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.

Comparison of the MICs Obtained by Gradient Concentration Strip and EUCAST Methods for Four Azole Drugs and Amphotericin B against Azole-Susceptible and -Resistant Aspergillus Section Fumigati Clinical Isolates.

Reference methods used to assess the drug susceptibilities of Aspergillus fumigatus isolates consisted of EUCAST and CLSI standardized broth microdilution techniques. Considering the increasing rate and the potential impact on the clinical outcome of azole resistance in A. fumigatus, more suitable techniques for routine testing are needed. The gradient concentration strip (GCS) method has been favorably evaluated for yeast testing. The aim of this study was to compare the CGS test with EUCAST broth microdilution for amphotericin B (AMB), posaconazole (PCZ), itraconazole (ITZ), voriconazole (VRZ), and isavuconazole (ISA). A total of 121 Aspergillus section Fumigati strains were collected, including 24 A. fumigatus sensu stricto strains that were resistant to at least one azole drug. MICs were determined using GCS and EUCAST methods. Essential agreement between the 2 methods was considered when MICs fell within ±1 dilution or ±2 dilutions of the 2-fold dilution scale. Categorical agreement was defined as the percentage of strains classified in the same category (susceptible, intermediate, or resistant) with both methods. Essential agreements with ±1 dilution and ±2 dilutions were 96.7, 93.4, 90.0, 89.3, and 95% and 100, 99.2, 100, 97.5, and 100% for AMB, PCZ, ITZ, VRZ, and ISA, respectively. Categorical agreements were 94.3, 86.1, 89.3, and 88.5% for AMB, PCZ, ITZ, and VRZ, respectively. Detection of resistance was missed with the GCS for one strain (4.1%) for PCZ and for 2 strains (8.3%) for ISA. Determination of ITZ MICs using the GCS allowed the detection of 91.7% of azole-resistant strains. The GCS test appears to be a valuable method for screening azole-resistant A. fumigatus clinical isolates.

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.

Dramatic improvement of severe cryptococcosis-induced immune reconstitution syndrome with adalimumab in a renal transplant recipient.

In solid organ transplant recipients, immune reconstitution inflammatory syndrome (IRIS) is a rare complication of cryptococcosis, which may require steroids in its most severe forms. Here, we report the case of a renal transplant recipient who developed severe cryptococcal meningitis-associated IRIS 1 week after immunosuppression reduction. High-dose steroids failed to improve the disease. Finally, a recombinant human monoclonal tumor necrosis factor-α (TNF-α) antagonist, adalimumab, was prescribed, and the patient rapidly experienced dramatic neurological improvement. No IRIS relapse occurred within 14 months following adalimumab discontinuation.

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.

Disseminated toxoplasmosis in non-allografted patients with hematologic malignancies: report of two cases and literature review.

Toxoplasmosis can be a severe opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS), and also among solid organ transplant and allogeneic hematopoietic stem cell transplant (HSCT) patients. Patients with low-grade or chronic hematologic malignancies are treated with increasing immunosuppressive regimens and, therefore, represent an emerging population at risk for opportunistic diseases. We report here two cases of disseminated toxoplasmosis occurring in non-allografted hematologic patients with chronic lymphoproliferations. A review of 44 cases from the literature reveals that toxoplasmosis occurs increasingly in indolent B cell lymphoproliferative disorders. Aggressive lymphoproliferations, adenosine analogs, autologous HSCT, and the absence of chemoprophylaxis are the main risk factors for opportunistic toxoplasmosis. The central nervous system is the main organ involved. Fever is only present in half of all cases. Latent Toxoplasma cysts reactivation (LTCR) is the most common, but primary infection occurs in about 20% of cases. Global mortality is over 50%.

Mucormycosis as a rare cause of severe gastrointestinal bleeding after multivisceral transplantation.

