Estimating poultry aspergillosis prevalence and diagnostic accuracy of histopathological and mycological culture in Côte d'Ivoire using Bayesian latent class analysis.

This study aimed to estimate the prevalence of poultry aspergillosis and evaluate the accuracy of histopathology (test under evaluation) and mycological culture (an imperfect reference test). Farms raising layer and breeder or broiler birds, with suspected aspergillosis cases, clinical or subclinical, were eligible and visited for sampling. After necropsy, histopathology and mycological culture examinations were conducted by two evaluators. A Bayesian latent class model was used to estimate the accuracy of histopathology when compared to the imperfect reference test, mycological culture. A total of 142 chicken farms, 96 laying and breeding hen farms, and 46 broiler farms were used for the study. True aspergillosis median prevalence was estimated at 63.7% (95% credibility intervals, CrI: 53.8%, 73.0%) in layers and breeders and at 65.2% (95% CrI: 50.2%, 78.3%) in the broiler farms' population. The median diagnostic sensitivity of histopathology and culture were estimated at, respectively, 98.8% (95% CrI: 94.6%, 100.0%) and 90.4% (95% CrI: 83.6%, 95.3%). Tests' diagnostic specificity was estimated at, respectively, 97.3% (95% CrI: 87.7%, 99.9%) and 95.7% (95% CrI: 91.8%, 98.2%). Both tests had very high and comparable positive predictive values, but, in a population where disease prevalence was 25%, histopathology had a higher negative predictive value than culture.

A High Proportion of Malaria Vector Biting and Resting Indoors despite Extensive LLIN Coverage in Côte d'Ivoire.

Malaria is still a leading cause mortality in Côte d'Ivoire despite extensive LLINs coverage. We present the results of an entomological survey conducted in a coastal and in an inland village with the aim to estimate Anopheles gambiae sensu lato (s.l.) female's abundance indoor/outdoor and Plasmodium falciparum infection rate and analyze the occurrence of blood-feeding in relation to LLINs use. Pyrethrum spray (PSC) and window exit traps (WT) collections were carried out to target endophagic/endophilic and endophagic/exophilic females, respectively. Data on LLINs use in sampled houses were collected. (1) high levels of malaria transmission despite LLINs coverage >70% (~1 An. gambiae s.l. predicted mean/person/night and ~5% Plasmodium falciparum infection rate); (2) 46% of females in the PSC sample were blood-fed, suggesting that they fed on an unprotected host inside the house; (3) 81% of females in WT were unfed, suggesting that they were leaving the house to find an available host. Model estimates that if everyone sleeps under LLINs the probability for a mosquito to bite decreases of 48% and 95% in the coastal and inland village, respectively. The results show a high proportion of mosquito biting and resting indoors despite extensive LLINs. The biological/epidemiological determinants of accounting for these results merit deeper investigations.

Ganaplacide (KAF156) plus lumefantrine solid dispersion formulation combination for uncomplicated Plasmodium falciparum malaria: an open-label, multicentre, parallel-group, randomised, controlled, phase 2 trial.

