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.

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.

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.

Species of Metarhizium anisopliae complex implicated in human infections: retrospective sequencing study.

OBJECTIVES: Fungi belonging to the Metarhizium anisopliae complex comprise ubiquitous arthropod pathogenic moulds used as mycopesticides. Rare cases of human infections due to M. anisopliae have been reported. We hypothesize misidentifications of fungal strains implicated in these cases or used in mycopesticides. METHODS: A review of the literature was conducted to identify previously published cases. We collected some of these previous described strains and reported new cases, and a French mycopesticide containing M. anisopliae. All identifications were performed based on elongation factor-1α gene sequencing. RESULTS: We report eight new cases of Metarhizium infection in humans (three from France and five from Australia). The strains isolated from these cases, and three others from already published cases and reported as M. anisopliae, were molecularly identified based on elongation factor-1α (Ef1-α) gene sequencing as follows: Metarhizium robertsii (six), Metarhizium guizhouense (three), Metarhizium brunneum (one) and Metarhizium pingshaense (one). CONCLUSIONS: In this study, we report new human cases of Metarhizium infections, and, based on Ef-1α gene sequencing, we demonstrate the misidentification of species in case reports. We also correct the species identification of a strain reported as M. anisopliae used in a commercially available mycopesticide. According to our results, none of the strains from the human infection reports reviewed belongs to the species M. anisopliae.

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.

Antifungal activity of sodium bicarbonate against fungal agents causing superficial infections.

Although sodium bicarbonate-NaHCO(3) (SB) has many domestic and medical, traditional and empirical uses, only little scientific documentation of its activity is available. The aims of this study were to investigate the antifungal activity of SB on the three fungal groups (yeasts, dermatophytes and molds) responsible for human skin and nail infections. We first evaluated the in vitro antifungal activity of SB on 70 fungal strains isolated from skin and nail infections: 40 dermatophytes, 18 yeasts and 12 molds. A concentration of 10 g/L SB inhibited the growth of 80% of all the fungal isolates tested on Sabouraud dextrose agar. The minimal inhibitory concentration 90 (MIC90) of SB measured on Sabouraud dextrose agar, Sabouraud dextrose broth and potato dextrose broth was 5 g/L for the yeasts, 20 g/L for the dermatophytes and 40 g/L for the molds. In a second step, we prospectively evaluated the ex vivo antifungal activity of SB on 24 infected (15 dermatophytes, 7 yeasts and 2 molds) clinical specimens (15 nails and 9 skin scrapings). The fungal growth was completely inhibited for 19 (79%) specimens and reduced for 4 (17%) specimens after 7 days of incubation on Sabouraud dextrose-chloramphenicol agar supplemented with 10 g/L of SB as compared to Sabouraud dextrose-chloramphenicol agar without SB. In conclusion, we documented the antifungal activity of SB on the most common agents of cutaneous fungal infection and onychomycosis, and we specified the effective concentrations for the different groups of pathogenic fungi. The mechanism of action of SB has yet to be explored.

[Fungal keratitis]. / Kératites fongiques.

Fungal keratitis (keratomycosis) is a rare but severe cause of infectious keratitis. Its incidence is constant, due to steroids or immunosuppressive treatments and contact lenses. Pathogens often invade corneas with chronic diseases of the ocular surface but fungal keratitis is also observed following injuries with plant foreign objects. The poor prognosis of these infections is related both to fungal virulence, decreased host defense, as well as delays in diagnosis. However, new antimycotic treatments allow better management and prognosis.

Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: 10.5-year analysis of susceptibilities of noncandidal yeast species to fluconazole and voriconazole determined by CLSI standardized disk diffusion testing.

