A.S. Badiane

A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum

Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry‐based method for simultaneously calculating both the parasitemia and the number of multiply‐infected erythrocytes. Staining with the DNA‐specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion. Am. J. Hematol., 2010.

Evaluation of the Illumigene Malaria LAMP: A Robust Molecular Diagnostic Tool for Malaria Parasites.

Isothermal nucleic acid amplification assays such as the loop mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to amplify the DNA. To further facilitate the use of LAMP assays in remote settings, simpler sample preparation methods and lyophilized reagents are required. The performance of a commercial malaria LAMP assay (Illumigene Malaria LAMP) was evaluated using two sample preparation workflows (simple filtration prep (SFP)) and gravity-driven filtration prep (GFP)) and pre-dispensed lyophilized reagents. Laboratory and clinical samples were tested in a field laboratory in Senegal and the results independently confirmed in a reference laboratory in the U.S.A. The Illumigene Malaria LAMP assay was easily implemented in the clinical laboratory and gave similar results to a real-time PCR reference test with limits of detection of ≤2.0 parasites/μl depending on the sample preparation method used. This assay reliably detected Plasmodium sp. parasites in a simple low-tech format, providing a much needed alternative to the more complex molecular tests for malaria diagnosis.

Inhibitory Humoral Responses to the Plasmodium falciparum Vaccine Candidate EBA-175 Are Independent of the Erythrocyte Invasion Pathway

ABSTRACT Plasmodium falciparum utilizes multiple ligand-receptor interactions for invasion. The invasion ligand EBA-175 is being developed as a major blood-stage vaccine candidate. EBA-175 mediates parasite invasion of host erythrocytes in a sialic acid-dependent manner through its binding to the erythrocyte receptor glycophorin A. In this study, we addressed the ability of naturally acquired human antibodies against the EBA-175 RII erythrocyte-binding domain to inhibit parasite invasion of ex vivo isolates, in relationship to the sialic acid dependence of these parasites. We have determined the presence of antibodies to the EBA-175 RII domain by enzyme-linked immunosorbent assay (ELISA) in individuals from areas of Senegal where malaria is endemic with high and low transmission. Using affinity-purified human antibodies to the EBA-175 RII domain from pooled patient plasma, we have measured the invasion pathway as well as the invasion inhibition of clinical isolates from Senegalese patients in ex vivo assays. Our results suggest that naturally acquired anti-EBA-175 RII antibodies significantly inhibit invasion of Senegalese parasites and that these responses can be significantly enhanced through limiting other ligand-receptor interactions. However, the extent of this functional inhibition by EBA-175 antibodies is not associated with the sialic acid dependence of the parasite strain, suggesting that erythrocyte invasion pathway usage by parasite strains is not driven by antibodies targeting the EBA-175/glycophorin A interaction. This work has implications for vaccine design based on the RII domain of EBA-175 in the context of alternative invasion pathways.

Acute kidney injury associated with Plasmodium malariae infection

According to current estimates, Plasmodium malariae is not very common in Senegal, as more than 98% of malaria cases are suspected to be due to Plasmodium falciparum. However, it is possible that other malarial species are being under-reported or misdiagnosed. This is a report of a case of P. malariae in a 30-year-old man previously hospitalized with acute kidney injury after treatment with quinine and re-hospitalized three months later. He was diagnosed with renal cortical necrosis post malaria treatment. Plasmodium malariae was identified with light microscope and confirmed using species-specific small-subunit rRNA (ssrRNA) amplification.The patient was treated for malaria with intravenous quinine for seven days, followed by three days of oral treatment; the bacterial infection was treated using ceftriaxone during the first hospitalization and ciprofloxacin associated with ceftriaxone the second time. He also had four rounds of dialysis after which he partially recovered the renal function. Given the complications that can be caused by P. malariae infection, it should be systematically looked for, even if the predominant species is P. falciparum in Senegal.

Burden of fungal infections in Senegal.

