Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Cryptosporidiosis after treatment with fingolimod: a case report and pharmacovigilance review.

BACKGROUND: Cryptosporidium sp. are common intracellular parasites responsible of severe diarrhea in T-cell-immunocompromised patients. We report the first case of a woman who contracted cryptosporidiosis after treatment with fingolimod, a drug labeled for multiple sclerosis and responsible for marked lymphopenia. CASE PRESENTATION: A 60-year-old woman was admitted for abdominal pain diarrhea and fever. The patient suffered from multiple sclerosis and had been treated with fingolimod from august 2017 to september 2018 time of occurrence of the first digestive symptoms. Stool culture was negative but parasitological examination was positive for Cryptosporidium sp. Blood biological examination profound lymphopenia of 240/mm3 [17 CD4/mm3 (7%) and 32 CD8/mm3 (14%)]. Fingolimod was stopped, and the patient was put on nitazoxanide 500 mg bid for 7 days. The diarrhea resolved and no relapse was observed. Six other cases were found in the Pharmacovigilance database. CONCLUSION: Physicians should be aware of this association and screen for Cryptosporidium in cases of diarrhea in patients treated with fingolimod. Patients should be aware of this risk and advise to take appropriate measures to avoid such contamination.

Multicenter Comparison of the Etest and EUCAST Methods for Antifungal Susceptibility Testing of Candida Isolates to Micafungin.

In vitro susceptibility of 933 Candida isolates, from 16 French hospitals, to micafungin was determined using the Etest in each center. All isolates were then sent to a single center for determination of MICs by the EUCAST reference method. Overall essential agreement between the two tests was 98.5% at ±2 log2 dilutions and 90.2% at ±1 log2 dilutions. Categorical agreement was 98.2%. The Etest is a valuable alternative to EUCAST for the routine determination of micafungin MICs in medical mycology laboratories.

Ruling out nosocomial transmission of Cryptosporidium in a renal transplantation unit: case report.

BACKGROUND: Cryptosporidium spp. is a ubiquitous parasite affecting humans as well as domestic and wild vertebrates, causing diarrhea in both immunocompetent and immunocompromised hosts worldwide. Its transmission occurs primarily by the fecal-oral route. In humans, C. parvum and C. hominis are the most prevalent species, whereas immunocompetent and immunocompromised individuals can also be infected by other zoonotic species. Renal transplant patients are prone to develop cryptosporidiosis, which can induce severe and life-threatening diarrhea. CASE PRESENTATION: We report here a series of nearly concomitant cases of acute symptomatic cryptosporidiosis in three renal transplant patients attending the Strasbourg University Hospital Nephrology Unit. The clinical presentation was persistent diarrhea and acute renal failure. The diagnosis was confirmed by microscopic stool examination using a modified Ziehl-Neelsen staining method and species identification by molecular tools. All patients were treated with nitazoxanide and recovered from diarrhea after 14 days of therapy. CONCLUSION: Genotypic species identification was not consistent with an epidemic context, thus underlining the need for genotyping to monitor at risk patients.

The reversed halo sign: pathognomonic pattern of pulmonary mucormycosis in leukemic patients with neutropenia?

BACKGROUND: Pulmonary mucormycosis (PM) is a life-threatening fungal infection with an increasing incidence among patients with acute leukemia. In some immunocompromised hosts, the reversed halo sign (RHS) has been described on the pulmonary computed tomographic (CT) scan of patients with mucormycosis. METHODS: This study reports a single-center experience with PM exclusively in patients with acute leukemia. Clinical records, laboratory results, and CT scans were retrospectively analyzed to evaluate the clinical usefulness of the RHS for the early identification and treatment of PM, with regard to outcomes in these patients. RESULTS: Between 2003 and 2012, 16 cases of proven PM were diagnosed among 752 consecutive patients receiving chemotherapy for acute myeloblastic or lymphoblastic leukemia. At the time PM was diagnosed, all patients but one were neutropenic. The study of sequential thoracic CT scans showed that during the first week of the disease, the RHS was observed in 15 of 16 patients (94%). Initially, other radiologic findings (multiple nodules and pleural effusion) were less frequent, but appeared later in the course of the disease (6% and 12% before vs 64% and 55% after the first week). After the diagnosis of PM, median overall survival was 25 weeks (range, 3-193 weeks), and 6 patients (38%) died before day 90. CONCLUSIONS: In the particular setting of neutropenic leukemia patients with pulmonary infection, the presence of the RHS on CT was a strong indicator of PM. It could allow the early initiation of appropriate therapy and thus improve the outcome.

