Validation of a New Web Application for Identification of Fungi by Use of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has emerged as a reliable technique to identify molds involved in human diseases, including dermatophytes, provided that exhaustive reference databases are available. This study assessed an online identification application based on original algorithms and an extensive in-house reference database comprising 11,851 spectra (938 fungal species and 246 fungal genera). Validation criteria were established using an initial panel of 422 molds, including dermatophytes, previously identified via DNA sequencing (126 species). The application was further assessed using a separate panel of 501 cultured clinical isolates (88 mold taxa including dermatophytes) derived from five hospital laboratories. A total of 438 (87.35%) isolates were correctly identified at the species level, while 26 (5.22%) were assigned to the correct genus but the wrong species and 37 (7.43%) were not identified, since the defined threshold of 20 was not reached. The use of the Bruker Daltonics database included in the MALDI Biotyper software resulted in a much higher rate of unidentified isolates (39.76 and 74.30% using the score thresholds 1.7 and 2.0, respectively). Moreover, the identification delay of the online application remained compatible with real-time online queries (0.15 s per spectrum), and the application was faster than identifications using the MALDI Biotyper software. This is the first study to assess an online identification system based on MALDI-TOF spectrum analysis. We have successfully applied this approach to identify molds, including dermatophytes, for which diversity is insufficiently represented in commercial databases. This free-access application is available to medical mycologists to improve fungal identification.

Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients.

Pneumocystis jirovecii pneumonia (PCP) is an acute and life-threatening lung disease caused by the fungus Pneumocystis jirovecii The presentation of PCP in HIV-positive patients is well-known and consists of a triad of dyspnea, fever, and cough, whereas the presentation of PCP in HIV-negative patients is atypical and consists of a sudden outbreak, O2 desaturation, and a rapid lethal outcome without therapy. Despite the availability of direct and indirect identification methods, the diagnosis of PCP remains difficult. The cycle threshold (CT) values obtained by quantitative PCR (qPCR) allow estimation of the fungal burden. The more elevated that the fungal burden is, the higher the probability that the diagnosis is pneumonia. The purposes of the present study were to evaluate the CT values to differentiate colonization and pneumonia in a population of immunocompromised patients overall and patients stratified on the basis of their HIV infection status. Testing of bronchoalveolar lavage (BAL) fluid samples from the whole population of qPCR-positive patients showed a mean CT value for patients with PCP of 28 (95% confidence interval [CI], 26 to 30) and a mean CT value for colonized patients of 35 (95% CI, 34 to 36) (P < 10(-3)). For the subgroup of HIV-positive patients, we demonstrated that a CT value below 27 excluded colonization and a CT value above 30 excluded PCP with a specificity of 100% and a sensitivity of 80%, respectively. In the subgroup of HIV-negative patients, we demonstrated that a CT value below 31 excluded colonization and a CT value above 35 excluded PCP with a specificity of 80% and a sensitivity of 80%, respectively. Thus, qPCR of BAL fluid samples is an important tool for the differentiation of colonization and pneumonia in P. jirovecii-infected immunocompromised patients and patients stratified on the basis of HIV infection status with different CT values.

A 6-year antifungal stewardship programme in a teaching hospital.

PURPOSE: To describe the antifungal stewardship programme in our hospital and to assess its impact on total antifungal prescriptions and their cost, and on the process of care measures regarding the diagnostic and therapeutic management of invasive aspergillosis and candidaemia. METHODS: We conducted a prospective observational study describing the multifaceted antifungal stewardship programme in place at our French teaching tertiary-care hospital since 2005. Several actions were implemented successively, including the systematic evaluation of all costly antifungal prescriptions (echinocandins, lipid formulations of amphotericin B, posaconazole and voriconazole). RESULTS: A total of 636 antifungal prescriptions were discussed by the antifungal management team from 2005 to 2010 inclusive, mainly from the haematology department (72 %). In 344/636 cases (54 %), a piece of advice was fed back to the physician in charge of the patient, with an 88 % compliance rate. Optimal standard of care was achieved for galactomannan antigen testing, performance of chest computed tomography (CT) scan and voriconazole therapeutic drug monitoring for invasive aspergillosis, with no combination therapies used since 2008. Regarding candidaemia, optimal standard of care was achieved for the timing of antifungal therapy, recommended first-line therapy, duration of therapy and the removal of central venous catheters. Total antifungal prescriptions (in defined daily doses, DDD) and their cost were contained between 2003 and 2010. CONCLUSIONS: The implementation of an antifungal stewardship programme was feasible, sustainable and well accepted. We observed an improved quality of care for some process of care measures, and antifungal use and cost were contained in our hospital.

