Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis).

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

Epidemiological and clinical study of microsporidiosis in French kidney transplant recipients from 2005 to 2019: TRANS-SPORE registry.

INTRODUCTION: Microsporidiosis is an emerging opportunistic infection in renal transplantation (RT) recipients. We aimed to describe its clinical presentation and treatment. MATERIALS AND METHODS: We collected microsporidiosis cases identified in RT recipients between 2005 and 2019 in six French centers from the Crystal, Divat and Astre prospective databases. RESULTS: We report 68 RT recipients with intestinal microsporidiosis; the patients were predominantly male (61.8%), with a median age of 58 (46-69) years. Infection occurred at a median time of 3 (0.8-6.8) years posttransplant. Only Enterocytozoon bieneusi was found. Microsporidiosis manifested as diarrhea (98.5% of patients) with weight loss (72.1%) and acute renal injury (57.4%) without inflammatory biological parameters. The therapeutic approaches were no treatment (N = 9), reduction of the immunosuppressive regimen (∆IS) (N = 22), fumagillin alone (N = 9), fumagillin and ∆IS (N = 19), and albendazole or nitazoxanide and ∆IS (N = 9). Overall clinical remission was observed in 60 patients (88.2%). We observed no acute kidney rejection, renal transplant failure, or death within 6 months after microsporidiosis. CONCLUSION: E. bieneusi is an underestimated opportunistic pathogen in RT recipients, and infection with E. bieneusi leads to diarrhea with important dehydration and acute renal injury. The treatment is based on the reduction of the immunosuppressive regimen and the administration of fumagillin if available.

Systemic efficacy on Cryptosporidium parvum infection of aminoxanide (RM-5061), a new amino-acid ester thiazolide prodrug of tizoxanide.

Cryptosporidiosis is a gastrointestinal illness with profuse diarrhoea. Although there are no other Food and Drug Administration (FDA)-approved alternatives for the treatment of cryptosporidiosis, nitazoxanide (NTZ) can be qualified as partially effective. In immunosuppressed conditions, severe and/or disseminated cryptosporidiosis may occur and patients should be treated parenterally. To achieve the goal of developing parenteral treatment for cryptosporidiosis, the current study was undertaken to investigate the in vitro and in vivo anticryptosporidial activity of aminoxanide. This new l-tert-leucyl thiazolide is a soluble prodrug of tizoxanide (TIZ), the main metabolite of NTZ. Confirming the good efficacy of aminoxanide in Cryptosporidium parvum-infected HCT-8 cells with a 50% inhibitory concentration of 1.55 µm (±0.21), in immunosuppressed C. parvum-infected Mongolian gerbils (Meriones unguiculatus), a 5-day treatment with a daily intramuscular dose of 100 mg kg−1 aminoxanide resulted in a 72.5% oocyst excretion inhibition, statistically equivalent to 75.5% in gerbils treated with a 4-fold lower oral dose of NTZ. Cryptosporidium parvum-induced intestinal pathology and inflammation were improved. Aminoxanide provides an injectable form of TIZ that NTZ was unable to do and is a promising drug for which optimization of the formulation should be further explored. These results represent a first promising step towards the goal of developing a parenteral treatment for cryptosporidiosis.

Detection methods and prevalence of transmission stages of Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp. in fresh vegetables: a review.

One of the ways of human parasitic infection is the accidental ingestion of vegetables contaminated with parasites, which represents a major human health hazard. This non-exhaustive review aims to evaluate studies carried out on five types of vegetables (lettuce, parsley, coriander, carrot and radish) since 2000, particularly the methods used for recovery, concentration, detection and identification of protozoan parasites such as Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp., and the results of each work. Various studies have determined the presence of pathogenic parasites in fresh vegetables with different rates; this variation in rate depends particularly on the detection method used which is related to each parasite and each vegetable type. The variation in parasitic prevalence in food could be due to different factors such as the geographical location, the size of analysed samples and the methods used for parasite detection.

Toxoplasma gondii Oocyst Infectivity Assessed Using a Sporocyst-Based Cell Culture Assay Combined with Quantitative PCR for Environmental Applications.