Senegal has a high rate of tuberculosis and a low HIV seropositivity rate and aspergilloma, life‐threatening fungal infections, dermatophytosis and mycetoma have been reported in this study. All published epidemiology papers reporting fungal infection rates from Senegal were identified. Where no data existed, we used specific populations at risk and fungal infection frequencies in each to estimate national incidence or prevalence. The results show that tinea capitis is common being found in 25% of children, ~1.5 million. About 191 000 Senegalese women get recurrent vaginal thrush, ≥4 times annually. We estimate 685 incident cases of chronic pulmonary aspergillosis (CPA) following TB and prevalence of 2160 cases. Asthma prevalence in adults varies from 3.2% to 8.2% (mean 5%); 9976 adults have allergic bronchopulmonary aspergillosis (ABPA) and 13 168 have severe asthma with fungal sensitisation (SAFS). Of the 59 000 estimated HIV‐positive patients, 366 develop cryptococcal meningitis; 1149 develop Pneumocystis pneumonia and 1946 develop oesophageal candidiasis, in which oral candidiasis (53%) and dermatophytosis (16%) are common. Since 2008–2010, 113 cases of mycetoma were diagnosed. In conclusion, we estimate that 1 743 507 (12.5%) people in Senegal suffer from a fungal infection, excluding oral candidiasis, fungal keratitis, invasive candidiasis or aspergillosis. Diagnostic and treatment deficiencies should be rectified to allow epidemiological studies.

Posttreatment HRP2 Clearance in Patients with Uncomplicated Plasmodium falciparum Malaria

Background The response to antimalarial treatment is assessed using serial microscopy. New techniques for accurate measurement of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen have allowed for monitoring of the antigen concentration over time, offering a potential alternative for assessing treatment response. Methods Posttreatment HRP2 concentrations were measured in samples obtained longitudinally from 537 individuals with P. falciparum malaria who were participating in efficacy trials in Angola, Tanzania, and Senegal. The HRP2 half-life was estimated using a first-order kinetics clearance model. The association between the HRP2 concentration 3 days after treatment and recrudescence of infection was assessed. Results Despite substantial variation in HRP2 concentrations among participants at baseline, concentrations consistently showed a first-order exponential decline. The median half-life of HRP2 was estimated to be 4.5 days (interquartile range [IQR], 3.3-6.6 days) in Angola, 4.7 days (IQR, 4.0-5.9 days) in Tanzania, and 3.0 days (IQR, 2.1-4.5 days) in Senegal. The day 3 HRP2 concentration was predictive of eventual recrudescence, with an area under the receiver operating characteristic curve of 0.86 (95% confidence interval, .73-.99). Conclusions Consistent HRP2 clearance dynamics following successful antimalarial treatment imply a common underlying mechanism of biological clearance. Patients who ultimately did not respond to treatment did not exhibit this same pattern of clearance, even in the absence of other indications of inadequate response to treatment.

Article original/Original articleChampignons non dermatophytiques et non candidosiques isolés au CHU Le Dantec de Dakar en 2014 : étude épidémiologique, clinique et mycologiqueNondermatophytic and noncandidal fungi isolated in Le Dantec University hospital of Dakar in 2014: Epidemiological, clinical and mycological study

In recent years, the incidence of superficial fungal infections involving nondermatophytic and noncandidal fungi increased considerably. The objective of this work was to analyze the epidemiological, clinical and mycological fungal infections due to nondermatophytic and noncandidal fungi diagnosed in the laboratory of parasitology-mycology of Le Dantec hospital in Dakar. With a retrospective study of the various cases of nondermatophytic and noncandidal fungi isolated in the laboratory of parasitology-mycology during the period of November 2013 to December 2014, we collected 22 cases of infections in 11 men and 11 women; age ranging from 17 to 75 years with a mean of 45.3 years (sex ratio=1): eight cases of intertrigo, seven cases of onychomycosis, four cases of palmoplantar keratoderma (KPP), a case of onychomycosis associated with interdigital intertrigo, a case of infectious myositis and one case of African histoplasmosis. We have isolated and identified a total of 22 nondermatophytic and noncandidal fungi: ten Fusarium, five Trichosporon, two Chrysosporium, two Geotrichum, one Rhodotorula, one Neoscytalidium dimidiatum and one Histoplasma capsulatum var. duboisii. So we are seeing the emergence of nondermatophytic and noncandidal increasingly isolated from superficial and local lesions. These fungi, generally contaminants or commensal, cause a problem regarding their direct involvement in pathological processes in which they are isolated. So we should respect the recommendations proposed for their involvement in pathological processes and, by a collaboration between clinician and biologist, demonstrate their real involvement through effective, targeted treatment.

