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.

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.

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.

RecQ helicases in the malaria parasite Plasmodium falciparum affect genome stability, gene expression patterns and DNA replication dynamics.

The malaria parasite Plasmodium falciparum has evolved an unusual genome structure. The majority of the genome is relatively stable, with mutation rates similar to most eukaryotic species. However, some regions are very unstable with high recombination rates, driving the generation of new immune evasion-associated var genes. The molecular factors controlling the inconsistent stability of this genome are not known. Here we studied the roles of the two putative RecQ helicases in P. falciparum, PfBLM and PfWRN. When PfWRN was knocked down, recombination rates increased four-fold, generating chromosomal abnormalities, a high rate of chimeric var genes and many microindels, particularly in known 'fragile sites'. This is the first identification of a gene involved in suppressing recombination and maintaining genome stability in Plasmodium. By contrast, no change in mutation rate appeared when the second RecQ helicase, PfBLM, was mutated. At the transcriptional level, however, both helicases evidently modulate the transcription of large cohorts of genes, with several hundred genes-including a large proportion of vars-showing deregulated expression in each RecQ mutant. Aberrant processing of stalled replication forks is a possible mechanism underlying elevated mutation rates and this was assessed by measuring DNA replication dynamics in the RecQ mutant lines. Replication forks moved slowly and stalled at elevated rates in both mutants, confirming that RecQ helicases are required for efficient DNA replication. Overall, this work identifies the Plasmodium RecQ helicases as major players in DNA replication, antigenic diversification and genome stability in the most lethal human malaria parasite, with important implications for genome evolution in this pathogen.

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.

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.

Identification of the centromeres of Leishmania major: revealing the hidden pieces.

Leishmania affects millions of people worldwide. Its genome undergoes constitutive mosaic aneuploidy, a type of genomic plasticity that may serve as an adaptive strategy to survive distinct host environments. We previously found high rates of asymmetric chromosome allotments during mitosis that lead to the generation of such ploidy. However, the underlying molecular events remain elusive. Centromeres and kinetochores most likely play a key role in this process, yet their identification has failed using classical methods. Our analysis of the unconventional kinetochore complex recently discovered in Trypanosoma brucei (KKTs) leads to the identification of a Leishmania KKT gene candidate (LmKKT1). The GFP-tagged LmKKT1 displays "kinetochore-like" dynamics of intranuclear localization throughout the cell cycle. By ChIP-Seq assay, one major peak per chromosome is revealed, covering a region of 4 ±2 kb. We find two largely conserved motifs mapping to 14 of 36 chromosomes while a higher density of retroposons are observed in 27 of 36 centromeres. The identification of centromeres and of a kinetochore component of Leishmania chromosomes opens avenues to explore their role in mosaic aneuploidy.

The nucleoporin Mlp2 is involved in chromosomal distribution during mitosis in trypanosomatids.

Nucleoporins are evolutionary conserved proteins mainly involved in the constitution of the nuclear pores and trafficking between the nucleus and cytoplasm, but are also increasingly viewed as main actors in chromatin dynamics and intra-nuclear mitotic events. Here, we determined the cellular localization of the nucleoporin Mlp2 in the 'divergent' eukaryotes Leishmania major and Trypanosoma brucei. In both protozoa, Mlp2 displayed an atypical localization for a nucleoporin, essentially intranuclear, and preferentially in the periphery of the nucleolus during interphase; moreover, it relocated at the mitotic spindle poles during mitosis. In T. brucei, where most centromeres have been identified, TbMlp2 was found adjacent to the centromeric sequences, as well as to a recently described unconventional kinetochore protein, in the periphery of the nucleolus, during interphase and from the end of anaphase onwards. TbMlp2 and the centromeres/kinetochores exhibited a differential migration towards the poles during mitosis. RNAi knockdown of TbMlp2 disrupted the mitotic distribution of chromosomes, leading to a surprisingly well-tolerated aneuploidy. In addition, diploidy was restored in a complementation assay where LmMlp2, the orthologue of TbMlp2 in Leishmania, was expressed in TbMlp2-RNAi-knockdown parasites. Taken together, our results demonstrate that Mlp2 is involved in the distribution of chromosomes during mitosis in trypanosomatids.

First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites.

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR-Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod-2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off-target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR-Cas9-mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis.

TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals.

TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.

RNA interference screen reveals a high proportion of mitochondrial proteins essential for correct cell cycle progress in Trypanosoma brucei.

