Nosocomial transmission of Aspergillus flavus in a neonatal intensive care unit: Long-term persistence in environment and interest of MALDI-ToF mass-spectrometry coupled with convolutional neural network for rapid clone recognition.

Aspergillosis of the newborn remains a rare but severe disease. We report four cases of primary cutaneous Aspergillus flavus infections in premature newborns linked to incubators contamination by putative clonal strains. Our objective was to evaluate the ability of matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) coupled to convolutional neural network (CNN) for clone recognition in a context where only a very small number of strains are available for machine learning. Clinical and environmental A. flavus isolates (n = 64) were studied, 15 were epidemiologically related to the four cases. All strains were typed using microsatellite length polymorphism. We found a common genotype for 9/15 related strains. The isolates of this common genotype were selected to obtain a training dataset (6 clonal isolates/25 non-clonal) and a test dataset (3 clonal isolates/31 non-clonal), and spectra were analysed with a simple CNN model. On the test dataset using CNN model, all 31 non-clonal isolates were correctly classified, 2/3 clonal isolates were unambiguously correctly classified, whereas the third strain was undetermined (i.e., the CNN model was unable to discriminate between GT8 and non-GT8). Clonal strains of A. flavus have persisted in the neonatal intensive care unit for several years. Indeed, two strains of A. flavus isolated from incubators in September 2007 are identical to the strain responsible for the second case that occurred 3 years later. MALDI-TOF is a promising tool for detecting clonal isolates of A. flavus using CNN even with a limited training set for limited cost and handling time.

Cutaneous aspergillosis is a rare but potentially fatal disease of the prematurely born infant. We described here several cases due to Aspergillus flavus and have linked them to environnemental strains using MLP genotyping and MALDI-TOF mass spectrometry coupled with artificial intelligence.

Characteristics and outcome according to underlying disease in non-AIDS patients with acute respiratory failure due to Pneumocystis pneumonia.

In the non-AIDS group, several underlying conditions and immune defects could lead to different PCP presentations. This study compared PCP presentation and outcome according to the underlying disease. A secondary analysis of a previously published prospective observational study including 544 PCP patients was done. Only non-AIDS patients were included. Underlying disease was defined as chronic lymphocytic leukemia (CLL), organ transplantation, solid cancer, allogeneic hematopoietic stem cell transplant (AHSCT), other hematological diseases, and immunosuppressive treatment. Clinical characteristics and outcomes were compared between groups. Multiple correspondent analyses compared clinical characteristics at diagnosis. Day 30 mortality was analyzed. Three hundred and twenty-one patients were included in the study. The underlying diseases were hematological malignancy (n = 75), AHSCT (n = 14), CLL (n = 19), solid organ transplant (n = 94), solid tumor (n = 39), and immunosuppressive treatment (n = 57). Compared with other underlying diseases, PCP related to CLL was closer to PCP related to AIDS presentation (long duration of symptoms before diagnosis, high level of dyspnea, and low oxygen saturation at diagnosis). Day 30 mortality was associated with underlying disease, oxygen flow, and shock at ICU admission. PCP presentations may vary according to the underlying reason for immunosuppression. Response to treatment and adjuvant steroid therapy should be analyzed regarding this result.

Pneumocystis Infection Outbreaks in Organ Transplantation Units in France: A Nation-Wide Survey.

The burden of nosocomial Pneumocystis infections in transplantation units in France was evaluated through a retrospective survey. Over 12 years, 16 outbreaks occurred, including 13 among renal transplant recipients (RTRs). We performed Pneumocystis jirovecii genotyping in 5 outbreaks, which suggested that specific strains may have been selected by RTRs.

Pulmonary adverse events related to idelalisib therapy: A single centre experience.

