Comparison of the Micronaut-AM System and the EUCAST Broth Microdilution Reference Method for MIC Determination of Four Antifungals against Aspergillus fumigatus.

The Antifungal Susceptibility Testing method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST) is a reference technique for the determination of the Minimum Inhibitory Concentration (MIC) of antifungals for Aspergillus fumigatus. However, it is time-consuming and requires expertise. Micronaut-AM (M-AM) is a fast, simple, time-saving, and ready-to-use new colorimetric method using an indicator (resazurin) to facilitate the visual reading. The aim of this retrospective study was to evaluate the performance of the M-AM system and compare it with the EUCAST broth microdilution reference method to determine the susceptibility of 77 A. fumigatus clinical strains to amphotericin B, itraconazole, voriconazole, and posaconazole. Overall, the essential agreements within ±2 dilutions were 100%, 62%, 58%, and 30% and the categorical agreements were 100%, 97%, 91%, and 87% for amphotericin B, itraconazole, voriconazole, and posaconazole, respectively. No categorical discrepancy was found for amphotericin B, but several categorical discordances were observed with azole antifungals. However, only 2 of the 16 azole-resistant strains confirmed by the cyp51A sequencing would have been misclassified by M-AM. The use of M-AM is probably suitable for the determination of the MICs of amphotericin B, but further evaluations are needed to confirm its usefulness for the determination of the MICs of azoles for A. fumigatus.

Genetic Profiling of Plasmodium ovale wallikeri Relapses With Microsatellite Markers and Whole-Genome Sequencing.

Like Plasmodium vivax, both Plasmodium ovale curtisi and Plasmodium ovale wallikeri have the ability to cause relapse in humans, defined as recurring asexual parasitemia originating from liver-dormant forms subsequent to a primary infection. Here, we investigated relapse patterns in P ovale wallikeri infections from a cohort of travelers who were exposed to the parasite in sub-Saharan Africa and then experienced relapses after their return to France. Using a novel set of 8 highly polymorphic microsatellite markers, we genotyped 15 P ovale wallikeri relapses. For most relapses, the paired primary and relapse infections were highly genetically related (with 12 being homologous), an observation that was confirmed by whole-genome sequencing for the 4 relapses we further studied. This is, to our knowledge, the first genetic evidence of relapses in P ovale spp.

Evaluation of two commercial kits and two laboratory-developed qPCR assays compared to LAMP for molecular diagnosis of malaria.

BACKGROUND: Malaria is an infectious disease considered as one of the biggest causes of mortality in endemic areas. This life-threatening disease needs to be quickly diagnosed and treated. The standard diagnostic tools recommended by the World Health Organization are thick blood smears microscopy and immuno-chromatographic rapid diagnostic tests. However, these methods lack sensitivity especially in cases of low parasitaemia and non-falciparum infections. Therefore, the need for more accurate and reliable diagnostic tools, such as real-time polymerase chain reaction based methods which have proven greater sensitivity particularly in the screening of malaria, is prominent. This study was conducted at the French National Malaria Reference Centre to assess sensitivity and specificity of two commercial malaria qPCR kits and two in-house developed qPCRs compared to LAMP. METHODS: 183 blood samples received for expertise at the FNMRC were included in this study and were subjected to four different qPCR methods: the Biosynex Ampliquick® Malaria test, the BioEvolution Plasmodium Typage test, the in-house HRM and the in-house TaqMan qPCRs. The specificity and sensitivity of each method and their confidence intervals were determined with the LAMP-based assay Alethia® Malaria as the reference for malaria diagnosis. The accuracy of species diagnosis of the Ampliquick® Malaria test and the two in-house qPCRs was also evaluated using the BioEvolution Plasmodium Typage test as the reference method for species identification. RESULTS: The main results showed that when compared to LAMP, a test with excellent diagnostic performances, the two in-house developed qPCRs were the most sensitive (sensitivity at 100% for the in-house TaqMan qPCR and 98.1% for the in-house HRM qPCR), followed by the two commercial kits: the Biosynex Ampliquick® Malaria test (sensitivity at 97.2%) and the BioEvolution Plasmodium Typage (sensitivity at 95.4%). Additionally, with the in-house qPCRs we were able to confirm a Plasmodium falciparum infection in microscopically negative samples that were not detected by commercial qPCR kits. This demonstrates that the var genes of P. falciparum used in these in-house qPCRs are more reliable targets than the 18S sRNA commonly used in most of the developed qPCR methods for malaria diagnosis. CONCLUSION: Overall, these results accentuate the role molecular methods could play in the screening of malaria. This may represent a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis, which could be essential for the detection and treatment of malaria carriers and even for the eradication of this disease.

Plasmodium ovale wallikeri and P. ovale curtisi Infections and Diagnostic Approaches to Imported Malaria, France, 2013-2018.

We retrospectively analyzed epidemiologic, clinical, and biologic characteristics of 368 Plasmodium ovale wallikeri and 309 P. ovale curtisi infections treated in France during January 2013­December 2018. P. ovale wallikeri infections displayed deeper thrombocytopenia and shorter latency periods. Despite similar clinical manifestations, P. ovale wallikeri­infected patients were more frequently treated with artemisinin-based combination therapy. Although the difference was not statistically significant, P. ovale wallikeri­infected patients were 5 times more frequently hospitalized in intensive care or intermediate care and had a higher proportion of severe thrombocytopenia than P. ovale curtisi­infected patients. Rapid diagnostic tests that detect aldolase were more efficient than those detecting Plasmodium lactate dehydrogenase. Sequence analysis of the potra gene from 90 P. ovale isolates reveals an insufficient polymorphism for relapse typing.