Fluconazole in vitro susceptibility test results determined by the CLSI M44-A disk diffusion method for 11,240 isolates of noncandidal yeasts were collected from 134 study sites in 40 countries from June 1997 through December 2007. Data were collected for 8,717 yeast isolates tested with voriconazole from 2001 through 2007. A total of 22 different species/organism groups were isolated, of which Cryptococcus neoformans was the most common (31.2% of all isolates). Overall, Cryptococcus (32.9%), Saccharomyces (11.7%), Trichosporon (10.6%), and Rhodotorula (4.1%) were the most commonly identified genera. The overall percentages of isolates in each category (susceptible, susceptible dose dependent, and resistant) were 78.0%, 9.5%, and 12.5% and 92.7%, 2.3%, and 5.0% for fluconazole and voriconazole, respectively. Less than 30% of fluconazole-resistant isolates of Cryptococcus spp., Cryptococcus albidus, Cryptococcus laurentii, Trichosporon beigelii/Trichosporon cutaneum, Rhodotorula spp., Rhodotorula rubra/Rhodotorula mucilaginosa, and Rhodotorula glutinis remained susceptible to voriconazole. Emerging resistance to fluconazole was documented among isolates of C. neoformans from the Asia-Pacific, Africa/Middle East, and Latin American regions but not among isolates from Europe or North America. This survey documents the continuing broad spectrum of activity of voriconazole against opportunistic yeast pathogens but identifies several of the less common species with decreased azole susceptibility. These organisms may pose a future threat to optimal antifungal therapy and emphasize the importance of prompt and accurate species identification.

Cellular and molecular physiopathology of congenital toxoplasmosis: the dual role of IFN-gamma.

Toxoplasma gondii is one of the few pathogens that can cross the placenta. Frequency and severity of transmission vary with gestational age. While the control of acquired toxoplasmosis is already well explored, the control of materno-foetal transmission of the parasite remains almost unknown. This is partly due to the lack of an animal model to study this process. This review summarises the studies which have been undertaken and shows that the mouse is a valuable model despite obvious differences to the human case. The paramount role of the cellular immune response has been shown by several experiments. However, IFN-gamma has a dual role in this process. While its beneficial effects in the control of toxoplasmosis are well known, it also seems to have transmission-enhancing effects and can also directly harm the developing foetus. The ultimate goal of these studies is to develop a vaccine which protects both mother and foetus. Therefore, it is useful to study the mechanisms of natural resistance against transmission during a secondary infection. In this setting, the process is more complicated, involving both cellular and also humoral components of the immune system. In summary, even if the whole process is far from being elucidated, important insights have been gained so far which will help us to undertake rational vaccine research.

Combination of caspofungin and an azole or an amphotericin B formulation in invasive fungal infections.

OBJECTIVES: Combination of caspofungin and another anti-fungal agent raise expectation of improved efficacy in severe fungal infections including failures to first line therapy. METHODS: We assessed the efficacy and safety of a combination therapy including caspofungin in 17 immunosuppressed or postoperative patients progressive despite standard anti-fungal therapy. RESULTS: The infections included aspergillosis (6), invasive candidiasis (9), mucormycosis (1) and Scedosporium pneumonia (1). Infections had failed one to four prior lines of treatment. The anti-fungal agent combined to caspofungin was either an amphotericin B formulation or an azole. There were 12 favourable responses (71%) and five failures. The survival rate at 3 months was 47%. Eleven patients died within 2-533 days. The causes of death included the initial fungal infection (4), relapse of the infection after switching to oral monotherapy (2), breakthrough aspergillosis (1), and the underlying condition (4). Clinical and renal tolerance were good. Significant hepatic abnormalities were recorded in eight (50%) of the 16 patients evaluable for biological tolerance. CONCLUSION: Caspofungin combined with an azole or with amphotericin B may be of interest in the treatment of serious fungal infections after failure of conventional therapy. Close monitoring of hepatic function is required. These approach should be evaluated in prospective trials.

Invasive aspergillosis in the hematologic and immunologic patient: new findings and key questions in leukemia.

Patients suffering from acute leukemia are at high risk for invasive aspergillosis and a large review and a recent clinical trial have shown that they represent the largest group of patients developing the disease. New host groups such as patients with multiple myeloma or low-grade lymphoproliferative disorders have contributed to an increase in the incidence of invasive aspergillosis over recent years. There are substantial differences in the diagnostic strategy and therapeutic outcome of disease between patients with a hematological malignancy and other host groups such as allogeneic hematopoietic stem cell transplant patients. Galactomannan detection ELISA test is more specific in adult patients with hematological malignancies than in hematopoietic stem cell transplantation recipients. As a result of possible improvement of the underlying immune deficiency upon recovery from neutropenia, survival is higher in leukemic patients with invasive aspergillosis than in other host groups. However, there is currently no evidence of an effective antifungal prophylaxis strategy against aspergillosis in leukemic patients. As these patients account for a majority of the aspergillosis cases, clinical trials on prophylaxis should not only be focused on allogeneic stem transplant recipients but also be designed for the patient with leukemia.

