Jacques Chandenier

The antibacterial and antifungal properties of trappin‐2 (pre‐elafin) do not depend on its protease inhibitory function

Trappin‐2 (also known as pre‐elafin) is an endogenous inhibitor of neutrophil serine proteases and is involved in the control of excess proteolysis, especially in inflammatory events, along with the structurally related secretory leucocyte proteinase inhibitor. Secretory leucocyte proteinase inhibitor has been shown to have antibacterial and antifungal properties, whereas recent data indicate that trappin‐2 has antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In the present study, we tested the antibacterial properties of trappin‐2 towards other respiratory pathogens. We found that trappin‐2, at concentrations of 5–20 μm, has significant activity against Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Branhamella catarrhalis and the pathogenic fungi Aspergillus fumigatus and Candida albicans, in addition to P. aeruginosa and S. aureus. A similar antimicrobial activity was observed with trappin‐2 A62D/M63L, a trappin‐2 variant that has lost its antiprotease properties, indicating that trappin‐2 exerts its antibacterial effects through mechanisms independent from its intrinsic antiprotease capacity. Furthermore, the antibacterial and antifungal activities of trappin‐2 were sensitive to NaCl and heparin, demonstrating that its mechanism of action is most probably dependent on its cationic nature. This enables trappin‐2 to interact with the membranes of target organisms and disrupt them, as shown by our scanning electron microscopy analyses. Thus, trappin‐2 not only provides an antiprotease shield, but also may play an important role in the innate defense of the human lungs and mucosae against pathogenic microorganisms.

Molecular identification of Pentatrichomonas hominis in two patients with gastrointestinal symptoms

The trichomonad species Pentatrichomonas hominis colonises the gastrointestinal tract and is generally considered as a commensal organism in humans. However, some studies have recognised an association between diarrhoea and P hominis infection in dogs and cats.1 2 In the present report, we have identified this species using molecular tools in two patients with gastrointestinal troubles. Our data suggest that P hominis is a possible zoonotic species with a significant potential of transmission by water and could be the causative agent of intestinal symptoms in children. An adult (case 1) was followed up for different pathologies including irritable bowel syndrome (IBS). Diarrhoeic stools of the patient were examined and were negative for intestinal parasites. Filter paper/slant culture technique for the recovery of Strongyloides stercoralis larval-stage nematodes from fresh faeces was performed. After 5 days, microscopic examination of the stool culture using merthiolate-iodine-formalin and RAL555 stains did not detect larval nematodes but numerous flagellates, provisionally identified as trichomonads (figure 1A,B). Although no treatment against trichomonads was administered to the patient, the stool examinations performed afterwards did not reveal trichomonads or other parasites. Figure 1 Cytological appearance of trichomonad cells in stool culture (case 1). (A) Merthiolate-iodine-formalin- and (B) RAL555-stained smears showing numerous trichomonad cells (arrows). Note the round shape of the micro-organisms. Typical microtubular cytoskeletal structures of trichomonads including flagella and axostyle–pelta complex are not visible. Bar=15 μm. A young child (case 2) presented with abdominal pain and loose stools without fever. A stool sample was examined by direct light …

Diagnosis of invasive pulmonary aspergillosis: updates and recommendations.

Invasive pulmonary aspergillosis is an opportunistic mycosis, difficult to diagnose, due to the environmental fungi of the genus Aspergillus. The diagnostic tools, even if more are available, are still limited in number and effectiveness. The current recommendations issued by the EORTC/MSG (European Organization of Research and Treatment of Cancer/Mycoses Study Group) and the ECIL (European Conference for Infection in Leukemia) suggest collecting epidemiological, radio-clinical, and biological data to support the diagnosis of aspergillosis with a strong presumption. Thus, medical imaging and serum galactomannan antigen currently constitute the basis of the screening approach, although they both have some limitations in specificity. (1→3)-β-D-glucans are pan-fungal serum markers with a very good negative predictive value. Real-time PCR lacks standardization, and fungal culture from respiratory specimens is sometimes not sensitive enough. Histology allows proving the diagnosis of aspergillosis, but biopsy is not always possible in immunodepressed patients. We present the various arguments for the diagnosis of invasive aspergillosis, with a particular emphasis on recent exploration techniques.