Toxoplasma gondii is a ubiquitous foodborne protozoan that can infect humans at low dose and displays different prevalences among countries in the world. Ingestion of food or water contaminated with small amounts of T. gondii oocysts may result in human infection. However, there are no regulations for monitoring oocysts in food, mainly because of a lack of standardized methods to detect them. The objectives of this study were (i) to develop a reliable method, applicable in biomonitoring, for the rapid detection of infectious oocysts by cell culture of their sporocysts combined with quantitative PCR (sporocyst-CC-qPCR) and (ii) to adapt this method to blue and zebra mussels experimentally contaminated by oocysts with the objective to use these organisms as sentinels of aquatic environments. Combining mechanical treatment and bead beating leads to the release of 84% ± 14% of free sporocysts. The sporocyst-CC-qPCR detected fewer than ten infectious oocysts in water within 4 days (1 day of contact and 3 days of cell culture) compared to detection after 4 weeks by mouse bioassay. For both mussel matrices, oocysts were prepurified using a 30% Percoll gradient and treated with sodium hypochlorite before cell culture of their sporocysts. This assay was able to detect as few as ten infective oocysts. This sporocyst-based CC-qPCR appears to be a good alternative to mouse bioassay for monitoring infectious T. gondii oocysts directly in water and also using biological sentinel mussel species. This method offers a new perspective to assess the environmental risk for human health associated with this parasite.IMPORTANCE The ubiquitous protozoan Toxoplasma gondii is the subject of renewed interest due to the spread of oocysts in water and food causing endemic and epidemic outbreaks of toxoplasmosis in humans and animals worldwide. Displaying a sensitivity close to animal models, cell culture represents a real alternative to assess the infectivity of oocysts in water and in biological sentinel mussels. This method opens interesting perspectives for evaluating human exposure to infectious T. gondii oocysts in the environment, where oocyst amounts are considered to be very small.

Evaluation of propidium monoazide-based qPCR to detect viable oocysts of Toxoplasma gondii.

Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)-qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99 °C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts' dye permeability. Different PMA concentrations (50 to 150 µM), incubation temperatures (22, 37, and 45 °C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA-qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22 °C with 100 µM PMA coupled to qPCR targeting a 123-bp sequence of the 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA-qPCR is able to assess the impact of heating on T. gondii oocysts' viability but underestimates the efficacy of this treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations.

Evaluation of voriconazole anti-Acanthamoeba polyphaga in vitro activity, rat cornea penetration and efficacy against experimental rat Acanthamoeba keratitis.

Background: Acanthamoeba keratitis (AK) is a sight-threatening infectious disease. Its effective and safe medical therapy remains highly debated. Recently, voriconazole, a monotriazole with noted in vitro activity against a large variety of fungi, has been successfully used both topically and systemically to treat human AK cases. Objectives: To measure anti-Acanthamoeba polyphaga in vitro activity, anti-rat AK efficiency and rat cornea penetration of eye-drop and oral voriconazole. Methods: A. polyphaga was maintained in axenic cultures. In vitro, amoebicidal and cysticidal activities of voriconazole were measured using an XTT assay. AK lesions of Sprague Dawley rats were scored from grade 0 to grade 3. For 21 days, from day 7 post-infection, voriconazole (1% solution) eye drops were instilled or voriconazole was administered by gavage (60 mg/kg/day). After killing, superficial corneal epithelium scrapings were cultured and analysed by PCR, and eye-globe histology was performed. Cornea and plasma concentrations were determined using 2D HPLC separation and tandem MS. Results: In vitro, voriconazole inhibited trophozoite proliferation with an IC50 value of 0.02 mg/L and an IC90 value of 2.86 mg/L; no cysticidal effect was found. In AK rats, eye drops reduced clinical worsening from day 7 to day 14 post-infection and oral voriconazole was not effective. Voriconazole cornea concentrations were directly dependent on the frequency of eye-drop instillations, which resulted in lower plasma concentrations, whilst oral voriconazole resulted in lower cornea concentrations. Conclusions: Present data underline the need for high-frequency eye-drop instillation regimens for efficient AK therapy.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

Human cryptosporidiosis in immunodeficient patients in France (2015-2017).