Management of systemic fungal infections: alternatives to itraconazole.

For many years, amphotericin B and flucytosine have been the only antifungal agents for invasive fungal infections. Amphotericin B was the standard of care for most of these infections. However, its use was often associated with low efficacy and poor tolerance. Fortunately, the antifungal armamentarium has increased during the past two decades with the addition of several new agents. In addition to itraconazole and fluconazole, lipid formulations of amphotericin B, voriconazole, caspofungin and micafungin have arrived on the market. Other agents are expected to be licensed shortly (anidulafungin, posaconazole). These various antifungal agents differ in their spectrum, pharmacokinetic profile, route of administration, efficacy in clinical trials, safety profile, drug-drug interactions and, importantly, their cost. There is no longer a unique standard agent for all or nearly all invasive fungal infections but a real choice among several agents. The characteristics of these new agents are reviewed to help clinicians in their decision to select an antifungal agent for their patients.

Epidemiological study of invasive pulmonary aspergillosis in a haematology unit by molecular typing of environmental and patient isolates of Aspergillus fumigatus.

In order to determine the possible relationship between environmental contamination by Aspergillus fumigatus and occurrence of invasive aspergillosis, a one-year prospective study was carried out in the haematology ward of Hautepierre Hospital, Strasbourg, France. During the study period, 21 environmental isolates and 26 clinical isolates of A. fumigatus were collected. Each was genotyped using a random amplification of polymorphic DNA (RAPD) technique. Thirty-four distinct profiles were identified by RAPD analysis, indicating the great genetic diversity of A. fumigatus isolated from infected patients and from the environment. For two patients, RAPD analysis demonstrated concurrent infection by at least two different strains. In two cases, a genetic similarity was noted between isolates obtained from a patient and from the environment.

[Allergic fungal sinusitis: ten cases]. / La Sinusite Fongique Allergique (SFA). A propos de 10 cas.

OBJECTIVES: Diagnosis, nosological individualization, and treatment of allergic fungal sinusitis remain difficult and controversial despite the increasing number of publications. We present ten cases of allergic fungal sinusitis and review the literature to highlight the main clinical, radiological, biological, immunoallergological, mycological, and therapeutics features. MATERIAL AND METHODS: This retrospective study included ten patients (six men and four women, mean age 45 years) with allergic fungal sinusitis diagnosed on the basis of all diagnostic criteria reported in the literature. RESULTS: Six patients had isolated allergic fungal sinusitis which was associated with allergic bronchopulmonary aspergillosis in the four others. Treatment combined endoscopic sinus surgery and corticosteroids, which provided good results in six patients and average results in three. Treatment failure was observed in one patient. CONCLUSION: As in the case of allergic bronchopulmonary aspergillosis, a set of clinical, radiological, histopathological, immunoallergological and mycological criteria is necessary for precise diagnosis and to avoid fungal drift. The most appropriate endoscopic sinus surgery and the best corticosteroid regimen remain to be determined.

Role of gamma interferon and T cells in congenital Toxoplasma transmission.

In the BALB/c mouse model, primary infection with Toxoplasma gondii during the second third of gestation leads to a high percentage of infected foetuses. However, immunity induced by infection contracted before pregnancy prevents parasites from crossing the placenta and completely protects the foetuses, as well as the pregnant women. In order to clarify the roles of CD4+, CD8+ T lymphocytes and IFN-gamma in this protection, pregnant BALB/c mice were treated with depleting monoclonal antibodies against CD4, CD8, IFN-gamma, or control antibody. Only the foetuses of the groups treated with anti-CD8 and anti-IFN-gamma antibodies developed congenital toxoplasmosis. The maternal production of IFN-gamma was depressed in the mice depleted of CD4 and CD8 cells (P < 0.001). Determination of the blood parasite load demonstrated that materno-foetal transmission of T. gondii correlates with maternal parasitaemia. Together, these results show that CD8+ T lymphocytes and IFN-gamma play an important role in protection against congenital toxoplasmosis during reinfection.

Acremonium strictum pulmonary infection in a leukemic patient successfully treated with posaconazole after failure of amphotericin B.