Current knowledge and practice of Candida auris screening in France: A nationwide survey from the French Society of Medical Mycology (SFMM).

Due to large outbreaks observed worldwide, Candida auris has emerged as a major threat to healthcare facilities. To prevent these phenomena, a systematic screening should be performed in patients transferred from regions where the pathogen is highly endemic. In this study, we recorded and analyzed French mycologists' current knowledge and practice regarding C. auris screening and diagnosis. Thirty-six centers answered an online questionnaire. Only 11 (30.6 %) participants were aware of any systematic screening for C. auris for patients admitted to their hospital. In the case of post-admission screening, axillae/groins (n = 21), nares (n = 7), rectum (n = 9), and mouth (n = 6) alone or various combinations were the body sites the most frequently sampled. Only six centers (8.3 %) reported using a commercially available plate allowing the differentiation of C. auris colonies from that of other Candida species, while five laboratories (13.8 %) had implemented a C. auris-specific qPCR. Considering the potential impact on infected patients and the risk of disorganization in the care of patients, it is crucial to remember to biologists and clinicians the utmost importance of systematic screening on admission.

Histological and molecular biology diagnosis of neurocysticercosis in a patient without history of travel to endemic areas: case report.

BACKGROUND: in endemic areas, neurocysticercosis appears mainly as a single, large, spherical and non-enhancing intracranial cyst. CASE PRESENTATION: an atypical case of neurocysticercosis (NCC) in a French Caucasian, without history of travel to endemic areas, was confirmed by histology and molecular speciation. Imaging was atypical, showing several hook-bearing scolices visible in the cyst, while the serology employed was non-contributary. CONCLUSIONS: NCC should be considered when multiple taeniid scolices are observed within the same cystic lesion.

Evaluation of five automated and one manual method for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid.

OBJECTIVES: Molecular detection of Toxoplasma gondii plays a crucial role in the prenatal and neonatal diagnosis of congenital toxoplasmosis (CT). Sensitivity of this diagnosis is partly related to the efficiency of parasite DNA extraction and amplification. DNA extraction methods with automated platforms have been developed. Therefore, it is essential to evaluate them in combination with adequate PCR amplification assays. METHODS: In this multisite study, we investigated the suitability of two recent automated procedures for the isolation of Toxoplasma DNA from amniotic fluid (AF) (Magtration system 12GC, PSS and Freedom EVO VacS, Tecan), compared with three other automated procedures (MagNAPure Compact, Roche, BioRobot EZ1, Qiagen and modified NucliSens easyMAG, bioMérieux) and with the manual DNA extraction QIAamp DNA Mini kit (Qiagen). Two Toxoplasma PCR assays targeting the '529-bp' repeat DNA element were used, based upon dual hybridization (FRET) or hydrolysis (TaqMan) probes. A total of 1296 PCRs were performed including 972 Toxoplasma PCRs. RESULTS: We showed variable efficacy (4.2%-100% positive results) among the DNA extraction procedures in isolating up to five T. gondii cells/mL in AF samples. Moreover, for a given DNA extraction method, variable results were obtained among the two Toxoplasma PCR assays for detecting up to five T. gondii cells/mL: when using TaqMan PCR, all the automated systems yielded more than 60% positive results. Nevertheless, when testing the DNA extracts in triplicate, four out of six extraction methods allowed a satisfactory detection of low amounts of T. gondii DNA (≥33% of positive results) independently of the PCR assay used. CONCLUSIONS: Despite the influence of the subsequent PCR method used, this study should help microbiologists in the choice of DNA extraction methods for the detection of T. gondii in amniotic fluid. The extraction method should be checked as adequate for the PCR assay used.

