Multicentric Analysis of the Species Distribution and Antifungal Susceptibility of Clinical Isolates from Aspergillus Section Circumdati.

The clinical involvement and antifungal susceptibility of Aspergillus section Circumdati are poorly known. We analyzed 52 isolates, including 48 clinical isolates, belonging to 9 species inside the section Circumdati. The whole section exhibited, by the EUCAST reference method, a poor susceptibility to amphotericin B, but species/series-specific patterns were observed for azole drugs. This underlines the interest in getting an accurate identification inside the section Circumdati to guide the choice of antifungal treatment in clinical practice.

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.

Etest ECVs/ECOFFs for Detection of Resistance in Prevalent and Three Nonprevalent Candida spp. to Triazoles and Amphotericin B and Aspergillus spp. to Caspofungin: Further Assessment of Modal Variability.

Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs), or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs, and ECVs for some fungal species. Although the concentration gradient strip bioMérieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We reevaluated and consolidated Etest data points from three previous studies and included new data. We defined ECOFFinder Etest ECVs for three sets of species-agent combinations: fluconazole, posaconazole, and voriconazole and 9 Candida spp.; amphotericin B and 3 nonprevalent Candida spp.; and caspofungin and 4 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, and South Africa) included (antifungal agent dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled, and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.), amphotericin B (3 less-prevalent Candida spp.), and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 non-WT, 4 of 6 species).

Multicentric Analysis of the Species Distribution and Antifungal Susceptibility of Cryptic Isolates from Aspergillus Section Fumigati.

The antifungal susceptibility of Aspergillus cryptic species is poorly known. We assessed 51 isolates, belonging to seven Fumigati cryptic species, by the EUCAST reference method and the concentration gradient strip (CGS) method. Species-specific patterns were observed, with high MICs for azole drugs, except for Aspergillus hiratsukae and Aspergillus tsurutae, and high MICs for amphotericin B for Aspergillus lentulus and Aspergillus udagawae Essential and categorical agreements between EUCAST and CGS results were between 53.3 and 93.3%.

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.

Candida bloodstream infection in patients with systemic autoimmune diseases.

OBJECTIVES: To describe the epidemiological, clinical and microbiological characteristics and mortality of patients with Candida bloodstream infection and systemic autoimmune diseases. METHODS: We performed a retrospective multicenter study of candidemia in adults with systemic autoimmune diseases between 2010 and 2016. RESULTS: Among 1040 patients with candidemia, 36 (3.5%) had a systemic autoimmune disease. The most common systemic autoimmune disease was rheumatoid arthritis (27.8%). The most common species was Candida albicans (66.7%). Twenty-two (61.1%) patients received a corticosteroid therapy and nine (25%) received an immunosuppressive therapy at the time of candidemia. The mortality rate was 27.8%. CONCLUSIONS: Systemic autoimmune diseases are not common in patients with candidemia. The unadjusted mortality rate was comparable to other candidemia studies in the general population.

Performance evaluation of different strategies based on microscopy techniques, rapid diagnostic test and molecular loop-mediated isothermal amplification assay for the diagnosis of imported malaria.

OBJECTIVES: Malaria is one of most common tropical diseases encountered in travellers and migrants. It requires an urgent and reliable diagnosis considering its potential severity. In this study, performance of five diagnostic assays were evaluated in a nonendemic region and compared prospectively to quantitative PCR (qPCR). METHODS: A prospective study was conducted at Toulouse Hospital from August 2017 to January 2018 and included all patients with initial Plasmodium screening. Thin and thick blood smears (TnS, TkS), quantitative buffy coat (QBC), rapid diagnostic tests (RDTs) and commercial loop-mediated isothermal amplification (LAMP) were independently performed on each blood sample and compared to our qPCR reference standard. RESULTS: The study encompassed 331 patients, mainly returning from Africa. qPCR detected 73 Plasmodium-positive samples (including 58 falciparum). Individually, LAMP had a 97.3% (71/73) sensitivity, far ahead of TnS (84.9%, 62/73), TkS (86.3%, 63/73), QBC (86.3%, 63/73) and RDT (86.3%, 63/73). RDT demonstrated a high sensitivity for falciparum (98.3%, 57/58) but missed all ovale, malariae and knowlesi infections. Specificity was excellent for all techniques (99.6-100%). The most sensitive diagnosis strategies were TnS + RDT (95.9%, 70/73), TnS + LAMP (97.3%, 71/73) and TnS + RDT + LAMP (100%, 73/73), about 10% higher than strategies using exclusively microscopy, TkS + TnS (87.7%, 64/73) or QBC + TnS (87.7%, 64/73). TnS remains necessary for Plasmodium species identification and quantification. Adding sequentially TnS only on LAMP-positive samples did not decrease TnS + LAMP strategy sensitivity. CONCLUSIONS: In nonendemic countries, the currently recommended microscopy-based strategies seem unsatisfactory for malaria diagnosis considering RDT and LAMP performance, two rapid and sensitive assays that require limited training.

