Could PLATELIA Toxo IgM be the new gold standard for the serological diagnosis of congenital toxoplasmosis: a French multicenter study.

To assess the performance of PLATELIA Toxo IgM (Bio-Rad) and Toxo ISAGA (BioMérieux) to detect anti-Toxoplasma IgM in infants at risk of congenital toxoplasmosis, a retrospective multicenter study was conducted comparing serological results obtained in the framework of routine diagnosis work-up for congenital toxoplasmosis. All infants born to mothers infected with T. gondii during pregnancy from 2010 to 2020 with at least 6 months of serological follow-up were included (n = 1,010). One thousand ten cases were included, of which 250 infants (24.75%) had congenital toxoplasmosis. A total of 1039 sera were included. The concordance between the two techniques was 96%, with kappa coefficient of 0.87, showing an almost perfect agreement between ISAGA and PLATELIA. Cumulative sensitivity and specificity were 73.2% and 99.5.% and 74.8% and 100% for ISAGA and PLATELIA, respectively. The mean time to detect IgM using ISAGA and PLATELIA tests was 6.9 ± 20.1 days and 5.6 ± 14.7 days, respectively not significant (ns). Finally, the sensitivity of ISAGA and PLATELIA to detect IgM antibodies in infected neonates at 5 days of life was 62% and 64%, respectively. Performances of PLATELIA Toxo IgM assay were comparable to the gold standard ISAGA. This enzyme-linked immunosorbent assay is suitable for routine serology for the diagnosis of congenital toxoplasmosis in newborns. IMPORTANCE This study will help clinical microbiologists to chose an alternative serological method for the neonatal diagnosis of congenital toxoplasmosis, once the gold standard technique ISAGA will be withdrawn next year.

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.

Human Outbreak of Trichinellosis Caused by Trichinella papuae Nematodes, Central Kampong Thom Province, Cambodia.

In September 2017, a severe trichinellosis outbreak occurred in Cambodia after persons consumed raw wild pig meat; 33 persons were infected and 8 died. We collected and analyzed the medical records for 25 patients. Clinical signs and symptoms included myalgia, facial or peripheral edema, asthenia, and fever. We observed increased levels of creatine phosphokinase and aspartate aminotransferase-, as well as eosinophilia. Histopathologic examination of muscle biopsy specimens showed nonencapsulated Trichinella larvae. A Trichinella excretory/secretory antigen ELISA identified Trichinella IgM and IgG. Biopsy samples were digested and larvae were isolated and counted. PCR for the 5S rDNA intergenic spacer region and a multiplex PCR, followed by sequencing identified the parasite as Trichinella papuae. This species was identified in Papua New Guinea during 1999 and in several outbreaks in humans in Thailand. Thus, we identified T. papuae nematodes in humans in Cambodia.

Multicentric Evaluation of the Bio-Evolution Toxoplasma gondii Assay for the Detection of Toxoplasma DNA in Immunocompromised Patients.

PCR-based methods are a key tool for the diagnosis of toxoplasmosis in immunocompromised patients. Laboratory-developed protocols lack standardization. This study aimed to assess the performances of a commercial kit for the detection of Toxoplasma DNA in different specimens drawn from immunocompromised patients. This multicentric retrospective study included 227 DNA specimens (157 blood specimens, 22 bronchoalveolar fluid [BALF] specimens, 39 cerebrospinal fluid [CSF] specimens, and 9 miscellaneous specimens) collected between 2010 and 2015 from 126 immunocompromised patients. The specimens were selected based on previous laboratory-developed quantitative PCR (qPCR) analyses targeting either the rep529 element or the B1 gene, and the results were classified as positive, negative, and "negative of interest," where the latter was defined as representing either the last specimen with a negative result before a positive one or the first with a negative result following a positive result(s). All specimens were secondary tested using the Bio-Evolution Toxoplasma DNA assay targeting the T. gondii rep529 element. We found a 95.6% concordance rate for qualitative results obtained with laboratory-developed qPCR techniques and the commercial kit. The rate reached 99.3% in comparisons of rep529-based laboratory-developed PCR methods and the commercial kit. The quantifications obtained with the commercial kit and the rep529 laboratory-developed PCRs were in very good agreement. Sensitivity and specificity of the commercial kit were calculated at 98.8% and 100%, respectively. The Bio-Evolution Toxoplasma DNA assay appears to be a valuable method for the detection of Toxoplasma DNA in blood, BALF, and CSF specimens from immunocompromised patients.

A common source for a trichinellosis outbreak reported in France and Serbia in 2017.