BACKGROUND: Emergence of drug resistance demands novel antimalarial drugs with new mechanisms of action. We aimed to identify effective and well tolerated doses of ganaplacide plus lumefantrine solid dispersion formulation (SDF) in patients with uncomplicated Plasmodium falciparum malaria. METHODS: This open-label, multicentre, parallel-group, randomised, controlled, phase 2 trial was conducted at 13 research clinics and general hospitals in ten African and Asian countries. Patients had microscopically-confirmed uncomplicated P falciparum malaria (>1000 and <150 000 parasites per µL). Part A identified the optimal dose regimens in adults and adolescents (aged ≥12 years) and in part B, the selected doses were assessed in children (≥2 years and <12 years). In part A, patients were randomly assigned to one of seven groups (once a day ganaplacide 400 mg plus lumefantrine-SDF 960 mg for 1, 2, or 3 days; ganaplacide 800 mg plus lumefantrine-SDF 960 mg as a single dose; once a day ganaplacide 200 mg plus lumefantrine-SDF 480 mg for 3 days; once a day ganaplacide 400 mg plus lumefantrine-SDF 480 mg for 3 days; or twice a day artemether plus lumefantrine for 3 days [control]), with stratification by country (2:2:2:2:2:2:1) using randomisation blocks of 13. In part B, patients were randomly assigned to one of four groups (once a day ganaplacide 400 mg plus lumefantrine-SDF 960 mg for 1, 2, or 3 days, or twice a day artemether plus lumefantrine for 3 days) with stratification by country and age (2 to <6 years and 6 to <12 years; 2:2:2:1) using randomisation blocks of seven. The primary efficacy endpoint was PCR-corrected adequate clinical and parasitological response at day 29, analysed in the per protocol set. The null hypothesis was that the response was 80% or lower, rejected when the lower limit of two-sided 95% CI was higher than 80%. This study is registered with EudraCT (2020-003284-25) and ClinicalTrials.gov (NCT03167242). FINDINGS: Between Aug 2, 2017, and May 17, 2021, 1220 patients were screened and of those, 12 were included in the run-in cohort, 337 in part A, and 175 in part B. In part A, 337 adult or adolescent patients were randomly assigned, 326 completed the study, and 305 were included in the per protocol set. The lower limit of the 95% CI for PCR-corrected adequate clinical and parasitological response on day 29 was more than 80% for all treatment regimens in part A (46 of 50 patients [92%, 95% CI 81-98] with 1 day, 47 of 48 [98%, 89-100] with 2 days, and 42 of 43 [98%, 88-100] with 3 days of ganaplacide 400 mg plus lumefantrine-SDF 960 mg; 45 of 48 [94%, 83-99] with ganaplacide 800 mg plus lumefantrine-SDF 960 mg for 1 day; 47 of 47 [100%, 93-100] with ganaplacide 200 mg plus lumefantrine-SDF 480 mg for 3 days; 44 of 44 [100%, 92-100] with ganaplacide 400 mg plus lumefantrine-SDF 480 mg for 3 days; and 25 of 25 [100%, 86-100] with artemether plus lumefantrine). In part B, 351 children were screened, 175 randomly assigned (ganaplacide 400 mg plus lumefantrine-SDF 960 mg once a day for 1, 2, or 3 days), and 171 completed the study. Only the 3-day regimen met the prespecified primary endpoint in paediatric patients (38 of 40 patients [95%, 95% CI 83-99] vs 21 of 22 [96%, 77-100] with artemether plus lumefantrine). The most common adverse events were headache (in seven [14%] of 51 to 15 [28%] of 54 in the ganaplacide plus lumefantrine-SDF groups and five [19%] of 27 in the artemether plus lumefantrine group) in part A, and malaria (in 12 [27%] of 45 to 23 [44%] of 52 in the ganaplacide plus lumefantrine-SDF groups and 12 [50%] of 24 in the artemether plus lumefantrine group) in part B. No patients died during the study. INTERPRETATION: Ganaplacide plus lumefantrine-SDF was effective and well tolerated in patients, especially adults and adolescents, with uncomplicated P falciparum malaria. Ganaplacide 400 mg plus lumefantrine-SDF 960 mg once daily for 3 days was identified as the optimal treatment regimen for adults, adolescents, and children. This combination is being evaluated further in a phase 2 trial (NCT04546633). FUNDING: Novartis and Medicines for Malaria Venture.

Practical example of multiple antibody screening for evaluation of malaria control strategies.

BACKGROUND: Ongoing efforts to fight Plasmodium falciparum malaria has reduced malaria in many areas, but new tools are needed to monitor further progress, including indicators of decreasing exposure to parasite infection. Sero-surveillance is considered promising to monitor exposure, transmission and immunity. METHODS: IgG responses to three antigen biomarkers were evaluated in a retrospective study involving: (i) surveys of 798 asymptomatic villagers from 2 Senegalese endemic settings conducted before 2002 and after the 2013 intensification of control measures, and (ii) in 105 symptomatic individuals from different settings in Côte d'Ivoire. Response to up to eight P. falciparum antigens, including recombinant MSP1p9 antigen and LSA141 peptide, were analysed using multiplex technology and responses to whole P. falciparum schizont extract (SE, local strain adapted to culture) were measured by ELISA. RESULTS: MSP1p9 and LSA141 IgG responses were shown to be relevant indicators monitoring immune status in the different study sites both from Côte d'Ivoire and Senegal. Between 2002 and 2013, individuals participating in both studies showed higher decline of sero-positivity in young (< 15 years: range 12% to 50%) than older (> 15 years: no decline to 15%) individuals from Dielmo and Ndiop. A mathematical sero-catalytic model from the complete Dielmo/Ndiop survey was used to reconstruct declining levels of sero-positivity in more detail, demonstrating that anti-SE seroprevalence levels most accurately reflected malaria exposure in the two villages. CONCLUSION: For standard screening of population immune status at sites envisaging elimination, the use of ELISA-based assays targeting selected antigens can contribute to provide important epidemiologic surveillance data to aid malaria control programmes.

