A brain cyst load-associated antigen is a Toxoplasma gondii biomarker for serodetection of persistent parasites and chronic infection.

BACKGROUND: Biomarker discovery remains a major challenge for predictive medicine, in particular, in the context of chronic diseases. This is true for the widespread protozoan Toxoplasma gondii which establishes long-lasting parasitism in metazoans, humans included. This microbe successively unfolds distinct genetic programs that direct the transition from high to low replicative potential inside host cells. As a slow-replicating cell, the T. gondii bradyzoite developmental stage persists enclosed in a cyst compartment within tissues including the nervous system, being held by a sustained immune equilibrium which accounts for the prolonged clinically silent phase of parasitism. Serological surveys indicate that nearly one third of the human population has been exposed to T. gondii and possibly host bradyzoites. Because any disruption of the immune balance drives the reverse transition from bradyzoite to fast replicating tachyzoite and uncontrolled growth of the latter, these people are at risk for life-threatening disease. While serological tests for discriminating recent from past infection are available, there is yet no immunogenic biomarker used in the serological test to allow ascertaining the presence of persistent bradyzoites. RESULTS: Capitalizing on genetically engineered parasites induced to produce mature bradyzoites in vitro, we have identified the BCLA/MAG2 protein being restricted to the bradyzoite and the cyst envelope. Using laboratory mice as relevant T. gondii host models, we demonstrated that BCLA/MAG2 drives the generation of antibodies that recognize bradyzoite and the enveloping cyst structure. We have designed an ELISA assay based on a bacterially produced BCLA recombinant polypeptide, which was validated using a large collection of sera from mice of different genetic backgrounds and infected with bcla+ or bcla-null cystogenic and non-cystogenic T. gondii strains. To refine the design of the ELISA assay, we applied high-resolution BCLA epitope mapping and identified a specific combination of peptides and accordingly set up a selective and sensitive ELISA assay which allowed the detection of anti-BCLA/MAG2 antibodies in the sera of human patients with various forms of toxoplasmosis. CONCLUSIONS: We brought proof of principle that anti-BCLA/MAG2 antibodies serve as specific and sensitive serological markers in the perspective of a combinatorial strategy for detection of persistent T. gondii parasitism.

Evaluation of ID Fungi Plates Medium for Identification of Molds by MALDI Biotyper.

MALDI-TOF mass spectrometry (MS) identification of pathogenic filamentous fungi is often impaired by difficulties in harvesting hyphae embedded in the medium and long extraction protocols. The ID Fungi Plate (IDFP) is a novel culture method developed to address such difficulties and improve the identification of filamentous fungi by MALDI-TOF MS. We cultured 64 strains and 11 clinical samples on IDFP, Sabouraud agar-chloramphenicol (SAB), and ChromID Candida agar (CAN2). We then compared the three media for growth, ease of harvest, amount of material picked, and MALDI-TOF identification scores after either rapid direct transfer (DT) or a long ethanol-acetonitrile (EA) extraction protocol. Antifungal susceptibility testing and microscopic morphology after subculture on SAB and IDFP were also compared for ten molds. Growth rates and morphological aspects were similar for the three media. With IDFP, harvesting of fungal material for the extraction procedure was rapid and easy in 92.4% of cases, whereas it was tedious on SAB or CAN2 in 65.2% and 80.3% of cases, respectively. The proportion of scores above 1.7 (defined as acceptable identification) were comparable for both extraction protocols using IDFP (P = 0.256). Moreover, rates of acceptable identification after DT performed on IDFP (93.9%) were significantly higher than those obtained after EA extraction with SAB (69.7%) or CAN2 (71.2%) (P = <0.001 and P = 0.001, respectively). Morphological aspects and antifungal susceptibility testing were similar between IDFP and SAB. IDFP is a culture plate that facilitates and improves the identification of filamentous fungi, allowing accurate routine identification of molds with MALDI-TOF-MS using a rapid-extraction protocol.

