Comparison of different lateral flow assays on bronchoalveolar lavage fluid for invasive aspergillosis screening in non-hematological patients.

Several lateral flow assays (LFA) capable of detecting Aspergillus fumigatus in serum and broncho-alveolar lavage fluid (BALF) within the hour, thereby potentially accelerating the screening process, are now commercially available. We prospectively compared three LFA targeting A. fumigatus on BALF collected from non-surgical intensive care patients between June 2022 and February 2023. The three LFA tested were Sõna Aspergillus galactomannan LFA (Immy), Fungadia Aspergillus antigen (Gadia), and AspLFD (OLM Diagnostics). We compared the results of these LFA with those of the galactomannan (GM) Platelia Aspergillus enzyme immunoassay (Bio-Rad), culture on Sabouraud medium and Aspergillus qPCR. We tested 97 BALF samples from 92 patients. In total 84 BALF samples tested negative with all three LFA, and four BALF samples tested positive with the AspLFD assay only (OLM). Only one BALF sample tested positive with the three LFA. In addition, three BALF samples tested positive only with the GM Platelia immunoassay. Four diagnosis of probable invasive aspergillosis were retained for the 92 patients tested. This prospective series included very few positive samples. From a practical point of view, the LFA from OLM presented the simplest protocol for use.

Use of the Mucorales qPCR on blood to screen high-risk hematology patients is associated with better survival.

Our objective was to determine whether the twice-weekly screening of high-risk hematology patients by Mucorales qPCR on serum affects the prognosis of mucormycosis. Results from all serum Mucorales qPCR tests performed on patients from the hematology unit from January 2017 to December 2022 were analyzed. Patients with positive results were classified as having proven, probable or 'PCR-only' mucormycosis. One-month mortality for the local cohort was compared with that of a national cohort of cases of mucormycosis collected by the French surveillance network for invasive fungal disease ('Réseau de surveillances des infections fongiques invasives en France' (RESSIF)) from 2012 to 2018. From 2017 to 2022, 7825 serum Mucorales qPCR tests were performed for patients from the hematology unit; 107 patients with at least one positive Mucorales qPCR (164 positive samples) were identified. Sixty patients (70 positive samples, median Cq = 40) had no radiological criteria for mucormycosis and were considered not to have invasive fungal disease (70/7825, 0.9% false positives). It was not possible to classify disease status for six patients (12 positive samples, median Cq = 38). Forty-one patients (82 positive samples, median Cq = 35) had a final diagnosis of mucormycosis. In comparison with the RESSIF cohort, the local cohort was independently associated with a 48% lower one-month all-cause mortality rate (age-, sex-, and primary disease-adjusted hazard ratio = 0.52; 95% confidence interval: 0.29-0.94; P 0.03). Proactive screening for invasive mold diseases in high-risk hematology patients, including twice-weekly Mucorales qPCR on serum, was associated with mucormycosis higher survival.

Assessing the role of individual foxes in environmental contamination with Echinococcus multilocularis through faecal samples.

Key parasite transmission parameters are difficult to obtain from elusive wild animals. For Echinococcus multilocularis, the causative agent of alveolar echinococcosis (AE), the red fox is responsible for most of the environmental contamination in Europe. The identification of individual spreaders of E. multilocularis environmental contamination is crucial to improving our understanding of the ecology of parasite transmission in areas of high endemicity and optimising the effectiveness of prevention and control measures in the field. Genetic faecal sampling appears to be a feasible method to gain information about the faecal deposition of individual animals. We conducted a 4 year faecal sampling study in a village that is highly endemic for E. multilocularis, to assess the feasibility of individual identification and sexing of foxes to describe individual infection patterns. Individual fox identification from faecal samples was performed by obtaining reliable genotypes from 14 microsatellites and one sex locus, coupled with the detection of E. multilocularis DNA, first using captive foxes and then by environmental sampling. From a collection of 386 fox stools collected between 2017 and 2020, tested for the presence of E. multilocularis DNA, 180 were selected and 124 samples were successfully genotyped (68.9%). In total, 45 unique individual foxes were identified and 26 associated with at least one sample which tested positive for E. multilocularis (Em(+)). Estimation of the population size showed the fox population to be between 29 and 34 individuals for a given year and 67 individuals over 4 years. One-third of infected individuals (9/26 Em(+) foxes) deposited 2/3 of the faeces which tested positive for E. multilocularis (36/60 Em(+) stools). Genetic investigation showed a significantly higher average number of multiple stools for females than males, suggesting that the two sexes potentially defecated unequally in the studied area. Three partially overlapping clusters of fox faeces were found, with one cluster concentrating 2/3 of the total E. multilocularis-positive faeces. Based on these findings, we estimated that 12.5 million E. multilocularis eggs were produced during the study period, emphasizing the high contamination level of the environment and the risk of exposure faced by the parasite hosts.

