based on predicted neutralisation titres from multiple antibody measurements in a longitudinal cohort, France, April 2020 to November 2021.

BackgroundThe risk of SARS-CoV-2 (re-)infection remains present given waning of vaccine-induced and infection-acquired immunity, and ongoing circulation of new variants.AimTo develop a method that predicts virus neutralisation and disease protection based on variant-specific antibody measurements to SARS-CoV-2 antigens.MethodsTo correlate antibody and neutralisation titres, we collected 304 serum samples from individuals with either vaccine-induced or infection-acquired SARS-CoV-2 immunity. Using the association between antibody and neutralisation titres, we developed a prediction model for SARS-CoV-2-specific neutralisation titres. From predicted neutralising titres, we inferred protection estimates to symptomatic and severe COVID-19 using previously described relationships between neutralisation titres and protection estimates. We estimated population immunity in a French longitudinal cohort of 905 individuals followed from April 2020 to November 2021.ResultsWe demonstrated a strong correlation between anti-SARS-CoV-2 antibodies measured using a low cost high-throughput assay and antibody response capacity to neutralise live virus. Participants with a single vaccination or immunity caused by infection were especially vulnerable to symptomatic or severe COVID-19. While the median reduced risk of COVID-19 from Delta variant infection in participants with three vaccinations was 96% (IQR: 94-98), median reduced risk among participants with infection-acquired immunity was only 42% (IQR: 22-66).ConclusionOur results are consistent with data from vaccine effectiveness studies, indicating the robustness of our approach. Our multiplex serological assay can be readily adapted to study new variants and provides a framework for development of an assay that would include protection estimates.

Declines in prevalence alter the optimal level of sexual investment for the malaria parasite Plasmodium falciparum.

Successful infectious disease interventions can result in large reductions in parasite prevalence. Such demographic change has fitness implications for individual parasites and may shift the parasite's optimal life history strategy. Here, we explore whether declining infection rates can alter Plasmodium falciparum's investment in sexual versus asexual growth. Using a multiscale mathematical model, we demonstrate how the proportion of polyclonal infections, which decreases as parasite prevalence declines, affects the optimal sexual development strategy: Within-host competition in multiclone infections favors a greater investment in asexual growth whereas single-clone infections benefit from higher conversion to sexual forms. At the same time, drug treatment also imposes selection pressure on sexual development by shortening infection length and reducing within-host competition. We assess these models using 148 P. falciparum parasite genomes sampled in French Guiana over an 18-y period of intensive intervention (1998 to 2015). During this time frame, multiple public health measures, including the introduction of new drugs and expanded rapid diagnostic testing, were implemented, reducing P. falciparum malaria cases by an order of magnitude. Consistent with this prevalence decline, we see an increase in the relatedness among parasites, but no single clonal background grew to dominate the population. Analyzing individual allele frequency trajectories, we identify genes that likely experienced selective sweeps. Supporting our model predictions, genes showing the strongest signatures of selection include transcription factors involved in the development of P. falciparum's sexual gametocyte form. These results highlight how public health interventions impose wide-ranging selection pressures that affect basic parasite life history traits.

Kinetics of the SARS-CoV-2 Antibody Avidity Response Following Infection and Vaccination.

Serological assays capable of measuring antibody responses induced by previous infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been critical tools in the response to the COVID-19 pandemic. In this study, we use bead-based multiplex assays to measure IgG and IgA antibodies and IgG avidity to five SARS-CoV-2 antigens (Spike (S), receptor-binding domain (RBD), Nucleocapsid (N), S subunit 2, and Membrane-Envelope fusion (ME)). These assays were performed in several cohorts of healthcare workers and nursing home residents, who were followed for up to eleven months after SARS-CoV-2 infection or up to six months after vaccination. Our results show distinct kinetic patterns of antibody quantity (IgG and IgA) and avidity. While IgG and IgA antibody levels waned over time, with IgA antibody levels waning more rapidly, avidity increased with time after infection or vaccination. These contrasting kinetic patterns allow for the estimation of time since previous SARS-CoV-2 infection. Including avidity measurements in addition to antibody levels in a classification algorithm for estimating time since infection led to a substantial improvement in accuracy, from 62% to 78%. The inclusion of antibody avidity in panels of serological assays can yield valuable information for improving serosurveillance during SARS-CoV-2 epidemics.

