Use of calcofluor-blue brightener for the diagnosis of Pneumocystis jirovecii pneumonia in bronchial-alveolar lavage fluids: A single-center prospective study.

The biological diagnosis of Pneumocystis jirovecii pneumonia (PjP) is based on the investigation of respiratory fluids by conventional staining methods and/or molecular biology. Diagnostic performance of an in-house technique based on calcofluor-blue brightener for the direct detection of P. jirovecii cysts was prospectively assessed in bronchial-alveolar lavage fluids (BALF) from patients with a suspected PjP infection over a three-year period in a single center: the diagnostic yield was compared to that of a commercial kit based on monoclonal immunofluorescence assay (IFA) on replicate smears. May-Grünwald Giemsa (MGG) staining and quantitative Polymerase Chain Reaction (qPCR) were also performed. The gold standard for each patient was the definitive diagnosis of PjP infection by an independent committee based on clinical, radiological, and biological data. Overall, 481 BALF were assessed: 42 were found to be positive for the detection of P. jirovecii by at least one laboratory technique, but only 35 were actually judged to be in agreement with the definitive diagnosis of PjP infection. The sensitivity of the calcofluor-blue brightener technique was 74.3% vs. 60.0%, 34.6%, and 82.9% for IFA, MGG, and qPCR, respectively; and its specificity was 99.6% vs. 99.3%, 100.0%, and 99.4% for IFA, MGG, and qPCR. No technique was shown to be statistically superior to calcofluor-blue brightener. Further validation of the test through multicenter studies is now required, but in light of its low cost and easy preparation, the use of calcofluor-blue brightener in BALF appears to be a valuable alternative method for the routine first-line diagnosis of PjP infection.

Epidemiological Outbreaks of Pneumocystis jirovecii Pneumonia Are Not Limited to Kidney Transplant Recipients: Genotyping Confirms Common Source of Transmission in a Liver Transplantation Unit.

Over a 5-month period, four liver transplant patients at a single hospital were diagnosed with Pneumocystis jirovecii pneumonia (PCP). This unusually high incidence was investigated using molecular genotyping. Bronchoalveolar lavage fluids (BALF) obtained from the four liver recipients diagnosed with PCP were processed for multilocus sequence typing (MLST) at three loci (SOD, mt26s, and CYB). Twenty-four other BALF samples, which were positive for P. jirovecii and collected from 24 epidemiologically unrelated patients with clinical signs of PCP, were studied in parallel by use of the same method. Pneumocystis jirovecii isolates from the four liver recipients all had the same genotype, which was different from those of the isolates from all the epidemiologically unrelated individuals studied. These findings supported the hypothesis of a common source of contamination or even cross-transmission of a single P. jirovecii clone between the four liver recipients. Hospitalization mapping showed several possible encounters between these four patients, including outpatient consultations on one particular date when they all possibly met. This study demonstrates the value of molecular genotyping of P. jirovecii isolated from clinical samples for epidemiological investigation of PCP outbreaks. It is also the first description of a common source of exposure to a single P. jirovecii clone between liver transplant recipients and highlights the importance of prophylaxis in such a population.

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.

Testing an innovative device against airborne Aspergillus contamination.

Aspergillus fumigatus is a major airborne nosocomial pathogen that is responsible for severe mycosis in immunocompromised patients. We studied the efficacy of an innovative mobile air-treatment device in eliminating A. fumigatus from the air following experimental massive contamination in a high-security room. Viable mycological particles were isolated from sequential air samples in order to evaluate the device's effectiveness in removing the fungus. The concentration of airborne conidia was reduced by 95% in 18 min. Contamination was reduced below the detection threshold in 29 min, even when the machine was at the lowest airflow setting. In contrast, during spontaneous settling with no air treatment, conidia remained airborne for more than 1 h. This indoor air contamination model provided consistent and reproducible results. Because the air purifier proved to be effective at eliminating a major contaminant, it may prove useful in preventing air-transmitted disease agents. In an experimental space mimicking a hospital room, the AirLyse air purifier, which uses a combination of germicidal ultraviolet C irradiation and titanium photocatalysis, effectively eliminated Aspergillus conidia. Such a mobile device may be useful in routine practice for lowering microbiological air contamination in the rooms of patients at risk.