Interlaboratory reproducibility of Etest amphotericin B and caspofungin yeast susceptibility testing and comparison with the CLSI method.

This study aimed to assess the interlaboratory reproducibility at four university hospital laboratories in the southeast region of France of the Etest technique for the determination of caspofungin (CAS) and amphotericin B (AMB) MICs and to compare it to the CLSI broth microdilution reference method. Consecutive clinical yeast isolates (n = 198) were included in the study. AMB and CAS MICs were read at 24 and 48 h. Interlaboratory reproducibility was estimated by using (i) an intraclass correlation coefficient (ICC), (ii) essential agreement (EA), and (iii) categorical agreement (CA). For Etest interlaboratory reproducibility for CAS, ICCs were 0.80 (95% confidence interval [CI], 0.76 to 0.84) and 0.81 (95% CI, 0.77 to 0.85) at 24 and 48 h, respectively. For AMB, the ICCs were 0.51 (95% CI, 0.43 to 0.58) and 0.69 (95% CI, 0.63 to 0.74) at 24 and 48 h, respectively. At 48 h, the between-center EAs ranged from 94.4 to 99.0% for both antifungals. For the comparison of the CLSI method and the Etest, the between-technique ICCs were 0.69 (95% CI, 0.63 to 0.74) and 0.62 (95% CI, 0.55 to 0.68) for CAS and AMB, respectively. The EAs ranged from 76.5 to 98.5% for CAS and from 90.3 to 97.4% for AMB according to the centers. CAs ranged from 87.9% to 91.4%, with four very major errors for 2 strains (1 Candida albicans strain and 1 Candida krusei strain), for CAS and from 97.5 to 99.5%, with four major errors, for AMB. In conclusion, the Etest showed a good interlaboratory reproducibility and a good correlation with the CLSI technique. It is well suited for the routine clinical laboratory and can thus be used to monitor clinical yeast isolates' in vitro susceptibilities in this setting.

First endogenous fungal endophthalmitis due to Fusarium dimerum: A severe eye infection contracted during induction chemotherapy for acute leukemia.

Endophthalmitis is a rare infection of the vitreous and/or aqueous. It can be bacterial or fungal. Exogenous endophthalmitis is the most common form and results from direct inoculation of a pathogen after eye surgery or penetrating trauma. Endophthalmitis can also be endogenous, secondary to disseminated infection. Fungal endophthalmitis is associated with poor prognosis and treatment is difficult given the low penetration of most of the antifungal agents available and the emergence of resistant filamentous fungi like Fusarium. To our knowledge, we describe the first endogenous fungal endophthalmitis due to Fusarium dimerum, a ubiquitous pathogen found in soil and plants. A 71-year-old woman, diagnosed with acute myeloid leukemia, was hospitalized for surveillance after induction chemotherapy. Prophylaxis by antibiotics and posaconazole was ongoing when she complained of pain and decreased vision in the left eye. A voluminous chorioretinal abscess developed and after multiple sterile aqueous humour samples, only vitrectomy allowed diagnosis with fungal hyphae seen on May-Grünwald Giemsa stained smear and positive cultures. The fungus was identified as Fusarium dimerum. The treatment, that included intravitreal injections of voriconazole and amphotericin B associated with systemic administration of voriconazole, allowed complete control of the infection. The source of this infection could not be confirmed despite the discovery of several possible infection sites including a periungual whitlow on the left hand and a lesion on a nail, from which samples were negative in microbiology laboratories. Unfortunately, damages of the retina were too important and the patient did not recover sight of her left eye.

[Disseminated fungal infection due to Magnusiomyces capitatus in a liver graft patient]. / Infection fongique disséminée à Magnusiomyces capitatus chez un patient transplanté hépatique.