Mucormycosis, an emerging fungal infection in solid organ transplant patients, is mostly located in rhino-orbito-cerebral, pulmonary, and cutaneous areas, or disseminated with poor prognosis. A 4-year-old girl with chronic intestinal pseudo-obstruction syndrome underwent a modified multivisceral transplantation, including half of the stomach, the duodeno-pancreas, the small bowel, and the right colon. On postoperative day 5, a digestive perforation was suspected. Surgical exploration found a small necrotic area on the native stomach, which was externally drained. The next day, massive gastric bleeding occurred. During the emergency laparotomy, 2 hemorrhagic ulcers were found and resected from the transplanted stomach. Pathology and fungal culture showed mucormycosis caused by Lichtheimia (formerly Absidia) ramosa in both the transplanted and native stomach. High-dose intravenous liposomal amphotericin B was immediately started. No other site of fungal infection was found. The child recovered, and 3 years after transplantation, is alive and well, off parenteral nutrition. The originality of this case is the very early presentation after transplantation, the unusual site, and the complete recovery after rapid medico-surgical management. The origin of the fungus and treatment are discussed.

Cerebral aspergillosis in the era of new antifungals: The CEREALS national cohort study Nationwide CEREbral Aspergillosis Lesional study (CEREALS).

BACKGROUND: Cerebral aspergillosis (CA) is a life-threatening disease for which diagnosis and management remain challenging. Detailed analyses from large cohorts are lacking. METHODS: We included 119 cases of proven (n = 54) or probable (n = 65) CA diagnosed between 2006 and 2018 at 20 French hospitals. Data were collected at baseline and during follow-up. Cerebral imaging was reviewed centrally by two neuroradiologists. RESULTS: The most frequent underlying conditions were hematological malignancy (40%) and solid organ transplantation (29%). Galactomannan was detected in the serum of 64% of patients. In 75% of cases, at least one of galactomannan, Aspergillus PCR, and ß-d-glucan was positive in the cerebrospinal fluid. Six-week mortality was 45%. Two distinct patterns of disease were identified according to presumed route of dissemination. Presumed haematogenous dissemination (n = 88) was associated with a higher frequency of impaired consciousness (64%), shorter time to diagnosis, the presence of multiple abscesses (70%), microangiopathy (52%), detection of serum galactomannan (69%) and Aspergillus PCR (68%), and higher six-week mortality (54%). By contrast, contiguous dissemination from the paranasal sinuses (n = 31) was associated with a higher frequency of cranial nerve palsy (65%), evidence of meningitis on cerebral imaging (83%), macrovascular lesions (61%), delayed diagnosis, and lower six-week mortality (30%). In multivariate analysis and in a risk prediction model, haematogenous dissemination, hematological malignancy and the detection of serum galactomannan were associated with higher six-week mortality. CONCLUSION: Distinguishing between hematogenous and contiguous dissemination patterns appears to be critical in the workup for CA, as they are associated with significant differences in clinical presentation and outcome.

Pneumocystosis: a network survey in the Paris area 2003-2008.

The aims of this network group were to collect epidemiological data of PcP cases in 14 hospitals in the Paris area and to determine the Di-Hydro Pteroate Synthase (DHPS) genotypes, genetic markers for possible sulfamide resistance. From January 1, 2003 to December 31, 2008, 993 (mean 166/year) PcP cases have been reported. Sixty-five percent of patients were HIV-positive. The median count of CD4 lymphocytes was 32/mm(3) (30 in HIV-positive patients, 152 in HIV-negative patients). In HIV-positive patients, PcP revealed the HIV infection in 39%. Among 304 PcP occurring in HIV known infected patients, no prophylaxis was prescribed for 64%; cotrimoxazole prophylaxis had been prescribed to 47 patients but only one of them had the right compliance. In HIV-negative patients (264), corticosteroids were prescribed in 59% and cytotoxic chemotherapies in 34%; 78% did not receive prophylaxis. One hundred sixty nine tumoral pathologies and 116 transplantations were notified. The mortality rate was 16% at day 14 (13% in HIV-positive patients, 26% in HIV-negative patients). Mutations in DHPS genes were detected in 18.5% of samples; 12.5% of patients were infected with several strains. The total annual number of cases has been stable for five years but the proportion of HIV-negative patients increased from 25% to 43%.