[Toxoplasmic cyst and heart transplant: a case report of serological reactivation in an acute graft rejection context]. / Kyste toxoplasmique et transplant cardiaque : cas d'une réactivation sérologique dans un contexte de rejet de greffe.

We describe the case of a serological reactivation in a Toxoplasma-seropositive subject, following a cardiac transplantation transmitting cysts contained in the myocardial tissue. In a context of acute graft rejection, primary chemoprophylaxis enables to avoid onset of opportunistic toxoplasmosis, emerging with immunodepletion performed by high-dose steroids. Then, we draw up a brief review of the bibliographical literature about pathophysiological mechanisms of toxoplasmic reactivation in heart transplants.

Effects of the antimalarial drugs ferroquine and artesunate on Plasmodium yoelii yoelii gametocytegenesis and vectorial transmission.

Chemistry still has a role in the management of malaria, alongside the mosquito netting soaked in insecticide that is used increasingly, as we continue to await the long anticipated vaccine. During its cycle, the hematozoon parasite develops through three major periods. The first, malarial infection, corresponds to the intrahepatic development of infective forms from the mosquito vector; this period is not sensitive to treatment and is often asymptomatic. The period of erythrocytic schizogony is the most urgent, and treatment activity is primordial. Finally, the phase of sexual reproduction, when gametocytes develop within the erythrocytes ensures the perpetuation of the species when these reach the blood-feeding female anopheles mosquitoes. The aim of our work was to study the effect on gametocytes of drugs known to be effective on the asexual blood forms of the protozoan and thus the potential repercussions on malaria transmission. This experimental study was conducted with an animal model whose parasite cycle and modes of transmission are close to those of human malaria: Plasmodium yoelii, maintained on Swiss mice, with the Anopheles stephensi vector (maintained in an animal facility at the National Museum of Natural History in Paris). Two drugs were tested: ferroquine (a chloroquine derivative with a ferrocene molecule at the lateral carbon chain that restores its efficacy against chloroquine-resistant strains) and artesunate (a derivative of artemisinin, from ginghao, a Chinese plant also known as artemisia annua, or sweet wormwood), a treatment of choice in the combined therapies recommended by WHO. The efficacy of these drugs, prescribed at doses subcurative for the asexual forms, were tested against gametocyte production, quantitatively by counting them in the blood and qualitatively by counting the quantity of oocysts developed on the mosquito's midgut, which are indicators of gametocyte activity. The mice that were parasite-infected and then treated served as their own controls: lots of 30 mosquitoes fed on each mouse before treatment and then 90  minutes and 5  hours after treatment. Quantitatively, the comparison of the blood parasite level and the gametocyte index shows that treated mice had a higher level of circulating gametocytes than untreated parasite infested mice, regardless of drug or dose (5 or 10  mg/kg). For artesunate at 5  mg/kg, we noted that the blood gametocyte level was almost double that of the controls. On the other hand, qualitatively, the first results obtained with optical and electronic microscopy showed morphologic alterations of the circulating gametocytes (pigment clumping and lateralisation within red blood cells) and reduced infectivity of the gametocytes for the mosquitoes that fed at 1 and 5  hours after treatment. We were able to demonstrate statistically that the infectivity of gametocytes, measured by the quantity of oocysts counted in the mosquito midgut, was reduced by 70% for those treated with ferroquine and by 85% for those from mice treated by artesunate. Complementary studies will seek to specify the populations (age) of gametocytes damaged by treatment and the importance and nature of their morphologic alterations.

Targeting Aspergillus fumigatus Crf Transglycosylases With Neutralizing Antibody Is Relevant but Not Sufficient to Erase Fungal Burden in a Neutropenic Rat Model.