First case of proven invasive pulmonary infection due to Trichoderma longibrachiatum in a neutropenic patient with acute leukemia.

Trichoderma species are saprophytic filamentous fungi that can be found all over the word. These fungi show increasing medical importance as opportunistic human pathogens, particularly in immunocompromised patients. Invasive infections due to Trichoderma are rare and definitive diagnosis is complex to achieve because of the lack of specific diagnosis tools. We report in this work the first proven case of invasive pulmonary infection due to T. longibrachiatum in a 69-year-old white male with hematologic malignancy. The patient was successfully treated initially with voriconazole alone followed by a combination of voriconazole and caspofungine.

[Retrospective study of 25 cases of pulmonary mucormycosis in acute leukaemia]. / Mucormycoses pulmonaires au cours des traitements de leucémies aiguës. Analyse rétrospective d'une série de 25 patients.

INTRODUCTION: In acute leukaemia (AL), the occurrence of pulmonary mucormycosis (PM), the incidence of which is increasing, as a result of chemotherapy induced marrow aplasia, remains a life threatening complication. METHODS: Analysis of clinical, biological and thoracic CT characteristics of patients with PM developing during the treatment of AL between 2000 and 2015. Day 0 (D0) was defined as the day with first CT evidence of PM. RESULTS: Among 1193 patients, 25 cases of PM were recorded during 2099 episodes of bone marrow aplasia. At time of diagnosis of PM, 24/25 patients had been neutropenic for a median of 12 days. None of the patients had diabetes mellitus. On initial CT (D0), the lesion was solitary in 20/25 cases and a reversed halo sign (RHS) was observed in 23/25 cases. From D1 to D7, D8 to D15 and after D15, RHS was seen in 100 %, 75 % and 27 % of cases, respectively. A tissue biopsy was positive in 17/18 cases. The detection of circulating Mucorales DNA in serum was positive in 23/24 patients and in 97/188 serum specimens between D-9 and D9. Bronchoalveolar lavage contributed to diagnosis in only 3/21 cases. The antifungal treatment was mainly based on liposomal amphotericin B combined with, or followed by, posaconazole. A pulmonary surgical resection was performed in 9/25 cases. At 3 months, 76 % of patients were alive and median overall survival was 14 months. CONCLUSION: In AL, early use of CT could improve the prognosis of PM. The presence of a RHS on CT suggests PM and is an indication for prompt antifungal treatment.

Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis, Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification.

OBJECTIVES: Besides the potential to identify a wide variety of gastrointestinal parasites, microscopy remains the reference standard in clinical microbiology for amoeba species identification and, especially when coupled with adhesin detection, to discriminate the pathogenic Entamoeba histolytica from its sister but non-pathogenic species Entamoeba dispar/Entamoeba moshkovskii. However, this approach is time-consuming, requires a high-level of expertise that can be jeopardized considering the low prevalence of gastrointestinal parasites in non-endemic countries. Here, we evaluated the CE-IVD-marked multiplex PCR (ParaGENIE G-Amoeba, Ademtech) targeting E. histolytica and E. dispar/E. moshkovskii and Giardia intestinalis. METHODS: This evaluation was performed blindly on a reference panel of 172 clinical stool samples collected prospectively from 12 laboratories and analysed using a standardized protocol relying on microscopy (and adhesin detection by ELISA for the detection of E. histolytica) including G. intestinalis (n = 37), various amoeba species (n = 55) including E. dispar (n = 15), E. histolytica (n = 5), as well as 17 other gastrointestinal parasites (n = 80), and negative samples (n = 37). RESULTS: This new multiplex PCR assay offers fast and reliable results with appropriate sensitivity and specificity for the detection of G. intestinalis and E. dispar/E. moshkovskii from stools (89.7%/96.9% and 95%/100%, respectively). Detection rate and specificity were greatly improved by the PCR assay, highlighting several samples misidentified by microscopy, including false-negative and false-positive results for both E. dispar/E. moshkovskii and E. histolytica. CONCLUSION: Given the clinical relevance of amoeba species identification, microbiologists should be aware of the limitations of using an algorithm relying on microscopy coupled with adhesin detection by ELISA.

