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.

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.

Evaluation of the PneumoGenius® PCR assay for the diagnosis of Pneumocystis pneumonia and the detection of Pneumocystis dihydropteroate synthase mutations in respiratory samples.

Pneumocystis pneumonia (PCP) is the most frequent fungal opportunistic infection defining AIDS in HIV-infected patients, and is of growing importance in HIV-negative patients. In this latter category of patients, the diagnosis mainly relies on real-time polymerase chain reaction (qPCR) detection of Pneumocystis jirovecii (Pj) on respiratory samples. The PneumoGenius® kit (PathoNostics) allows the simultaneous detection of Pj mitochondrial large subunit (mtLSU) and dihydropteroate synthase (DHPS) polymorphisms, which could be of interest to anticipate therapeutic failure. This study aimed at evaluating its clinical performance on 251 respiratory specimens (239 patients), (i) for P. jirovecii detection in clinical samples, and (ii) for DHPS polymorphisms detection in circulating strains. Patients were classified according to modified European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, as having proven PCP (n = 62), probable PCP (n = 87), Pneumocystis colonization (n = 37), and no PCP (n = 53). Compared with in-house qPCR, the sensitivity of PneumoGenius® assay for P. jirovecii detection reached 91.9% (182/198), the specificity was excellent (100%, 53/53) and the global concordance was 93.6% (235/253). A total of four diagnoses of proven/probable PCP were missed by the PneumoGenius® assay, reaching a 97.5% sensitivity (157/161) in this sub-group. The 12 other 'false-negative' results were obtained in patients diagnosed as colonized using the in-house PCR. DHPS genotyping was successful for 147/182 samples with PneumoGenius® and revealed dhps mutation in 8 samples, which were all confirmed by sequencing. In conclusion, PneumoGenius® assay missed the detection of low-burden PCP. This lower sensitivity for PCP diagnosis can be balanced by a higher specificity (P. jirovecii colonization less frequently detected) and the efficient detection of DHPS hot spot mutations.

The diagnosis of Pneumocystis pneumonia (PCP) relies on DNA detection of P. jirovecii in respiratory samples. In this study, we show that the commercial assay PneumoGenius® has a lower sensitivity than our in-house qPCR for PCP diagnosis, but provides accurate results for DHPS genotyping.

Endogenous IL-33 Accelerates Metacestode Growth during Late-Stage Alveolar Echinococcosis.

During the course of the infectious disease alveolar echinococcosis (AE), the larval stage of Echinococcus multilocularis develops in the liver, where an initial Th1/Th17 immune response may allow its elimination in resistant individuals. In patients susceptible to infection and disease, the Th2 response initiates later, inducing tolerance to the parasite. The role of interleukin 33 (IL-33), an alarmin released during necrosis and known to drive a Th2 immune response, has not yet been described during AE. Wild-type (WT) and IL-33-/- C57BL/6J mice were infected by peritoneal inoculation with E. multilocularis metacestodes and euthanized 4 months later, and their immune response were analyzed. Immunofluorescence staining and IL-33 enzyme-linked immunosorbent assay (ELISA) were also performed on liver samples from human patients with AE. Overall, metacestode lesions were smaller in IL-33-/- mice than in WT mice. IL-33 was detected in periparasitic tissues, but not in mouse or human serum. In infected mice, endogenous IL-33 modified peritoneal macrophage polarization and cytokine profiles. Th2 cytokine concentrations were positively correlated with parasite mass in WT mice, but not in IL-33-/- mice. In human AE patients, IL-33 concentrations were higher in parasitic tissues than in distant liver parenchyma. The main sources of IL-33 were CD31+ endothelial cells of the neovasculature, present within lymphoid periparasitic infiltrates together with FOXP3+ Tregs. In the murine model, periparasitic IL-33 correlated with accelerated parasite growth putatively through the polarization of M2-like macrophages and release of immunosuppressive cytokines IL-10 and transforming growth factor ß1 (TGF-ß1). We concluded that IL-33 is a key alarmin in AE that contributes to the tolerogenic effect of systemic Th2 cytokines. IMPORTANCE Infection with the metacestode stage of Echinococcus multilocularis, known as alveolar echinococcosis, is the most severe cestodosis worldwide. However, less than 1% of exposed individuals, in which the immune system is unable to control the parasite, develop the disease. The factors responsible for this interindividual variability are not fully understood. In this in vivo study comparing wild-type and IL-33-/- infected mice, together with data from human clinical samples, we determined that IL-33, an alarmin released following tissue injury and involved in the pathogenesis of cancer and asthma, accelerates the progression of the disease by modulating the periparasitic microenvironment. This suggests that targeting IL-33 could be of interest for the management of patients with AE, and that IL-33 polymorphisms could be responsible for increased susceptibility to AE.