Multicentre study to determine the Etest epidemiological cut-off values of antifungal drugs in Candida spp. and Aspergillus fumigatus species complex.

OBJECTIVES: To determine the Etest-based epidemiological cut-off values (ECVs) for antifungal agents against the most frequent yeast and Aspergillus fumigatus species isolated in 12 French hospitals. METHODS: For each antifungal agent, the Etest MICs in yeast and A. fumigatus isolates from 12 French laboratories were retrospectively collected from 2004 to 2018. The ECVs were then calculated using the iterative statistical method with a 97.5% cut-off. RESULTS: Forty-eight Etest ECVs were determined for amphotericin B, caspofungin, micafungin, anidulafungin, fluconazole, voriconazole, posaconazole and itraconazole, after pooling and analysing the MICs of 9654 Candida albicans, 2939 Candida glabrata SC, 1458 Candida parapsilosis SC, 1148 Candida tropicalis, 575 Candida krusei, 518 Candida kefyr, 241 Candida lusitaniae, 131 Candida guilliermondii and 1526 Aspergillus fumigatus species complex isolates. These ECVs were 100% concordant (identical or within one two-fold dilution) with the previously reported Etest-based ECVs (when available), and they were concordant in 76.1% of cases with the Clinical and Laboratory Standards Institute ECVs and in 81.6% of cases with the European Committee on Antimicrobial Susceptibility Testing ECVs. CONCLUSIONS: On the basis of these and other previous results, we recommend the determination of method-dependent ECVs. Etest ECVs should not be used instead of breakpoints, but may be useful to identify non-wild-type isolates with potential resistance to antifungal agents, and to indicate that an isolate may not respond as expected to the standard treatment.

Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF).

The main objective of this study was to assess the diagnostic performance of a set of three Mucorales quantitative PCR assays in a retrospective multicentre study. Mucormycosis cases were recorded thanks to the French prospective surveillance programme (RESSIF network). The day of sampling of the first histological or mycological positive specimen was defined as day 0 (D0). Detection of circulating DNA was performed on frozen serum samples collected from D-30 to D30, using quantitative PCR assays targeting Rhizomucor, Lichtheimia, Mucor/Rhizopus. Forty-four patients diagnosed with probable (n = 19) or proven (n = 25) mucormycosis were included. Thirty-six of the 44 patients (81%) had at least one PCR-positive serum. The first PCR-positive sample was observed 9 days (range 0-28 days) before diagnosis was made using mycological criteria and at least 2 days (range 0-24 days) before imaging. The identifications provided with the quantitative PCR assays were all concordant with culture and/or PCR-based identification of the causal species. Survival rate at D84 was significantly higher for patients with an initially positive PCR that became negative after treatment initiation than for patients whose PCR remained positive (48% and 4%, respectively; p <10-6). The median time for complete negativity of PCR was 7 days (range 3-19 days) after initiation of l-AmB treatment. Despite some limitations due to the retrospective design of the study, we showed that Mucorales quantitative PCR could not only confirm the mucormycosis diagnosis when other mycological arguments were present but could also anticipate this diagnosis. Quantification of DNA loads may also be a useful adjunct to treatment monitoring.

Risk factors of Pneumocystis pneumonia in solid organ recipients in the era of the common use of posttransplantation prophylaxis.