Unusual presentation of chromoblastomycosis due to Cladophialophora carrionii in a renal and pancreas transplant recipient patient successfully treated with posaconazole and surgical excision.

Chromoblastomycosis is a chronic, tropical and subtropical, subcutaneous mycosis caused by inoculation of dematiaceous molds. This disease is uncommonly reported in patients who have undergone solid organ transplantation. We describe a case of chromoblastomycosis caused by Cladophialophora carrionii that occurred 7 years after transplantation in a 58-year-old male renal and pancreatic transplant recipient. Diagnosis was based on histopathology and isolation of multiple colonies of the dematiaceous mold in pure culture. Identification was achieved by sequencing of the internal transcribed spacer regions of the rRNA. The patient was successfully treated with posaconazole and surgical excision of a residual lesion.

Post-traumatic Curvularia sp. arthritis in an immunocompetent adult.

A 44-year-old woman, victim of a road accident in Mali was diagnosed with left knee arthritis. Joint effusion aspiration and subcutaneous surgical biopsies were positive for a melanized asexual ascomycete. Using microscopy and molecular biology, the fungus was identified as Curvularia sp. In vitro antifungal susceptibility was determined by the EUCAST broth microdilution reference technique and by E-test. The patient was treated with liposomal amphotericin B before posaconazole relay. Mycological samples obtained 10 days after starting the antifungal therapy by liposomal amphotericin B were negative in culture. Curvularia spp. are environmental fungi which can under certain conditions be pathogenic for humans.

Diagnostic accuracy of serum (1,3)-beta-d-glucan for neonatal invasive candidiasis: systematic review and meta-analysis.

OBJECTIVES: Neonatal invasive candidiasis (NIC) is a leading cause of infection-related morbidity and mortality in preterm neonates. Several studies have shown that (1,3)-Beta-d-glucan (BDG) was accurate in detecting invasive fungal infection in adults, but studies in neonates are scarce. The aim was to obtain summary estimates of the accuracy of BDG detection in serum for the diagnosis of NIC. METHODS: We searched Medline, Embase, Clinicaltrials.gov, and Google Scholar (inception to July 2019). We checked the reference lists of included studies, clinical guidelines, and review articles. We included studies that assessed the accuracy of BDG against a reference standard that defined groups of patients with ordinal levels of NIC probability (e.g. proven, probable, possible) and included fungal blood culture. Participants were neonates suspected of having NIC. The intervention was BDG measurement in serum (Fungitell® assay). We assessed risk of bias and applicability using QUADAS-2. We used bivariate meta-analysis to produce summary estimates of diagnostic accuracy at prespecified positivity thresholds of 80 and 120 pg/mL. This study was registered with PROSPERO (CRD42018089545). RESULTS: We included eight studies (465 participants). Of these, two were judged at low overall risk of bias. There was substantial variability across studies in the reference standards used. At a positivity threshold of 80 pg/mL, summary estimates of sensitivity and specificity of BDG were 89% (95% CI: 80-94%) and 60% (53-66%), respectively; summary sensitivity for detecting proven cases of NIC was 99% (93-100%). At a positivity threshold of 120 pg/mL, summary estimates of sensitivity and specificity were 81% (71-88%) and 80% (67-88%), respectively. CONCLUSIONS: Because of high sensitivity, BDG seems promising to rule-out NIC. It might be too early to recommend its use because of the scarcity of reliable clinical data, heterogeneity in case definitions, and unstable accuracy estimates.

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

Microsporidiosis in solid organ transplant recipients: two Enterocytozoon bieneusi cases and review.