The Burden of Pneumocystis Pneumonia Infection among HIV Patients in Ethiopia: A Systematic Review.

Pneumocystis pneumonia (PCP) is a leading cause of death among patients with AIDS worldwide, but its burden is difficult to estimate in low- and middle-income countries, including Ethiopia. This systematic review aimed to estimate the pooled prevalence of PCP in Ethiopia, the second most densely populated African country. The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines were used to review published and unpublished studies conducted in Ethiopia. Studies that reported on the prevalence of PCP among HIV-infected patients were searched systematically. Variations between the studies were assessed by using forest plot and I-squared heterogeneity tests. Subgroup and sensitivity analyses were carried out when I2 > 50. The pooled estimate prevalence with 95% CI was computed using a random-effects model of analysis. Thirteen articles, comprising studies of 4847 individuals living with HIV, were included for analysis. The pooled prevalence of PCP was 5.65% (95% CI [3.74-7.56]) with high heterogeneity (I2 = 93.6%, p < 0.01). To identify the source of heterogeneity, subgroup analyses were conducted by study design, geographical region, diagnosis methods, and year of publication. PCP prevalence differed significantly when biological diagnostic methods were used (32.25%), in studies published before 2010 (32.51%), in cross-sectional studies (8.08%), and in Addis Ababa (14.05%). PCP prevalence differences of 3.25%, 3.07%, 3.23%, and 2.29% were recorded in studies based on clinical records, published since 2017, follow-up studies, and north-west Ethiopian studies, respectively. The prevalence of PCP is probably underestimated, as the reports were mainly based on clinical records. An expansion of biological diagnostic methods could make it possible to estimate the exact burden of PCP in Ethiopia.

Alveolar echinococcosis in immunocompromised hosts.

BACKGROUND: Alveolar echinococcosis (AE) results of an infection with the larval stage of Echinococcus multilocularis. It has been increasingly described in individuals with impaired immune responsiveness. OBJECTIVES: This narrative review aims at describing the presentation of AE according to the type of immune impairment, based on retrospective cohorts and case reports. Implications for patient management and future research are proposed accordingly. SOURCES: Targeted search was conducted in PubMed using ((alveolar echinococcosis) OR (multilocularis)) AND ((immunosuppressive) OR (immunodeficiency) OR (AIDS) OR (solid organ transplant) OR (autoimmunity) OR (immune deficiency)). Only publications in English were considered. CONTENT: Seventeen publications were found, including 13 reports of 55 AE in immunocompromised patients (AE/IS) and 4 retrospective studies of 755 AE immunocompetent patients and 115 AE/IS (13%). The cohorts included 9 (1%) solid organ transplantation (SOT) recipients, 2 (0.2%) HIV patients, 41 (4.7%) with chronic inflammatory/autoimmune diseases (I/AID) and 72 (8.3%) with malignancies. SOT, I/AID and malignancies, but not HIV infection, were significantly associated with AE (odds ratios of 10.8, 1.6, 5.9, and 1.3, respectively). Compared to AE immunocompetent patients, AE/IS was associated with earlier diagnosis (PNM stages I-II: 49/85 (58%) vs. 137/348 (39%), p < 0.001), high rate of atypical imaging (24/50 (48%) vs. 106/375 (28%), p < 0.01), and low sensitivity of serology (19/77 (25%) vs. 265/329 (81%), p < 0.001). Unusually extensive or disseminated infections were described in SOT and I/AID patients. IMPLICATIONS: Patients who live in endemic areas should benefit from serology before onset of a long-term immunosuppressive therapy, even if the cost-benefit ratio has to be evaluated. Physicians should explain AE to immunocompromised patients and think about AE when finding a liver lesion. Further research should address gaps in knowledge of AE/IS. Especially, extensive and accurate records of AE cases have to be collected by multinational registries.