Pneumocystis pneumonia (PCP) in solid organ transplant (SOT) recipients becomes rare in the immediate posttransplantation period thanks to generalized prophylaxis. We aimed to identify the predictive factors for PCP in the era of universal prophylaxis and to propose a strategy for preventing PCP beyond the first year after transplantation. In a retrospective case-control study, 33 SOT cases with PCP diagnosed between 2004 and 2010 were matched with two controls each to identify risk factors for PCP by uni- and multivariate analysis. All the patients benefited from 6 months of posttransplantation trimethoprim-sulfamethoxazole prophylaxis. Most PCP in SOT patients occurred during the second year posttransplantation (33%). By univariate analysis, age, nonuse of tacrolimus, total and CD4 lymphocyte counts, gamma-globulin concentration and cytomegalovirus (CMV) infection appeared to be PCP risk factors. In the final multivariate analysis, age (adjusted odds ratio [OR] 3.7, 95% confidence interval [CI]: 1.3-10.4), CMV infection (OR: 5.2, 95% CI: 1.8-14.7) and total lymphocyte count (OR: 3.9, 95% CI: 1.4-10.7) were found to be independently associated with PCP. The second year posttransplantation appeared to be the new period of highest risk of PCP. Age, CMV viremia and lymphocytes were the most pertinent predictive criteria to evaluate the risk of PCP in clinical practice.

Contribution of molecular diagnosis to the management of cutaneous leishmaniasis in travellers.

Cutaneous leishmaniasis is one of the most frequent skin diseases occurring after travelling in endemic areas. Optimal management requires identification of the species of Leishmania involved. In this study we aimed to evaluate the use of molecular diagnosis as routine, in comparison with direct examination and culture. Thirty positive diagnoses were carried out between 2007 and 2013. Classical PCR enabled 11 positive cases to be identified that were found to be negative by conventional methods. Sequencing led to the identification of eight different species. Routine use of PCR and sequencing appears very efficient in the management of cutaneous leishmaniasis.

Disseminated Ochroconis gallopava infection in a heart transplant patient.

Ochroconis gallopava is an emerging cause of mycosis in solid organ transplant recipients. Herein, we report a rare case of disseminated O. gallopava infection that involved lung, subcutaneous area, brain and peritoneum in a heart transplant recipient. Despite voriconazole therapy, the patient died 2 months after diagnosis.

Simultaneous cutaneous infection due to Paecilomyces lilacinus and Alternaria in a heart transplant patient.

Paecilomyces lilacinus is an emerging pathogen in immunocompromised patients. We report here a case of cutaneous hyphomycosis in a 63-year-old heart transplant recipient caused by the simultaneous presence of 2 molds: Paecilomyces lilacinus and Alternaria alternata. The infection was successfully treated with local voriconazole followed by oral terbinafine.

Eumycetoma caused by Diaporthe phaseolorum (Phomopsis phaseoli): a case report and a mini-review of Diaporthe/Phomopsis spp invasive infections in humans.

Diaporthe phaseolorum (syn. Phomopsis phaseoli) is a frequent fungal parasite of plants, present on all continents around the world. It has rarely been involved in human diseases. We report a case of eumycetoma with osteomyelitis of the forefoot caused by this fungus and diagnosed by molecular biology. The patient had positive HTLV-1 serology and was a farmer from French Guiana who walked barefoot. He was successfully treated with long-term oral itraconazole (400 mg/day). A review of the literature underlines the essential roles of plants and host immunosuppression in this infection and the favourable outcome with a triazole antifungal treatment.

[Quality controls in medical mycology]. / Les contrôles de qualité en mycologie médicale.

The Quality Management System in medical mycology refers to the systematic monitoring with internal and external quality controls: it needs to be organized in the laboratory. ISO 15189 standard is not precise in how to demonstrate the correctness of tests, in terms of frequency and requirements for quality controls QC. That's why the COFRAC, the French Accreditation Committee has published guides to which we should refer. The laboratory has to apply internal Quality Control Programs. They consist of various tests to check the reagents including the culture media. Reference strains have to be provided and preparations of homemade reagents are needed, because few are commercialized. Maintaining the competence of the technical staff through identification of unknown strains is also required. In the fungal serology field, home made antibodies with pooled sera or antigen controls are needed. This monitoring has to follow the recommandations from the Cofrac technical guide LAB GTA 06. For quantitative analysis, the Levey-Jennings chart is a graph with quality control data plotted on to give a visual indication. Some external QC references, besides the national quality control AFSSAPS, are available. Data evaluation, corrective actions in case of out of range results and preventive actions have to be determined in the Quality System documents and presented in the annual management review.

Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis.