Neutralising antibody responses to SARS-CoV-2 omicron among elderly nursing home residents following a booster dose of BNT162b2 vaccine: A community-based, prospective, longitudinal cohort study.

Background: The protective immunity against omicron following a BNT162b2 Pfizer booster dose among elderly individuals (ie, those aged >65 years) is not well characterised. Methods: In a community-based, prospective, longitudinal cohort study taking place in France in which 75 residents from three nursing homes were enrolled, we selected 38 residents who had received a two-dose regimen of mRNA vaccine and a booster dose of Pfizer BNT162b2 vaccine. We excluded individuals that did not receive three vaccine doses or did not have available sera samples. We measured anti-S IgG antibodies and neutralisation capacity in sera taken 56 (28-68) and 55 (48-64) days (median (range)) after the 2nd and 3rd vaccine doses, respectively. Antibodies targeting the SARS-CoV-2 Spike protein were measured with the S-Flow assay as binding antibody units per milliliter (BAU/mL). Neutralising activities in sera were measured as effective dilution 50% (ED50) with the S-Fuse assay using authentic isolates of delta and omicron BA.1. Findings: Among the 38 elderly individuals recruited to the cohort study between November 23rd, 2020 and April 29th, 2021, with median age of 88 (range 72-101) years, 30 (78.95%) had been previously infected with SARS-CoV-2. After three vaccine doses, serum neutralising activity was lower against omicron BA.1 (median ED50 of 774.5, range 15.0-34660.0) than the delta variant (median ED50 of 4972.0, range 213.7-66340.0), and higher among previously infected (ie, convalescent; median ED50 against omicron: 1088.0, range 32.6-34660.0) compared with infection-naive residents (median ED50 against omicron: 188.4, range 15.0-8918.0). During the French omicron wave in December 2021-January 2022, 75% (6/8) of naive residents were infected, compared to 25% (7/30) of convalescent residents (P=0.0114). Anti-Spike antibody levels and neutralising activity against omicron BA.1 after a third BNT162b2 booster dose were lower in those with breakthrough BA.1 infection (n=13) compared with those without (n=25), with a median of 1429.9 (range 670.9-3818.3) BAU/mL vs 2528.3 (range 695.4-8832.0) BAU/mL (P=0.029) and a median ED50 of 281.1 (range 15.0-2136.0) vs 1376.0 (range 32.6-34660.0) (P=0.0013), respectively. Interpretation: This study shows that elderly individuals who received three vaccine doses elicit neutralising antibodies against the omicron BA.1 variant of SARS-CoV-2. Elderly individuals who had also been previously infected showed higher neutralising activity compared with naive individuals. Yet, breakthrough infections with omicron occurred. Individuals with breakthrough infections had significantly lower neutralising titers compared to individuals without breakthrough infection. Thus, a fourth dose of vaccine may be useful in the elderly population to increase the level of neutralising antibodies and compensate for waning immunity. Funding: Institut Pasteur, Fondation pour la Recherche Médicale (FRM), European Health Emergency Preparedness and Response Authority (HERA), Agence nationale de recherches sur le sida et les hépatites virales - Maladies Infectieuses Emergentes (ANRS-MIE), Agence nationale de la recherche (ANR), Assistance Publique des Hôpitaux de Paris (AP-HP) and Fondation de France.

Kinetics of the Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Response and Serological Estimation of Time Since Infection.

BACKGROUND: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a complex antibody response that varies by orders of magnitude between individuals and over time. METHODS: We developed a multiplex serological test for measuring antibodies to 5 SARS-CoV-2 antigens and the spike proteins of seasonal coronaviruses. We measured antibody responses in cohorts of hospitalized patients and healthcare workers followed for up to 11 months after symptoms. A mathematical model of antibody kinetics was used to quantify the duration of antibody responses. Antibody response data were used to train algorithms for estimating time since infection. RESULTS: One year after symptoms, we estimate that 36% (95% range, 11%-94%) of anti-Spike immunoglobulin G (IgG) remains, 31% (95% range, 9%-89%) anti-RBD IgG remains, and 7% (1%-31%) of anti-nucleocapsid IgG remains. The multiplex assay classified previous infections into time intervals of 0-3 months, 3-6 months, and 6-12 months. This method was validated using data from a seroprevalence survey in France, demonstrating that historical SARS-CoV-2 transmission can be reconstructed using samples from a single survey. CONCLUSIONS: In addition to diagnosing previous SARS-CoV-2 infection, multiplex serological assays can estimate the time since infection, which can be used to reconstruct past epidemics.