Baseline Ex Vivo and Molecular Responses of Plasmodium falciparum Isolates to Piperaquine before Implementation of Dihydroartemisinin-Piperaquine in Senegal.

Dihydroartemisinin-piperaquine, which was registered in 2017 in Senegal, is not currently used as the first-line treatment against uncomplicated malaria. A total of 6.6% to 17.1% of P. falciparum isolates collected in Dakar in 2013 to 2015 showed ex vivo-reduced susceptibility to piperaquine. Neither the exonuclease E415G mutation nor the copy number variation of the plasmepsin II gene (Pfpm2), associated with piperaquine resistance in Cambodia, was detected in Senegalese parasites.

Are k13 and plasmepsin II genes, involved in Plasmodium falciparum resistance to artemisinin derivatives and piperaquine in Southeast Asia, reliable to monitor resistance surveillance in Africa?

Mutations in the propeller domain of Plasmodium falciparum kelch 13 (Pfk13) gene are associated with artemisinin resistance in Southeast Asia. Artemisinin resistance is defined by increased ring survival rate and delayed parasite clearance half-life in patients. Additionally, an amplification of the Plasmodium falciparum plasmepsin II gene (pfpm2), encoding a protease involved in hemoglobin degradation, has been found to be associated with reduced in vitro susceptibility to piperaquine in Cambodian P. falciparum parasites and with dihydroartemisinin-piperaquine failures in Cambodia. The World Health Organization (WHO) has recommended the use of these two genes to track the emergence and the spread of the resistance to dihydroartemisinin-piperaquine in malaria endemic areas. Although the resistance to dihydroartemisinin-piperaquine has not yet emerged in Africa, few reports on clinical failures suggest that k13 and pfpm2 would not be the only genes involved in artemisinin and piperaquine resistance. It is imperative to identify molecular markers or drug resistance genes that associate with artemisinin and piperaquine in Africa. K13 polymorphisms and Pfpm2 copy number variation analysis may not be sufficient for monitoring the emergence of dihydroartemisinin-piperaquine resistance in Africa. But, these markers should not be ruled out for tracking the emergence of resistance.

Low polymorphisms in pfact, pfugt and pfcarl genes in African Plasmodium falciparum isolates and absence of association with susceptibility to common anti-malarial drugs.

BACKGROUND: Resistance to all available anti-malarial drugs has emerged and spread including artemisinin derivatives and their partner drugs. Several genes involved in artemisinin and partner drugs resistance, such as pfcrt, pfmdr1, pfK13 or pfpm2, have been identified. However, these genes do not properly explain anti-malarial drug resistance, and more particularly clinical failures observed in Africa. Mutations in genes encoding for Plasmodium falciparum proteins, such as P. falciparum Acetyl-CoA transporter (PfACT), P. falciparum UDP-galactose transporter (PfUGT) and P. falciparum cyclic amine resistance locus (PfCARL) have recently been associated to resistance to imidazolopiperazines and other unrelated drugs. METHODS: Mutations on pfugt, pfact and pfcarl were characterized on 86 isolates collected in Dakar, Senegal and 173 samples collected from patients hospitalized in France after a travel in African countries from 2015 and 2016 to assess their potential association with ex vivo susceptibility to chloroquine, quinine, lumefantrine, monodesethylamodiaquine, mefloquine, dihydroartemisinin, artesunate, doxycycline, pyronaridine and piperaquine. RESULTS: No mutations were found on the genes pfugt and pfact. None of the pfcarl described mutations were identified in these samples from Africa. The K784N mutation was found in one sample and the K734M mutation was identified on 7.9% of all samples for pfcarl. The only significant differences in ex vivo susceptibility according to the K734M mutation were observed for pyronaridine for African isolates from imported malaria and for doxycycline for Senegalese parasites. CONCLUSION: No evidence was found of involvement of these genes in reduced susceptibility to standard anti-malarial drugs in African P. falciparum isolates.