Toxoplasmosis in patients with an autoimmune disease and immunosuppressive agents: A multicenter study and literature review.

BACKGROUND: Cases of Toxoplasma reactivation or more severe primary infection have been reported in patients receiving immunosuppressive (IS) treatment for autoimmune diseases (AID). The purpose of this study was to describe features of toxoplasmosis occurring in patients with AID treated by IS therapy, excluded HIV-positive and transplant patients. METHODS: A multicenter descriptive study was conducted using data from the French National Reference Center for Toxoplasmosis (NRCT) that received DNA extracts or strains isolated from patients, associated with clinical data. Other cases were retrieved through a questionnaire sent to all French parasitology and internal medicine departments. Furthermore, a systematic literature review was conducted. RESULTS: 61 cases were collected: 25 retrieved by the NRCT and by a call for observations and 36 from a literature review. Half of the cases were attributed to reactivation (50.9%), and most of cases (49.2%) were cerebral toxoplasmosis. The most common associated AID were rheumatoid arthritis (28%) and most frequent treatments were antimetabolites (44.3%). Corticosteroids were involved in 60.7% of cases. Patients had a favorable outcome (50.8%) but nine did not survive. For 12 cases, a successful Toxoplasma strain characterization suggested the possible role of this parasitic factor in ocular cases. CONCLUSION: Although this remains a rare condition, clinicians should be aware for the management of patients and for the choice of IS treatment.

Evaluation of Commercial Concentration Methods for Microscopic Diagnosis of Protozoa and Helminths in Human Stool Samples in a Non-Endemic Area.

The diagnosis of intestinal parasitic infections still widely relies on microscopic examination of stools and requires reliable reagents and staff expertise. The ParaFlo® assays (Eurobio Ingen) are ready-to-use concentration methods for parasite egg detection, and they could improve reagent traceability and ease of manipulation. Ninety-three stool samples were analyzed with the ParaFlo® concentration methods and then compared with routine microscopic methods for protozoa and helminth detection: seventy-eight were analyzed with ParaFlo® Bailenger and in-house Thebault or Bailenger concentrations, and fifty-five were analyzed with ParaFlo®DC and the in-house merthiolate-formalin diphasic concentration (DC) method. Fully concordant results were obtained for 75%, 70%, and 69% of samples when comparing ParaFlo® DC and in-house DC, ParaFlo® Bailenger and in-house Bailenger, and ParaFlo® Bailenger and Thebault, respectively. The performances of the ParaFlo® assays did not differ statistically from that obtained with their in-house counterparts (Bailenger and DC) for the detection of protozoa, but ParaFlo® Bailenger performed significantly poorer than the Thebault method (p < 0.001). No statistical differences were observed between the commercial and in-house methods for helminth detection. These marketed concentration methods could be used in routine if combined with other techniques for protozoa detection.

Treatment outcome of imported cutaneous leishmaniasis among travelers and migrants infected with Leishmania major and Leishmania tropica: a retrospective study in European centers 2013 to 2019.

OBJECTIVES: Cutaneous leishmaniasis (CL) in Asia, Northern, and Sub-Saharan Africa is mainly caused by Leishmania major and Leishmania tropica. We describe and evaluate the treatment outcome of CL among travelers and migrants in Europe. METHODS: We conducted a retrospective study of parasitological confirmed CL cases caused by L. major and L. tropica during 2013-2019 in Europe. Data were collected from medical records and databases within the LeishMan network. RESULTS: Of 206 included cases of CL, 75 were identified as L. major and 131 as L. tropica. Of patients with L. tropica infection, 80% were migrants, whereas 53% of patients with L. major infection had been visiting friends and relatives. Among patients with L. tropica, 48% were younger than 15 years. Pentavalent antimony cured 73% (L. major) and 78% (L. tropica) of patients. The cure rate for intralesional administration was 86% and 67% for systemic, on L. tropica. Liposomal amphotericin B had a cure rate of 44-63%. CONCLUSION: L. major infections were mostly found in individuals visiting friends and relatives, whereas L. tropica were mainly identified in migrants. No patients with L. major relapsed. Pentavalent antimony, liposomal amphotericin B, and cryotherapy had cure rates in accordance with previous studies.