Genetic diversity of Echinococcus multilocularis specimens isolated from Belgian patients with alveolar echinococcosis using EmsB microsatellites analysis.

The genetic diversity of Echinococcus multilocularis (E. multilocularis) specimens isolated from patients with alveolar echinococcosis (AE), is a major field of investigation to correlate with sources of infection, clinical manifestations and prognosis of the disease. Molecular markers able to distinguish samples are commonly used worldwide, including the EmsB microsatellite. Here, we report the use of the EmsB microsatellite polymorphism data mining for the retrospective typing of Belgian specimens of E. multilocularis infecting humans. A total of 18 samples from 16 AE patients treated between 2006 and 2021 were analyzed through the EmsB polymorphism. Classification of specimens was performed through a dendrogram construction in order to compare the similarity among Belgian samples, some human referenced specimens on the EWET database (EmsB Website for the Echinococcus Typing) and previously published EmsB profiles from red foxes circulating in/near Belgium. According to a comparison with human European specimens previously genotyped in profiles, the 18 Belgian ones were classified into three EmsB profiles. Four specimens could not be assigned to an already known profile but some are near to EWET referenced samples. This study also highlights that some specimens share the same EmsB profile with profiles characterized in red foxes from north Belgium, the Netherlands, Luxembourg and French department near to the Belgian border. Furthermore, Belgian specimens present a genetic diversity and include one profile that don't share similarities with the ones referenced in the EWET database. However, at this geographical scale, there is no clear correlation between EmsB profiles and geographical location. Further studies including additional clinical samples and isolates from foxes and rodents of south Belgium are necessary to better understand the spatial and temporal circumstances of human infections but also a potential correlation between EmsB profiles and parasite virulence.

Invasive fungal diseases in patients with autoimmune diseases: a case series from the French RESSIF network.

OBJECTIVES: We aimed to describe patients with autoimmune diseases (AID) developing invasive fungal disease (IFD) and identify factors associated with short-term mortality. METHODS: We analysed cases of IFD associated with AID from the surveillance network of invasive fungal diseases (Réseau de surveillance des infections fongiques invasives, RESSIF) registry of the French national reference centre for invasive mycoses. We studied association of AID-specific treatments with 30-day mortality. We analysed total lymphocyte and CD4-T cell counts in patients with Pneumocystis jirovecii pneumonia (PCP). RESULTS: From 2012 to 2018, 549 individuals with IFD and AID were included, mainly with PCP (n=227, 41.3%), fungemia (n=167, 30.4%) and invasive aspergillosis (n=84, 15.5%). Rheumatoid arthritis (RA) and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) were the most frequent AID in PCP (n=55 and 25, respectively) and invasive aspergillosis (n=15 and 10, respectively), inflammatory bowel diseases (IBDs) were predominant in fungemia (n=36). At IFD diagnosis, 365 (66.5%) patients received glucocorticoids (GCs), 285 (51.9%) immunosuppressants, 42 (7.7%) tumor necrosis factor (TNF)-α blockers, 75 (13.7%) other biologics. Mortality at 30 days was 28.1% (143/508). Fungemia and high-dose GCs were independently associated with higher 30-day mortality. In PCP patients, lymphopenia <1500/mm3 was frequent (132/179, 73.7%) even if CD4+T cell count exceeded 200/mm3 in 56/78 patients (71.8%) (median 472.5/mm3, IQR 160-858). CONCLUSION: IFD associated with AID occurs primarily in RA, AAV and IBD, especially when treated with GCs and immunosuppressants. Mortality is high, especially for patients on high-dose GCs. Lymphopenia may help identify risk of PCP, but normal CD4+T cell count does not rule out the risk. Further studies are needed to assess the individual risk factors for IFD.

Improving Diagnosis of Pulmonary Mucormycosis: Leads From a Contemporary National Study of 114 Cases.