Cryptosporidiosis is a common disease in children and immunodeficient individuals. In 2006, a national network was set up on the surveillance of human cryptosporidiosis in France. Since January 2015, the 41 tertiary care hospitals and the 3 private laboratories of the French National Network on the surveillance of human cryptosporidiosis have been able to declare confirmed cases of cryptosporidiosis online. Between 2015 and 2017, 210 cases of cryptosporidiosis were declared in immunodeficient patients in France; Cryptosporidium parvum and Cryptosporidium hominis represented 66% and 22% of cases, respectively. A peak was observed in autumn. Cryptosporidiosis occurred mainly in a context of solid organ transplantation (SOT) (49%) and of HIV infection (30%). In SOT recipients, cryptosporidiosis appeared more frequently in the first 6 months post transplantation. Regarding cases declared in SOT recipients, mycophenolate mofetil was used in 68%. A mortality rate of 6% was observed. Present results underline the importance of screening for cryptosporidiosis in immunocompromised patients suffering from diarrhea, especially in the course of major cell mediated immunodeficiency or even systematic screening before SOT. Exclusive Cryptosporidium free water feeding could be suggested during major cell mediated immunodeficiency.

Toward the Standardization of Mycological Examination of Sputum Samples in Cystic Fibrosis: Results from a French Multicenter Prospective Study.

Fungal respiratory colonization of cystic fibrosis (CF) patients emerges as a new concern; however, the heterogeneity of mycological protocols limits investigations. We first aimed at setting up an efficient standardized protocol for mycological analysis of CF sputa that was assessed during a prospective, multicenter study: "MucoFong" program (PHRC-06/1902). Sputa from 243 CF patients from seven centers in France were collected over a 15-month period and submitted to a standardized protocol based on 6 semi-selective media. After mucolytic pretreatment, sputa were plated in parallel on cycloheximide-enriched (ACT37), erythritol-enriched (ERY37), benomyl dichloran-rose bengal (BENO37) and chromogenic (CAN37) media incubated at 37 °C and on Sabouraud-chloramphenicol (SAB27) and erythritol-enriched (ERY27) media incubated at 20-27 °C. Each plate was checked twice a week during 3 weeks. Fungi were conventionally identified; time for detection of fungal growth was noted for each species. Fungal prevalences and media performances were assessed; an optimal combination of media was determined using the Chi-squared automatic interaction detector method. At least one fungal species was isolated from 81% of sputa. Candida albicans was the most prevalent species (58.8%), followed by Aspergillus fumigatus (35.4%). Cultivation on CAN37, SAB27, ACT37 and ERY27 during 16 days provided an optimal combination, detecting C. albicans, A. fumigatus, Scedosporium apiospermum complex and Exophiala spp. with sensitivities of 96.5, 98.8, 100 and 100%. Combination of these four culture media is recommended to ensure the growth of key fungal pathogens in CF respiratory specimens. The use of such consensual protocol is of major interest for merging results from future epidemiological studies.

Giardia duodenalis induces paracellular bacterial translocation and causes postinfectious visceral hypersensitivity.

Irritable bowel syndrome (IBS) is the most frequent functional gastrointestinal disorder. It is characterized by abdominal hypersensitivity, leading to discomfort and pain, as well as altered bowel habits. While it is common for IBS to develop following the resolution of infectious gastroenteritis [then termed postinfectious IBS (PI-IBS)], the mechanisms remain incompletely understood. Giardia duodenalis is a cosmopolitan water-borne enteropathogen that causes intestinal malabsorption, diarrhea, and postinfectious complications. Cause-and-effect studies using a human enteropathogen to help investigate the mechanisms of PI-IBS are sorely lacking. In an attempt to establish causality between giardiasis and postinfectious visceral hypersensitivity, this study describes a new model of PI-IBS in neonatal rats infected with G. duodenalis At 50 days postinfection with G. duodenalis (assemblage A or B), long after the parasite was cleared, rats developed visceral hypersensitivity to luminal balloon distension in the jejunum and rectum, activation of the nociceptive signaling pathway (increased c-fos expression), histological modifications (villus atrophy and crypt hyperplasia), and proliferation of mucosal intraepithelial lymphocytes and mast cells in the jejunum, but not in the rectum. G. duodenalis infection also disrupted the intestinal barrier, in vivo and in vitro, which in turn promoted the translocation of commensal bacteria. Giardia-induced bacterial paracellular translocation in vitro correlated with degradation of the tight junction proteins occludin and claudin-4. The extensive observations associated with gut hypersensitivity described here demonstrate that, indeed, in this new model of postgiardiasis IBS, alterations to the gut mucosa and c-fos are consistent with those associated with PI-IBS and, hence, offer avenues for new mechanistic research in the field.

