Multicenter evaluation of the Toxoplasma gondii Real-TM (Sacace) kit performance for the molecular diagnosis of toxoplasmosis.

The molecular detection of Toxoplasma gondii DNA is a key tool for the diagnosis of disseminated and congenital toxoplasmosis. This multicentric study from the Molecular Biology Pole of the French National Reference Center for toxoplasmosis aimed to evaluate Toxoplasma gondii Real-TM PCR kit (Sacace). The study compared the analytical and clinical performances of this PCR assay with the reference PCRs used in proficient laboratories. PCR efficiencies varied from 90% to 112%; linearity zone extended over four log units (R2 > 0.99) and limit of detection varied from 0.01 to ≤1 Tg/mL depending on the center. Determined on 173 cryopreserved DNAs from a large range of clinical specimens, clinical sensitivity was 100% [106/106; 95 confidence interval (CI): 96.5%-100%] and specificity was 100% (67/67; 95 CI: 94.6%-100%). The study revealed two potential limitations of the Sacace PCR assay: the first was the inconsistency of the internal control (IC) when added to the PCR mixture. This point was not found under routine conditions when the IC was added during the extraction step. The second is a lack of practicality, as the mixture is distributed over several vials, requiring numerous pipetting operations. Overall, this study provides useful information for the molecular diagnosis of toxoplasmosis; the analytical and clinical performances of the Sacace PCR kit were satisfactory, the kit having sensitivity and specificity similar to those of expert center methods and being able to detect low parasite loads, at levels where multiplicative analysis gives inconsistently positive results. Finally, the study recommends multiplicative analysis in particular for amniotic fluids, aqueous humor, and other single specimens.

Molecular diagnosis of visceral leishmaniasis: a French study comparing a reference PCR method targeting kinetoplast DNA and a commercial kit targeting ribosomal DNA.

IMPORTANCE: PCR revolutionized the direct diagnosis of infectious diseases, especially protozooses, where the infectious load is usually low. Commercial PCR methods are available and offer many advantages, including convenience and batch tracking as part of a quality system. For most parameters, the performance of commercial methods is at least as good as that of finely optimized methods developed in expert laboratories. This comparison work has not been done for the molecular diagnosis of visceral leishmaniasis. Leishmania sp. has a unique organelle, the kinetoplast, which corresponds to the mitochondrial DNA. It is organized into a large number of minicircles, which has made it a target for the development of diagnostic PCR. The quanty Leishmaniae, Clonit kit targeting ribosomal DNA was compared to a widely used laboratory-developed method based on kinetoplast DNA. This reference method gave significantly better results, probably due to the difference in the number of repeats of the PCR targets.

Assessment of Droplet Digital PCR for the Detection and Absolute Quantification of Toxoplasma gondii: A Comparative Retrospective Study.

Accurate tools for Toxoplasma gondii detection and quantification can be valuable for the early and effective management of toxoplasmosis. Droplet digital PCR (ddPCR) is a next-generation end-point PCR technique with high performance. The objective of the study was to evaluate the performance of ddPCR for the detection and absolute quantification of T. gondii. From January 2019 to October 2020, DNA samples collected at the Laboratory of Parasitology and Mycology of Pitié-Salpêtrière Hospital in Paris were retrospectively analyzed by ddPCR and real-time quantitative PCR (qPCR). To detect T. gondii with the best sensitivity possible, the REP-529 multicopy target was used. For absolute quantification of T. gondii, a specific single-copy target of α-tubulin was designed. T. gondii detection by ddPCR and qPCR was strongly correlated (R2 = 0.93), with a total concordance of 96.7% (n = 145/150). Quantification of T. gondii using ddPCR was successful for 15 of 35 samples showing a parasite load ≥170 copies/mL of DNA eluate using the α-tubulin target. The qPCR REP-529 quantification based on a standard curve was approximate and dependent on the strain genotype, which led to an estimate of parasite copy number 14- to 160-fold superior to the ddPCR result. In total, ddPCR is an effective molecular method for T. gondii detection that shows equivalent performance to qPCR. For robust T. gondii quantification, ddPCR is clearly more accurate than semiquantitative qPCR methods.

Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth.

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1-3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.

Leishmaniasis epidemiology in endemic areas of metropolitan France and its overseas territories from 1998 to 2020.

BACKGROUND: In France, leishmaniasis is endemic in the Mediterranean region, in French Guiana and to a lesser extent, in the French West Indies. This study wanted to provide an updated picture of leishmaniasis epidemiology in metropolitan France and in its overseas territories. METHODOLOGY/PRINCIPAL FINDINGS: Leishmaniasis cases were collected by passive notification to the French National Reference Centre for Leishmaniases (NRCL) in Montpellier from 1998 to 2020 and at the associated Centre in Cayenne (French Guiana) from 2003 to 2020. In metropolitan France, 517 autochthonous leishmaniasis cases, mostly visceral forms due to Leishmania infantum (79%), and 1725 imported cases (French Guiana excluded), mainly cutaneous leishmaniasis from Maghreb, were recorded. A slight decrease of autochthonous cases was observed during the survey period, from 0.48 cases/100,000 inhabitants per year in 1999 (highest value) to 0.1 cases/100,000 inhabitants per year in 2017 (lowest value). Conversely, imported cases increased over time (from 59.7 in the 2000s to 94.5 in the 2010s). In French Guiana, 4126 cutaneous and mucocutaneous leishmaniasis cases were reported from 2003 to 2020. The mean incidence was 103.3 cases per 100,000 inhabitants/year but varied in function of the year (from 198 in 2004 to 54 in 2006). In Guadeloupe and Martinique (French West Indies), only sporadic cases were reported. CONCLUSIONS/SIGNIFICANCE: Because of concerns about disease expansion and outbreaks in other Southern Europe countries, and leishmaniasis monitoring by the NRCL should be continued and associated with a more active surveillance.

Using haematophagous fly blood meals to study the diversity of blood-borne pathogens infecting wild mammals.

Many emerging infectious diseases originate from wild animals, so there is a profound need for surveillance and monitoring of their pathogens. However, the practical difficulty of sample acquisition from wild animals tends to limit the feasibility and effectiveness of such surveys. Xenosurveillance, using blood-feeding invertebrates to obtain tissue samples from wild animals and then detect their pathogens, is a promising method to do so. Here, we describe the use of tsetse fly blood meals to determine (directly through molecular diagnostic and indirectly through serology), the diversity of circulating blood-borne pathogens (including bacteria, viruses and protozoa) in a natural mammalian community of Tanzania. Molecular analyses of captured tsetse flies (182 pools of flies totalizing 1728 flies) revealed that the blood meals obtained came from 18 different vertebrate species including 16 non-human mammals, representing approximately 25% of the large mammal species present in the study area. Molecular diagnostic demonstrated the presence of different protozoa parasites and bacteria of medical and/or veterinary interest. None of the six virus species searched for by molecular methods were detected but an ELISA test detected antibodies against African swine fever virus among warthogs, indicating that the virus had been circulating in the area. Sampling of blood-feeding insects represents an efficient and practical approach to tracking a diversity of pathogens from multiple mammalian species, directly through molecular diagnostic or indirectly through serology, which could readily expand and enhance our understanding of the ecology and evolution of infectious agents and their interactions with their hosts in wild animal communities.

Molecular Diagnosis of Toxoplasmosis: Multicenter Evaluation of the Toxoplasma RealCycler Universal PCR Assay on 168 Characterized Human Samples.