BACKGROUND: Pulmonary mucormycosis (PM) is a life-threatening invasive mold infection. Diagnosis of mucormycosis is challenging and often delayed, resulting in higher mortality. RESEARCH QUESTION: Are the disease presentation of PM and contribution of diagnosis tools influenced by the patient's underlying condition? STUDY DESIGN AND METHODS: All PM cases from six French teaching hospitals between 2008 and 2019 were retrospectively reviewed. Cases were defined according to updated European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria with the addition of diabetes and trauma as host factors and positive serum or tissue PCR as mycologic evidence. Thoracic CT scans were reviewed centrally. RESULTS: A total of 114 cases of PM were recorded, including 40% with disseminated forms. Main underlying conditions were hematologic malignancy (49%), allogeneic hematopoietic stem cell transplantation (21%), and solid organ transplantation (17%). When disseminated, main dissemination sites were the liver (48%), spleen (48%), brain (44%), and kidneys (37%). Radiologic presentation included consolidation (58%), pleural effusion (52%), reversed halo sign (26%), halo sign (24%), vascular abnormalities (26%), and cavity (23%). Serum quantitative polymerase chain reaction (qPCR) was positive in 42 (79%) of 53 patients and BAL in 46 (50%) of 96 patients. Results of transthoracic lung biopsy were diagnostic in 8 (73%) of 11 patients with noncontributive BAL. Overall 90-day mortality was 59%. Patients with neutropenia more frequently displayed an angioinvasive presentation, including reversed halo sign and disseminated disease (P < .05). Serum qPCR was more contributive in patients with neutropenia (91% vs 62%; P = .02), and BAL was more contributive in patients without neutropenia (69% vs 41%; P = .02). Serum qPCR was more frequently positive in patients with a > 3 cm main lesion (91% vs 62%; P = .02). Overall, positive qPCR was associated with an early diagnosis (P = .03) and treatment onset (P = .01). INTERPRETATION: Neutropenia and radiologic findings influence disease presentation and contribution of diagnostic tools during PM. Serum qPCR is more contributive in patients with neutropenia and BAL examination in patients without neutropenia. Results of lung biopsies are highly contributive in cases of noncontributive BAL.

Complete mitochondrial exploration of Echinococcus multilocularis from French alveolar echinococcosis patients.

Alveolar echinococcosis (AE) is a parasitosis that is expanding worldwide, including in Europe. The development of genotypic markers is essential to follow its spatiotemporal evolution. Sequencing of the commonly used mitochondrial genes cob, cox1, and nad2 shows low discriminatory power, and analysis of the microsatellite marker EmsB does not allow nucleotide sequence analysis. We aimed to develop a new method for the genotyping of Echinococcus multilocularis based on whole mitochondrial genome (mitogenome) sequencing, to determine the genetic diversity among 30 human visceral samples from French patients, and compare this method with those currently in use. Sequencing of the whole mitochondrial genome was carried out after amplification by PCR, using one uniplex and two multiplex reactions to cover the 13,738 bp of the mitogenome, combined with Illumina technology. Thirty complete mitogenome sequences were obtained from AE lesions. One showed strong identity with Asian genotypes (99.98% identity) in a patient who had travelled to China. The other 29 mitogenomes could be differentiated into 13 haplotypes, showing higher haplotype and nucleotide diversity than when using the cob, cox1, and nad2 gene sequences alone. The mitochondrial genotyping data and EmsB profiles did not overlap, probably because one method uses the mitochondrial genome and the other the nuclear genome. The pairwise fixation index (Fst) value between individuals living inside and those living outside the endemic area was high (Fst = 0.222, P = 0.002). This is consistent with the hypothesis of an expansion from historical endemic areas to peripheral regions.

Whispers of change in preschool asthma phenotypes: Findings in the French ELFE cohort.

RATIONALE: Early life asthma phenotyping remains an unmet need in pediatric asthma. In France, severe pediatric asthma phenotyping has been done extensively; however, phenotypes in the general population remain underexplored. Based on the course and severity of respiratory/allergic symptoms, we aimed to identify and characterize early life wheeze profiles and asthma phenotypes in the general population. METHODS: ELFE is a general population based birth cohort; which recruited 18,329 newborns in 2011, from 320 maternity units nationwide. Data was collected using parental responses to modified versions of ISAAC questionnaire on eczema, rhinitis, food allergy, cough, wheezing, dyspnoea and sleep disturbance due to wheezing at 3 time points: post-natal (2 months), infancy (age 1) and pre-school (age 5). We built a supervised trajectory for wheeze profiles and an unsupervised approach was used for asthma phenotypes. Chi squared (χ2) test or fisher's exact test was used as appropriate (p < 0.05). RESULTS: Wheeze profiles and asthma phenotypes were ascertained at age 5. Supervised wheeze trajectory of 9161 children resulted in 4 wheeze profiles: Persistent (0.8%), Transient (12.1%), Incident wheezers at age 5 (13.3%) and Non wheezers (73.9%). While 9517 children in unsupervised clusters displayed 4 distinct asthma phenotypes: Mildly symptomatic (70%), Post-natal bronchiolitis with persistent rhinitis (10.2%), Severe early asthma (16.9%) and Early persistent atopy with late onset severe wheeze (2.9%). CONCLUSION: We successfully determined early life wheeze profiles and asthma phenotypes in the general population of France.

Alveolar echinococcosis in solid organ transplant recipients: a case series from two national cohorts.