[Current situation of malaria in the world]. / Situation du paludisme dans le monde.

Current situation of malaria in the world. In 2016, 216 million indigenous malaria cases were estimated in the world. Most of them were in Africa -90%-, followed by South-East Asia -7%- and Eastern Mediterranean Region. Almost half of the world population remains at risk of malaria in 2016. Of the 91 countries reporting indigenous malaria cases, 15 countries carried 80% of the global malaria burden. In 2016, 445 000 malaria deaths were estimated worldwide, and 91% were in Africa. Almost 66% of these deaths involved children under five year-old, a population that is particularly vulnerable to this disease. Despite significant funding for malaria control in 2015, the number of indigenous malaria cases has progressively increased since 2013 after a significant decrease between 2000 and 2013 and the number of malaria-related deaths has stagnated since 2015. Emergence of resistance to most antimalarial drugs used in prevention or treatment has been observed. Malaria control and elimination require sustained long-term efforts.

Situation du paludisme dans le monde. En 2016, 216 millions de cas de paludisme autochtone ont été estimés au niveau mondial, dont 90 % en Afrique, 7 % en Asie du Sud-Est et 2 % dans la région Méditerranée orientale. Près de la moitié de la population mondiale reste exposée au risque de paludisme en 2016. Quinze pays sur 91 ayant déclaré des cas de paludisme autochtone concentrent 80 % des cas enregistrés au niveau mondial. Le nombre de décès dus au paludisme à travers le monde a été estimé à 445 000 en 2016, et 91 % d'entre eux sont survenus en Afrique. Près des deux tiers de ces décès concernaient des enfants de moins de 5 ans, population particulièrement vulnérable face à cette maladie. En dépit de financements considérables dédiés à la lutte contre le paludisme en 2015, le nombre de cas de paludisme autochtone augmente progressivement depuis 2013 après une diminution importante entre 2000 et 2013, et le nombre de décès liés au paludisme stagne depuis 2015. Des émergences de résistance à la plupart des antipaludiques utilisés en prévention ou en traitement ont été observées. Le contrôle du paludisme et son élimination nécessitent des efforts soutenus à long terme.

Absence of a High Level of Duplication of the Plasmepsin II Gene in Africa.

Resistance to piperaquine has been associated with the amplification of the plasmepsin II gene in Cambodia. None of the 175 African isolates that we analyzed had plasmepsin II gene amplification (piperaquine 50% inhibitory concentration ranged from 0.94 to 137.5 nM), suggesting there is a low prevalence of piperaquine reduced susceptibility in Africa. Additionally, the few isolates with reduced susceptibility to piperaquine did not harbor amplification of the plasmepsin II gene.

The D113N mutation in the RING E3 ubiquitin protein ligase gene is not associated with ex vivo susceptibility to common anti-malarial drugs in African Plasmodium falciparum isolates.

BACKGROUND: Plasmodium falciparum resistance to artemisinin-based combination therapy has emerged and spread in Southeast Asia. In areas where artemisinin resistance is emerging, the efficacy of combination is now based on partner drugs. In this context, the identification of novel markers of resistance is essential to monitor the emergence and spread of resistance to these partner drugs. The ubiquitylation pathway could be a possible target for anti-malarial compounds and might be involved in resistance. Polymorphisms in the E3 ubiquitin-protein ligase (PF3D7_0627300) gene could be associated with decreased in vitro susceptibility to anti-malarial drugs. METHODS: Plasmodium falciparum isolates were collected from patients hospitalized in France with imported malaria from a malaria-endemic country from January 2015 to December 2016 and, more particularly, from African French-speaking countries. In total, 215 isolates were successfully sequenced for the E3 ubiquitin-protein ligase gene and assessed for ex vivo susceptibility to anti-malarial drugs. RESULTS: The D113N mutation in the RING E3 ubiquitin-protein ligase gene was present in 147 out of the 215 samples (68.4%). The IC50 values for the ten anti-malarial drugs were not significantly different between the wild-type and mutant parasites (p values between 0.225 and 0.933). There was no significant difference in terms of the percentage of parasites with decreased susceptibility between the D113 wild-type and the 133N mutated P. falciparum strains (p values between 0.541 and 1). CONCLUSION: The present data confirmed the absence of the association between polymorphisms in the RING E3 ubiquitin-protein ligase gene and the ex vivo susceptibility to common anti-malarial drugs in African P. falciparum isolates.