Microsporidiosis first came to prominence as an opportunistic infection in patients with acquired immunodeficiency syndrome. Microsporidia are now emerging pathogens responsible for severe diarrhea during solid organ transplantation. Two main clinical entities can be identified: infection by Enterocytozoon bieneusi, causing diarrhea with limited treatment options; and infection by Encephalitozoon intestinalis, which may disseminate and usually responds to albendazole treatment. We describe here 2 cases of microsporidiosis caused by E. bieneusi in a renal and a liver transplant recipient, respectively, in whom complete clinical efficacy of a short course of fumagillin therapy was obtained. Long-term microbiological eradication was assessed using classical methods and monitored using a real-time quantitative polymerase chain reaction-based method. Both patients experienced drug-induced thrombocytopenia, which resolved after withdrawal of the treatment. We also review the 18 other previously reported cases of microsporidiosis in transplant recipients. In case of persistent diarrhea in solid organ transplant patients, microsporidiosis should be considered. Based on the present experience, treating E. bieneusi infection with 7 days of fumagillin therapy is adequate to eradicate E. bieneusi in this context.

Detection of circulating Aspergillus fumigatus DNA by real-time PCR assay of large serum volumes improves early diagnosis of invasive aspergillosis in high-risk adult patients under hematologic surveillance.

Detection of galactomannan antigen (GMA) in serum is the standard assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematological disorders. Detection of Aspergillus DNA in serum has been proposed, but its sensitivity is lower than that of GMA when small serum volumes (SSV) are used. In this study, we investigated whether extraction of DNA from large serum volumes (LSV) improves diagnostic yield. In a 13-month prospective study, we compared the performances of twice-weekly screening of serum for GMA by an enzyme immunoassay and weekly screening for Aspergillus fumigatus DNA by a real-time PCR (RT-PCR) assay of 1.0 ml (LSV) or 100 mul (SSV) of serum. We included 124 patients (138 treatment episodes), with 17 episodes of EORTC (European Organization for Research and Treatment of Cancer)/MSG (Mycoses Study Group)-documented IA. In all, 1,870 samples were screened for GMA. The sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV, respectively) of GMA for IA were 88.2%, 95.8%, 75%, and 98.3%, respectively. We screened 938 samples for Aspergillus DNA by using LSV; 404 of these samples were also tested with SSV. The Se, Sp, PPV, and NPV of RT-PCR were 100%, 96.7%, 81%, and 100%, respectively, with LSV and 76.5%, 96.7%, 81.3%, and 95.6%, respectively, with SSV. DNA detection gave a positive result when performed on LSV in two cases of IA where the GMA assay result remained negative. Furthermore, in four IA cases, DNA was detected earlier than GMA. The use of LSV for extraction improved the performance of the RT-PCR, which appears highly sensitive and specific for the early diagnosis of IA in high-risk patients with hematological disorders.

[Coccidioidomycosis: an imported invasive fungal disease in France]. / Coccidioïdomycose : une maladie d'importation d'actualité en France.

Coccidioidomycosis is an endemic mycosis in the southwest of United States resulting from the inhalation of arthrospores present in desert soil. The authors present a case of uncomplicated pulmonary coccidioidomycosis in a healthy woman, acquired during a recent trip to California. The initial clinical presentation first suggested a diagnosis of community-acquired pneumonia, then of tuberculosis. The diagnosis was finally reached with blood tests and mycological culture of broncho-alveolar lavage fluid. The final identification of Coccidioides immitis was made by molecular analysis. Clinical resolution of the infection was obtained after three months of posaconazole treatment. Coccidioidomycosis is a major cause of pneumonia. Its diagnosis requires specific investigation such as mycological culture, histology, blood tests and molecular biology helps to identify the species. The progression of the disease as well as the associated immunocellular deficit are strictly correlated with the onset of complications and late relapses despite an adequate initial treatment using antifungal molecules and/or surgery.

Could we predict airborne Aspergillus contamination during construction work?