Alveolar echinococcosis (AE) is a severe parasitic infection caused by the ingestion of Echinococcus multilocularis eggs. While higher incidence and faster evolution have been reported in immunosuppressed patients, no studies have been performed specifically on AE in transplant patients. We searched for all de novo AE cases diagnosed between January 2008 and August 2018 in solid organ transplant (SOT) recipients included in the Swiss Transplant Cohort Study and the FrancEchino Registry. Eight cases were identified (kidney = 5, lung = 2, heart = 1, liver = 0), half of which were asymptomatic at diagnosis. AE diagnosis was difficult due to the low sensitivity (60%) of the standard screening serology (Em2+) and the frequently atypical radiological presentations. Conversely, Echinococcus Western blot retained good diagnostic performances and was positive in all eight cases. Five patients underwent surgery, but complete resection could only be achieved in one case. Moreover, two patients died of peri-operative complications. Albendazole was initiated in seven patients and was well tolerated. Overall, AE regressed in one, stabilized in three, and progressed in one case, and had an overall mortality of 37.5% (3/8 patients). Our data suggest that AE has a higher mortality and a faster clinical course in SOT recipients; they also suggest that the parasitic disease might be due to the reactivation of latent microscopic liver lesions through immune suppression. Western blot serology should be preferred in this population. Finally, surgery should be considered with caution, because of its low success rate and high mortality, and conservative treatment with albendazole is well tolerated.

Title: Échinococcose alvéolaire chez les receveurs d'une greffe d'organe solide : une série de cas de deux cohortes nationales. Abstract: L'échinococcose alvéolaire (EA) est une maladie parasitaire grave causée par l'ingestion d'œufs d'Echinococcus multilocularis. Bien qu'une plus haute incidence et une évolution plus rapide aient été rapportées chez les patients immunodéprimés, aucune étude n'a été conduite spécifiquement sur cette maladie chez les patients transplantés. Nous avons donc listé tous les cas d'échinococcose alvéolaire apparus de novo entre janvier 2008 et août 2018 chez les patients transplantés d'organe solide inclus dans la cohorte Swiss Transplant Cohort Study et le registre FrancEchino. Huit patients ont été identifiés (rein = 5, poumon = 2, cœur = 1, foie = 0), dont la moitié était asymptomatique au moment du diagnostic. Le diagnostic était compliqué par la basse sensibilité (60 %) de la sérologie standard de dépistage (Em2+) et par les présentations radiologiques atypiques des lésions. Les performances diagnostiques du Western Blot n'étaient toutefois pas affectées et ce test était positif chez tous les patients. Sur les cinq patients opérés, une résection complète n'a été possible que dans un cas, tandis que deux patients sont décédés dans les suites de l'opération. L'albendazole a été introduit chez 7 patients et a été bien toléré. Dans l'ensemble, l'EA s'est stabilisée dans 3 cas, a régressé dans un cas et a progressé dans un autre cas, avec une mortalité de 37,5 % (3/8 patients). Nos résultats suggèrent une mortalité plus élevée et une évolution plus rapide de l'EA chez les patients transplantés. Ils suggèrent aussi que la maladie parasitaire pourrait être due à la réactivation de lésions hépatiques microscopiques latentes à la faveur de l'immunosuppression. Le Western Blot devrait être préféré dans cette population. Finalement, la chirurgie devrait être envisagée avec prudence, étant donnés son faible taux de réussite, le nombre élevé de décès peri-opératoires et la bonne tolérance au traitement conservateur par albendazole.

Real-time multiplex PCR for human echinococcosis and differential diagnosis.

Molecular identification of rare human infectious pathogens appears to be one of the most relevant current methods for rapid diagnosis and management of patients. PCR techniques, in particular real-time quantitative PCR, are best suited for the detection of DNA from the pathogens, even at low concentrations. Echinococcosis infections are due to helminths of the Echinococcus genus, with closely related species involved in parasitic lesions affecting animals and, accidentally, humans. We developed a multiplex qPCR (MLX qPCR) assay allowing for the detection of four Echinococcus species involved in Europe in alveolar echinococcosis (AE) and cystic echinococcosis (CE) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi, and E. canadensis), based on short mitochondrial targets. A collection of 81 fresh and formalin-fixed paraffin-embedded tissues (FFPE) of AE and CE lesions was assembled. The qPCR assays were performed in triplex for Echinococcus spp. detection, associated with a qPCR inhibitor control. A duplex qPCR was also designed to enable diagnosis of two other dead-end helminthiases (cysticercosis (Taenia solium), and toxocariasis (Toxocara cati and T. canis)). The sensitivity of the qPCR was assessed and ranged from 1 to 5 × 10-4 ng/µL (seven PCR assays positive), corresponding to 37-42 cycles for quantifiable DNA. The specificity was 100% for all the targets. This multiplex qPCR, adapted to low amounts of DNA can be implemented in the laboratory for the rapid molecular diagnosis of Echinococcosis species.