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.

Surveillance of leishmaniasis cases from 15 European centres, 2014 to 2019: a retrospective analysis.

BackgroundSurveillance of human leishmaniasis in Europe is mostly limited to country-specific information from autochthonous infections in the southern part. As at the end of 2021, no integrated analysis has been performed for cases seen across centres in different European countries.AimTo provide a broad perspective on autochthonous and imported leishmaniasis cases in endemic and non-endemic countries in Europe.MethodsWe retrospectively collected records from cutaneous, mucosal and visceral leishmaniasis cases diagnosed in 15 centres between 2014 and 2019. Centres were located in 11 countries: Belgium, France, Germany, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Data on country of infection, reason for travelling, infecting species, age and sex were analysed.ResultsWe obtained diagnostic files from 1,142 cases, of which 76%, 21% and 3% had cutaneous, visceral, and mucosal disease, respectively. Of these, 68% were men, and 32% women, with the median age of 37 years (range: 0-90) at diagnosis. Visceral leishmaniasis was mainly acquired in Europe (88%; 167/190), while cutaneous leishmaniasis was primarily imported from outside Europe (77%; 575/749). Sixty-two percent of cutaneous leishmaniasis cases from outside Europe were from the Old World, and 38% from the New World. Geographic species distribution largely confirmed known epidemiology, with notable exceptions.ConclusionsOur study confirms previous reports regarding geographic origin, species, and traveller subgroups importing leishmaniasis into Europe. We demonstrate the importance of pooling species typing data from many centres, even from areas where the aetiology is presumably known, to monitor changing epidemiology.

Fungal infections in mechanically ventilated patients with COVID-19 during the first wave: the French multicentre MYCOVID study.

BACKGROUND: Patients with severe COVID-19 have emerged as a population at high risk of invasive fungal infections (IFIs). However, to our knowledge, the prevalence of IFIs has not yet been assessed in large populations of mechanically ventilated patients. We aimed to identify the prevalence, risk factors, and mortality associated with IFIs in mechanically ventilated patients with COVID-19 under intensive care. METHODS: We performed a national, multicentre, observational cohort study in 18 French intensive care units (ICUs). We retrospectively and prospectively enrolled adult patients (aged ≥18 years) with RT-PCR-confirmed SARS-CoV-2 infection and requiring mechanical ventilation for acute respiratory distress syndrome, with all demographic and clinical and biological follow-up data anonymised and collected from electronic case report forms. Patients were systematically screened for respiratory fungal microorganisms once or twice a week during the period of mechanical ventilation up to ICU discharge. The primary outcome was the prevalence of IFIs in all eligible participants with a minimum of three microbiological samples screened during ICU admission, with proven or probable (pr/pb) COVID-19-associated pulmonary aspergillosis (CAPA) classified according to the recent ECMM/ISHAM definitions. Secondary outcomes were risk factors of pr/pb CAPA, ICU mortality between the pr/pb CAPA and non-pr/pb CAPA groups, and associations of pr/pb CAPA and related variables with ICU mortality, identified by regression models. The MYCOVID study is registered with ClinicalTrials.gov, NCT04368221. FINDINGS: Between Feb 29 and July 9, 2020, we enrolled 565 mechanically ventilated patients with COVID-19. 509 patients with at least three screening samples were analysed (mean age 59·4 years [SD 12·5], 400 [79%] men). 128 (25%) patients had 138 episodes of pr/pb or possible IFIs. 76 (15%) patients fulfilled the criteria for pr/pb CAPA. According to multivariate analysis, age older than 62 years (odds ratio [OR] 2·34 [95% CI 1·39-3·92], p=0·0013), treatment with dexamethasone and anti-IL-6 (OR 2·71 [1·12-6·56], p=0·027), and long duration of mechanical ventilation (>14 days; OR 2·16 [1·14-4·09], p=0·019) were independently associated with pr/pb CAPA. 38 (7%) patients had one or more other pr/pb IFIs: 32 (6%) had candidaemia, six (1%) had invasive mucormycosis, and one (<1%) had invasive fusariosis. Multivariate analysis of associations with death, adjusted for candidaemia, for the 509 patients identified three significant factors: age older than 62 years (hazard ratio [HR] 1·71 [95% CI 1·26-2·32], p=0·0005), solid organ transplantation (HR 2·46 [1·53-3·95], p=0·0002), and pr/pb CAPA (HR 1·45 [95% CI 1·03-2·03], p=0·033). At time of ICU discharge, survival curves showed that overall ICU mortality was significantly higher in patients with pr/pb CAPA than in those without, at 61·8% (95% CI 50·0-72·8) versus 32·1% (27·7-36·7; p<0·0001). INTERPRETATION: This study shows the high prevalence of invasive pulmonary aspergillosis and candidaemia and high mortality associated with pr/pb CAPA in mechanically ventilated patients with COVID-19. These findings highlight the need for active surveillance of fungal pathogens in patients with severe COVID-19. FUNDING: Pfizer.