Idelalisib is a potent and selective inhibitor of the PI3Kδ approved since September 2014 for the treatment of several types of B cell malignancies. Pulmonary adverse events related to idelalisib are an emerging serious adverse event. We report here a single centre cohort of 16 patients who initiated idelalisib as routine treatment. Five of them experienced severe pulmonary adverse events related to idelalisib therapy. Comparison of the 5 patients with severe pulmonary events versus the 11 patients without identified no predisposing factors. Severe pulmonary adverse events were related to infectious pneumonia and/or to a drug-induced pneumonitis. The mechanisms of idelalisib-associated pneumonitis are unknown but consistent with the drug-induced pneumonitis described with mTOR inhibitors. Indeed, by inhibiting PI3Kδ, idelalisib also inhibits the mTOR pathway. Clinicians should be aware that any idelalisib-treated patient who presents with pulmonary symptoms should be evaluated for pneumonitis. Corticosteroids should be considered in addition to anti-infective therapy in case of severe pneumonitis or persistent pulmonary symptoms despite adequate antibiotic therapy.

Severe post-artesunate delayed onset anaemia responding to corticotherapy: a case report.

Delayed onset haemolysis occurring post-artesunate and post-artemisinin combination therapy is secondary to delayed clearance of infected erythrocytes spared by pitting during treatment. We report a case of severe post-treatment delayed haemolytic anaemia with a positive direct antiglobulin test and a positive response to corticosteroid therapy, suggesting an associated immune mechanism.

Donor Derived Candida stellimalicola in a Clinical Specimen: Preservation Fluid Contamination During Pancreas Procurement.

We report here a case of possible donor-derived Candida stellimalicola infection after pancreas transplantation. Candida stellimalicola, an environmental non-filamentous yeast, was isolated from both the peritoneal fluid of the graft donor and the preservation fluid of the transplanted pancreas. Interestingly, this strain exhibited high minimum inhibitory concentrations to azoles. These results justified the use of echinocandins as therapy instead of fluconazole. This switch permitted a favorable outcome. To our knowledge, this is the first report of C. stellimalicola from clinical samples and therefore the first reported case of a possible human infection. This case report highlights the need for standardized microbiological procedures in solid organ transplant settings. Moreover, it underlines the importance of using molecular identification technique when routine techniques do not allow successful identification of the pathogen. It is of utmost importance to determine sensitivity profile, even in the absence of species-level identification, because resistance to fluconazole is not uncommon, especially in emergent species.

A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study.

This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.

Posttraumatic mucormycosis: a nationwide study in France and review of the literature.

Data on clinical, mycologic characteristics, and outcome of posttraumatic mucormycosis are scarce and often limited to case reports. From the French nationwide "RetroZygo" study, we compared posttraumatic mucormycosis cases with other forms of mucormycosis. We also reviewed reports of posttraumatic mucormycosis in the English-language literature from 1993 to 2013. We included all proven or probable cases for which underlying condition, route of infection, surgical and antifungal treatments, and outcome were detailed. From our cohort, posttraumatic mucormycosis (n = 16) differed significantly from other forms (n = 85) by rarity of underlying disease (31.2% vs 81%, p < 0.0001), frequency of cutaneous localization (87% vs 7%, p < 0.0001), short time before diagnosis (4.5 vs 21 d, p = 0.0002), species involved (Apophysomyces elegans complex and Saksenaea vasiformis), surgical requirement (93.7% vs 47%, p = 0.0006) and better survival (87.5% vs 47.6% at day 90, p = 0.03). We studied 122 cases of posttraumatic mucormycosis through our literature review. Most frequently reported traumas were traffic (37%), domestic accidents (15.1%), or natural disasters (13.4%). Mucormycosis occurred after extensive soft-tissue damage in 47.5% cases, with symptoms occurring a median of 9.5 days after trauma with necrosis being reported in 76.2% cases. Dissemination was found in 9% of patients, and bacterial coinfection in 41%. Nineteen percent of cases occurred in the Middle East or in India where Apophysomyces elegans complex was the predominant species recovered. Awareness of mucormycosis as a cause of posttrauma soft-tissue infection is warranted, especially in cases of soil-contaminated wounds. Survival is higher than in other forms of mucormycosis, but morbidity remains high.