Title: PCR multiplex en temps-réel pour le diagnostic de l'échinococcose humaine et diagnostic différentiel. Abstract: L'identification moléculaire des pathogènes infectieux humains rares semble être l'une des méthodes actuelles les plus pertinentes pour un diagnostic et une prise en charge rapides des patients. Les techniques de PCR, en particulier la PCR quantitative en temps réel, sont bien adaptées à la détection d'ADN de pathogènes, même pour de faibles concentrations. Les infections à échinocoque sont dues à des helminthes du genre Echinococcus, des espèces étroitement apparentées, impliquées dans des lésions parasitaires affectant les animaux et accidentellement l'homme. Une qPCR multiplex (MLX qPCR), permettant la détection de quatre espèces d'Echinococcus impliquées en Europe dans l'échinococcose alvéolaire (EA) et kystique (EK) (Echinococcus multilocularis, E. granulosus sensu stricto, E. ortleppi et E. canadensis), basée sur de courtes cibles mitochondriales a été développée ici. Une collection a été constituée de 81 tissus frais ou fixés en paraffine (FFPE) de lésions d'EA et EK. Les essais de qPCR ont été réalisées en triplex pour la détection d'Echinococcus spp., associés à une qPCR de contrôle d'inhibition. Une PCR duplex a été développée pour le diagnostic de deux autres helminthiases en impasse chez l'Homme (cysticercose (Taenia solium), et toxocarose (Toxocara cati et T. canis). La sensibilité de la qPCR a été évaluée et s'échelonne de 1 à 5 × 10−4 ng/µl (sept essais de qPCR positifs), correspondant à 37 à 42 cycles pour l'ADN quantifiable. La spécificité était de 100 % pour toutes les cibles. Cette qPCR multiplex, adaptée à de faibles quantités d'ADN peut être mise en œuvre au laboratoire pour un diagnostic moléculaire rapide des espèces d'Echinococcus.

Development of a cockroach (Blattella germanica) qPCR for the objective measurement of exposure at home.

Cockroach allergens have a greater impact on asthma morbidity than those from dust mites, cats, and dogs. The American cockroach (Periplaneta americana) and the German cockroach (Blattella germanica) are most frequently responsible for sensitization. The worldwide prevalence of allergic sensitization has been estimated at 2 to 26 % and is influenced by unfavorable socioeconomic conditions. Exposure is generally measured by determining antigen levels in dust or through insect trapping. We developed a real-time quantitative PCR (qPCR) method to provide an objective measurement of B. germanica levels in dwellings. The specificity of the qPCR primers and TaqMan® hydrolysis probe was validated in silico with 18S rRNA sequences. No amplification was observed for other species of cockroaches, with the exception of Blattella nipponica, which is not common indoors. From 2018 to 2021, exposure to B. germanica was detected and quantified in 27 of 389 dwellings in Bourgogne-Franche-Comté (mean = 333.8; median = 9.1 and maximum = 5304 copy number equivalents) and in 236 of 3193 ELFE cohort dwellings in mainland France in 2011 (mean = 15.6; median < 1 and maximum = 1275 copy number equivalents). The distribution of dwellings testing positive for cockroaches (7 %) differed among the 12 regions of France: <1 % in two regions, between 1 and 5 % in eight regions, 16.5 % in two regions and 35 % around Paris. Exposure measurements by the EDC sampling and qPCR methods are effective ways to assess the exposure to cockroaches in dwellings. A knowledge of the level of exposure to cockroaches is particularly important for asthmatic patients, particularly those not allergic to other common antigens.

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.

Unveiling the incidences and trends of the neglected zoonosis cystic echinococcosis in Europe: a systematic review from the MEmE project.

The neglected zoonosis cystic echinococcosis affects mainly pastoral and rural communities in both low-income and upper-middle-income countries. In Europe, it should be regarded as an orphan and rare disease. Although human cystic echinococcosis is a notifiable parasitic infectious disease in most European countries, in practice it is largely under-reported by national health systems. To fill this gap, we extracted data on the number, incidence, and trend of human cases in Europe through a systematic review approach, using both the scientific and grey literature and accounting for the period of publication from 1997 to 2021. The highest number of possible human cases at the national level was calculated from various data sources to generate a descriptive model of human cystic echinococcosis in Europe. We identified 64 745 human cystic echinococcosis cases from 40 European countries. The mean annual incidence from 1997 to 2020 throughout Europe was 0·64 cases per 100 000 people and in EU member states was 0·50 cases per 100 000 people. Based on incidence rates and trends detected in this study, the current epicentre of cystic echinococcosis in Europe is in the southeastern European countries, whereas historical endemic European Mediterranean countries have recorded a decrease in the number of cases over the time.