Disseminated fungal infections due to Magnusiomyces capitatus are rare, occurring exclusively in immunocompromised patients. We report the first case in a liver transplant patient with chronic rejection and portal thrombosis who had a M. capitatus fungemia with a refractory septic shock. Despite an antibacterial and antifungal treatment with caspofungin empirical treatment, the patient died from multiple organ failure. Subsequently, mycological examinations of blood cultures, bronchoalveolar lavage fluid and urine were positive to M. capitatus identified by mass spectrometry and confirmed by sequencing respectively. The stain was resistant to caspofungin and fluconazole. The best treatment appears to be the combination of amphotericin B and voriconazole or amphotericin B and 5 fluorocytosine.

Human trichinellosis due to Trichinella britovi in southern France after consumption of frozen wild boar meat.

Six patients were infected with Trichinella britovi in southern France following consumption of frozen wild boar meat, which had been frozen at -35 degrees C for 7 days. Microscopic examination of a sample of frozen wild boar muscle revealed the presence of rare encapsulated Trichinella larvae, identified as T. britovi. People eating wild boar must follow individual prophylactic rules such as efficient cooking of meat (at least 65 degrees C at the core for 1 minute) as recommended by the International Commission on Trichinellosis, or freezing exceeding four weeks at -20 degrees C.

Human visceral leishmaniasis in Alpes-Maritimes, France: epidemiological characteristics for the period 1985-1992.

In 8 years (1985-1992), 65 cases of human visceral leishmaniasis (HVL) have been diagnosed in the department of Alpes-Maritimes, France, 56 of them having been infected locally. The annual frequency has increased from 3 cases in 1985 to 15 cases in 1992. There is a low rate of paediatric cases (29%) and a predominance of males among adult cases (85%). Since 1986, 19 cases of co-infection with Leishmania and human immunodeficiency virus 1 have been reported, corresponding to 40% of adult cases and to 30% of the total cases. The frequency of co-infections is stable at about 3 per annum. Isoenzymatic identification of the strains isolated from patients confirmed Leishmania infantum zymodeme MON-1 as responsible for most if not all HVL in the department of Alpes-Maritimes; 42 of the 44 strains isolated belonged to that zymodeme.

Use of the leishmanin skin test and western blot analysis for epidemiological studies in visceral leishmaniasis areas: experience in a highly endemic focus in Alpes-Maritimes (France).

Fifty unselected subjects living in Alpes-Maritimes, France, a high risk area for visceral leishmaniasis due to Leishmania infantum, were examined simultaneously by the leishmanin skin test and the Western blot technique in 1993; 32% and 38%, respectively, gave a positive reaction. The concordance of the 2 methods was 82%. Thus, in this high risk area, a large proportion of inhabitants had been exposed to the parasite. The use of these 2 tests should permit the detection of potential cases of reactivated leishmaniasis in prospective follow-up investigations.

Detection by Western blot of four antigens characterizing acute clinical leishmaniasis due to Leishmania infantum.

Western blot analysis of sera from 32 patients with acute clinical leishmaniasis due to Leishmania infantum showed the simultaneous presence of antibodies against 4 antigens with molecular masses of 18, 21, 23, 31 kDa. The simultaneous presence of these 4 antigens was specific to the clinical disease and it was not detected in 47 sera from asymptomatic individuals living in the leishmaniasis endemic area of Alpes-Maritimes (southern France) or in 37 sera from patients with other protozoan infections.

Medical treatment of a pacemaker endocarditis due to Candida albicans and to Candida glabrata.

We describe a case of pacemaker infection due to two fungal species: Candida albicans and C. glabrata. Transthoracic echocardiography showed a large vegetation on the intraventricular wires. Because of severe underlying diseases, surgery was believed to be contraindicated. The patient was treated using high dose of fluconazole, resulting in clinical improvement and negative blood cultures. However, 2 months later, the patient underwent a fatal stroke. At autopsy, a large vegetation was found only all along the wires. Postmortem culture of the infected material was positive for both C. albicans and C. glabrata.

[Fungal myocarditis in acquired immunodeficiency syndrome]. / Myocardites fungiques au cours du syndrome d'immunodéficience acquise.