An estimation of burden of serious fungal infections in France

OBJECTIVE OF THE STUDY An estimation of burden of serious fungal diseases in France is essential data to inform public health priorities on the importance of resources and research needed on these infections. In France, precise data are available for invasive fungal diseases but estimates for several other diseases such as chronic and immunoallergic diseases are by contrast less known. MATERIALS AND METHODS A systematic literature search was conducted using the Web of Science Platform. Published epidemiology papers reporting fungal infection rates from France were identified. Where no data existed, we used specific populations at risk and fungal infection frequencies in those populations to estimate national incidence or prevalence, depending on the condition. RESULTS The model predicts high prevalences of severe asthma with fungal sensitization episodes (189 cases/100,000 adults per year), of allergic bronchopulmonary aspergillosis (145/100,000) and of chronic pulmonary aspergillosis (5.24/100,000). Besides, estimated incidence for invasive aspergillosis is 1.8/100,000 annually based on classical high risk factors. Estimates for invasive mucormycosis, pneumocystosis and cryptococcosis are 0.12/100,000, 1/100,000 and 0.2/100,000, respectively. Regarding invasive candidiasis, more than 10,000 cases per year are estimated, and a much higher number of recurrent vaginal candidiasis is probable but must be confirmed. Finally, this survey was an opportunity to report a first picture of the frequency of tinea capitis in France. CONCLUSION Using local and literature data of the incidence or prevalence of fungal infections, approximately 1,000,000 (1.47%) people in France are estimated to suffer from serious fungal infections each year.

Two cases of opportunistic parasite infections in patients receiving alemtuzumab

Two cases are reported of rare digestive opportunistic parasites in patients being treated with alemtuzumab for lymphoid haematological malignancies. In both patients, classical biological examinations were insufficient to reach the diagnosis. Only specific parasitological techniques enabled diagnoses of cryptosporidiosis and microsporidiosis, respectively. In both cases, cellular immune reconstitution was sufficient to eradicate these opportunistic infections. In this context, parasitological diagnosis is often underestimated by medical practitioners, so immunologists and oncohaematologists need to be aware of this kind of opportunistic pathogen.

Fatal algaemia in patient with chronic lymphocytic leukemia.

To the Editor: Prototheca species are achlorophyllic lower algae, ubiquitous in nature, which can cause human infections, particularly in immunocompromised patients (1). Human protothecosis is mostly caused by P. wickerhamii and P. zopfii. Although such infections are infrequent, they can manifest themselves clinically as cutaneous lesions, olecranon bursitis, and, even more rarely, as disseminated or systemic infections (1). These infections occur in severely immunocompromised patients, such as persons with AIDS, or patients undergoing extensive treatment, such as cancer treatment or organ transplantation (1–4). We describe a fatal case of P. wickerhamii algaemia in a patient with chronic lymphocytic leukemia. In July 2007, a 79-year-old man, who had been monitored since 1993 for stage C chronic lymphocytic leukemia (5), was hospitalized July 13–20 for a depressive syndrome with fever, asthenia, and weight loss (3 kg over 2 months). Blood and urinary cultures on admission were sterile. The patient was hospitalized again on July 30 for fever (39°C), anorexia, and diarrhea, with ≈7 stools per day. He had lost 10 kg in 2 weeks. Blood cultures for bacteria (in BD Bactec Plus Aerobic/F and BD Bactec Lytic Anaerobic/F vials; Becton Dickinson, Le Pont de Claix, France) and fungi (BD Bactec Mycosis IC/F; Becton Dickinson) and stool cultures for bacteria were negative. Blood cultures were incubated in a Bactec 9240 instrument (Becton Dickinson). Aspergillus fumigatus was found in a bronchoalveolar lavage specimen, but no Aspergillus galactomannan antigen was detected in blood. The patient was treated with piperacillin-tazobactam, ciprofloxacin, acyclovir, voriconazole, and loperamide. Voriconazole (400 mg/day) was used from day 17 to day 27. On day 21, Cryptosporidium parvum was detected on parasitologic stool examination. Symptoms persisted on day 26, with strong asthenia and deterioration of general state. At that time, the leukocyte count was 178 × 109/L with 3.56 × 109/L polymorphonuclear neutrophils and 172 × 109/L lymphocytes. Three peripheral blood samples were cultured for detection of bacteria and fungi. On day 27, septic shock developed in the patient. A blood culture showed an Escherichia coli strain susceptible to piperacillin-tazobactam, aminoglycosides, and quinolones. Amikacin was added to the treatment regimen. Nonetheless, the patient died on day 28. Two blood cultures for bacteria in aerobic vials grew the day of the patient’s death, but tests of blood cultures for fungus remained negative. After Gram staining, gram-positive spherical unicellular organisms were observed (Figure). After 48 hours of incubation, creamy, yeast-like colonies grew on chocolate agar (bioMerieux, Marcy l’Etoile, France), but not on Sabouraud agar containing gentamicin and chloramphenicol (Becton Dickinson). Microscopy and the API 20C AUX system (bioMerieux) identified P. wickerhamii. Figure Gram-positive spherical unicellular organisms in a blood culture from a 79-year-old man with chronic lymphocytic leukemia. Magnification ×1,000. Sequencing the 18S rDNA with the primers Pw18SF 5′-TCAAAAAGTCCCGGCTAATCTCGTGC-3′ and Pw18SR 5′-CGCTTTCGTGCCTCAATGTCAGTGTT-3′ confirmed the identification. The sequence of the amplified product was compared with sequences published in the database of the National Center for Biotechnology Information (Bethesda, MD, USA). The most likely identification, according to BLAST analysis (www.ncbi.nlm.nih.gov/blast/Blast.cgi), was P. wickerhamii. In vitro susceptibility tests were performed by the Etest method (AB Biodisk, Solna, Sweden), on RPMI agar. P. wickerhamii was found to be susceptible to amphotericin B and posaconazole, with MICs of 0.047 μg/mL and 0.012 μg/mL, respectively. By contrast, it was resistant to fluconazole (MIC>256 μg/mL), voriconazole (MIC>32 μg/mL), and caspofungin (MIC>32 μg/mL). It was also susceptible to gentamicin (MIC = 0.25 μg/mL) but resistant to amikacin (MIC>24 μg/mL). However, the patient died before the algae were detected in the blood culture vials. In this case, antifungal treatment based on voriconazole use was empiric and ineffective. Some authors have described a successful treatment on localized protothecosis with voriconazole (6). Amphotericin B currently seems to be most effective agent, although the best treatment remains a matter of debate (1,4,7). Although in vitro susceptibility test results are not necessarily well correlated with results obtained in vivo, the low MIC of posaconazole reported here may be of interest in clinical practice (8). In the laboratory, use of selective yeast media, such as Sabouraud plus gentamicin, or Mycosis IC/F vials for blood culture, which contain chloramphenicol and tobramycin, may make it difficult to detect Prototheca spp., which are susceptible to these antimicrobial drugs.- In patients with algaemia, Prototheca spp. are often associated with bacteria, viruses, or yeasts which cause co-infections (1), as in this case, in which the alga was associated with E. coli. This association is probably the result of disseminated protothecosis in severely immunocompromised patients, and the alga may cross digestive or cutaneous barriers. Reasons for septic shock or death are unclear for most associations of pathogens (2,4). Prototheca spp. are found in various reservoirs, including the environment, animals, and food (1). In the case described here, the infection may have originated from a contaminated well used to obtain water for the patient’s kitchen garden. However, we were unable to test this hypothesis. Disseminated protothecosis is currently rare but, due to the algae’s ubiquitous nature, increasing use of immunosuppressive therapy, and increasing incidence of hematologic malignancy, Prototheca spp. may emerge as opportunistic pathogens. Prototheca spp. should also be considered as an emerging cause of systemic infection in immunocompromised patients.