Pneumocystis jirovecii pneumonia in patients with or without AIDS, France.

Pneumocystis jirovecii pneumonia (PCP) in patients without AIDS is increasingly common. We conducted a prospective cohort study of consecutive patients with proven PCP; of 544 patients, 223 (41%) had AIDS (AIDS patients) and 321 (59%) had other immunosuppressive disorders (non-AIDS patients). Fewer AIDS than non-AIDS patients required intensive care or ventilation, and the rate of hospital deaths--17.4% overall--was significantly lower for AIDS versus non-AIDS patients (4% vs. 27%; p<0.0001). Multivariable analysis showed the odds of hospital death increased with older age, receipt of allogeneic bone marrow transplant, immediate use of oxygen, need for mechanical ventilation, and longer time to treatment; HIV-positive status or receipt of a solid organ transplant decreased odds for death. PCP is more often fatal in non-AIDS patients, but time to diagnosis affects survival and is longer for non-AIDS patients. Clinicians must maintain a high index of suspicion for PCP in immunocompromised patients who do not have AIDS.

Diversity of the bacterial and fungal microflora from the midgut and cuticle of phlebotomine sand flies collected in North-Western Iran.

BACKGROUND: Phlebotomine sand flies are the vectors of the leishmaniases, parasitic diseases caused by Leishmania spp. Little is known about the prevalence and diversity of sand fly microflora colonizing the midgut or the cuticle. Particularly, there is little information on the fungal diversity. This information is important for development of vector control strategies. METHODOLOGY/PRINCIPAL FINDINGS: FIVE SAND FLY SPECIES: Phlebotomus papatasi, P. sergenti, P. kandelakii, P. perfiliewi and P. halepensis were caught in Bileh Savar and Kaleybar in North-Western Iran that are located in endemic foci of visceral leishmaniasis. A total of 35 specimens were processed. Bacterial and fungal strains were identified by routine microbiological methods. We characterized 39 fungal isolates from the cuticle and/or the midgut. They belong to six different genera including Penicillium (17 isolates), Aspergillus (14), Acremonium (5), Fusarium (1), Geotrichum (1) and Candida (1). We identified 33 Gram-negative bacteria: Serratia marcescens (9 isolates), Enterobacter cloacae (6), Pseudomonas fluorescens (6), Klebsiella ozaenae (4), Acinetobacter sp. (3), Escherichia coli (3), Asaia sp. (1) and Pantoea sp. (1) as well as Gram-positive bacteria Bacillus subtilis (5) and Micrococcus luteus (5) in 10 isolates. CONCLUSION/SIGNIFICANCE: Our study provides new data on the microbiotic diversity of field-collected sand flies and for the first time, evidence of the presence of Asaia sp. in sand flies. We have also found a link between physiological stages (unfed, fresh fed, semi gravid and gravid) of sand flies and number of bacteria that they carry. Interestingly Pantoea sp. and Klebsiella ozaenae have been isolated in Old World sand fly species. The presence of latter species on sand fly cuticle and in the female midgut suggests a role for this arthropod in dissemination of these pathogenic bacteria in endemic areas. Further experiments are required to clearly delineate the vectorial role (passive or active) of sand flies.

[Prevention of fungal infections related to the water supply in French hospitals: proposal for standardization of methods]. / Surveillance mycologique de l'eau pour la prévention des mycoses invasives dans les établissements de santé: propositions de standardisation des méthodologies.