The authors report 8 cases of fungal myocarditis discovered at autopsy of a series of 118 patients with AIDS. The cardiac disease was symptomatic and responsible for death in 2 cases. Antemortem diagnosis of fungal infection was made on examination of bronchioloalveolar lavage, the cerebrospinal fluid or gastrointestinal biopsy. The demonstration of intramyocardial pathogens was postmortem in all cases. The organism was Candida albicans in 3 cases, Cryptococcus neoformans in 3 cases, and Aspergillus fumigatus in 2 cases. The lesions were not confined to the heart; multi-visceral fungal involvement was diagnosed at autopsy in all cases. Cryptococcal and Candida myocarditis have already been described in most autopsy series in AIDS, but Aspergillus myocarditis is very rare. These fungal myocarditis are usually clinically latent or masked by neurological or respiratory symptoms.

[Cytological study of the duodenal mucosa during chronic diarrhea in HIV-positive patients]. / Etude cytologique de la muqueuse duodénale au cours des diarrhées chroniques chez le sujet VIH positif.

The cytologic findings of duodenal smears in diarrheic HIV-positive patients were compared with results of histologic and ultrastructural studies. This study included 50 diarrheic patients undergoing upper gastrointestinal endoscopy. Duodenal biopsies were taken with touch preparations. Smears were stained with Giemsa and then with Ziehl-Neelsen or the PAS method. The biopsy specimens were then processed in a standard fashion for histologic and electron microscopy examination. Cytologic findings included 8 cases of intestinal microsporidiosis, 4 cases of cryptosporidiosis, and 3 cases of atypical mycobacteriosis. Histologic and ultrastructural studies confirmed these diagnoses and showed 8 cases of CMV enteritis, of which 4 were associated with other enteric pathogens. Duodenal cytology complements endoscopic biopsy in the work-up of diarrhea in AIDS patients and may provide a rapid diagnosis especially in the case of intestinal protozoonosis or atypical intestinal mycobacteriosis.

[Childhood visceral leishmaniasis in the Alpes-Maritimes from 1985 to 1992]. / La leishmaniose viscérale infantile dans les alpes-maritimes de 1985 a 1992.

Here, we present an analysis of clinical and biological data from 15 infantile visceral leishmaniasis recorded between 1985 and 1992 in the French department of Alpes-Maritimes. The association of fever, splenomegalia, tricytopenia remains the most constant and predictive of diagnosis (90% of cases). Bone marrow smear affirms diagnosis in 93% of cases. Leishmania infantum MON-1 is the causative agent. Except for one case of advanced stade with severe hepatic lesions and one case of clinical resistance to Glucantime, prognosis is favourable under treatment with antimony derivatives. The long term results for children considered cured after initial treatment is satisfactory. In the department, the number of cases of combined ages has increased regularly since 1985. The number of infantile forms has increased but now represents nevertheless no more than 28% of total cases. On the other hand, cases are increasing in adults and 40% of those adult cases are in HIV positive patients.

[Visceral leishmaniasis caused by a dermotropic strain of Leishmania infantum in an AIDS patient]. / Leishmaniose viscérale causée par une souche dermotrope de Leishmania infantum chez un sidéen.

A case of fatal visceral leishmaniasis due to a dermotropic strain of Leishmania infantum (Zymodeme MON-24) is related in an AIDS patient in spite of a specific treatment.

[Delayed diagnosis of neurocysticercosis: two case reports]. / Neurocysticercose de diagnostic tardif: à propos de deux cas.

INTRODUCTION: Neurocysticercosis is the most frequently encountered cerebral parasitic infection worldwide. It is due to infection of the central nervous system by Taenia solium larval form. According to the location of the cysts, parenchymal and extra-parenchymal forms may be identified, with different clinical expressions. EXEGESIS: We report two cases of neurocysticercosis, one with typical parenchymal involvement and the second with extra-parenchymal involvement revealed by increased intra-cranial pressure. In both cases, the diagnosis was established over 10 years after the onset of symptoms. CONCLUSION: Neurocysticercosis is very frequent in non-Islamic developing countries, and its incidence is increasing in industrialized nations in relation to tourism and immigration from highly endemic areas. Symptoms usually appear several years after infection and this accounts for the frequent delays before the diagnosis is established.

[Rhino-orbito-cerebral mucormycosis caused by Rhizopus oryzae.A typical case in a cirrhotic patient]. / Mucormycose rhino-orbito-cérébrale due à Rhizopus oryzae. Un cas typique survenu chez une patiente cirrhotique.