Real-time PCR plays a crucial role in the diagnosis of toxoplasmosis. In this multicenter study, the Toxoplasma RealCycler Universal assay was assessed for the diagnosis of toxoplasmosis by eight reference laboratories. DNAs from diverse clinical samples were included: 141 characterized samples from patients with different clinical forms of proven toxoplasmosis and 27 from patients without toxoplasmosis were tested in duplicate with the commercial assay. Final diagnosis was affirmed by each center by analysis of clinical settings and biological follow-up. Calibrated Toxoplasma gondii standards and 11 external quality control samples were also included. Discrepant results observed after the first run of commercial PCR were controlled by both reference and commercial PCR assays. Using the commercial assay, the detection threshold varied from 0.01 to 1 tachyzoites/mL, depending on the center. The relationship between crossing point and DNA concentration was linear over 4 log units (r2 > 0.99), and PCR efficiencies were satisfactory (89% to 104%). The results of the 11 external quality control samples were concordant after one retesting, but those for 3 clinical samples remained discrepant. Sensitivity and specificity were calculated at 97.8% (95% CI, 97.8%-100%) and 100% (95% CI, 87.2%-100%), respectively. Provided that PCRs are performed at least in duplicate to detect low parasitic loads, Toxoplasma RealCycler Universal PCR showed suitable performances to diagnose the different forms of toxoplasmosis.

High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani.

Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.

The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis, cell division and virulence in the mammalian host.

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.

Multicenter Comparative Assessment of the TIB MolBiol Toxoplasma gondii Detection Kit and Four Laboratory-Developed PCR Assays for Molecular Diagnosis of Toxoplasmosis.

Toxoplasmosis can be a life-threatening infection, particularly during pregnancy and in immunocompromised patients. The biological diagnosis of toxoplasmosis is challenging and has been revolutionized by molecular detection methods. This article summarizes the data of a multicenter study involving four centers to assess the performances of a commercial PCR assay as compared with four in-house PCR assays using Toxoplasma gondii standards, 20 external quality control specimens, and 133 clinical samples. This clinical cohort includes well-characterized clinical samples corresponding to different clinical situations: confirmed congenital toxoplasmosis (44 samples), toxoplasmosis in immunocompromised patients (25 samples), and chorioretinitis (5 samples). Furthermore, 59 samples from patients without toxoplasmosis were included as negative controls. The analytical sensitivities of the five methods tested were very similar; and the limit of Toxoplasma DNA detection was around 0.01 T. gondii genome per reaction for all the methods. The overall concordance between the commercial PCR and the four in-house PCR assays was 97.7% (130/133). The clinical sensitivity and specificity were >98% and could be increased for the commercial kit when PCR was performed in multiplicate to detect low parasitic loads. In conclusion, the commercial PCR assay shows suitable performances to diagnose the different clinical forms of toxoplasmosis.

Evaluation of Two Commercial Kits on the Automated ELITe InGenius PCR Platform for Molecular Diagnosis of Toxoplasmosis.

Molecular diagnosis of toxoplasmosis is essential for establishing the diagnosis of congenital contaminations and for primary infection or reactivation of immunocompromised patients. An integrated extraction and real-time PCR-based system is of particular interest in this context. Commercial kits for automated extraction and amplification steps are now available. Herein, we assessed two commercial PCR assays for this diagnosis, those of Bio-Evolution and Elitech, on the ELITe InGenius platform. The Bio-Evolution assay showed a specificity and a sensitivity of 100% on clinical samples, but a lower analytical detection threshold than the Elitech assay. The latter showed a specificity of 100% and a sensitivity of 96%. The SP1000 cartridges, which allow DNA extraction from 1 mL of template, showed interesting performances on amniotic fluid samples. Overall, the two kits had good performances on the InGenius platform, which offers a turn-key solution suitable for the molecular diagnosis of toxoplasmosis.

Impact of pre-analytic step duration on molecular diagnosis of toxoplasmosis for five types of biological samples.