BACKGROUND: Trypanosomatid parasites possess a single mitochondrion which is classically involved in the energetic metabolism of the cell, but also, in a much more original way, through its single and complex DNA (termed kinetoplast), in the correct progress of cell division. In order to identify proteins potentially involved in the cell cycle, we performed RNAi knockdowns of 101 genes encoding mitochondrial proteins using procyclic cells of Trypanosoma brucei. RESULTS: A major cell growth reduction was observed in 10 cases and a moderate reduction in 29 other cases. These data are overall in agreement with those previously obtained by a case-by-case approach performed on chromosome 1 genes, and quantitatively with those obtained by "high-throughput phenotyping using parallel sequencing of RNA interference targets" (RIT-seq). Nevertheless, a detailed analysis revealed many qualitative discrepancies with the RIT-seq-based approach. Moreover, for 37 out of 39 mutants for which a moderate or severe growth defect was observed here, we noted abnormalities in the cell cycle progress, leading to increased proportions of abnormal cell cycle stages, such as cells containing more than 2 kinetoplasts (K) and/or more than 2 nuclei (N), and modified proportions of the normal phenotypes (1N1K, 1N2K and 2N2K). CONCLUSIONS: These data, together with the observation of other abnormal phenotypes, show that all the corresponding mitochondrial proteins are involved, directly or indirectly, in the correct progress or, less likely, in the regulation, of the cell cycle in T. brucei. They also show how post-genomics analyses performed on a case-by-case basis may yield discrepancies with global approaches.

Multicentric comparative assessment of the bio-evolution Toxoplasma gondii detection kit with eight laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis.

The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents.

Molecular diagnosis of toxoplasmosis in immunocompromised patients: a 3-year multicenter retrospective study.

Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of Toxoplasma gondii DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (n = 337 samples), cerebrospinal fluid (n = 101 samples), and aqueous humor (n = 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV(+); the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (P < 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.

Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays.

The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 10(4) to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was ≥ 6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections.

Novel insights into genome plasticity in Eukaryotes: mosaic aneuploidy in Leishmania.

Leishmania are unicellular eukaryotes that have many markedly original molecular features compared with other uni- or multicellular eukaryotes like yeasts or mammals. Genome plasticity in this parasite has been the subject of many publications, and has been associated with drug resistance or adaptability. Aneuploidy has been suspected by several authors and it is now confirmed using state-of-the-art technologies such as high-throughput DNA sequencing. The analysis of genome contents at the single cell level using fluorescence in situ hybridization (FISH) has brought a new light on the genome organization: within a cell population, every chromosome, in every cell, may be present in at least two ploidy states (being either monosomic, disomic or trisomic), and the chromosomal content varies greatly from cell to cell, thus generating a constitutive intra-strain genomic heterogeneity, here termed 'mosaic aneuploidy'. Mosaic aneuploidy deeply affects the genetics of these organisms, leading, for example, to an extreme degree of intra-strain genomic diversity, as well as to a clearance of heterozygous cells in the population without however affecting genetic heterogeneity. Second, mosaic aneuploidy might be considered as a powerful strategy evolved by the parasite for adapting to modifications of environment conditions as well as for the emergence of drug resistance. On the whole, mosaic aneuploidy may be considered as a novel mechanism for generating phenotypic diversity driven by genomic plasticity.

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.

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.

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.

Comparative assessment of a commercial kit and two laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis.

Toxoplasmosis is a worldwide infection that may cause severe disease and is regarded as a serious health problem in France. Detection of the parasite by molecular methods is crucial for diagnosing the disease. The extreme diversity of methods and performances of Toxoplasma PCR assays makes the use of commercial PCR kits an attractive alternative, as they offer a chance for standardization. We compared the performances of three molecular methods for the detection of Toxoplasma gondii DNA in amniotic fluid: a commercial method using nested PCR and two laboratory-developed methods, one using conventional PCR and the other one real-time PCR. This evaluation was based upon a T. gondii DNA serial dilution assay, three amniotic fluid samples spiked with T. gondii at different concentrations, and a clinical cohort of 33 amniotic fluid samples. The T. gondii DNA serial dilution assay showed a much lower sensitivity for the commercial kit than for the laboratory-developed methods. Moreover, out of 12 proven congenital toxoplasmosis cases, 91.7% were detected by the laboratory-developed assays, whereas only 50% were detected by the commercial kit. A lack of sensitivity of the method, partly due to the presence of PCR inhibitors, was the main drawback of the commercial method. This study emphasizes that commercial PCR diagnostic kits do not systematically perform better than carefully optimized laboratory-developed methods. There is a need for thorough evaluation of such kits by proficient groups, as well as for performance standards that commercial kits can be tested against to improve confidence in those selected by health care providers.