Long-Term Rasamsonia argillacea Complex Species Colonization Revealed by PCR Amplification of Repetitive DNA Sequences in Cystic Fibrosis Patients.

The aim of this work was to document molecular epidemiology of Rasamsonia argillacea species complex isolates from cystic fibrosis (CF) patients. In this work, 116 isolates belonging to this species complex and collected from 26 CF patients and one patient with chronic granulomatous disease were characterized using PCR amplification assays of repetitive DNA sequences and electrophoretic separation of amplicons (rep-PCR). Data revealed a clustering consistent with molecular species identification. A single species was recovered from most patients. Rasamsonia aegroticola was the most common species, followed by R. argillacea sensu stricto and R. piperina, while R. eburnea was not identified. Of 29 genotypes, 7 were shared by distinct patients while 22 were patient specific. In each clinical sample, most isolates exhibited an identical genotype. Genotyping of isolates recovered from sequential samples from the same patient confirmed the capability of R. aegroticola and R. argillacea isolates to chronically colonize the airways. A unique genotype was recovered from two siblings during a 6-month period. In the other cases, a largely dominant genotype was detected. Present results which support the use of rep-PCR for both identification and genotyping for the R. argillacea species complex provide the first molecular evidence of chronic airway colonization by these fungi in CF patients.

Semi-automated repetitive sequence-based PCR amplification for species of the Scedosporium apiospermum complex.

PURPOSE: The Scedosporium apiospermum species complex usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), but little is known about the molecular epidemiology of the airway colonization. METHODS: Polymerase chain reaction (PCR) amplification of repetitive sequences (rep-PCR) was applied to the retrospective analysis of a panel of isolates already studied by random amplification of polymorphic DNA (RAPD) and comprising 63 isolates recovered from sputa from 9 CF patients. Results were compared to those obtained previously by RAPD, and herein by beta-tubulin (TUB) gene sequencing and Multilocus Sequence Typing (MLST). RESULTS: Within the panel of isolates studied,S. apiospermum sensu stricto and Scedosporium boydii, as expected, were the predominant species with 21 and 36 isolates, respectively. Four isolates from one patient were identified as Scedosporium aurantiacum, whereas two isolates belonged to the Pseudallescheria ellipsoidea subgroup of S. boydii rep-PCR analysis of these isolates clearly differentiated the three species and P. ellipsoidea isolates, whatever the rep-PCR kit used, and also permitted strain differentiation. When using the mold primer kit, results from rep-PCR were in close agreement with those obtained by MLST. For both S. apiospermum and S. boydii, 8 genotypes were differentiated by rep-PCR and MLST compared to 10 by RAPD. All S. aurantiacum isolates shared the same RAPD genotype and exhibited the same rep-PCR profile and sequence type. CONCLUSIONS: These results illustrate the efficacy of rep-PCR for both species identification within the S. apiospermum complex and genotyping for the two major species of this complex.Abstract presentation: Part of this work was presented during the 18th Congress of the International Society for Human and Animal Mycology, Berlin (Germany), June 2012.S. Giraud, C. Godon, A. Rougeron, J.P. Bouchara and L. Favennec are members of the ECMM/ISHAM working group on Fungal respiratory infections in Cystic Fibrosis(Fri-CF).

First description of azole-resistant Aspergillus fumigatus due to TR46/Y121F/T289A mutation in France.

Azole resistance in Aspergillus fumigatus is an emerging public health concern. Recently, a novel fungicide-driven mutation in the cyp51A gene and its promoter, TR46/Y121F/T289A, leading to high-level resistance to voriconazole has been identified in The Netherlands, Belgium, Germany, Denmark, Tanzania, and India in both clinical and environmental samples. Here we report the first description of A. fumigatus carrying this mutation in France, in a cystic fibrosis patient, underlining the need for extensive monitoring of Aspergillus resistance.

Cryptococcus neoformans Infections Differ Among Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Individuals: Results From a Nationwide Surveillance Program in France.

Among 1107 cryptococcosis cases from the French surveillance network (2005-2020), the proportion of HIV-seronegative individuals has recently surpassed that of HIV-seropositive individuals. We observed marked differences in patient characteristics, disease presentations, cryptococcal antigen results, infecting species, and mortality according to HIV serostatus.

Features of cryptococcosis among 652 HIV-seronegative individuals in France: a cross-sectional observational study (2005-2020).