Towards a re-emergence of chloroquine sensitivity in Côte d'Ivoire?

BACKGROUND: Resistance of Plasmodium falciparum to anti-malarial drugs has hampered efforts to eradicate malaria. Recent reports of a decline in the prevalence of chloroquine-resistant P. falciparum in several countries, including Malawi and Zambia, is raising the hope of reintroducing chloroquine in the near future, ideally in combination with another anti-malarial drug for the treatment of uncomplicated malaria. In Côte d'Ivoire, the decrease in the clinical efficacy of chloroquine, in addition to a high proportion of clinical isolates carrying the Thr-76 mutant allele of the pfcrt gene, had led to the discontinuation of the use of chloroquine in 2004. Previous studies have indicated the persistence of a high prevalence of the Thr-76 mutant allele despite the withdrawal of chloroquine as first-line anti-malarial drug. This present study is conducted to determine the prevalence of the Thr-76T mutant allele of the Pfcrt gene after a decade of the ban on the sale and use of chloroquine in Côte d'Ivoire. RESULTS: Analysis of the 64 sequences from all three study sites indicated a prevalence of 15% (10/64) of the Thr-76 mutant allele against 62% (40/64) of the Lys-76 wild-type allele. No mutation of the allele Thr-76 was observed at Anonkoua Kouté while this mutant allele was in 31% (5/16) and 25% (5/20) of isolate sequences from Port-Bouët and Ayamé respectively. CONCLUSION: More than a decade after the discontinuation of the use of chloroquine in Côte d'Ivoire, the proportion of parasites sensitive to this anti-malarial seems to increase in Anonkoua-kouté, Port-bouët and Ayamé.

Antiplasmodial activity of two medicinal plants against clinical isolates of Plasmodium falciparum and Plasmodium berghei infected mice.

Malaria is an infectious and deadly parasitic disease, associated with fever, anaemia and other ailments. Unfortunately the upsurge of plasmodium multidrug resistant constrained researchers to look for new effective drugs. Medicinal plants seem to be an unquenchable source of bioactive principles in the treatment of various diseases. The aim of this study was to assess the antiplasmodial activity of two Ivorian medicinal plants. The in vitro activity was evaluated against clinical isolates and Plasmodium falciparum K1 multidrug resistant strain using the fluorescence based SYBR green I assay. The in vivo bioassay was carried out using the classical 4 day suppressive and curative tests on Plasmodium berghei infected mice. Results showed that the in vitro bioassay of both plant extracts were found to exhibit a promising and moderate antiparasitic effects on clinical isolates (5 µg/mL < IC50 < 15 µg/mL) and Plasmodium falciparum multidrug resistant K1 strain (15 µg/mL < IC50 < 50 µg/mL). Furthermore, the in vivo antiplasmodial screening of both extracts showed a significant decrease in parasitemia, which was dose-dependent. Body temperature in mice treated with both extracts at experimental doses increased, compared to the negative control group and was dose-dependent. As for mice body weight a significant decrease (p < 0.001) was noticed in the negative control group compared to tested groups of animals. The hydroethanolic stem bark extract of Anthocleista djalonensis A Chev and leaves extract of Ziziphus mauritiana Lam exhibited anti-malarial activities. Therefore, the bioactive compounds of both plant extracts need to be investigated.

Longitudinal analysis of antibody responses in symptomatic malaria cases do not mirror parasite transmission in peri-urban area of Cote d'Ivoire between 2010 and 2013.

BACKGROUND: In the agenda towards malaria eradication, assessment of both malaria exposure and efficacy of anti-vectorial and therapeutic strategies is a key component of management and the follow-up of field interventions. The simultaneous use of several antigens (Ags) as serological markers has the potential for accurate evaluation of malaria exposure. Here we aimed to measure the longitudinal evolution of the background levels of immunity in an urban setting in confirmed clinical cases of malaria. METHODS: A retrospective serological cross-sectional study on was carried out using 234 samples taken from 2010 to 2013 in peri-urban sentinel facility of Cote d'Ivoire. Antibody responses to recombinant proteins or BSA-peptides, 8 Plasmodium falciparum (PfAMA1, PfMSP4, PfMSP1, PfEMP1-DBL1α1-PF13, PfLSA1-41, PfLSA3-NR2, PfGLURP and PfCSP), one P. malariae (PmCSP) and one Anopheles gambiae salivary (gSG6-P1) antigens were measured using magnetic bead-based multiplex immunoassay (MBA). Total anti- P. falciparum IgG responses against schizont lysate from african 07/03 strain (adapted to culture) and 3D7 strain was measured by ELISA. RESULTS: High prevalence (7-93%) and levels of antibody responses to most of the antigens were evidenced. However, analysis showed only marginal decreasing trend of Ab responses from 2010 to 2013 that did not parallel the reduction of clinical malaria prevalence following the implementation of intervention in this area. There was a significant inverse correlation between Ab responses and parasitaemia (P<10-3, rho = 0.3). The particular recruitment of asymptomatic individuals in 2011 underlined a high background level of immunity almost equivalent to symptomatic patients, possibly obscuring observable yearly variations. CONCLUSION: The use of cross-sectional clinical malaria surveys and MBA can help to identify endemic sites where control measures have unequal impact providing relevant information about population immunity and possible decrease of transmission. However, when immunity is substantially boosted despite observable clinical decline, a larger cohort including asymptomatic recruitment is needed to monitor the impact of control measures on level of immunity.