Humoral immunity to SARS-CoV-2 and seasonal coronaviruses in children and adults in north-eastern France.

BACKGROUND: Children are underrepresented in the COVID-19 pandemic and often experience milder disease than adolescents and adults. Reduced severity is possibly due to recent and more frequent seasonal human coronaviruses (HCoV) infections. We assessed the seroprevalence of SARS-CoV-2 and seasonal HCoV specific antibodies in a large cohort in north-eastern France. METHODS: In this cross-sectional seroprevalence study, serum samples were collected from children and adults requiring hospital admission for non-COVID-19 between February and August 2020. Antibody responses to SARS-CoV-2 and seasonal HCoV (229E, HKU1, NL63, OC43) were assessed using a bead-based multiplex assay, Luciferase-Linked ImmunoSorbent Assay, and a pseudotype neutralisation assay. FINDINGS: In 2,408 individuals, seroprevalence of SARS-CoV-2-specific antibodies was 7-8% with three different immunoassays. Antibody levels to seasonal HCoV increased substantially up to the age of 10. Antibody responses in SARS-CoV-2 seropositive individuals were lowest in adults 18-30 years. In SARS-CoV-2 seronegative individuals, we observed cross-reactivity between antibodies to the four HCoV and SARS-CoV-2 Spike. In contrast to other antibodies to SARS-CoV-2, specific antibodies to sub-unit 2 of Spike (S2) in seronegative samples were highest in children. Upon infection with SARS-CoV-2, antibody levels to Spike of betacoronavirus OC43 increased across the whole age spectrum. No SARS-CoV-2 seropositive individuals with low levels of antibodies to seasonal HCoV were observed. INTERPRETATION: Our findings underline significant cross-reactivity between antibodies to SARS-CoV-2 and seasonal HCoV, but provide no significant evidence for cross-protective immunity to SARS-CoV-2 infection due to a recent seasonal HCoV infection. In particular, across all age groups we did not observe SARS-CoV-2 infected individuals with low levels of antibodies to seasonal HCoV. FUNDING: This work was supported by the « URGENCE COVID-19 ¼ fundraising campaign of Institut Pasteur, by the French Government's Investissement d'Avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (Grant No. ANR-10-LABX-62-IBEID), and by the REACTing (Research & Action Emerging Infectious Diseases), and by the RECOVER project funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No. 101003589, and by a grant from LabEx IBEID (ANR-10-LABX-62-IBEID).

Ecology, evolution, and epidemiology of zoonotic and vector-borne infectious diseases in French Guiana: Transdisciplinarity does matter to tackle new emerging threats.

French Guiana is a European ultraperipheric region located on the northern Atlantic coast of South America. It constitutes an important forested region for biological conservation in the Neotropics. Although very sparsely populated, with its inhabitants mainly concentrated on the Atlantic coastal strip and along the two main rivers, it is marked by the presence and development of old and new epidemic disease outbreaks, both research and health priorities. In this review paper, we synthetize 15 years of multidisciplinary and integrative research at the interface between wildlife, ecosystem modification, human activities and sociodemographic development, and human health. This study reveals a complex epidemiological landscape marked by important transitional changes, facilitated by increased interconnections between wildlife, land-use change and human occupation and activity, human and trade transportation, demography with substantial immigration, and identified vector and parasite pharmacological resistance. Among other French Guianese characteristics, we demonstrate herein the existence of more complex multi-host disease life cycles than previously described for several disease systems in Central and South America, which clearly indicates that today the greater promiscuity between wildlife and humans due to demographic and economic pressures may offer novel settings for microbes and their hosts to circulate and spread. French Guiana is a microcosm that crystallizes all the current global environmental, demographic and socioeconomic change conditions, which may favor the development of ancient and future infectious diseases.