Refractory invasive pulmonary aspergillosis due to Aspergillus flavus detected with the combination of two in-house Aspergillus qPCR.

We present a case of probable invasive pulmonary aspergillosis due to Aspergillus flavus, in a female patient treated for an acute myeloid leukemia. Two weeks after an allogenic stem cell transplantation a probable invasive pulmonary aspergillosis was diagnosed based on thoracic imaging combined with positive galactomannan antigen and positive in-house mitochondrial Aspergillus qPCR in serum. Although an antifungal treatment was initiated, Aspergillus qPCR and galactomannan antigen remained positive in serum and worsening of the thoracic lesions was observed. The discordance between the negativity of the in-house ribosomal Aspergillus qPCR (specific to A. fumigatus) and the positivity of the in-house mitochondrial Aspergillus qPCR (targeting A. fumigatus and some other Aspergillus) allowed the suspicion of a thermophilic Aspergillus species that was not A. fumigatus. No strain was obtained in culture but the involvement of A. flavus was confirmed using a specific A. flavus qPCR. This case illustrated the usefulness of our original strategy combining two different in-house Aspergillus qPCRs, in addition to galactomannan assay, to diagnose invasive aspergillosis in hematology patients.

Pilot Study Using Recombinant Antigens r-PROE and r-IGLL1 for the Serodiagnosis of Feather Duvet Lung

Background: Feather duvet lung (FDL) is an underestimated form of acute and chronic hypersensitivity pneumonitis. Serological tests for FDL need to be validated. We investigated the ability of recombinant pigeon Proproteinase E (r-PROE) and Immunoglobulin-lambda-like-polypeptide-1 (r-IGLL1) proteins to support the serological diagnosis of FDL, and propose them as a serological tool for clinicians to differentiate cases from FDL and Bird fancier's lung (BFL). Methods: Specific IgG antibodies against r-PROE and r-IGLL1, analyzed with ELISA, were measured in patients diagnosed with FDL (n=31), BFL (n=15) controls exposed (n=15) and unexposed to feathers (n=15). Results: The sensitivity and specificity of the r-PROE ELISA for the serological diagnosis of FDL cases versus exposed and unexposed controls were 74.2% and 86.7% respectively, with an index threshold of 0.5 (AUC: 0.89). In addition, this serological test was effective to support the serological diagnosis of FDL and BFL cases with significantly different thresholds. The r-IGLL1 ELISA was only effective for the serological diagnosis of BFL. Also, these two serological tests were useful for the diagnosis of both chronic and acute forms. Conclusions: The new diagnostic test for FDL using r-PROE protein should help to detect overt and hidden cases of FDL. The combination of both test will help the clinician in distinguish between the etiology of birds or feathers duvet. (AU)

Active Surveillance Program to Increase Awareness on Invasive Fungal Diseases: the French RESSIF Network (2012 to 2018).

The French National Reference Center for Invasive Mycoses and Antifungals leads an active and sustained nationwide surveillance program on probable and proven invasive fungal diseases (IFDs) to determine their epidemiology in France. Between 2012 and 2018, a total of 10,886 IFDs were recorded. The incidence increased slightly over time (2.16 to 2.36/10,000 hospitalization days, P = 0.0562) in relation with an increase of fungemia incidence (1.03 to 1.19/10,000, P = 0.0023), while that of other IFDs remained stable. The proportion of ≥65-year-old patients increased from 38.4% to 45.3% (P < 0.0001). Yeast fungemia (n = 5,444) was due mainly to Candida albicans (55.6%) with stable proportions of species over time. Echinocandins became the main drug prescribed (46.7% to 61.8%), but global mortality rate remained unchanged (36.3% at 1 month). Pneumocystis jirovecii pneumonia (n = 2,106) was diagnosed mostly in HIV-negative patients (80.7%) with a significantly higher mortality than in HIV-positive patients (21.9% versus 5.4% at 1 month, P < 0.0001). Invasive aspergillosis (n = 1,661) and mucormycosis (n = 314) were diagnosed mostly in hematology (>60% of the cases) with a global mortality rate of 42.5% and 59.3%, respectively, at 3 months and significant changes in diagnosis procedure over time. More concurrent infections were also diagnosed over time (from 5.4% to 9.4% for mold IFDs, P = 0.0115). In conclusion, we observed an aging of patients with IFD with a significant increase in incidence only for yeast fungemia, a trend toward more concurrent infections, which raises diagnostic and therapeutic issues. Overall, global survival associated with IFDs has not improved despite updated guidelines and new diagnostic tools. IMPORTANCE The epidemiology of invasive fungal diseases (IFDs) is hard to delineate given the difficulties in ascertaining the diagnosis that is often based on the confrontation of clinical and microbiological criteria. The present report underlines the interest of active surveillance involving mycologists and clinicians to describe the global incidence and that of the main IFDs. Globally, although the incidence of Pneumocystis pneumonia, invasive aspergillosis, and mucormycosis remained stable over the study period (2012 to 2018), that of yeast fungemia increased slightly. We also show here that IFDs seem to affect older people more frequently. The most worrisome observation is the lack of improvement in the global survival rate associated with IFDs despite the increasing use of more sensitive diagnostic tools, the availability of new antifungal drugs very active in clinical trials, and a still low/marginal rate of acquired in vitro resistance in France. Therefore, other tracks of improvement should be investigated actively.