Aspergillus fumigatus is an airborne opportunistic fungal pathogen responsible for severe infections. Among them, invasive pulmonary aspergillosis has become a major concern as mortality rates exceed 50% in immunocompromised hosts. In parallel, allergic bronchopulmonary aspergillosis frequently encountered in cystic fibrosis patients, is also a comorbidity factor. Current treatments suffer from high toxicity which prevents their use in weakened subjects, resulting in impaired prognostic. Because of their low toxicity and high specificity, anti-infectious therapeutic antibodies could be a new alternative to conventional therapeutics. In this study, we investigated the potential of Chitin Ring Formation cell wall transglycosylases of A. fumigatus to be therapeutic targets for therapeutic antibodies. We demonstrated that the Crf target was highly conserved, regardless of the pathophysiological context; whereas the CRF1 gene was found to be 100% conserved in 92% of the isolates studied, Crf proteins were expressed in 98% of the strains. In addition, we highlighted the role of Crf proteins in fungal growth, using a deletion mutant for CRF1 gene, for which a growth decrease of 23.6% was observed after 48 h. It was demonstrated that anti-Crf antibodies neutralized the enzymatic activity of recombinant Crf protein, and delayed fungal growth by 12.3% in vitro when added to spores. In a neutropenic rat model of invasive pulmonary aspergillosis, anti-Crf antibodies elicited a significant recruitment of neutrophils, macrophages and T CD4 lymphocytes but it was not correlated with a decrease of fungal burden in lungs and improvement in survival. Overall, our study highlighted the potential relevance of targeting Crf cell wall protein (CWP) with therapeutic antibodies.

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 microm, 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.

Pulmonary toxoplasmosis in immunocompromised patients with interstitial pneumonia: a single-centre prospective study assessing PCR-based diagnosis.

AIMS: Pulmonary toxoplasmosis has become a very rare parasitic infection since the advent of highly active antiretroviral therapies. It is generally diagnosed by the direct microscopic observation of Toxoplasma gondii tachyzoites in bronchoalveolar lavage fluid (BALF). The aim of this study was to assess possible improvements in diagnostic performance associated with the use of real-time PCR. METHODS: This prospective study was carried out on BALFs obtained from immunocompromised patients over a 2-year period. We systematically compared the results of conventional staining with those of molecular detection. RESULTS: Two cases of pulmonary toxoplasmosis were diagnosed for a total of 336 samples. PCR did not detect any additional cases and was more time-consuming than conventional staining. CONCLUSIONS: Conventional staining is a reliable technique and is probably the most appropriate method for experienced microbiology laboratories, whereas T. gondii-specific PCR may be useful for laboratories with less experience in parasitology. TRIAL REGISTRATION NUMBER: 2015_030, May 27th 2015.

Is procalcitonin increased in cases of invasive amoebiasis? A retrospective, observational study.

Procalcitonin (PCT) levels are commonly used for diagnostic guidance in routine bacterial infections. By contrast, little data are currently available regarding PCT in parasitic diseases, and its role in cases of invasive amoebiasis has not yet been described. For this purpose, 35 adult patients with a proven diagnosis of invasive or digestive amoebiasis were included in a 4-year study period. Serum PCT was retrospectively assessed. Results were analysed with regard to the usual inflammatory biomarkers, like C-reactive protein (CRP). PCT was significantly higher in patients with proven invasive amoebiasis than in digestive amoebiasis (mean value: 4.03 µg/L versus 0.07 µg/L, respectively; P < 0.001), but the SD was greater than with CRP, and the effect was less than that demonstrated in bacterial infections. By contrast, PCT was not shown to be elevated during digestive amoebiasis.

Ivermectin and filariasis.

Ivermectin, a parasiticide that long ago proved its worth in veterinary medicine, became one of the most effective tools for control programs against human filarial diseases in the 1980s. It is provided at no cost, is effective against microfilariae (blocking their transmission) and can be administered annually as a single oral dose with virtually no side-effects: these considerations led the WHO to officially declare eradicable two endemic filarial diseases (among the major endemic diseases worldwide), onchocerciasis and lymphatic filariasis.

Gastrointestinal symptoms resembling ulcerative proctitis caused by larvae of the drone fly Eristalis tenax.

We report a case of facultative intestinal myiasis due to larvae of the drone fly Eristalis tenax, also named the rat-tailed maggots. The development of larvae in the lower bowel was responsible for non-specific gastrointestinal symptoms that resembled ulcerative proctitis. The diagnosis was established upon the observation of four spontaneously excreted mobile larvae. The definite identification of the E. tenax species was made possible by scanning electron microscopy. The clinical outcome was satisfactory.

Unusual multiple large abscesses of the liver: interest of the radiological features and the real-time PCR to distinguish between bacterial and amebic etiologies.

We report a rare case of amebiasis generating 19 large liver abscesses. Such a quantity of abscesses is rare, especially when occurring in a young casual traveler without any immunodeficiency disorders. A possible co-infection was excluded. By contrast, the amebic etiology was confirmed by means of serology and real-time PCR.