[French recommendations on control measures to reduce the infectious risk in immunocompromised patients]. / Quelles mesures pour maîtriser le risque infectieux chez les patients immunodéprimés ? Recommandations formalisées d'experts.

The increase use of immunosuppressive treatments in patients with solid cancer and/or inflammatory diseases requires revisiting our practices for the prevention of infectious risk in the care setting. A review of the literature by a multidisciplinary working group at the beginning of 2014 wished to answer the following 4 questions to improve healthcare immunocompromised patients: (I) How can we define immunocompromised patients with high, intermediate and low infectious risk, (II) which air treatment should be recommended for this specific population? (III) What additional precautions should be recommended for immunocompromised patients at risk for infection? (IV) Which global environmental control should be recommended? Based on data from the literature and using the GRADE method, we propose 15 recommendations that could help to reduce the risk of infection in these exposed populations.

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.

Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of Candida species.

Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas™ and Bruker Biotyper™, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level (1359/1383 for Andromas™; 1360/1383 for Bruker Biotyper™) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management.

[Detection of Aspergillus antigen galactomannan using ELISA method: validation of the performances of the method for accreditation]. / Validation d'une méthode Elisa pour la recherche de l'antigène aspergillaire galactomannane en vue de l'accréditation.

Diagnosis of invasive aspergillosis for patients with high risk of infection is based on the monitoring of Aspergillus antigenemia assessed by the detection of galactomannan in serum by a sandwich-type ELISA (Biorad(®)). The validation of the method was displayed according to the guide COFRAC SH GTA 04. The internal quality control system settled, involves two quality control samples made of pools of sera (negative and positive). The repeatability of the measurements, as estimated by the coefficients of variation (CV), obtained by two different technicians was found from 9 to 13.7% for the positive control. The CV of the negative control, for which the provider indicates it is not useful in the analytical process, was found from 7.1 to 30%. In our experience it could be an indicator of environmental contamination. The evaluation of the intermediary fidelity was 15.7% for the positive control and 22.5% for the negative one. In the lack of reference material (International Standard) and recommendation from scientific societies, performances obtained will be discussed according to the results reported in the technical form of the supplier and those obtained by 39 laboratories participating in the only available external quality assessment program organized in France by ProBioQual(®) where the CV of reproducibility are 44.7% of unit (mean index 0.131) for the negative control and 18% (mean index 1.089) for the positive one in 2011.

[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.

[Survey on the medical mycology processes to the members of the French Society for Medical Mycology]. / Enquête sur les conditions analytiques d'un examen de mycologie médicale auprès des membres de la Société française de mycologie médicale.

AIM OF THE STUDY: The objective of the survey was to describe the practices of clinical laboratories in terms of cultures in medical mycology. We have implemented this project within the members of the French Society for Medical Mycology (SFMM) to evaluate the analytical processes of the mycological examination in our laboratories. This preliminary study would help to suggest the future French guidelines. MATERIALS AND METHODS: A questionnaire regarding the processing of mycology analysis was sent to the 227 members of the SFMM in 2009. The data involved 21 types of samples, direct microscopic examination with or without colouring and the reagents, the number of culture media, the types of media (Sabouraud, Sabouraud antibiotic, Sabouraud cycloheximide and chromogenic medium), temperature and duration of the incubation (days) and the existence of a first result before the end of the incubation period. The analytical processes were compared to an accredited laboratory according to EN ISO 15189. RESULTS: A great heterogeneity was observed in the 36 forms from 27 (75%) laboratories belonging to university hospitals among the 38 existing in France. As for deep samples, two microscopic exams were performed, only one was usually done. A more sensitive technique was preferred to the wet-mount for some samples. Routine samples are often inoculated on a chromogenic media. For deep samples two medium are inoculated (chromogenic media, Sabouraud and antibiotics). If the temperature of incubation is unique, 30°C was chosen. A temperature of 37°C was preferred for samples where Candida spp. is selected. When there are two temperatures of incubation, 27°C and 37°C were preferred. CONCLUSION: Each biologist can compare his proceedings to the other laboratories and to a laboratory already accredited. The question is to find the best strategies for each medical mycology specimen. They will aid the process of accreditation according to EN ISO 15189, which now applies in all laboratories in Europe.