Asymptomatic and symptomatic SARS-CoV-2 infections elicit polyfunctional antibodies.

Many SARS-CoV-2-infected individuals remain asymptomatic. Little is known about the extent and quality of their antiviral humoral response. Here, we analyze antibody functions in 52 asymptomatic infected individuals, 119 mildly symptomatic, and 21 hospitalized patients with COVID-19. We measure anti-spike immunoglobulin G (IgG), IgA, and IgM levels with the S-Flow assay and map IgG-targeted epitopes with a Luminex assay. We also evaluate neutralization, complement deposition, and antibody-dependent cellular cytotoxicity (ADCC) using replication-competent SARS-CoV-2 or reporter cell systems. We show that COVID-19 sera mediate complement deposition and kill infected cells by ADCC. Sera from asymptomatic individuals neutralize the virus, activate ADCC, and trigger complement deposition. Antibody levels and functions are lower in asymptomatic individuals than they are in symptomatic cases. Antibody functions are correlated, regardless of disease severity. Longitudinal samplings show that antibody functions follow similar kinetics of induction and contraction. Overall, asymptomatic SARS-CoV-2 infection elicits polyfunctional antibodies neutralizing the virus and targeting infected cells.

Multiplex assays for the identification of serological signatures of SARS-CoV-2 infection: an antibody-based diagnostic and machine learning study.

BACKGROUND: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces an antibody response targeting multiple antigens that changes over time. This study aims to take advantage of this complexity to develop more accurate serological diagnostics. METHODS: A multiplex serological assay was developed to measure IgG and IgM antibody responses to seven SARS-CoV-2 spike or nucleoprotein antigens, two antigens for the nucleoproteins of the 229E and NL63 seasonal coronaviruses, and three non-coronavirus antigens. Antibodies were measured in serum samples collected up to 39 days after symptom onset from 215 adults in four French hospitals (53 patients and 162 health-care workers) with quantitative RT-PCR-confirmed SARS-CoV-2 infection, and negative control serum samples collected from healthy adult blood donors before the start of the SARS-CoV-2 epidemic (335 samples from France, Thailand, and Peru). Machine learning classifiers were trained with the multiplex data to classify individuals with previous SARS-CoV-2 infection, with the best classification performance displayed by a random forests algorithm. A Bayesian mathematical model of antibody kinetics informed by prior information from other coronaviruses was used to estimate time-varying antibody responses and assess the sensitivity and classification performance of serological diagnostics during the first year following symptom onset. A statistical estimator is presented that can provide estimates of seroprevalence in very low-transmission settings. FINDINGS: IgG antibody responses to trimeric spike protein (Stri) identified individuals with previous SARS-CoV-2 infection with 91·6% (95% CI 87·5-94·5) sensitivity and 99·1% (97·4-99·7) specificity. Using a serological signature of IgG and IgM to multiple antigens, it was possible to identify infected individuals with 98·8% (96·5-99·6) sensitivity and 99·3% (97·6-99·8) specificity. Informed by existing data from other coronaviruses, we estimate that 1 year after infection, a monoplex assay with optimal anti-Stri IgG cutoff has 88·7% (95% credible interval 63·4-97·4) sensitivity and that a four-antigen multiplex assay can increase sensitivity to 96·4% (80·9-100·0). When applied to population-level serological surveys, statistical analysis of multiplex data allows estimation of seroprevalence levels less than 2%, below the false-positivity rate of many other assays. INTERPRETATION: Serological signatures based on antibody responses to multiple antigens can provide accurate and robust serological classification of individuals with previous SARS-CoV-2 infection. This provides potential solutions to two pressing challenges for SARS-CoV-2 serological surveillance: classifying individuals who were infected more than 6 months ago and measuring seroprevalence in serological surveys in very low-transmission settings. FUNDING: European Research Council. Fondation pour la Recherche Médicale. Institut Pasteur Task Force COVID-19.

Prevalence of Plasmodium spp. in the Amazonian Border Context (French Guiana-Brazil): Associated Factors and Spatial Distribution.