Clinical value of serology for the diagnosis of strongyloidiasis in travelers and migrants: A 4-year retrospective study using the Bordier IVD® Strongyloides ratti ELISA assay.

Strongyloides stercoralis serology is a sensitive method for strongyloidiasis diagnosis, but it is prone to cross-reactions with other helminthiases. This four-year retrospective study aimed at estimating the performance of the Bordier IVD® Strongyloides ratti ELISA assay in a non-endemic country (France). The study included all patients tested for strongyloidiasis in our center between 2015 and 2019, by both serology and stool examination. Cases were defined using an algorithm considering serological results, microscopic examination of stools, and other biological, clinical or epidemiological data. The study included 805 stools from 341 patients (70% migrants, 20% travelers, 10% without travel to a highly endemic area). Thirty patients (8.8%) had positive serology, 9 had microscopically proven strongyloidiasis, and 11 and 10 were classified as probable and possible strongyloidiasis, respectively. Performances of microscopy and serology were compared, considering proven and probable strongyloidiasis as true infections. The sensitivity, specificity, positive predictive value and negative predictive value of serology were 100%, 97%, 67% and 100%, respectively, and those of microscopic examination of stools were 45% (p < 0.01), 100% (p < 0.01), 100% (p = 0.079) and 96% (p < 0.001), respectively. Eosinophilia did not help in discriminating true-positive from false-positive results. Overall, these results underline the high value of the S. stercoralis serologic assay, compared to stool examination. The systematic use of this technique for screening purposes in travelers or migrants, or before onset of immunosuppressive therapy, could help to improve patient management and epidemiological knowledge.

TITLE: Utilité clinique de la sérologie pour le diagnostic de la strongyloïdose chez les voyageurs et les migrants : une étude rétrospective de 4 ans utilisant le test ELISA Strongyloides ratti Bordier IVD®. ABSTRACT: La sérologie de Strongyloides stercoralis est une méthode sensible pour le diagnostic de la strongyloïdose, mais elle est sujette à des réactions croisées avec d'autres helminthes. Cette étude rétrospective sur 4 ans visait à estimer les performances du test ELISA Strongyloides ratti Bordier IVD® dans un pays non endémique (la France). L'étude a inclus tous les patients testés pour la strongyloïdose dans notre centre entre 2015 et 2019, à la fois par sérologie et examen des selles. La définition des cas a été faite à l'aide d'un algorithme tenant compte des résultats sérologiques, de l'examen microscopique des selles et d'autres données biologiques, cliniques ou épidémiologiques. L'étude a inclus 805 selles de 341 patients (70 % de migrants, 20 % de voyageurs, 10 % sans voyage dans une zone de forte endémie). Trente patients (8,8 %) avaient une sérologie positive, 9 avaient une strongyloïdose prouvée au microscope, et 11 et 10 ont été classés respectivement comme strongyloïdose probable et possible. Les performances de la microscopie et de la sérologie ont été comparées, en considérant les strongyloïdoses avérées et probables comme de véritables infections. La sensibilité, la spécificité, la valeur prédictive positive et la valeur prédictive négative de la sérologie étaient de 100 %, 97 %, 67 % et 100 %, respectivement, et celles de l'examen microscopique des selles étaient de 45 % (p < 0,01), 100 % (p < 0,01), 100 % (p = 0,079) et 96 % (p < 0,001), respectivement. L'éosinophilie n'a pas aidé à distinguer les vrais positifs des faux positifs. Dans l'ensemble, ces résultats soulignent la valeur élevée du test sérologique de S. stercoralis, par rapport à l'examen des selles. L'utilisation systématique de cette technique à des fins de dépistage chez les voyageurs ou les migrants, ou avant le début d'un traitement immunosuppresseur, pourrait contribuer à améliorer la prise en charge des patients et les connaissances épidémiologiques.