Paranasal sinus fungus ball: epidemiology, clinical features and diagnosis. A retrospective analysis of 173 cases from a single medical center in France, 1989-2002.

Over the last decade, we have observed a high frequency of Aspergillus rhinosinusitis in french medical centers. The epidemiological data, clinical presentations, radiology, mycology and histology results of 173 consecutive patients with paranasal sinus fungus balls who were admitted from 1989 to 2002 have been reviewed. The most common symptoms included purulent nasal discharges and nasal obstructions, with the maxillary sinus being the most common site of infection (152 cases, 87.8%). Computed tomography scans (CT scan) were performed in 92% (159/173) of the cases and heterogeneous opacities were observed in 132 patients (83%). Histology examinations were performed in all cases and proved positive in 162 patients. Fungi were recovered, mainly Aspergillus fumigatus, from samples of 50 patients, while specimens from the remaining 123 patients were negative. Since no specific clinical sign could be found, a diagnosis of fungus ball is frequently made after a long term symptomatic period. CT scan findings of metallic or calcified densities within an opacified sinus cavity are highly suggestive of a fungus ball, but mycological and histological studies are essential to confirm the diagnosis. Treatment consisted of functional endonasal sinus surgery and was successful in 172 out of 173 cases.

[Mycologic surveillance of the environment for preventive invasive aspergillosis. Proposals for standardization of the methodologies and implementation]. / Surveillance mycologique de l'environnement pour la prévention de l'aspergillose invasive. Propositions de standardisation des méthodologies et des modalités d'application.

A MAJOR RISK: The infection of immunodepressed patients by Aspergillus-type fungi increases morbidity and mortality, particularly in hematology units or during solid organ transplantation. Although present diagnostic means benefit from the progress over the last years, they remain limited and chemoprophylaxis protocols have still not demonstrated significant efficacy. THE NEED FOR RECOMMENDATIONS: Today, the handling of environmental risks is the only strategy that has proved its efficacy and usefulness. On the basis of administrative recommendations and data from the literature, a multicentric and pluri-disciplinary task force, grouping clinicians, microbiologists and hygienists, has assessed different methods and has proposed recommendations for the standardization and optimization of fungal surveillance of the environment.

Effect of matrix metalloprotease inhibitors on the 95 kDa metallopeptidase of Candida albicans.

A 95 kDa metallopeptidase of Candida albicans could be involved in the process of dissemination of the yeast. Matrix metalloproteases (MMPs) are also responsible for collagen breakdown in inflammatory and malignant processes. We tested six compounds on the C. albicans enzyme. Doxycycline, gentamicin, cefalothin, galardin, and elaidic and oleic acids are known for their capacity to inhibit some MMPs. Amongst these agents, only oleic acid was able to markedly inhibit the purified metallopeptidase at very low concentrations. Moreover, this fatty acid inhibited the secretion of the enzyme in the culture medium without altering the yeast viability.

[Imported cutaneous leishmaniasis caused by Leishmania infantum confirmed with immunoassay]. / Leishmaniose cutanée autochtone due à Leishmania infantum confirmée par immunoempreinte.

BACKGROUND: Localized cutaneous leishmaniasis acquired in France is rarely reported. Diagnosis usually relies on detection of leishmania on smears or by culture. CASE REPORT: A 9-year-old child living outside endemic French Mediterranean areas had long-lasting crusted papules on the face for several months. Although the lesions were suggestive of cutaneous leishmaniasis, smears and culture were negative for Leishmania. Skin biopsy showed epithelioid and giant cell granuloma, but Leishman bodies were absent. Western Blot analysis of the patient's serum revealed antibodies directed against Leishmania infantum antigens, thus confirming the diagnosis of cutaneous leishmaniasis. Intralesional injections of meglumine antimoniate yielded complete regression of lesions. DISCUSSION: Localized cutaneous leishmaniasis in France is caused by Leishmania infantum and may be diagnosed outside endemic Mediterranean areas, following transmission from a sandfly bite during summer holidays in Southern France. Serum analysis by Western Blot assay distinguishes between clinically active and asymptomatic Leishmania infections, the latter being common in endemic areas. Western Blot analysis is useful for the diagnosis of cutaneous leishmaniasis when parasites cannot be detected by direct techniques.