Sustained Effectiveness of a Fixed-Dose Combination of Artesunate and Amodiaquine in 480 Patients with Uncomplicated Plasmodium falciparum Malaria in Côte d'Ivoire.

The objective of this study was to monitor the effectiveness of artesunate-amodiaquine fixed-dose combination tablets (ASAQ Winthrop®) in the treatment of uncomplicated Plasmodium falciparum malaria in Côte d'Ivoire. Two enrolment periods (November 2009 to May 2010 and March to October 2013) were compared using an identical design. Subjects with proven monospecific P. falciparum infection according to the WHO diagnostic criteria were eligible. 290 patients during each period received a dose of ASAQ Winthrop tablets appropriate for their age. The primary outcome measure was PCR-corrected adequate clinical and parasitological response at Day 28 in the per protocol population (255 in Period 1 and 240 in Period 2). This was achieved by 95.7% of patients during Period 1 and 96.3% during Period 2. Over 95% of patients were afebrile at Day 3 and complete parasite clearance was achieved at Day 3 in >99% of patients. Nineteen adverse events in nineteen patients were considered as possibly related to treatment, principally vomiting, abnormal liver function tests, and pruritus. There was no evidence for loss of effectiveness over the three-year period in spite of strong drug pressure. This trial was registered in the US Clinical Trials Registry (clinical.trials.gov) under the identifier number NCT01023399.

Analysis of antibody profiles in symptomatic malaria in three sentinel sites of Ivory Coast by using multiplex, fluorescent, magnetic, bead-based serological assay (MAGPIX™).

BACKGROUND: Advances in malaria control have reduced the burden of disease resulting from exposure to parasite infections. The consequences on naturally acquired immunity are unclear. A magnetic bead-based immunoassay (MBA) to assess antibody levels in populations living in endemic areas was previously evaluated. In this study, the effect of clinical attacks on immunity was analysed in three sentinel sites of Ivory Coast. METHODS: Recombinant proteins or peptides derived from liver or blood stage antigens of Plasmodium falciparum (CSP, LSA141, LSA3, SALSA, PF13-DBL1α1, GLURP, AMA1, MSP1p19, MSP4p20), the CSP of Plasmodium malariae and the salivary glands antigen of Anopheles gambiae (gSG6) were covalently linked to a colour-coded microsphere (Luminex™ beads) for the multiplex assay. ELISA was used for whole parasite extract antigen. Blood samples (n = 94) of patients consulting for symptomatic malaria attacks and living in three different malaria endemic settings (rural and periurban) were analysed. RESULTS: Highly variable seroprevalence of antibody responses against parasite antigens was found ranging from 3 (gSG6) to 97% (MSP4p20). A marked prevalence and significantly higher level of antibodies was found in patients from the rural site (Korhogo), those harbouring the lowest level of parasitaemia. The use of whole schizont extract could not discriminate immunity level, contrary to parasite-derived recombinant proteins or peptides. Prevalence of responders to LSA141 and levels of antibodies to PF13 were significantly different between the three settings. Moreover, the post-treatment clearance of parasites was clearly associated with a significantly higher level of antibody response for almost 50% of the parasite antigens tested. CONCLUSION: The multiplex MBA-Magpix technology assay provides an accurate high throughput monitoring of parasite-specific antibodies during symptomatic malaria. The levels of antibody responses may provide a risk criterion with respect to the degree of parasitic infection. Additionally, they can be used as an indicator in the implementation of malaria prevention and local control strategies.