OBJECTIVES: The aims of this study were to assess the risk of fungal infections related to the water supply in several hospitals and to clarify the appropriate methodology in order to standardize the technical conditions of the controls and develop guidelines. It was conducted in 10 university hospital centers across the country from February 2004 through March 2005. METHOD: A preliminary study allowed us to optimize the mycological analysis. The study was conducted under the same conditions as for bacteriological controls: water filtration through a cellulose acetate membrane cultured on agar. Departments with the highest patient risk were selected, including hematology, organ transplantation, and burn units. We selected 98 sites and sampled both water and water-related surfaces at each: three one-liter water samples (the first flow, cold and hot water) and two or three surface samples (inside the tap, pommel of the shower and siphon). At each site, a form was filled to specify its location in the unit, any water treatment (chlorine or other), filtering, and temperature. Water from taps equipped with sterilized filtration was sampled without the filter. RESULTS: There was a significant difference (p=0.039) in the number of positive cultures between the three types of water sampled: hot water (>50 degrees C) was colonized less often than first flow or cold water. Only 4% of the hot-water samples had positive cultures, compared to the 52% of the cold-water samples. Except in two hospitals with generalized contamination of the water pipes (one with Exophiala spp and the other with Fusarium spp), colonization was usually slight. Cold water was more colonized than hot water, but 79% of the samples yielded fewer than 5CFU/L. Dematiaceous hyphomycetes were isolated; Aspergillus spp were rare. The number of CFU in surface samples (that is, biofilms) was higher (mean=15 CFU per sample) but surfaces were positive less often than water (13% compared with 43% of all water samples). Sampling from siphons was productive more often than from taps (23%), but the molds isolated differed from those in the related water. Relations to bacterial flora and P. aeruginosa were also studied, together with the effects of chemical treatment. CONCLUSION: Current regulations require only bacteriological survey. The absence of knowledge about the threshold of contamination at which there is a risk of nosocomial invasive fungal infections makes it difficult to impose routine monitoring. Mycological surveys of water are required during hospital renovation, plumbing work, pipe maintenance and when air samples are negative during nosocomial infection investigations.

Epidemiological investigation and typing of Candida glabrata clinical isolates by FTIR spectroscopy.

Candida glabrata has emerged as one of the leading agents of fungal infections and strain typing is essential for epidemiological investigation that is generally achieved by molecular techniques. In this work, we studied twenty-nine C. glabrata strains isolated from different patients, using a phenotypic approach based on Fourier Transform Infrared (FTIR) spectroscopy, which has been in a previous study successfully applied as a rapid typing method for Candida albicans. A two-step procedure was used for the analysis. The first step included sixteen strains for the internal validation phase, which aimed at finding the spectral windows that would provide the best differentiation between strains. In this phase, hierarchical cluster analysis (HCA) carried out using three spectral windows (900-1200, 1540-1800, 2800-3000 cm(-1)) allowed to obtain the best classification, where each patient strains could be clustered together. A genotypic technique based on randomly amplified polymorphic DNA-analysis (RAPD) confirmed these results. In a second step, the external validation phase, thirteen other clinical strains of C. glabrata isolated from multiple sites in four ICU patients, were tested by FTIR spectroscopy. The analysis was based on the spectral regions previously found in the first step. HCA classification of the strains gave four groups, one group per patient. These results suggest that no inter-human transmission took place. This study shows the potential of FTIR approach for typing of C. glabrata with several advantages compared to other techniques. FTIR typing is fast, effective, and reagent free. Moreover, it is applicable to all micro-organisms and requires a small quantity of biomass.

FTIR spectroscopy in medical mycology: applications to the differentiation and typing of Candida.

The incidence of fungal infections, in particular candidiasis and aspergillosis, has considerably increased during the last three decades. This is mainly due to advances in medical treatments and technologies. In high risk patients (e.g. in haematology or intensive care), the prognosis of invasive candidiasis is relatively poor. Therefore, a rapid and correct identification of the infectious agent is important for an efficient and prompt therapy. Most clinical laboratories rely on conventional identification methods that are based on morphological, physiological and nutritional characteristics. However, these have their limitations because they are time-consuming and not always very accurate. Moreover, molecular methods may be required to determine the genetic relationship between the infectious strains, for instance in Candida outbreaks. In addition, the latter methods require time, expensive consumables and highly trained staff to be performed adequately. In this study, we have applied the FTIR spectroscopic approach to different situations encountered in routine mycological diagnosis. We show the potentials of this phenotypic approach, used in parallel with routine identification methods, for the differentiation of 3 frequently encountered Candida species (C. albicans, C. glabrata and C. krusei) by using both suspensions and microcolonies. This approach, developed for an early discrimination, may help in the initial choice of antifungal treatment. Furthermore, we demonstrate the feasibility of the method for intraspecies comparison (typing) of 3 Candida species (C. albicans, C. glabrata and C. parapsilosis), particularly when an outbreak is suspected.