To implement future malaria elimination strategies in French Guiana, a characterization of the infectious reservoir is recommended. A cross-sectional survey was conducted between October and December 2017 in the French Guianese municipality of St Georges de l'Oyapock, located along the Brazilian border. The prevalence of Plasmodium spp. was determined using a rapid diagnostic test (RDT) and a polymerase chain reaction (PCR). Demographic, house locations, medical history, and biological data were analyzed. Factors associated with Plasmodium spp. carriage were analyzed using logistic regression, and the carriage localization was investigated through spatial cluster analysis. Of the 1,501 samples analyzed with PCR, positive results totaled 90 and 10 for Plasmodium vivax and Plasmodium falciparum, respectively. The general PCR prevalence was 6.6% [5.3-7.9], among which 74% were asymptomatic. Only 13/1,549 were positive by RDT. In multivariate analysis, participants older than 15 years, living in a remote neighborhood, with a prior history of malaria, anemia, and thrombocytopenia were associated with an increased odds of Plasmodium spp. carriage. High-risk clusters of P. vivax carriage were detected in the most remote neighborhoods on the village outskirts and two small foci in the village center. We also detected a hot spot for both P. vivax and P. falciparum symptomatic carriers in the northwestern part of the village. The present study confirms a wide-scale presence of asymptomatic P. falciparum and P. vivax carriers in this area. Although they were more often located in remote areas, their geographic distribution was spatially heterogeneous and complex.

Emergence of Plasmodium vivax Resistance to Chloroquine in French Guiana.

In South America, Plasmodium vivax resistance to chloroquine was recently reported in Brazil and Bolivia. The objective of this study was to collect data on chloroquine resistance in French Guiana by associating a retrospective evaluation of therapeutic efficacy with an analysis of recurrent parasitemia from any patients. Patients with P. vivax infection, confirmed by microscopy and a body temperature of ≥37.5°C, were retrospectively identified at Cayenne Hospital between 2009 and 2015. Follow-up and treatment responses were performed according to the World Health Organization protocol. Parasite resistance was confirmed after dosage of a plasma concentration of chloroquine and microsatellite characterization. The pvmdr1 and pvcrt-o genes were analyzed for sequence and gene copy number variation. Among the 172 patients followed for 28 days, 164 presented adequate clinical and parasitological responses. Eight cases of treatment failures were identified (4.7%; n = 8/172), all after 14 days. The therapeutic efficacy of chloroquine was estimated at 95.3% (95% confidence interval [CI], 92.5 to 98.1%; n = 164/172). Among the eight failures, five were characterized: two cases were true P. vivax chloroquine resistance (1.2%; 95% CI, 0 to 2.6%; n = 2/172), and three cases were found with subtherapeutic concentrations of chloroquine. No particular polymorphism in the Plasmodium vivaxpvmdr1 and pvcrt-o genes was identified in the resistant parasites. This identified level of resistance of P. vivax to chloroquine in French Guiana does not require a change in therapeutic recommendations. However, primaquine should be administered more frequently to limit the spread of resistance, and there is still a need for a reliable molecular marker to facilitate the monitoring of P. vivax resistance to chloroquine.

Antiplasmodial activities of dyes against Plasmodium falciparum asexual and sexual stages: Contrasted uptakes of triarylmethanes Brilliant green, Green S (E142), and Patent Blue V (E131) by erythrocytes.

The search for safe antimalarial compounds acting against asexual symptom-responsible stages and sexual transmission-responsible forms of Plasmodium species is one of the major challenges in malaria elimination programs. So far, among current drugs approved for human use, only primaquine has transmission-blocking activity. The discovery of small molecules targeting different Plasmodium falciparum life stages remains a priority in antimalarial drug research. In this context, several independent studies have recently reported antiplasmodial and transmission-blocking activities of commonly used stains, dyes and fluorescent probes against P. falciparum including chloroquine-resistant isolates. Herein we have studied the antimalarial activities of dyes with different scaffold and we report that the triarylmethane dye (TRAM) Brilliant green inhibits the growth of asexual stages (IC50 ≤ 2 µM) and has exflagellation-blocking activity (IC50 ≤ 800 nM) against P. falciparum reference strains (3D7, 7G8) and chloroquine-resistant clinical isolate (Q206). In a second step we have investigated the antiplasmodial activities of two polysulfonated triarylmethane food dyes. Green S (E142) is weakly active against P. falciparum asexual stage (IC50 ≃ 17 µM) whereas Patent Blue V (E131) is inactive in both antimalarial assays. By applying liquid chromatography techniques for the culture supernatant analysis after cell washings and lysis, we report the detection of Brilliant green in erythrocytes, the selective uptake of Green S (E142) by infected erythrocytes, whereas Patent Blue V (E131) could not be detected within non-infected and 3D7-infected erythrocytes. Overall, our results suggest that two polysulfonated food dyes might display different affinity with transporters or channels on infected RBC membrane.