Bird fancier's lung serodiagnosis by automated r-IgLL1 ELISA.

CONTEXT: Bird fancier's lung (BFL) is the most prevalent form of hypersensitivity pneumonitis (HP) worldwide. The current techniques used for the serological diagnosis of BFL all use crude extracts from feathers, droppings, and blooms as test antigens, which is associated with a lack of standardization and variability of the results. An antigenic protein, immunoglobulin lambda-like polypeptide-1 (IgLL1), isolated from pigeon droppings, was recently identified to be associated with BFL. We used genetic engineering to produce IgLL1 as a recombinant antigen. AIM: We aimed to prospectively validate the use of an automated ELISA based on recombinant IgLL1 protein (r-IgLL1) as the test antigen for the serological diagnosis of BFL. METHODS: Immunoprecipitation (IP) techniques (immunodiffusion (ID), immunoelectrophoresis (IEP)) and ELISA using r-IgLL1 were performed concomitantly over 10 months on 634 sera from patients with a BFL serodiagnosis request. Questionnaires were sent to obtain details on the avian exposure, clinical data, and final diagnosis. Concordance, sensitivity (Se), and specificity (Sp) of the two techniques were compared. RESULTS: In total, 72 completed questionnaires were returned with 18 cases of BFL diagnosed and 54 of non-BFL. The concordance between the ELISA and ID+IEP precipitation techniques was 71%. The combination of immunoprecipitation techniques showed a Se of 78% and a Sp of 67%. The ELISA using r-IgLL1 showed a Se of 89% and a Sp of 91%. The automated r-IgLL1 ELISA test is sufficiently efficient to be used alone for the diagnosis of patients exposed solely to Columbidae. In cases of other avian exposure, the Se and Sp of the r-IgLL1 ELISA used for screening combined with the immunodiffusion test for confirmation were 89% and 93%, respectively. CONCLUSIONS: The automated ELISA using r-IgLL1 is a promising tool for BFL serodiagnosis. Replacing immunodiffusion by the automated ELISA using r-IgLL1 as a screening technique will be the basis of our future strategy for BFL serodiagnosis.

Pilot Study Using Recombinant Antigens r-PROE and r-IGLL1 for the Serodiagnosis of Feather Duvet Lung.

BACKGROUND: Feather duvet lung (FDL) is an underestimated form of acute and chronic hypersensitivity pneumonitis. Serological tests for FDL need to be validated. We investigated the ability of recombinant pigeon Proproteinase E (r-PROE) and Immunoglobulin-lambda-like-polypeptide-1 (r-IGLL1) proteins to support the serological diagnosis of FDL, and propose them as a serological tool for clinicians to differentiate cases from FDL and Bird fancier's lung (BFL). METHODS: Specific IgG antibodies against r-PROE and r-IGLL1, analyzed with ELISA, were measured in patients diagnosed with FDL (n=31), BFL (n=15) controls exposed (n=15) and unexposed to feathers (n=15). RESULTS: The sensitivity and specificity of the r-PROE ELISA for the serological diagnosis of FDL cases versus exposed and unexposed controls were 74.2% and 86.7% respectively, with an index threshold of 0.5 (AUC: 0.89). In addition, this serological test was effective to support the serological diagnosis of FDL and BFL cases with significantly different thresholds. The r-IGLL1 ELISA was only effective for the serological diagnosis of BFL. Also, these two serological tests were useful for the diagnosis of both chronic and acute forms. CONCLUSIONS: The new diagnostic test for FDL using r-PROE protein should help to detect overt and hidden cases of FDL. The combination of both test will help the clinician in distinguish between the etiology of birds or feathers duvet.

Molecular diagnosis of alveolar echinococcosis in patients based on frozen and formalin-fixed paraffin-embedded tissue samples.