Successful treatment with fumagillin of the first pediatric case of digestive microsporidiosis in a liver-kidney transplant

We report the first successful use, to our knowledge, of fumagillin alone in a pediatric patient to cure intestinal microsporidiosis in a liver‐kidney transplanted child. Detection of Enterocytozoon bieneusi in stool became negative from the first post‐therapeutic control, while digestive symptoms disappeared in 4 days. During a 9‐month follow‐up, polymerase chain reaction and direct examinations remained negative for microsporidia in her feces. No major undesirable effects were noted during the anti‐microsporidial therapy.

A Nebulized Intra-tracheal Rat Model of Invasive Pulmonary Aspergillosis

Animal models are particularly useful for the study of many infectious diseases, including those caused by fungi. Invasive pulmonary aspergillosis is most frequently studied in mouse models. We present here an animal model of this disease based on undernourished immunocompromised rats infected with Aspergillus fumigatus spores by intra-tracheal nebulisation.

Subcutaneous phaeohyphomycosis due to Phialemoniopsis ocularis successfully treated by voriconazole.

We report a case of subcutaneous infection in a 67 year-old Cambodian man who presented with a 5-month history of swelling of the right foot. Histopathology was compatible with phaeohyphomycosis and the hyphomycete Phialemoniopsis ocularis was identified by the means of morphological and molecular techniques. The patient responded well to a 6-month oral treatment with voriconazole alone.

Proteomic demonstration of the recurrent presence of inter-alpha-inhibitor H4 heavy-chain during aspergillosis induced in an animal model

Invasive pulmonary aspergillosis remains a matter of great concern in oncology/haematology, intensive care units and organ transplantation departments. Despite the availability of various diagnostic tools with attractive features, new markers of infection are required for better medical care. We therefore looked for potential pulmonary biomarkers of aspergillosis, by carrying out two-dimensional (2D) gel electrophoresis comparing the proteomes of bronchial-alveolar lavage fluids (BALF) from infected rats and from control rats presenting non-specific inflammation, both immunocompromised. A bioinformatic analysis of the 2D-maps revealed significant differences in the abundance of 20 protein spots (ANOVA P-value<0.01; q-value<0.03; power>0.8). One of these proteins, identified by mass spectrometry, was considered of potential interest: inter-alpha-inhibitor H4 heavy-chain (ITIH4), characterised for the first time in this infectious context. Western blotting confirmed its overabundance in all infected BALF, particularly at early stages of murine aspergillosis. Further investigations were carried on rat serum, and confirmed that ITIH4 levels increased during experimental aspergillosis. Preliminary results in human samples strengthened this trend. To our knowledge, this is the first description of the involvement of ITIH4 in aspergillosis.