Chromoblastomycosis Due to a Never-before-Seen Dematiaceous Fungus in a Kidney Transplant Patient.

Chromoblastomycosis is a neglected fungal infection of the epidermis and subcutaneous tissue that predominates in tropical areas and results from the traumatic inoculation of environmental dematiaceous filamentous fungi. We describe the case of an immunosuppressed patient diagnosed with foot chromoblastomycosis due to an uncommon dematiaceous fungus. A 52-year-old Congolese kidney transplant woman presented with a painful lesion located on the foot. No trauma to the lower limbs was reported during the previous months. She lived in France and had not returned to the Congo over the previous eight years. Histology and mycological examination from skin biopsy revealed swollen dark filaments associated with dematiaceous muriform cells, pathognomonic of chromoblastomycosis. Cultures grew with dark pigmented colonies, yielding poor microscopic features. The phylogenetic analysis confirmed that the isolate was a member of Kirschsteiniotheliales (Dothideomycetes) and unrelated to the Chaetotyriales, of which most species commonly responsible for chromoblastomycosis belong. As there was no bone spreading, excision surgery of the entire lesion followed by liposomal amphotericin B therapy resulted in complete healing after six months. This original case illustrates the potential diversity of environmental dematiaceous fungi responsible for phaeohyphomycosis, especially chromoblastomycosis, and the need to send samples to mycology labs for appropriate diagnosis.

A review of significance of Aspergillus detection in airways of ICU COVID-19 patients.

It is now well known that patients with SARS-CoV-2 infection admitted in ICU and mechanically ventilated are at risk of developing invasive pulmonary aspergillosis (IPA). Nevertheless, symptomatology of IPA is often atypical in mechanically ventilated patients, and radiological aspects in SARS-CoV-2 pneumonia and IPA are difficult to differentiate. In this context, the significance of the presence of Aspergillus in airway specimens (detected by culture, galactomannan antigen or specific PCR) remains to be fully understood. To decipher the relevance of the detection of Aspergillus, we performed a comprehensive review of all published cases of respiratory Aspergillus colonisation and IPA in COVID-19 patients. The comparison of patients receiving or not antifungal treatment allowed us to highlight the most important criteria for the decision to treat. The comparison of surviving and non-surviving patients made it possible to unveil criteria associated with mortality that should be taken into account in the treatment decision.

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.

Influenza- and COVID-19-Associated Pulmonary Aspergillosis: Are the Pictures Different?

Invasive pulmonary aspergillosis (IPA) in intensive care unit patients is a major concern. Influenza-associated acute respiratory distress syndrome (ARDS) and severe COVID-19 patients are both at risk of developing invasive fungal diseases. We used the new international definitions of influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) to compare the demographic, clinical, biological, and radiological aspects of IAPA and CAPA in a monocentric retrospective study. A total of 120 patients were included, 71 with influenza and 49 with COVID-19-associated ARDS. Among them, 27 fulfilled the newly published criteria of IPA: 17/71 IAPA (23.9%) and 10/49 CAPA (20.4%). Kaplan-Meier curves showed significantly higher 90-day mortality for IPA patients overall (p = 0.032), whereas mortality did not differ between CAPA and IAPA patients. Radiological findings showed differences between IAPA and CAPA, with a higher proportion of features suggestive of IPA during IAPA. Lastly, a wide proportion of IPA patients had low plasma voriconazole concentrations with a higher delay to reach concentrations > 2 mg/L in CAPA vs. IAPA patients (p = 0.045). Severe COVID-19 and influenza patients appeared very similar in terms of prevalence of IPA and outcome. The dramatic consequences on the patients' prognosis emphasize the need for a better awareness in these particular populations.