Confirmed diagnosis of alveolar echinococcosis (AE) is based on pathological criteria and molecular evidence. This parasite-borne disease, caused by the cestode Echinococcus multilocularis, sparingly involves humans as a dead-end host. In humans, the parasite mainly colonizes the liver but can colonize any organ and cause atypical forms, often difficult to characterize clinically. Moreover, molecular methods may be suitable to make the diagnosis of AE in cases of atypical forms, extra-hepatic localizations, or immunosuppressed patients. The aim of this study was to determine the most relevant published PCR techniques, for diagnosis of AE in patients and adopt the best strategy for molecular diagnosis depending on the nature of the tested sample. In this study, we evaluated nine end-point PCR assays and one real-time PCR assay (qPCR), targeting mitochondrial genes, using a total of 89 frozen or formalin-fixed paraffin-embedded (FFPE) samples from either 48 AE or 9 cystic echinococcosis patients. Targeted fragment-genes ranged from 84 to 529 bp. Six PCR assays were able to amplify the DNA of 100% of the frozen AE-samples and for one PCR, 69.8% of the FFPE AE-samples. The 16S rrnL PCR (84 bp) was positive in PCR for 77% of the AE samples and in qPCR for 86.5%. The sensitivity of the PCR assays was higher for fresh samples and FFPE samples stored for less than 5 years. The qPCR assay further increased sensitivity for the tested samples, confirming the need for the development of an Echinococcus spp. qPCR to improve the molecular diagnosis of echinococcoses.

TITLE: Diagnostic moléculaire de l'échinococcose alvéolaire chez les patients à partir d'échantillons de tissus congelés et fixés au formol et inclus en paraffine. ABSTRACT: La confirmation diagnostique de l'échinococcose alvéolaire (EA) est basée sur des critères anatomo-pathologiques et moléculaires. Cette maladie d'origine parasitaire, causée par le cestode Echinococcus multilocularis, implique sporadiquement l'homme, impasse parasitaire. Chez l'homme, le parasite colonise principalement le foie mais peut coloniser tout organe et causer des formes atypiques, souvent difficiles à caractériser cliniquement. En outre, les méthodes moléculaires permettent de réaliser le diagnostic de l'EA dans les formes atypiques, les localisations extra-hépatiques ou chez les patients immunodéprimés. Le but de cette étude était de déterminer les techniques PCR publiées les plus pertinentes, pour le diagnostic de l'EA chez les patients et adopter la meilleure stratégie par diagnostic moléculaire en fonction de la nature de l'échantillon testé. Dans cette étude nous avons évalué neuf PCR en point-final et une PCR-temps-réel (qPCR), ciblant des gènes mitochondriaux, utilisant 89 échantillons congelés ou fixés en paraffine (FFPE) de patients EA (n = 48) ou présentant une échinococcose kystique (n = 9). Les fragments de gènes ciblés allaient de 84 à 529 pb. Six tests PCR ont permis d'amplifier l'ADN de 100 % des échantillons EA congelés, et pour une PCR, 69,8 % des échantillons EA-FFPE. La PCR 16S rrnL (84 pb) était positive en PCR pour 77 % des échantillons EA et en qPCR pour 86,5 %. La sensibilité des tests PCR était plus importante pour les échantillons congelés et les FFPE stockés moins de 5 ans. Le test qPCR a permis d'augmenter la sensibilité pour les échantillons testés, confirmant le besoin de développement d'une qPCR Echinococcus spp. pour améliorer le diagnostic moléculaire des échinococcoses.

An overview of using fungal DNA for the diagnosis of invasive mycoses.

INTRODUCTION: Fungal PCR has undergone considerable standardization and, together with the availability of commercial assays, external quality assessment schemes, and extensive performance validation data, is ready for widespread use for the screening and diagnosis of invasive fungal disease (IFD). AREAS COVERED: Drawing on the experience and knowledge of the leads of the various working parties of the Fungal PCR initiative, this review will address general considerations concerning the use of molecular tests for the diagnosis of IFD, before focusing specifically on the technical and clinical aspects of molecular testing for the main causes of IFD and recent technological developments. EXPERT OPINION: For infections caused by Aspergillus, Candida, and Pneumocystis jirovecii, PCR testing is recommended, and combination with serological testing will likely enhance the diagnosis. For other IFD (e.g. mucormycosis), molecular diagnostics represent the only non-classical mycological approach toward diagnoses, and continued performance validation and standardization have improved confidence in such testing. The emergence of antifungal resistance can be diagnosed, in part, through molecular testing. Next-generation sequencing has the potential to significantly improve our understanding of fungal phylogeny, epidemiology, pathogenesis, mycobiome/microbiome, and interactions with the host, while identifying novel and existing mechanisms of antifungal resistance and novel diagnostic/therapeutic targets.