INTRODUCTION: Toxoplasma-PCR is essential to diagnose ocular, cerebral, disseminated and congenital toxoplasmosis. This multicenter study evaluated the impact of sample storage duration at +4°C on PCR assay performances in order to propose guidelines for the storage of samples during shipment or/and before PCR. MATERIALS AND METHODS: Five matrices, amniotic (AF), cerebrospinal (CSF), and bronchoalveolar lavage fluids (BALF), whole blood (WB) and buffy coat (BC), were artificially spiked with different amounts of Toxoplasma gondii (20, 100, 500 tachyzoites per mL of sample) or with previously infected THP1 cells. DNA extractions were performed at day 0 and after 2, 4 and 7 days of storage at +4°C. Each extract was amplified at least twice by real-time PCR. RESULTS: A total of 252 spiked samples was studied. No increase of crossing point was observed and all samples were positive for AF, BALF, BC and infected THP1-spiked WB after up to 7 days at 4°C. For CSF spiked with 20 parasites/mL, only 50% of PCR reactions were positive at D7 (p<0.05). For WB spiked with type II parasites, all reactions remained positive at D7 but amplifications were significantly delayed from D2; and for WB spiked with RH strain, the proportion of positive reactions decreased at D7. CONCLUSION: The storage of clinical samples at +4°C is compatible with the molecular detection of T. gondii parasites. Provided that PCR assays are performed in duplicate, storage of samples is possible up to 7 days. However, from the fifth day onwards, and for samples susceptible to contain low parasitic loads, we recommend to perform the PCR in multiplicate.

Gene Editing in Trypanosomatids: Tips and Tricks in the CRISPR-Cas9 Era.

Gene editing in trypanosomatids has long been proven difficult. The development of CRISPR-Cas9 has improved this issue, opening the way to a better understanding of biological processes and drug-resistance mechanisms, and screening of drug targets. Different strategies have now been developed: either PCR- or plasmid-based, differing mainly in the nature of the donor DNA and the single guide RNA transcription. Here we review the main genetic tools available for Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei for gene tagging, single-base editing, and deletion of nonessential and essential genes. We discuss the main advantages and challenges of different strategies and how to choose 'the right cut' depending on the importance of untranslated regions. These considerations allow selection of the most accurate gene editing approach for a given functional analysis.

Rapid diagnostic tests failing to detect infections by Plasmodium falciparum encoding pfhrp2 and pfhrp3 genes in a non-endemic setting.

BACKGROUND: Rapid diagnostic tests (RDTs) detecting the histidine-rich protein 2 (PfHRP2) have a central position for the management of Plasmodium falciparum infections. Yet, variable detection of certain targeted motifs, low parasitaemia, but also deletion of pfhrp2 gene or its homologue pfhrp3, may result in false-negative RDT leading to misdiagnosis and delayed treatment. This study aimed at investigating the prevalence, and understanding the possible causes, of P. falciparum RDT-negative infections at Montpellier Academic Hospital, France. METHODS: The prevalence of falsely-negative RDT results reported before and after the introduction of a loop-mediated isothermal amplification (LAMP) assay, as part as the malaria screening strategy in January 2017, was analysed. Negative P. falciparum RDT infections were screened for pfhrp2 or pfhrp3 deletion; and exons 2 were sequenced to show a putative genetic diversity impairing PfHRP2 detection. RESULTS: The overall prevalence of P. falciparum negative RDTs from January 2006 to December 2018 was low (3/446). Whereas no cases were reported from 2006 to 2016 (0/373), period during which the malaria diagnostic screen was based on microscopy and RDT, prevalence increased up to 4.1% (3/73) between 2017 and 2018, when molecular detection was implemented for primary screening. Neither pfhrp2/3 deletion nor major variation in the frequency of repetitive epitopes could explain these false-negative RDT results. CONCLUSION: This paper demonstrates the presence of pfhrp2 and pfhrp3 genes in three P. falciparum RDT-negative infections and reviews the possible reasons for non-detection of HRP2/3 antigens in a non-endemic setting. It highlights the emergence of falsely negative rapid diagnostic tests in a non-endemic setting and draws attention on the risk of missing malaria cases with low parasitaemia infections using the RDT plus microscopy-based strategy currently recommended by French authorities. The relevance of a novel diagnostic scheme based upon a LAMP assay is discussed.