Anti-Toxoplasma IgG assays: What performances for what purpose? A systematic review.

Chronic infection with Toxoplasma gondii is attested by the detection of specific anti-Toxoplasma IgG. A wide panel of serologic methods is currently marketed, and the most suitable method should be chosen according to the laboratory resources and the screened population. This systematic review of evaluation studies aimed at establishing an overview of the performances, i.e. sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of marketed anti-Toxoplasma IgG assays, and discussing their technical characteristics to guide further choice for routine diagnostic use. According to PRISMA guidelines, the search performed in PubMed and Web of Science databases recovered 826 studies, of which 17 were ultimately included. Twenty commercial anti-Toxoplasma IgG assays were evaluated, in comparison with an accepted reference method. Most of them were enzyme-immunoassays (EIAs, n = 12), followed by agglutination tests (n = 4), immunochromatographic tests (n = 3), and a Western-Blot assay (WB, n = 1). The mean sensitivity of IgG assays ranged from 89.7% to 100% for standard titers and from 13.4% to 99.2% for low IgG titers. A few studies pointed out the ability of some methods, especially WB to detect IgG early after primary infection. The specificity of IgG assays was generally high, ranging from 91.3% to 100%; and higher than 99% for most EIA assays. The PPV was not a discriminant indicator among methods, whereas significant disparities (87.5%-100%) were reported among NPVs, a key-parameter assessing the ability to definitively rule out a Toxoplasma infection in patients at-risk for opportunistic infections.

TITLE: Les tests pour la détection d'IgG anti-Toxoplasma : quelles performances pour quel usage ? Une revue systématique. ABSTRACT: L'infection chronique à Toxoplasma gondii est attestée par la détection d'IgG anti-Toxoplasma spécifiques. Un large panel de méthodes sérologiques est actuellement commercialisé, et le choix d'une méthode doit être adapté aux ressources du laboratoire ainsi qu'à la population ciblée. Cette revue systématique des études d'évaluation visait à établir une vue d'sensemble des performances, c'est-à-dire la sensibilité, la spécificité, la valeur prédictive positive (VPP) et la valeur prédictive négative (VPN) des kits commercialisés pour la détection d'IgG anti-Toxoplasma, et à discuter leurs caractéristiques techniques pour guider le choix pour un usage diagnostique de routine. Selon les directives PRISMA, la recherche effectuée dans les bases de données PubMed et Web of Science a permis de retrouver 826 études, dont 17 ont été définitivement incluses. Vingt dosages commerciaux d'IgG anti-Toxoplasma ont été évalués, en comparaison avec une méthode de référence. La plupart des tests étaient des méthodes de dosage immuno-enzymatique (n = 12), d'agglutination (n = 4), immunochromatographiques (n = 3) et de Western-Blot (n = 1). La sensibilité moyenne des dosages IgG variait de 89,7 à 100 % pour les titres standards et de 13,4 % à 99,2 % pour les faibles titres d'IgG. Quelques études ont souligné la capacité de certaines méthodes, en particulier le Western-Blot, à détecter les IgG au cours d'une primo-infection. La spécificité des tests IgG était généralement élevée, allant de 91,3 % à 100, et supérieure à 99 % pour la plupart des tests immuno-enzymatiques. La VPP n'était pas un indicateur discriminant entre les méthodes, alors que des disparités significatives (87,5 % à 100 %) ont été rapportées entre les VPN, un paramètre-clé reflétant la capacité d'un test à éliminer formellement une toxoplasmose chez les patients à risque d'infections opportunistes.