[In vitro susceptibility of P. falciparum isolates from Abidjan (Côte d'Ivoire) to quinine, artesunate and chloroquine]. / Sensibilité in vitro de P. falciparum à la quinine, l'artésunate et la chloroquine à Abidjan (Côte d'Ivoire).

INTRODUCTION: Malaria is still a major public health problem in Côte d'Ivoire. Both treatment and control there are hampered by the spread of resistance to common antimalarial drugs, especially in the south where multidrug-resistant malaria is highly prevalent. Recent treatment guidelines require in vitro tests and the adaptation of drug policies according to local resistance rates. In addition to performing clinical assays in the field, we sought to establish a national map of drug resistance by using in vitro tests with clinical surveys. These make it possible to detect changes in susceptibility and are expected to prevent the emergence of resistance against the most recently introduced combined therapy. MATERIALS AND METHODS: Isolates of Plasmodium falciparum. Isolates of P. falciparum were collected from symptomatic adults and paediatric patients seen at Anonkoua-Kouté Hospital or at the Pasteur Institute of Côte d'Ivoire. Venous blood samples were collected in heparinized vacutainer tubes (Becton Dickinson, Rutherford, NJ, USA). Giemsa-stained thin and thick blood smears were examined for infection by P. falciparum and parasite density was determined. Only blood samples with a parasite density>4,000 parasites/microL of blood were used. When parasite density exceeded 10,000 parasites/microL, freshly washed uninfected red blood cells were added to adjust parasite density to this level. All drug susceptibility assays were performed within 48 h after blood samples were taken. DRUGS: Stock solutions of chloroquine, quinine and artesunate were prepared in methanol. The final concentration of methanol did not exceed 0.05%. The concentrations of the solutions tested ranged from 12.5 to 1,600 nM for chloroquine, 25 to 2 400 nM for quinine and 0.12 to 100 nM for artesunate. In vitro assays The in vitro drug sensitivity of the Ivorian isolates was assessed with a standard 48-h isotope test. Briefly, fresh blood samples were washed three times with RPMI 1640 medium (GibcoTM, Invitrogen Corporation, France) and centrifuged (1,500xg, 5 minutes). The parasites were then tested directly without culture adaptation. If parasitemia > 0.5%, fresh uninfected erythrocytes were added to adjust it to 0.3%. The infected erythrocytes (1.5% hematocrit, 0.1-1% parasitemia) were suspended in complete RPMI medium supplemented with 10% decomplemented human AB+ serum (Biomedia, France) and buffered with 25 mM/L HEPES and 25 mM/L NaHCO3. The mixture was distributed (200 microL per well) into 96-well test plates pre-coated with antimalarial agents. Each plate included two drug-free control wells and one control well without parasites. The culture plates were incubated for 48 h at 37 degrees C in a 5% CO2 atmosphere. [3H]Hypoxanthine (0.5 mCi/well; Amersham Biosciences, France) was used to assess parasite growth. Each isolate was tested once in duplicate in the microplates with serial drug dilutions. Drug response was quantified by monitoring [3H] hypoxanthine uptake in a Wallac MicroBeta Trilux counter (Perkin-Elmer, France). DATA ANALYSIS: The IC50 values (defined as the drug concentration that resulted in a level of 3H-hypoxanthine uptake 50% lower than that measured in the drug-free control wells) were determined by nonlinear regression analysis of the plot of the concentration logarithm against growth inhibition. Data were adapted to fit the log-probit model (Excel, Microsoft; Redmond, WA, USA). The threshold IC50 values for in vitro resistance to chloroquine, quinine and artésunate have previously been estimated to be >100 nM, >800 nM and >19.81 nM respectively. RESULTS: In all 23, 21 and 19 P. falciparum isolates grew satisfactorily in quinine, artésunate and chloroquine, respectively, and yielded interpretable results for these drugs. The geometric mean IC50 for quinine was 272.12 nM with values ranging from 2.08 to 660.28. For artésunate, the IC50 values ranged from 0.03 to 43.84 nM and the geometric mean was 7.49 nM. The IC50 values for chloroquine ranged from 17.71 to 359.19 nM, with a geometric mean for the 23 isolates of 93.72 nM. The proportions of resistant isolates were 26.10% for chloroquine (IC50>100 nM), 9.5% for artesunate (IC50>9.66 nM) and 0% for quinine. No multidrug-resistant isolates (resistant to more than three drugs) were found. CONCLUSION: The decreased susceptibility to artesunate of isolates collected in Abidjan justifies an improved surveillance program for drug resistance to malaria in Côte d'Ivoire.