OBJECTIVES: We aimed to describe features and outcomes of cryptococcosis among HIV-seronegative individuals in a large surveillance network for cryptococcosis in France. METHODS: We included incident cases of cryptococcosis in HIV-seronegative individuals from 2005 to 2020. We compared patient characteristics, disease presentations, cryptococcal antigen results, and induction antifungal treatments according to underlying disease. We examined factors associated with 90-day mortality. Among patients with disseminated infections, we investigated whether receipt of flucytosine and polyene combination was associated with lower mortality. RESULTS: Among 652 individuals, 209 (32.1%) had malignancy, 130 (19.9%) were solid-organ transplant recipients, 204 (31.3%) had other immunocompromising conditions, and 109 (16.7%) had no reported underlying factor. The commonest presentations were disseminated infections (63.3%, 413/652) and isolated pulmonary infections (25.3%, 165/652). Solid-organ transplant patients were most likely to have disseminated infections and a positive serum cryptococcal antigen result. Patients with malignancy were older and less likely to receive a flucytosine-containing regimen for disseminated infections than others (58.7%, 78/133 vs. 73.2%, 194/265; p 0.029). The crude 90-day case-fatality ratio was 27.2% (95% CI, 23.5%-31.1%). Age ≥60 years (aOR: 2.75 [1.78-4.26]; p < 0.001), meningitis/fungaemia (aOR: 4.79 [1.80-12.7]; p 0.002), and malignancy (aOR: 2.4 [1.14-5.07]; p 0.02) were associated with higher 90-day mortality. Receipt of flucytosine and polyene combination was associated with lower 90-day mortality (aOR: 0.40 [0.23-0.71]; p 0.002) in multivariable analysis and inverse probability of treatment weighted analysis (aOR: 0.45 [0.25-0.80]; p 0.006). DISCUSSION: HIV-seronegative individuals with cryptococcosis comprise a wide range of underlying conditions with different presentations and outcomes, requiring a tailored approach to diagnosis and management.

Activity of halogeno-thiazolides against Cryptosporidium parvum in experimentally infected immunosuppressed gerbils (Meriones unguiculatus).

Nitazoxanide and three halogeno-thiazolides, RM-4850, RM-4865, and RM-5038, were tested against Cryptosporidium parvum in experimentally infected immunosuppressed Mongolian gerbils. Daily 400-mg/kg doses of the four test drugs for 5 to 8 consecutive days produced similar reductions of oocyst shedding. Using early-infected gerbils, a shorter 4-day treatment with RM-5038 reduced oocyst shedding by 95%, compared to 47% for nitazoxanide (P = 0.02), suggesting that RM-5038 is more effective than nitazoxanide under the experimental conditions used.

In vivo molecular microimaging of pulmonary aspergillosis.

The early diagnosis of invasive pulmonary aspergillosis (IPA) is challenging. Fibered confocal fluorescence microscopy (FCFM) is a new technique that allows in vivo imaging of the lung microstructure during bronchoscopy. In this study, we investigated the ability of FCFM to detect a fluorescent peptide-tracer bound to Aspergillus fumigatus in experimental IPA in 13 immunosuppressed, non-neutropenic rats. Subpleural IPA microabscesses were imaged through a transthoracic window using FCFM in vivo after i.v. injection of the c(CGGRLGPFC)-NH2([FITC]) peptide (n = 7) or saline. Results were compared to 10 immunosuppressed, non-infected rats and to six immunosuppressed Geosmithia argillacea-infected rats with and without i.v. injection of the peptide. The peptide in vitro specifically labeled A. fumigatus grown under biofilm growth conditions but not G. argillacea. In vivo, FCFM showed a local infiltration of fluorescent host cells in both Aspergillus and Geosmithia infections. Lung/inner thoracic wall fluorescence intensity ratio (FI) did not differ before and after peptide administration on healthy lung areas, on non-specific inflammatory areas, or on Geosmithia micro-abscesses. In contrast, FI increased from 1.05 without peptide to 1.83 after peptide injection on Aspergillus micro-abscesses (p < 0.0001). In peptide-injected rats, FI from IPA foci was higher than from non-specific inflammation or from Geosmithia abscesses (p ≤ 0.002). Using c(CGGRLFPC)-NH2([FITC]) peptide, FCFM allows the in vivo specific imaging of pulmonary aspergillosis. These data provide the basis for the in vivo diagnosis of human pulmonary aspergillosis using alveolar confocal endomicroscopy.