A severely neutropenic patient with chronic lymphocytic leukemia developed a diffuse bilateral pulmonary infection while receiving a therapeutic daily dosage of intravenous amphotericin B for Candida glabrata esophagitis. Computed tomography of the chest showed numerous lung nodules, ground glass areas and a pleural effusion. Biopsy of one nodule demonstrated hyaline septate hyphae. Multiple sputum cultures grew Acremonium strictum. Increasing the dose of amphotericin B and the addition of itraconazole did not resolve the infection. Change of treatment to posaconazole given orally at 200 mg four times/d resulted in progressive improvement leading finally to cure after 24 weeks of therapy. Treatment with posaconazole was clinically and biologically well tolerated.

Chronic disseminated Trichosporon asahii infection in a leukemic child.

We report a case of chronic disseminated Trichosporon asahii infection in a leukemic child. Administration of amphotericin B lipid complex resulted in rapid control and improvement of the initial infection but failed to prevent the development of chronic disseminated disease. Cure was achieved after treatment was changed to a 20-month course of itraconazole. This case report demonstrates that Trichosporon species can cause chronic disseminated disease with a pattern similar to that of disease caused by Candida species.

Treatment of 21 cases of invasive mucormycosis with amphotericin B colloidal dispersion.

The aim of this study was to review the characteristics and outcome of 21 patients with invasive mucormycosis treated with amphotericin B colloidal dispersion (ABCD) in five phase I and phase II studies. Mucormycosis is an increasing concern in immunocompromised patients, in whom mortality exceeds 60%. The standard treatment has been amphotericin B combined with surgical debridement. Twenty-one patients with invasive mucormycosis treated with ABCD, a lipid complex of amphotericin B and cholesteryl sulfate, were identified. Patients were given ABCD on the basis of pre-existing renal insufficiency, development of nephrotoxicity during amphotericin B therapy, or fungal infection that failed to respond to amphotericin B. Response could be evaluated in 20 patients, all of whom had bone marrow or organ transplantation, haematologic malignancies, or diabetes. Infection was disseminated in six patients and localised to the sinuses, lower respiratory tract, or skin in the other patients. ABCD was given at a mean dose of 4.8 mg/kg per infusion for a mean duration of 37 days. Twelve of 20 patients responded to ABCD therapy. Response rates were similar when patients were treated with ABCD alone (4/7) and ABCD combined with surgery (8/13), with more complete response obtained in the latter group. No difference in response rate was observed in leukaemic patients (3/5) or transplant recipients (6/10) compared to diabetics (3/5). No renal or hepatic toxicity was observed. These results compare favourably with the results of standard treatment and suggest that ABCD combined with surgery may be a useful therapy in patients with mucormycosis.

[Visceral candidiasis]. / Candidoses viscérales.

Invasive candidiasis is a frequent infection in compromised patient. Several risk factors have been identified and include neutropenia, broad-spectrum antibiotherapy, colonisation with a Candida spp. and presence of central venous catheter. Candidemia is the most common clinical aspect. Diagnosis is based on positive culture of blood, skin biopsy or fine needle aspiration of a deep-seated lesion. Serology is not helpful in severely immunocompromised patients. Prophylaxis is based on strict hygiene and, in neutropenic patients, oral fluconazole. Treatment of an invasive candidiasis depends on the localisation of the infection, of its acute or chronic evolution, on the species involved and on underlying condition. Amphotericin B deoxycholate or in lipid formulation and fluconazole are the antifungal drugs of choice. Removal of a central venous catheter should always be discussed in candidemia.

Efficacy of amphotericin B lipid complex in the treatment of invasive fungal infections in immunosuppressed paediatric patients.

The safety and efficacy of amphotericin B lipid complex (ABLC) were evaluated in a retrospective study of 46 paediatric patients with invasive infections. The study included a large proportion of patients who were refractory to or intolerant of conventional antifungal therapy. The mean age of the children was 9.7 +/- 4.8 years. Primary underlying conditions included mainly haematopoietic stem cell transplantation, leukaemia and lung transplantation. The mean daily dose given was 4.11 mg/kg for a mean duration of 38.7 days. At the end of therapy, 38 of 46 (83%) patients responded successfully to treatment with ABLC, including 18 of 23 (78%) with aspergillosis and 17 of 19 (89%) with candidiasis. ABLC was well tolerated, with a low incidence of adverse events. The mean creatinine value was 74.5 microl/mol/l at baseline and 78.2 micromol/l at the end of therapy. These results support the use of ABLC in the treatment of invasive fungal infections in children, including patients who have previously failed, or are intolerant of, traditional antifungal regimens.