Serological diagnosis of toxoplasmosis: evaluation of the commercial test recomLine Toxoplasma IgG immunoblot (Mikrogen) based on recombinant antigens.

BACKGROUND: IgG detection to determine immune status to Toxoplasma gondii infection and seroconversion mainly relies on ELISA techniques and, if necessary, on a confirmatory test, Western blot. This study evaluated the performance of the recomLine Toxoplasma IgG immunoblot (IB-recomLine) (Mikrogen) as a confirmatory test on a large number of sera. A total of 171 sera were selected (113 patients) and had previously been analyzed by two ELISA tests, ARCHITECT (Abbott) and VIDAS (bioMérieux) ± LDBIO-Toxo II IgG Western blot (WB-LDBIO) (LDBio). The sera were classified into three groups: group 1 included 50 sera without difficulty in interpreting the IgG results (patients with documented past infection or uninfected); group 2 included 47 sera with difficulty in interpreting the ELISA results; and group 3 included 74 sequential sera from 25 pregnant women with seroconversion. RESULTS: In group 1, overall IgG agreements were 94% and 90% with ARCHITECT and VIDAS, respectively. In group 2, low agreement was observed between IB-recomLine and WB-LDBIO, with eight false-positive and 13 false-negative results. In group 3, 4/13 seroconversions were detected earlier with IB-recomLine compared to other tests. CONCLUSIONS: IB-recomLine allowed for earlier diagnosis of toxoplasmic seroconversion compared to both ELISA tests and WB-LDBIO but led to insufficient performance to confirm the immune status when ELISA results were discordant or equivocal.

Title: Diagnostic sérologique de la toxoplasmose : évaluation du test commercial recomLine Toxoplasma IgG immunoblot (Mikrogen) basé sur des antigènes recombinants. Abstract: Contexte : La détection des IgG pour déterminer le statut immunitaire vis-à-vis de l'infection à Toxoplasma gondii et de la séroconversion repose principalement sur les techniques ELISA et, si nécessaire, sur un test de confirmation, le Western blot. Cette étude a évalué les performances de l'immunoblot recomLine Toxoplasma IgG (IB-recomLine) (Mikrogen) en tant que test de confirmation sur un grand nombre de sérums. Un total de 171 sérums ont été sélectionnés (113 patients) et ont été préalablement analysés par deux tests ELISA, ARCHITECT (Abbott) et VIDAS (bioMérieux) ± LDBIO-Toxo II IgG Western blot (WB-LDBIO) (LDBio). Les sérums ont été classés en trois groupes : le groupe 1 comprenait 50 sérums sans difficulté d'interprétation des résultats IgG (patients avec antécédents d'infection documentée ou non infectés); le groupe 2 comprenait 47 sérums avec des difficultés d'interprétation des résultats ELISA; le groupe 3 comprenait 74 sérums séquentiels de 25 femmes enceintes séroconverties. Résultats : Dans le groupe 1, les concordances globales des IgG étaient respectivement de 94 % et 90 % avec ARCHITECT et VIDAS. Dans le groupe 2, une faible concordance a été observée entre IB-recomLine et WB-LDBIO, avec huit faux positifs et 13 faux négatifs. Dans le groupe 3, 4/13 séroconversions ont été détectées plus tôt avec IB-recomLine par rapport aux autres tests. Conclusions : IB-recomLine a permis un diagnostic plus précoce de la séroconversion toxoplasmique par rapport aux tests ELISA et WB-LDBIO, mais a conduit à des performances insuffisantes pour confirmer le statut immunitaire lorsque les résultats ELISA étaient discordants ou équivoques.