Evaluation of PCR To Monitor Plasmodium falciparum Treatment Efficacy in a Nonendemicity Setting.

Adequate clinical and parasitological response (ACPR) after malaria treatment remains challenging to assess in settings of malaria nonendemicity. Biological evaluation of parasitological clearance relies on microscopic investigation of thick blood smears, which is a specific technique that not all diagnosis laboratories are able to perform. Rapid diagnosis tests (RDTs) and molecular biology techniques are proposed as alternatives to microscope conventional techniques; however, their performance for treatment efficacy evaluation is controversial. We present here a retrospective comparative study for RDT and PCR (nested and high-resolution-melting quantitative PCR [HRM-qPCR]) evaluation of ACPR in a nonendemicity context. Blood samples from 133 patients presenting a Plasmodium falciparum monoinfection were included. Samples obtained at the time of diagnosis and at 3, 7, and 28 days after diagnosis were investigated. Histidine-rich protein 2 (HRP-2)-based RDT results remained positive in 51% of cases 28 days after diagnosis and appropriate therapeutic management. Parasite DNA was detected by the two PCR techniques (nested PCR and HRM-qPCR) in 12% and 10% of samples 28 days after treatment initiation, respectively. No therapeutic failure was recorded in the studied patients. Persistence of positive signal might reflect the presence of circulating asexual parasites or persistence of HRP-2 and parasitic DNA in patient's peripheral blood after parasitic clearance.

Disseminated toxoplasmosis associated with hemophagocytic syndrome after kidney transplantation: A case report and review.

Disseminated toxoplasmosis is infrequent after kidney transplant transmission but life-threatening because of a lack of diagnostic suspicion as well as specific chemoprophylaxis recommendations. Solid organ transplantation has resulted in few cases of disseminated toxoplasmosis presenting with associated hemophagocytic syndrome. Herein, we report, within the context of a donor/receiver mismatch, a case of a toxoplasmosis associated with hemophagocytic syndrome in a kidney transplant recipient. Molecular and serological investigations confirmed Toxoplasma gondii transmission through the kidney graft.

Identification of Plasmodium falciparum and host factors associated with cerebral malaria: description of the protocol for a prospective, case-control study in Benin (NeuroCM).

INTRODUCTION: In 2016, an estimated 216 million cases and 445 000 deaths of malaria occurred worldwide, in 91 countries. In Benin, malaria causes 26.8% of consultation and hospitalisation motif in the general population and 20.9% in children under 5 years old.The goal of the NeuroCM project is to identify the causative factors of neuroinflammation in the context of cerebral malaria. There are currently very few systematic data from West Africa on the aetiologies and management of non-malarial non-traumatic coma in small children, and NeuroCM will help to fill this gap. We postulate that an accurate understanding of molecular and cellular mechanisms involved in neuroinflammation may help to define efficient strategies to prevent and manage cerebral malaria. METHODS AND ANALYSIS: This is a prospective, case-control study comparing cerebral malaria to uncomplicated malaria and non-malarial non-traumatic coma. This study takes place in Benin, precisely in Cotonou for children with coma and in Sô-Ava district for children with uncomplicated malaria. We aim to include 300 children aged between 24 and 71 months and divided in three different clinical groups during 12 months (from December 2017 to November 2018) with a 21 to 28 days follow-up for coma. Study data, including clinical, biological and research results will be collected and managed using CSOnline-Ennov Clinical. ETHICS AND DISSEMINATION: Ethics approval for the NeuroCM study has been obtained from Comité National d'Ethique pour la Recherche en santé of Benin (n°67/MS/DC/SGM/DRFMT/CNERS/SA; 10/17/2017). NeuroCM study has also been approved by Comité consultatif de déontologie et d'éthique of Institut de Recherche pour le Développement (IRD; 10/24/2017). The study results will be disseminated through the direct consultations with the WHO's Multilateral Initiative on Malaria (TDR-MIM) and Roll Back Malaria programme, through scientific meetings and peer-reviewed publications in scientific or medical journals, and through guidelines and booklets.

Roles of APOL1 G1 and G2 variants in sickle cell disease patients: kidney is the main target.

In African-American patients with sickle cell disease (SCD), APOL1 G1 and G2 variants are associated with increased risk of sickle cell nephropathy (SCN). To determine the role of APOL1 variants in SCD patients living in Europe, we genotyped 152 SCD patients [aged 30·4 (24·3-36·4) years], mainly of Sub-Saharan African ancestry, for APOL1 G1 and G2 and for variants of four genes with kidney tropism (GSTM1, GSTT1, GSTP1, and HMOX1). Homozygous or double-heterozygous APOL G1 and G2 genotypes were strongly associated with end stage renal disease (P = 0·003) and worse Kidney Disease: Improving Global Outcomes stages (P = 0·001). Further, these genotypes were associated in an age-dependent manner with lower estimated glomerular filtration rate (eGFR, P = 0·008), proteinuria (P = 0·009) and albuminuria (P < 0·001) but not with other SCD complications. Compared to APOL1 G1/wild type (WT), the APOL1 G2/WT genotype was associated with a lower eGFR (P = 0·04) in an age-dependent manner, suggesting that the G2/WT patients are likely to have worse kidney prognosis. Other genes variants analysed were not associated with SCN or other SCD complications. Our data indicate that APOL1 screening should be considered for the management of SCD patients, including those of non-African-American origin, as those with homozygous or double heterozygous variants are clearly at higher risk of SCN.