Possible role of catheters in Saccharomyces boulardii fungemia.

Four cases of Saccharomyces boulardii fungemia, a very rare side effect of Saccharomyces boulardii therapy, are reported. The clinical impact of Saccharomyces boulardii infection appeared to be moderate. However, even though organ involvement was never demonstrated, septic shock with no other etiology was observed in one of our patients. All patients had an indwelling vascular catheter. Contamination of the air, environmental surfaces, and hands following the opening of a packet suggests that catheter contamination may have been a source of infection. To prevent catheter contamination it is recommended that packets or capsules of Saccharomyces boulardii be opened with gloves, outside the patient's room.

Case report and review. Onychomycosis due to Candida parapsilosis.

We report a case of onychomycosis with melanonychia due to Candida parapsilosis alone. Candida parapsilosis is now identified in the great majority of candidal onychomycosis, mainly in association with other yeasts. The criteria allowing the distinction between invasion and colonization, the risk factors and the treatment of C. parapsilosis onychomycosis are discussed.

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix.

Among potential virulence factors of Candida albicans, enzymes seem to play an important role. Many studies concern the secreted aspartic proteinases (saps), and the degradation of some components of the subendothelial extracellular matrix by the isoenzyme sap2 has been proved. Nevertheless, other proteolytic enzymes could be involved in the pathogenicity of the yeast. We studied the degradation of four constitutive proteins of the extracellular matrix: type I and IV collagens, laminin and fibronectin, by a 95-kDa metallopeptidase, localised in the cell wall of C. albicans. Each of these constituents was incubated with the purified enzyme and its degradation products analysed by an electrophoretic method. We observed that type I collagen and fibronectin were totally degraded by the enzyme whereas type IV collagen and laminin were only partially degraded. The C. albicans metallopeptidase may play a role in the degradation of the subendothelial extracellular matrix components. This enzyme could facilitate the migration of the yeast in the tissues after crossing the endothelial layer, allowing the fungal invasion of target organs.

[Subconjunctival localization of a Wuchereria bancrofti adult female]. / Localisation sous-conjonctivale d'une femelle adulte de Wuchereria bancrofti.

The authors describe a case of conjunctival localization of a living adult Wuchereria bancrofti female observed in a 6 year old native Haitian girl, two years after her arrival in France. The adult was surgically removed from the conjunctiva. Microfilariae were evidenced in blood samples obtained at midnight. This is the first case of sub-conjunctival localization of W. bancrofti. This case stresses the necessity to identify the filaria by studying the microfilariae in blood samples obtained at different times of the nycthemere and/or by observing the adult after surgical extraction. The presence of a Loa, a Dirofilaria, a Mansonella, or a Wuchereria calls for different medical therapies.

[Comparison of two techniques for detection of anti-Plasmodium falciparum antibodies: Falciparum-spot IF (Biomérieux) and Malaria IgG Celisa (BMD). Preliminary results]. / Comparaison de deux techniques de détection des anticorps anti-Plasmodium falciparum: Falciparum-spot IF (Biomérieux) et Malaria IgG Celisa (BMD). Résultats préliminaires.

We compared a new Elisa assay to detect malaria antibodies: Malaria IgG Celisa (BMD) with the IFAT technique Falciparum-spot IF (Biomérieux): sensitivity, specificity, predictive positive and negative values were 81%, 99%, 95%, 95%, respectively. Eight patients had positive thick blood smear out of 23 performed. For these eight confirmed acute malaria cases, the Elisa assay was negative in five instances. For two recent malaria attacks both Elisa and IFI were negative. With blood donors, two sera were IFAT positive and Elisa negative; 16 were IFAT doubtful and Elisa negative. Doubtful results rose up to 13.5% by IFAT against 1.5% by Elisa assay. We preferred kappa coefficient instead of chi 2 test for data analysis, which measures the concordance degree between the two techniques. Here concordance is moderate. Choosing an Elisa assay to detect the transmission of malaria for at-risk blood donors collides with the method sensitivity compared with IFAT as reference.