Champignons non dermatophytiques et non candidosiques isolés au CHU Le Dantec de Dakar en 2014 : étude épidémiologique, clinique et mycologique

In recent years, the incidence of superficial fungal infections involving nondermatophytic and noncandidal fungi increased considerably. The objective of this work was to analyze the epidemiological, clinical and mycological fungal infections due to nondermatophytic and noncandidal fungi diagnosed in the laboratory of parasitology-mycology of Le Dantec hospital in Dakar. With a retrospective study of the various cases of nondermatophytic and noncandidal fungi isolated in the laboratory of parasitology-mycology during the period of November 2013 to December 2014, we collected 22 cases of infections in 11 men and 11 women; age ranging from 17 to 75 years with a mean of 45.3 years (sex ratio=1): eight cases of intertrigo, seven cases of onychomycosis, four cases of palmoplantar keratoderma (KPP), a case of onychomycosis associated with interdigital intertrigo, a case of infectious myositis and one case of African histoplasmosis. We have isolated and identified a total of 22 nondermatophytic and noncandidal fungi: ten Fusarium, five Trichosporon, two Chrysosporium, two Geotrichum, one Rhodotorula, one Neoscytalidium dimidiatum and one Histoplasma capsulatum var. duboisii. So we are seeing the emergence of nondermatophytic and noncandidal increasingly isolated from superficial and local lesions. These fungi, generally contaminants or commensal, cause a problem regarding their direct involvement in pathological processes in which they are isolated. So we should respect the recommendations proposed for their involvement in pathological processes and, by a collaboration between clinician and biologist, demonstrate their real involvement through effective, targeted treatment.

Distribution of erythrocyte binding antigen 175 (EBA-175) alleles and ABO blood groups in a hypoendemic area in Senegal

INTRODUCTION The study was conducted to determine for the first time the association between the erythrocyte binding antigen 175 (EBA-175) alleles and ABO blood groups in malaria patients living in Thies, a hypoendemic area in Senegal. METHODOLOGY In 2007, the EBA-175 alleles and blood group types were determined by nested PCR and the Simonin test respectively in blood samples obtained from uncomplicated Plasmodium falciparum malaria positive patients. RESULTS AND CONCLUSION In total, 129 patients were enrolled in the study. The EBA-175 genotyping showed a prevalence of 67.45% for the F-allele, 27.90% for the C-allele and 4.65% of mixed C+F infection. The distribution of the ABO blood group type showed 59.8% for the O group, 19.7% for the A group, 17.2% for the B group, and 3.3% for the AB group. No correlation was noted between the EBA-175 alleles and either the blood group type or parasitemia.

Evaluation of CareStart™ Malaria HRP2/pLDH (Pf/pan) Combo Test in a malaria low transmission region of Senegal

BackgroundThis study was initiated from the observation that prevalence of malaria obtained with rapid diagnostic test (RDT) (CareStart™Malaria HRP2/pLDH Combo Test) was higher than in microscopy in a malaria low transmission area of Senegal. PCR was then performed to evaluate the performance of the RDT compared to microscopy in clinical settings.MethodsThe study included 215 patients suspected of malaria in two peri-urban area of Dakar. Finger-pick blood samples were tested using RDT (CareStart™Malaria HRP2/pLDH Combo Test). Venous blood samples were collected for light microscopy and PCR (gold standard). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated as performance characteristics.ResultsConsidering PCR as the gold standard, CareStart™RDT showed high sensitivity (97.3%) and specificity (94.1%) with PPV and NPV of 97.3 and 94.1%, respectively, while microscopy had a sensitivity and specificity of 93.2 and 100%, respectively, and PPV and NPV of 100 and 87.2%, respectively.ConclusionsMalaria CareStart™RDT test demonstrated a superior sensitivity compared to microscopy, which is the gold standard for malaria diagnosis. CareStart™RDT could be a useful tool in individuals suspected of malaria even in areas where prevalence is low.