Molecular diagnosis of toxoplasmosis: recent advances and a look to the future.

INTRODUCTION: Toxoplasmosis is a globally distributed parasitic infection that can be particularly severe when opportunistic or congenital. Its diagnosis requires accurate and rapid techniques that rely mainly on serology and molecular methods. AREAS COVERED: The aim of this review was to discuss the positioning of the molecular diagnosis of toxoplasmosis according to the different clinical situations possibly resulting from infection with T. gondii, and to detail recent developments in this technique. The English and French literature were searched with the following keywords: 'Toxoplasmosis', "Molecular diagnosis" and 'PCR'. EXPERT OPINION: Molecular techniques have revolutionized the diagnosis of toxoplasmosis, and practices have considerably evolved over the past decades. However, there is still a high degree of inter-laboratory heterogeneity which impairs comparisons between results and studies. Efforts to standardize practices are underway.

MALDI-TOF MS in a Medical Mycology Laboratory: On Stage and Backstage.

The implementation of MALDI-TOF MS in medical microbiology laboratories has revolutionized practices and significantly reduced turnaround times of identification processes. However, although bacteriology quickly benefited from the contributions of this technique, adjustments were necessary to accommodate the specific characteristics of fungi. MALDI-TOF MS is now an indispensable tool in clinical mycology laboratories, both for the identification of yeasts and filamentous fungi, and other innovative uses are gradually emerging. Based on the practical experience of our medical mycology laboratory, this review will present the current uses of MALDI-TOF MS and the adaptations we implemented, to allow their practical execution in a daily routine. We will also introduce some less mainstream applications, like those for fungemia, or even still under development, as is the case for the determination of sensitivity to antifungal agents or typing methods.

Absence of Association between Methylene Blue Reduced Susceptibility and Polymorphisms in 12 Genes Involved in Antimalarial Drug Resistance in African Plasmodium falciparum.

Half the human population is exposed to malaria. Plasmodium falciparum antimalarial drug resistance monitoring and development of new drugs are major issues related to the control of malaria. Methylene blue (MB), the oldest synthetic antimalarial, is again a promising drug after the break of its use as an antimalarial drug for more than 80 years and a potential partner for triple combination. Very few data are available on the involvement of polymorphisms on genes known to be associated with standard antimalarial drugs and parasite in vitro susceptibility to MB (cross-resistance). In this context, MB susceptibility was evaluated against 482 isolates of imported malaria from Africa by HRP2-based ELISA chemosusceptibility assay. A total of 12 genes involved in antimalarial drug resistance (Pfcrt, Pfdhfr, Pfmdr1, Pfmdr5, Pfmdr6, PfK13, Pfubq, Pfcarl, Pfugt, Pfact, Pfcoronin, and copy number of Pfpm2) were sequenced by Sanger method and quantitative PCR. On the Pfmdr1 gene, the mutation 86Y combined with 184F led to more susceptible isolates to MB (8.0 nM vs. 11.6 nM, p = 0.03). Concerning Pfmdr6, the isolates bearing 12 Asn repetitions were more susceptible to MB (4.6 nM vs. 11.6 nM, p = 0.005). None of the polymorphisms previously described as involved in antimalarial drug resistance was shown to be associated with reduced susceptibility to MB. Some genes (particularly PfK13, Pfugt, Pfact, Pfpm2) did not present enough genetic variability to draw conclusions about their involvement in reduced susceptibility to MB. None of the polymorphisms analyzed by multiple correspondence analysis (MCA) had an impact on the MB susceptibility of the samples successfully included in the analysis. It seems that there is no in vitro cross-resistance between MB and commonly used antimalarial drugs.