Plasmodium vivax multidrug resistance-1 gene polymorphism in French Guiana.

BACKGROUND: Plasmodium vivax malaria is a major public health problem in French Guiana. Some cases of resistance to chloroquine, the first-line treatment used against P. vivax malaria, have been described in the Brazilian Amazon region. The aim of this study is to investigate a possible dispersion of chloroquine-resistant P. vivax isolates in French Guiana. The genotype, polymorphism and copy number variation, of the P. vivax multidrug resistance gene-1 (pvmdr1) have been previously associated with modification of the susceptibility to chloroquine. METHODS: The pvmdr1 gene polymorphism was evaluated by sequencing and copy number variation was assessed by real-time PCR, in P. vivax isolates obtained from 591 symptomatic patients from 1997 to 2013. RESULTS: The results reveal that 1.0% [95% CI 0.4-2.2] of French Guiana isolates carry the mutations Y976F and F1076L, and that the proportion of isolates with multiple copies of pvmdr1 has significantly decreased over time, from 71.3% (OR = 6.2 [95% CI 62.9-78.7], p < 0.0001) in 1997-2004 to 12.8% (OR = 0.03 [95% CI 9.4-16.9], p < 0.0001) in 2009-2013. A statistically significant relationship was found between Guf-A (harboring the single mutation T958M) and Sal-1 (wild type) alleles and pvmdr1 copy number. CONCLUSIONS: Few P. vivax isolates harboring chloroquine-resistant mutations in the pvmdr1 gene are circulating in French Guiana. However, the decrease in the prevalence of isolates carrying multiple copies of pvmdr1 might indicate that the P. vivax population in French Guiana is evolving towards a decreased susceptibility to chloroquine.

Prevalence of Plasmodium spp. in illegal gold miners in French Guiana in 2015: a hidden but critical malaria reservoir.

BACKGROUND: Malaria is endemic in French Guiana, an overseas territory of France on the Guiana Shield. Since 2005, notified malaria cases are decreasing. However, new data show that malaria affects many Brazilian gold miners working illegally in French Guiana, the majority of whom are not counted in official data. In addition, one major concern is the usual practice of improper self-treatment in this mining population, raising fear of the development of anti-malarial resistance. This prospective study, conducted in 2015, aimed to estimate the prevalence of Plasmodium spp. in illegal gold miners working in French Guiana. METHODS: The recruitment of gold miners was carried out in resting sites along the French Guiana-Suriname border, where they go for supplies, medical care or leisure. After recording agreement, three malaria diagnostic methods were performed: rapid diagnostic test, microscopy and PCR. RESULTS: Among 421 persons recruited in the study, malaria prevalence, detected by nested-PCR, was 22.3 % (CI [18.3-26.3], n = 94/421) of which 84 % were asymptomatic. CONCLUSIONS: This significant malaria reservoir in a mobile and illegal population with difficult access to a health care system raises the threat of artemisinin resistance and puts the population of the Guiana Shield at risk of new transmission foci while countries of the region aim at malaria elimination. Even though French legislation may hamper dealing with this population, France must face the reality of malaria in illegal gold miners in order to meet its commitment to malaria elimination.

Malaria Hyperendemicity and Risk for Artemisinin Resistance among Illegal Gold Miners, French Guiana.

To assess the prevalence of malaria among illegal gold miners in the French Guiana rainforest, we screened 205 miners during May-June 2014. Malaria prevalence was 48.3%; 48.5% of cases were asymptomatic. Patients reported self-medication with artemisinin-based combination therapy. Risk for emergence and spread of artemisinin resistance among gold miners in the rainforest is high.