Trichinellosis is a rare parasitic zoonosis in the European Union. Meat from backyard pigs was the common source for a trichinellosis outbreak caused by Trichinella spiralis, which occurred in France and Serbia in the beginning of 2017. An epidemiological study was conducted in France and Serbia to determine the extent of the outbreak, to identify its source and to implement control measures. Three cases were exposed in Serbia and brought back to France pork delicatessen which they shared with relatives and friends. Around 47 individuals were exposed to the parasitised meat in France and Serbia and 20 cases of trichinellosis were reported (nine in France and 11 in Serbia). Nine of them were female. The diagnosis was delayed, in part because the parasitosis was not known by most physicians, which led to complications in the French cases such as facial paralysis and pulmonary embolism. Health alerts and survey networks are indispensable at a European level to control the disease.

Lung Involvement in Chronic Schistosomiasis with Bladder Squamous Cell Carcinoma.

We report a case of chronic Schistosoma haematobium infection with pseudometastatic pulmonary nodules and high-grade squamous cell carcinoma in a 30-year-old man in Mali. Lung biopsies revealed chronic pulmonary involvement of S. haematobium and ruled out lung metastases.

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis.

Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal.

Molecular diagnosis of Pseudoterranova decipiens s.s in human, France.

BACKGROUND: Anisakis and Pseudoterranova are the main genera involved in human infections caused by nematodes of the Anisakidae family. Species identification is complicated due to the lack of differential morphological characteristics at the larval stage, thus requiring molecular differentiation. Pseudoterranova larvae ingested through raw fish are spontaneously eliminated in most cases, but mechanical removal by means of endoscopy might be required. To date, only very few cases of Pseudoterranova infection have been reported in France. CASE PRESENTATION: A 19-year-old woman from Northeastern France detected, while brushing her teeth, a larva exiting through her mouth. The patient who presented with headache, diarrhea, and abdominal cramps reported having eaten baked cod. The worm was a fourth-stage larva with a size of 22 × 0.9 mm, and molecular biology identified it as Pseudoterranova decipiens sensu stricto (s. s.). In a second P. decipiens infection case, occurring a few months later, a worm exited through the patient's nose after she had eaten raw sea bream. CONCLUSION: These two cases demonstrate that Pseudoterranova infection is not uncommon among French patients. Therefore, molecular techniques should be more widely applied for a better characterization of anisakidosis epidemiology in France.

Contribution of neonatal amniotic fluid testing to diagnosis of congenital toxoplasmosis.

We evaluated the molecular diagnosis of congenital toxoplasmosis (CT) on neonatal amniotic fluid samples from 488 mother-child pairs. Maternal infection during pregnancy was diagnosed and dated or could not be ruled out. Forty-six cases of CT were defined according to the European Research Network on CT classification system and case definitions. Neonatal amniotic fluid testing had an overall sensitivity of 54% (95% confidence interval [95% CI], 39 to 69%) and a specificity of 100% (95% CI, 99 to 100%). Its sensitivity was 33% (95% CI, 13 to 59%) when antenatal diagnosis was positive and 68% (95% CI, 48 to 84%) when antenatal diagnosis was negative or lacking. This difference in sensitivity may have been due to treatment of antenatally diagnosed cases. Relative to postnatal serology, neonatal amniotic fluid testing allowed an earlier diagnosis to be made in 26% of the cases (95% CI, 9 to 51%).

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.

In vitro growth, cytopathic effects and clearance of monolayers by clinical isolates of Balamuthia mandrillaris in human skin cell cultures.

Balamuthia mandrillaris is a free-living ameba (FLA) that has been isolated or its DNA identified in soil, dust and water. It causes a fatal central nervous system infection in humans and animals. Although it is environmental as Acanthamoeba and Naegleria fowleri, the two other free-living amebae that also cause CNS infections in humans and other animals, Balamuthia does not feed on bacteria as the other FLA. In the laboratory, it can be grown on a variety of mammalian cell cultures. In this study we examined the ability of three different Balamuthia isolates to grow on several different human skin cell cultures including the WT/A keratinocyte cell cultures. A corneal isolate of Acanthamoeba castellanii was used for comparison.

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.

Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites.

BACKGROUND INFORMATION: The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized. RESULTS: Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt-interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab-interactome using putative parasite Rab-effectors identified by homology to Ypt-effectors. We demonstrate its potential by wet-bench testing three predictions; that casein kinase-1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP-dependent protein kinase A (PfPKA-C) is a PfRab5A and PfRab7 effector. CONCLUSIONS: The establishment of a shared set of physical Ypt/PfRab-effector proteins sheds light on a core set Plasmodium Rab-interactants shared with yeast. The PfRab-interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA-C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab-recruited kinases potentially play a role in early and late endosome function in malaria parasites.

A Trichinella britovi outbreak in the Northern Alps of France: investigation by a local survey network.