BACKGROUND: Aspergillus fumigatus is a major opportunistic pathogen causing nosocomial infection. Hospital outbreaks of invasive aspergillosis have been associated with demolition and building construction. This study was designed to examine the impact of meteorologic factors and different periods of work on outdoor fungal airborne concentrations. METHODS: The study was conducted at Necker Enfants Malades Hospital, a 650-bed teaching care hospital recently involved in a large construction program, including renovation, construction, and demolition. During the work phases, prospective external air samplings were performed 3 times a week, and meteorologic parameters were collected every day. RESULTS: Two hundred and one samples were collected. Aspergillus spp were found in 80.1% of samples, with a median concentration of 16 colony forming units (CFU)/m3. A significant increase in the colony count of molds occurred after demolition. In the multivariate analysis, factors associated with overall fungi concentration were the type of work construction and temperature. Elevated Aspergillus spp concentrations (>20 CFU/m3) were associated with higher temperature. CONCLUSIONS: Our findings underline the importance of environmental surveillance. According to our results we suggest that demolition work should be performed during the winter and fall seasons.

The intraspecies diversity of C. albicans triggers qualitatively and temporally distinct host responses that determine the balance between commensalism and pathogenicity.

The host immune status is critical for preventing opportunistic infections with Candida albicans. Whether the natural fungal diversity that exists between C. albicans isolates also influences disease development remains unclear. Here, we used an experimental model of oral infection to probe the host response to diverse C. albicans isolates in vivo and found dramatic differences in their ability to persist in the oral mucosa, which inversely correlated with the degree and kinetics of immune activation in the host. Strikingly, the requirement of interleukin (IL)-17 signaling for fungal control was conserved between isolates, including isolates with delayed induction of IL-17. This underscores the relevance of IL-17 immunity in mucosal defense against C. albicans. In contrast, the accumulation of neutrophils and induction of inflammation in the infected tissue was strictly strain dependent. The dichotomy of the inflammatory neutrophil response was linked to the capacity of fungal strains to cause cellular damage and release of alarmins from the epithelium. The epithelium thus translates differences in the fungus into qualitatively distinct host responses. Altogether, this study provides a comprehensive understanding of the antifungal response in the oral mucosa and demonstrates the relevance of evaluating intraspecies differences for the outcome of fungal-host interactions in vivo.

Unexpected persistence of extended-spectrum ß-lactamase-producing Enterobacteriaceae in the faecal microbiota of hospitalised patients treated with imipenem.

Imipenem is active against extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) but favours the intestinal emergence of resistance. The effects of imipenem on intestinal microbiota have been studied using culture-based techniques. In this study, the effects were investigated in patients using culture and metagenomic techniques. Seventeen hospitalised adults receiving imipenem were included in a multicentre study (NCT01703299, http://www.clinicaltrials.gov). Most patients had a history of antibiotic use and/or hospitalisation. Stools were collected before, during and after imipenem treatment. Bacterial and fungal colonisation was assessed by culture, and microbiota changes were assessed using metagenomics. Unexpectedly, high colonisation rates by imipenem-susceptible ESBL-E before treatment (70.6%) remained stable over time, suggesting that imipenem intestinal concentrations were very low. Carriage rates of carbapenem-resistant Gram-negative bacilli (0-25.0%) were also stable over time, whereas those of yeasts (64.7% before treatment) peaked at 76.5% during treatment and decreased thereafter. However, these trends were not statistically significant. Yeasts included highly diverse colonising Candida spp. Metagenomics showed no global effect of imipenem on the bacterial taxonomic profiles at the sequencing depth used but demonstrated specific changes in the microbiota not detected with culture, attributed to factors other than imipenem, including sampling site or treatment with other antibiotics. In conclusion, culture and metagenomics were highly complementary in characterising the faecal microbiota of patients. The changes observed during imipenem treatment were unexpectedly limited, possibly because the microbiota was already disturbed by previous antibiotic exposure or hospitalisation.