Evaluation of the Allplex™ GI-Helminth(I) Assay, the first marketed multiplex PCR for helminth diagnosis.

Molecular biology has been gaining more importance in parasitology. Recently, a commercial multiplex PCR assay detecting helminths was marketed: the Allplex™ GI-Helminth(I) Assay. It targets Ancylostoma spp., Ascaris spp., Enterobius vermicularis, Hymenolepis spp., Necator americanus, Strongyloides spp., Taenia spp. and Trichuris trichiura, but also the two most common microsporidia genera in human health, i.e. Enterocytozoon spp. and Encephalitozoon spp. This study aimed to evaluate and compare the Allplex™ GI-Helminth(I) Assay to classical diagnostic methods, based on a cohort of 110 stool samples positive for helminths (microscopy) or for microsporidia (PCR). Samples were stored at -80 °C until analysis by the Allplex™ GI-Helminth(I) Assay. False-negatives were re-tested with bead-beating pretreatment. Without mechanical lysis, concordance and agreement between microscopy and Allplex™ GI-Helminth(I) Assay ranged from 91% to 100% and from 0.15 to 1.00, respectively depending on the target. Concordance was perfect for Taenia spp. (n = 5) and microsporidia (n = 10). False-negative results were observed in 54% (6/13), 34% (4/11) and 20% (7/35) of cases, for hookworms, E. vermicularis and Strongyloides spp. detection, respectively. For these targets, pretreatment improved the results, but only slightly. Trichuris trichiura detection was critically low without pretreatment, as only 9% (1/11) of the samples were positive, but detection reached 91% (10/11) with bead-beating pretreatment. Mechanical lysis was also needed for Ascaris spp. and Hymenolepis spp. to reduce false-negative results from 1/8 to 1/21, respectively, to none for both. Overall, with an optimized extraction process, the Allplex™ GI-Helminth(I) Assay allows the detection of numerous parasites with roughly equivalent performance to that of microscopy, except for hookworms.

TITLE: Évaluation du kit Allplex™ GI-Helminth(I) Assay, la première PCR multiplex commercialisée pour le diagnostic des helminthes. ABSTRACT: La biologie moléculaire a maintenant une place importante en parasitologie. Le kit Allplex™ GI-Helminth(I) Assay est le premier panel multiplex commercialisé détectant des helminthes : Ancylostoma spp., Ascaris spp., Enterobius vermicularis, Hymenolepis spp., Necator americanus, Strongyloides spp., Taenia spp. et Trichuris trichiura, mais également les deux genres de Microsporidies les plus fréquents en santé humaine, Enterocytozoon spp. et Encephalitozoon spp. Cette étude a comparé la PCR Allplex™ GI-Helminth(I) Assay aux techniques diagnostiques usuelles, sur une banque préservée à −80 °C, comprenant 110 échantillons de selles positifs à helminthes (microscopie) ou à microsporidies (PCR). Les faux négatifs ont été retestés après prétraitement par broyage en billes. Sans lyse mécanique, la concordance et l'accord entre la microscopie et le test Allplex™ GI-Helminth(I) Assay variaient respectivement de 91 % à 100 % et de 0,15 à 1,00, selon la cible. La concordance était parfaite pour Taenia spp. (n = 5) et les microsporidies (n = 10). Des faux négatifs ont été observés pour la détection des ankylostomes, E. vermicularis et Strongyloides spp. à des taux respectifs de 54 % (6/13), 34 % (4/11) et 20 % (7/35). Pour ces cibles, le prétraitement a peu amélioré les résultats. La détection de T. trichiura était défectueuse sans prétraitement, avec 9 % (1/11) de positifs, mais a atteint 91 % (10/11) après prétraitement par broyage en billes. La lyse mécanique était également nécessaire pour Ascaris spp. et Hymenolepis spp. pour réduire les faux négatifs de 1/8 et 1/21, respectivement, à aucun pour les deux. Au total, avec une optimisation de l'étape d'extraction, le test Allplex™ GI-Helminth(I) Assay permet la détection de nombreux parasites avec des performances proches de celles de la microscopie, excepté pour les ankylostomes.

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.