We describe a small family outbreak of trichinellosis caused by the consumption of raw ham from a wild boar (Sus scrofa) hunted in the northern Alps of France in February 2022. Out of the six people, aged 3-69 years, who consumed the meat, three were confirmed cases, and three were suspected cases. Eosinophilia detected in four people was the hallmark that drove the diagnosis. Three patients presented with myalgia, two with intense and prolonged chest pain, and one with elevated troponin. One patient presented with dermographism during treatment. Anti-Trichinella IgG were detected in three symptomatic individuals after about ten weeks. One patient had negative serology and no symptoms, but was on long-term corticosteroid therapy. Trichinella britovi larvae (8.3 larvae/g) were detected in the wild boar meat remnants. Trichinellosis is rare in France, but this family outbreak is reminiscent of the circulation of this pathogen in wild animals, highlighting the need to inform hunters about the risk of infection linked to the consumption of raw meat of game animals, and about the need for veterinary inspection of game meat. The consumption of raw meat outside controlled circuits is a practice not devoid of risks, which justifies raising the awareness of hunters, doctors, and medical biologists.

Title: Un foyer de Trichinella britovi dans les Alpes du Nord françaises : investigation par un réseau local de prospection. Abstract: Nous décrivons une épidémie familiale de trichinellose causée par la consommation de jambon cru d'un sanglier (Sus scrofa) chassé dans le nord des Alpes françaises en février 2022. Sur les six personnes âgées de 3 à 69 ans qui ont consommé la viande, trois étaient des cas confirmés, et trois étaient des cas suspects. L'éosinophilie détectée chez quatre personnes a permis d'évoquer le diagnostic. Trois patients présentaient des myalgies, et deux des douleurs thoraciques intenses et prolongées dont un avec une troponine élevée. Un patient a présenté un dermographisme pendant le traitement. Des IgG anti-Trichinella ont été détectées chez trois individus symptomatiques après environ dix semaines. Un des patients avait une sérologie négative et aucun symptôme mais était sous corticothérapie au long cours. Des larves de Trichinella britovi (8,3 larves/g) ont été détectées dans les restes du jambon de sanglier incriminé. La trichinellose est rare en France, mais cette épidémie familiale rappelle la circulation de cet agent pathogène chez les animaux sauvages, qui nécessite d'informer les chasseurs sur les risques d'infections liés à la consommation de viande crue de gibier, et de préconiser un contrôle vétérinaire des viandes de gibier. La consommation de viande crue en dehors des circuits contrôlés est une pratique non dénuée de risques, qui justifie une sensibilisation des chasseurs, médecins et biologistes médicaux.

Neurocysticercosis Diagnosis in a Non-Endemic Country: France.

Diagnosing neurocysticercosis (NCC) is difficult due to its variable clinical presentations and the different imaging techniques used to detect brain damage. This study aimed to evaluate the use of cerebrospinal fluid serology and PCR for diagnosing biological neurocysticercosis in a non-endemic country. We tested samples from patients living in France with suspected NCC and confirmed that 45 of the patients presented with the disease. A total of 89% of patients had previously traveled to countries where the disease was endemic. The sensitivity of Western blots compared to ELISA was not significantly different (80% vs. 60%) (p > 0.05), and neither was the sensitivity of Western blots vs. PCR (78% vs. 56%) (p > 0.05). The PCR sensitivity was 78% and 47% in definitive NCC and in probable NCC. PCR tests using cerebrospinal fluid should be considered as a diagnostic criterion for identifying NCC.

Gradient concentration strip-specific epidemiological cut-off values of antifungal drugs in various yeast species and five prevalent Aspergillus species complexes.

OBJECTIVES: To determine the epidemiological cut-off values (ECVs) of ten antifungal agents in a wide range of yeasts and Aspergillus spp. using gradient concentration strips. METHODS: The minimum inhibitory concentrations for amphotericin B, anidulafungin, caspofungin, micafungin, flucytosine, fluconazole, itraconazole, isavuconazole, posaconazole, and voriconazole, determined with gradient concentration strips at 35 French microbiology laboratories between 2002 and 2020, were retrospectively collected. Then, the ECVs were calculated using the iterative method and a cut-off value of 97.5%. RESULTS: Minimum inhibitory concentrations were available for 17 653 clinical isolates. In total, 48 ECVs (including 32 new ECVs) were determined: 29 ECVs for frequent yeast species (e.g. Candida albicans and itraconazole/flucytosine, and Candida glabrata species complex [SC] and flucytosine) and rare yeast species (e.g. Candida dubliniensis, Candida inconspicua, Saccharomyces cerevisiae, and Cryptococcus neoformans) and 19 ECVs for Aspergillusflavus SC, Aspergillusfumigatus SC, Aspergillusnidulans SC, Aspergillusniger SC, and Aspergillusterreus SC. CONCLUSIONS: These ECVs can be added to the already available gradient concentration strip-specific ECVs to facilitate minimum inhibitory concentration interpretation and streamline the identification of nonwild type isolates.