FTIR spectroscopy as a potential tool to analyse structural modifications during morphogenesis of Candida albicans.

Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950-1,185 cm(-1)), protein (1,480-1,720 cm(-1)), and the fatty acids (2,840-3,000 cm(-1)) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as beta-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.

Rapid identification of Candida species by FT-IR microspectroscopy.

Due to the continuous increase of human candidiasis and the great diversity of yeasts of the Candida genera, it is indispensable to identify this yeast as early as possible. Early identification enables an early diagnostic and patient-adapted anti-fungal therapy, thus reducing morbidity and mortality related to these infections. In view of this, we have in this study investigated microcolonies using a method based on Fourier transform-infrared microspectroscopy (FTIRM) for a rapid and early identification of the most frequent Candida species encountered in human pathology. FTIR spectroscopy is a whole-cell "fingerprinting" method by which microorganisms can be identified. By exploiting the huge discriminating capacity of this technique, we identified 6 species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, and Candida kefyr) from a collection of 57 clinical strains of Candida, isolated from hospitalised patients. Data obtained on 10- to 18-h-old microcolonies were compared to cultures of 24 h. Our results clearly show the efficiency and the robustness of FTIR (micro)spectroscopy in identifying species with a classification rate of 100% for both microcolonies and 24-h cultures. FTIR microspectroscopy is thus a promising clinical approach, because compared to conventional and molecular techniques, it is time and money saving, has great identification and discriminating potentials, and is amenable to an automated high-throughput routine system.

Use of co-immunoelectrodiffusion to detect presumed disease-associated precipitating antibodies, and time-course value of specific isotypes in bird-breeder's disease.

BACKGROUND AND OBJECTIVE: The practical value of immunological diagnosis of bird-breeder's disease (BBD) is controversial, because of difficulties in distinguishing active disease patients from simple contact subjects. The aim of this study was to determine the diagnostic and prognostic value of (a) presumed disease-associated antibodies precipitating pigeon antigens (immunoglobulin A (IgAp) and P2 component), (b) characterization of specific isotypes (IgG, IgM, and IgA), and (c) antibody kinetics after antigen eradication. METHODS: 405 subjects (775 sera) in contact with birds were studied [by means of co-immunoelectrodiffusion (Co-IED) and enzyme-linked immunofiltration (ELIFA)] with soluble extracts of pigeon droppings and squab crop milk. These patients were divided into two groups based on the final clinical evaluation of the patients' physicians, which was taken as the gold standard (positive in 90 and negative in 315 cases). RESULTS: On the basis of this gold standard, the detection of presumed disease-associated precipitating antibodies by Co-IED had a specificity of 95.5%, a sensitivity of 98.7%, an accuracy of 98%, and positive and negative predictive values of 95.5% and 98.7%, respectively. Most of the patients with a final positive diagnosis of BBD had specific IgG, IgM, and IgA antibodies by ELIFA. After antigen eradication, anti IgAp and/or P2 antibodies disappeared more rapidly than other precipitating systems. CONCLUSION: Identification by Co-IED of precipitating immune complexes IgAp and/or P2 significantly reinforces the intrinsic credibility of immunological diagnosis of BBD. Compared to these presumed disease-associated precipitating antibodies, detection and time course of specific IgM, IgA antibodies, provided no additional diagnostic value or prognostic arguments to judge disease activity after antigen eradication.