The rhino-orbito-cerebral mucormycosis is an acute rapidly progressive fungal infection. This disease is caused by a zygomycetes fungus, most often from the Rhizopus genus. This fungus is saprophitic of the nasal cavity and paranasal sinuses. It becomes pathogenic in some particular conditions, specially during diabetes mellitus. Histopathological study is the only method allowing the diagnosis, by revealing the tissue invasion by characteristic hyphae. Mycologic study allows a definitive identification of the fungus. The authors report a case of rhino-orbito-cerebral mucormycosis in a 44 year-old woman with cirrhosis. She presented an acute blindness and ophthalmoplegia. Despite of a rapid histologic diagnosis from the nasal and ethmoidal biopsies, the patient died 3 days after.

[Disseminated infection due to Scedosporium apiospermum in liver transplantation. Differential diagnosis from invasive aspergillosis]. / Infection disséminée à scedosporium apiospermum chez un transplanté hépatique. Un diagnostic différentiel de l'aspergillose invasive.

A case of fatal disseminated infection due to Scedosporium apiospermum occurring after liver transplantation is reported. Diagnosis was made at autopsy, as numerous fungal colonies were found in the lungs, heart, brain, kidney, spleen and liver. Scedosporium apiospermum was identified in pulmonary, cerebral and myocardial specimens by Sabouraud's glucose agar cultures. Infections due to Scedosporium (S. apiospermum ou Pseudallescheria boydii) occur in immunocompromised hosts, particularly after organ transplantation, and realize severe invasive fungal infection. Scedosporiosis is much more rare than aspergillosis and can be only identified by mycological study. This diagnosis is rarely performed during life time and allows an effective treatment by imidazoles.

[Diagnosis of aspergillosis and other invasive filamentous fungal infections in hematology]. / Diagnostic de l'aspergillose et des autres infections invasives à champignons filamenteux en hématologie.

OBJECTIVE: Invasive filamentous fungal infections (FFI) are difficult to diagnose in the department of hematology; a variety of arguments are necessary to confirm the fungal origin. Our study evaluated prospectively, in a large population in South Eastern France, the diagnostic techniques used ante and post mortem (mycology, anatomopathology, serology and aspergillosis antigens) to prove an FFI. METHODS: Two hundred and twenty eight neutropenic patients with obvious, probable, highly probable or presumed FFI were selected. Since completion of this study, a new classification of aspergillosis, with 3 instead of 4 levels of diagnosis, has been defined by the ICAAC in San Francisco but has not yet been published. Mycological or histological examinations were been performed on broncho-alveolar washings (BAW), puncture liquids or biopsies. Serology used precipitation, hemagglutination, immunofluorescence or ELISA techniques. Aspergillosis antigens were researched using the Pastorex method (agglutination of latex particles), launched at the beginning of the study. RESULTS: Invasive aspergillosis was diagnosed in 43.4% of patients, a non-aspergilla FFI in 6.5%, and no fungi was isolated in the cultures of 50% of patients. Ante mortem, 160 patients underwent BAW with a total of 175 samples. Among the latter, 41.7% mycological examinations were positive (presence of mycelium filaments and/or positive cultures) with 68.7% of Aspergillus fumigatus. Of the 48 puncture liquids, 25% of direct examinations and 29% of cultures were positive. Biopsies were taken from 59 patients and explored for mycology: 63.8% of direct examinations and 46.8% of cultures were positive; for those explored histologically, 76.7% revealed the presence of mycelium filaments. Post mortem, 17 patients out of 79 deceased underwent a total of 24 autopsy or biologic sampling. For 5 patients, FFI was diagnosed post mortem. Aspergillus serology and antigen explorations were conducted respectively in 202 and 182 patients and were positive in 31% and 33%. The presence of antibodies appears to be a good prognostic factor compared with the presence of antigens. CONCLUSION: Biological diagnosis of FFI relies on the multiplicity of examinations and renewal of mycological sampling.

[Blastomycosis localized in the brain stem]. / Blastomycose localisée au tronc cérébral.

A 33-year old male Tunisian developed, without any apparent cause, an abscess of the brainstem which was shown by various mycological investigations to be due to a Blastomyces dermatitidis infection. This case is of interest not only because of the exceptional location of the abscess in the brainstem, but also because of its treatment: this was the first case where the fluconazole-flucytosine combination was used against blastomycosis with fully satisfactory results after a 28-month follow-up.