Reduced fungal contamination of the indoor environment with the Plasmair system (Airinspace).

Aspergillus spp. and other moulds cause life-threatening opportunistic infections in immunocompromised patients. Indoor contamination and construction work that liberate fungal spores are a major source of nosocomial aspergillosis. Dijon hospital is a tertiary care institution in northeast France undergoing construction work beside high-risk clinical units. To determine the impact of this activity, a surveillance programme was implemented one year before building work began in order to establish baseline levels of contamination. Air and surface fungal contamination in adult and paediatric haematology units were prospectively examined following use, or not, of a new air-treatment system with mobile Plasmair units (Airinspace). There were significant reductions in overall fungal contamination for the Plasmair treated rooms for air and surface samples in both clinical units. Plasmair treatment also significantly reduced A. fumigatus in the air. These data suggest that Plasmair units may provide an efficient method of reducing indoor fungal contamination in hospitals.

Prospective survey of indoor fungal contamination in hospital during a period of building construction.

An 18-month survey of indoor fungal contamination was conducted in one haematology unit during a period of construction work. Air was sampled with a portable Air System Impactor and surfaces with contact Sabouraud plates. During this survey the mean concentration of viable fungi in air was 4.2 cfu/m(3) and that for surfaces was 1.7 cfu/plate. At the beginning of construction work, there were increases in airborne fungal spores (from 3.0 to 9.8 cfu/m(3)) in the unit, but concentrations did not exceed 10 cfu/m(3) during the 18-month period. The most frequently recovered airborne fungi were Penicillium spp. (27-38%), Aspergillus spp. (25%) and Bjerkandera adusta, a basidiomycete identified with molecular tools (7-12%). Blastomycetes accounted for more than 50% of the fungal flora on surfaces. Investigating the impact of a new air-treatment system (mobile Plasmair units), there were significant reductions in fungal contamination for the Plasmer -treated rooms, and in these rooms we observed the same level of fungal load whether construction work was in progress or not.

Emergency and elective pulmonary surgical resection in haematological patients with invasive fungal infections: a report of 50 cases in a single centre.

Invasive fungal infections (IFI) remain life-threatening complications in haematological patients. The aim of the study was to present the experience of a single centre in the surgical treatment of pulmonary IFI. Between 1992 and 2014, 50 haematological patients with IFI underwent pulmonary resection. In 27 cases it was an emergency procedure to avoid haemoptysis (if the lesion threatened pulmonary vessels). The remaining 23 patients underwent elective surgery before new chemotherapy or stem-cell transplantation. Among these patients (median age: 54 years; range: 5-70 years), 92% had acute leukaemia and 68% were on haematological first-line therapy (receiving induction or consolidation chemotherapies). Invasive pulmonary aspergillosis and pulmonary mucormycosis were diagnosed in 37 and 12 patients, respectively. One patient had IFI due to Trichoderma longibrachiatum. All of the patients received antifungal agents. In the month preceding IFI diagnosis, 94% of patients had been neutropenic. At the time of surgery, 30% of patients were still neutropenic and 54% required platelet transfusions. Lobectomy or segmentectomy were performed in 80% and 20% of cases, respectively. Mortality at 30 and 90 days post-surgery was 6% and 10%, respectively. After surgery, median overall survival was 21 months; median overall survival was similar between patients with emergency or elective surgery and between the types of IFI (invasive pulmonary aspergillosis or pulmonary mucormycosis). However, overall survival was far better in haematological first-line patients or in those achieving a haematological complete response than in other patients (p <0.001). In pulmonary IFI, lung resection could be an effective complement to medical treatment in selected haematological patients.

Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice.

[A case of free-living Amoebae keratitis in a non contact lens wearer]. / Une kératite à amibes libres chez un non porteur de lentilles de contact.

Free living amoebae keratitis is a rare but severe infection due to ubiquitous protozoa of the genus Acanthamoeba. Most cases occur in contact lens wearers. In the present paper, we report a case of Acanthamoeba keratitis secondary to a vegetal injury of the cornea in a patient who did not wear contact lens. This case emphasizes the fact that the visual outcome is dependent on early treatment and outlines the need for a rapid diagnosis of amoebic keratitis. The diagnosis is based essentially on culture of trophozoïtes and cysts of the parasite from a corneal scrape or a biopsy specimen. The treatment is long, difficult and often a failure. Successful management of amoebic keratitis infection thus requires constant dialogue between the physician and the clinical microbiologist, a quality sample and efficient laboratory tests.

Comparison of MALDI-TOF mass spectra with microsatellite length polymorphisms in Candida albicans.

Candida albicans is the most frequent yeast involved in human infections. Its population structure can be divided into several genetic clades, some of which have been associated with antifungal susceptibility. Therefore, detecting and monitoring fungal clones in a routine laboratory setting would be a major epidemiological advance. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra results are now widely used as bar codes to identify microorganisms in clinical microbiology laboratories. This study aimed at testing MALDI-TOF mass spectra bar codes to identify clades among a set of C. albicans isolates. Accordingly, 102 clinical strains were genotyped using 10 microsatellite markers and analyzed via MALDI-TOF mass spectrometry. The mass spectra were compared with a reference spectral library including 33 well-characterized collection strains, using a Microflex(TM) system and Biotyper(TM) software, to test the capacity of the spectrum of a given isolate to match with the reference mass spectrum of an isolate from the same genetic clade. Despite high confidence species identification, the spectra failed to significantly match with the corresponding clade (p = 0.74). This was confirmed with the MALDI-TOF spectra similarity dendrogram, in which the strains were dispersed irrespective of their genetic clade. Various attempts to improve intra-clade spectra recognition were unsuccessful. In conclusion, MALDI-TOF mass spectra bar code analysis failed to reliably recognize genetically related C. albicans isolates. Further studies are warranted to develop alternative MALDI-TOF mass spectra analytical approaches to identify and monitor C. albicans clades in the routine clinical laboratory.

Decrease in Candida albicans strains with reduced susceptibility to fluconazole following changes in prescribing policies.

This study was undertaken to identify prescribing policies likely to favour or limit fluconazole resistance within a clinical department. Fluconazole exposure within the infectious diseases and clinical haematology units was investigated, and data were compared with in vitro susceptibility of Candida albicans isolates obtained in these units. Fluconazole utilization was determined by the number of fluconazole treatment-days per 100 hospitalization days (penetration index). In the infectious diseases unit, separate evaluations for low-dose fluconazole (50 mg) prescribed as intermittent or prolonged treatment, and for higher-dosing schedules (fluconazole 200 mg) were made. Susceptibility of C. albicans isolates was surveyed in a broth microdilution assay by measuring the inhibitory concentration 50% (IC50). The penetration index (PI) for fluconazole 50mg declined from 1992 to 1977 in infectious diseases (P= 0.0048). In the meantime, total usage of fluconazole increased, due to increased prescribing of fluconazole 200 mg (P = 0.0724). The IC50 of C. albicans isolates tested in infectious diseases decreased between 1994 and 1996 from 7.33 mg/ml to 1.64 mg/ml (P = 0.0075). In clinical haematology, declines in C. albicans IC50 and fluconazole PI were not significant (P = 0.35 and P = 0.07, respectively). These data suggest that prolonged or repeated exposure to low-dose fluconazole, rather than total cumulative use, was associated with fluconazole resistance in the infectious diseases unit. Moreover, restoration of a normal ecology was observed when low-dose prolonged or intermittent prescriptions were reduced.