Universal highly efficient conditional knockout system in Leishmania, with a focus on untranscribed region preservation.

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.

Evolutionary Divergence of Enzymatic Mechanisms for Tubulin Detyrosination.

The two related members of the vasohibin family, VASH1 and VASH2, encode human tubulin detyrosinases. Here we demonstrate that, in contrast to VASH1, which requires binding of small vasohibin binding protein (SVBP), VASH2 has autonomous tubulin detyrosinating activity. Moreover, we demonstrate that SVBP acts as a bona fide activator of both enzymes. Phylogenetic analysis of the vasohibin family revealed that regulatory diversification of VASH-mediated tubulin detyrosination coincided with early vertebrate evolution. Thus, as a model organism for functional analysis, we used Trypanosoma brucei (Tb), an evolutionarily early-branched eukaryote that possesses a single VASH and encodes a terminal tyrosine on both α- and ß-tubulin tails, both subject to removal. Remarkably, although detyrosination levels are high in the flagellum, TbVASH knockout parasites did not present any noticeable flagellar abnormalities. In contrast, we observed reduced proliferation associated with profound morphological and mitotic defects, underscoring the importance of tubulin detyrosination in cell division.

Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0%), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden's index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

A single amino acid substitution (H451Y) in Leishmania calcium-dependent kinase SCAMK confers high tolerance and resistance to antimony.

BACKGROUND: For almost a century, antimonials have remained the first-line drugs for the treatment of leishmaniasis. However, little is known about their mode of action and clinical resistance mechanisms. OBJECTIVES: We have previously shown that Leishmania nicotinamidase (PNC1) is an essential enzyme for parasite NAD+ homeostasis and virulence in vivo. Here, we found that parasites lacking the pnc1 gene (Δpnc1) are hypersusceptible to the active form of antimony (SbIII) and used these mutant parasites to better understand antimony's mode of action and the mechanisms leading to resistance. METHODS: SbIII-resistant WT and Δpnc1 parasites were selected in vitro by a stepwise selection method. NAD(H)/NADP(H) dosages and quantitative RT-PCR experiments were performed to explain the susceptibility differences observed between strains. WGS and a marker-free CRISPR/Cas9 base-editing approach were used to identify and validate the role of a new resistance mutation. RESULTS: NAD+-depleted Δpnc1 parasites were highly susceptible to SbIII and this phenotype could be rescued by NAD+ precursor or trypanothione precursor supplementation. Δpnc1 parasites could become resistant to SbIII by an unknown mechanism. WGS revealed a unique amino acid substitution (H451Y) in an EF-hand domain of an orphan calcium-dependent kinase, recently named SCAMK. When introduced into a WT reference strain by base editing, the H451Y mutation allowed Leishmania parasites to survive at extreme concentrations of SbIII, potentiating the rapid emergence of resistant parasites. CONCLUSIONS: These results establish that Leishmania SCAMK is a new central hub of antimony's mode of action and resistance development, and uncover the importance of drug tolerance mutations in the evolution of parasite drug resistance.

The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing.

DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2-7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2-7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, demonstrating their relevance as OBI1 substrates for origin firing. Our results identify a ubiquitin signalling pathway involved in origin activation and provide a candidate protein for selecting the origins to be fired.

Molecular diagnosis of toxoplasmosis at the onset of symptomatic primary infection: A straightforward alternative to serological examinations.

Myopericarditis is a rare but well-documented clinical presentation of primary Toxoplasma gondii infection in immunocompetent patients. Here, early detection of Toxoplasma DNA in the peripheral blood by PCR allowed the diagnosis of acute toxoplasmosis while serological tests were negative. Additional serological evaluations 2 weeks later confirmed the diagnosis and showed that cardiac manifestations occurred before seroconversion. This highlights the importance of a second serological control in the case of a suspected active infection. Overall, we show here that PCR testing for Toxoplasma is a sensitive and straightforward alternative to serological examinations.