The aim of this study was to determine the incidence of congenital toxoplasmosis (CT) and to assess the performances of prenatal and neonatal diagnoses. From 1994-2005, in Toulouse University Hospital, France, amniocentesis was performed on 352 pregnant women who were infected during pregnancy. All women were treated with spiramycin and pyrimethamine-sulfadoxine when prenatal diagnosis was positive. Among the 275 foetuses with follow-up, 66 (24%) were infected. The transmission rates of Toxoplasma gondii were 7%, 24% and 59% in the first, second and third trimesters, respectively. The sensitivity and specificity of PCR on amniotic fluid (AF) were 91% and 99.5%, respectively. One case was diagnosed by mouse inoculation with AF and six cases were diagnosed by neonatal or postnatal screening. The sensitivity and specificity of PCR on placentas were 52% and 99%, respectively. The sensitivity of tests for the detection of specific IgA and IgM in cord blood was 53% and 64%, respectively, and specificity values were 91% and 92%. In conclusion, PCR performed on AF had the highest levels of sensitivity and specificity for the diagnosis of CT. This permits an early diagnosis of most cases and should be recommended.

Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis

The aim of this study was to determine the incidence of congenital toxoplasmosis (CT) and to assess the performances of prenatal and neonatal diagnoses. From 1994-2005, in Toulouse University Hospital, France, amniocentesis was performed on 352 pregnant women who were infected during pregnancy. All women were treated with spiramycin and pyrimethamine-sulfadoxine when prenatal diagnosis was positive. Among the 275 foetuses with follow-up, 66 (24 percent) were infected. The transmission rates of Toxoplasma gondii were 7 percent, 24 percent and 59 percent in the first, second and third trimesters, respectively. The sensitivity and specificity of PCR on amniotic fluid (AF) were 91 percent and 99.5 percent, respectively. One case was diagnosed by mouse inoculation with AF and six cases were diagnosed by neonatal or postnatal screening. The sensitivity and specificity of PCR on placentas were 52 percent and 99 percent, respectively. The sensitivity of tests for the detection of specific IgA and IgM in cord blood was 53 percent and 64 percent, respectively, and specificity values were 91 percent and 92 percent. In conclusion, PCR performed on AF had the highest levels of sensitivity and specificity for the diagnosis of CT. This permits an early diagnosis of most cases and should be recommended.

PCR-based methods to the diagnosis of imported malaria.

Rapid and precise diagnosis of malaria is needed to take care febrile patient returning from endemic areas. Since the first description of the diagnosis of Plasmodium infection by polymerase-chain-reaction (PCR), the role of this kind of molecular method in the laboratory diagnosis of imported malaria is still a topical question. PCR-based assays were found to be more sensitive and more specific than all conventional methods. The highest contribution of the molecular diagnosis is that a PCR negative result would ascertain the lack of any malaria infection, thus quickly orienting the investigations toward other aetiology. This technique should be now considered as the gold standard for the diagnosis of imported malaria.

[Fungal keratitis caused by Scedosporium apiospermum]. / Kératite fongique à Scedosporium apiospermum.

INTRODUCTION: We report a case of posttraumatic keratomycosis caused by Scedosporium apiospermum that was treated with oral and topical voriconazole and penetrating keratoplasty. CASE REPORT: A patient was admitted to the hospital with a corneal abscess of his right eye due to trauma while gardening. No improvement was noted with topical fortified antibiotics (vancomycin, gentamicin, and cefazolin) and antimycotic (amphotericin B 1%) and oral itraconazole (200 mg/day). Fungal cultures of corneal scrapings revealed growth of Scedosporium apiospermum, a strain for which the main antifungals showed high minimal inhibitory concentrations (MICs), whereas the MIC of voriconazole was 0.125 microg/mL. Despite some improvement with topical 1% voriconazole and oral voriconazole (200 mg/day) treatment, a therapeutic penetrating keratoplasty was performed because of the high risk of corneal perforation. The graft remained clear without fungal recurrence with topical 2% cyclosporine A, dexamethasone, and voriconazole treatment. CONCLUSION: Scedosporium apiospermum is an uncommon cause of mycotic keratitis in humans. Prognosis is generally poor because of delayed diagnosis and resistance to conventional antifungals. Voriconazole is a triazole broad-spectrum antifungal agent. In conjunction with its oral administration, topical application of voriconazole extends the current armamentarium of antifungal agents for keratomycosis.