Relationship between Fungal Colonisation of the Respiratory Tract in Lung Transplant Recipients and Fungal Contamination of the Hospital Environment.

BACKGROUND: Aspergillus colonisation is frequently reported after lung transplantation. The question of whether aspergillus colonisation is related to the hospital environment is crucial to prevention. METHOD: To elucidate this question, a prospective study of aspergillus colonisation after lung transplantation, along with a mycological survey of the patient environment, was performed. RESULTS: Forty-four consecutive patients were included from the day of lung transplantation and then examined weekly for aspergillus colonisation until hospital discharge. Environmental fungal contamination of each patient was followed weekly via air and surface sampling. Twelve patients (27%) had transient aspergillus colonisation, occurring 1-13 weeks after lung transplantation, without associated manifestation of aspergillosis. Responsible Aspergillus species were A. fumigatus (6), A. niger (3), A. sydowii (1), A. calidoustus (1) and Aspergillus sp. (1). In the environment, contamination by Penicillium and Aspergillus was predominant. Multivariate analysis showed a significant association between occurrence of aspergillus colonisation and fungal contamination of the patient's room, either by Aspergillus spp. in the air or by A.fumigatus on the floor. Related clinical and environmental isolates were genotyped in 9 cases of aspergillus colonisation. For A. fumigatus (4 cases), two identical microsatellite profiles were found between clinical and environmental isolates collected on distant dates or locations. For other Aspergillus species, isolates were different in 2 cases; in 3 cases of aspergillus colonisation by A. sydowii, A. niger and A. calidoustus, similarity between clinical and environmental internal transcribed spacer and tubulin sequences was >99%. CONCLUSION: Taken together, these results support the hypothesis of environmental risk of hospital acquisition of aspergillus colonisation in lung transplant recipients.

Use and limits of (1-3)-ß-d-glucan assay (Fungitell), compared to galactomannan determination (Platelia Aspergillus), for diagnosis of invasive aspergillosis.

This study was undertaken to examine the performance of the Fungitell ß-glucan (BG) assay, to compare it with that of the galactomannan (GM) test for the diagnosis of invasive aspergillosis (IA) in patients with hematological malignancies, and to examine the rates of false-positive BG and GM test results due to ß-lactam antibiotics among sera of patients with Gram-positive or Gram-negative bacteremia and selected sera with false-positive results from the GM test. Serum samples from 105 patients with proven (n = 14) or probable (n = 91) IA, 97 hematology patients at risk for invasive fungal infections, 50 healthy blood donors, and 60 patients with bacteremia were used to study the sensitivities and specificities of the assays. The GM test was more specific than the BG assay (97% versus 82%, respectively; P = 0.0001) and the BG assay was more sensitive than the GM test (81% versus 49%, respectively; P < 0.0001) for IA diagnosis. The study of 49 separate batches of ß-lactam antibiotics showed high and very similar rates of false-positive results for the GM and BG assays (29 and 33%, respectively; P = 0.82) but with an almost complete lack of concordance between the 2 assays. For patients with bacteremia, the rate of false-positive results was much higher with the BG test than with the GM test (37% versus 2%, respectively; P < 0.0001), with no significant difference between Gram-positive and Gram-negative bacteremia. In conclusion, the BG test may be useful for the diagnosis of IA because of its high sensitivity in comparison with the GM test, but the overall benefit of this assay remains limited because of its inadequate specificity and its cost.

Performance of the BioPlex 2200 flow immunoassay in critical cases of serodiagnosis of toxoplasmosis.

The BioPlex 2200 automated analyzer (Bio-Rad Laboratories, Hercules, CA) is a recently developed multiplex analyzer that enables the detection of anti-Toxoplasma, -rubella, and -cytomegalovirus antibodies in the same assay. The aim of this study was to compare this new technology (using the BioPlex 2200 ToRC IgG/IgM kit) in critical cases of serodiagnosis of toxoplasmosis (acute, chronic, or congenital infections and cases with discrepant results) to the technologies used in our routine practice, i.e., the Platelia IgG/IgM enzyme-linked immunosorbent assays (ELISAs) (Bio-Rad Laboratories) and the Toxo-Screen direct agglutination assay (bioMérieux, Lyon, France). Overall, most cases of false-positive/negative results obtained with the Platelia IgG or Toxo-Screen assay were corrected by the BioPlex 2200 ToRC IgG (87.5%). Furthermore, the analysis of 35 sequences of sera showed a trend toward a more rapid decrease of IgM titers by BioPlex 2200 than by Platelia. These results for IgM detection can be explained by a weaker detection of residual IgM. Indeed, among 23 serum samples from patients with probable past infection with long-lasting IgM (Platelia M positive and IgG avidity index, ≥0.5), the BioPlex 2200 Toxoplasma IgM assay was positive for only 11 serum samples. In our panel of critical cases comprising 156 serum and 6 cord blood samples from 103 patients with acute, chronic, or congenital infection, the BioPlex 2200 IgG assay was a sensitive (97.8%) and specific (91.3%) method for IgG detection. The high specificity (97.4%) of IgM detection combined with the shorter kinetics of IgM titers may considerably reduce the number of residual IgM detections, thus yielding more precise diagnoses of acute infections.

Evidence of airborne excretion of Pneumocystis carinii during infection in immunocompetent rats. Lung involvement and antibody response.

To better understand the role of immunocompetent hosts in the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected Sprague Dawley rats was quantified by means of a real-time PCR assay, in parallel with the kinetics of P. carinii loads in lungs and specific serum antibody titres. Pneumocystis-free Sprague Dawley rats were intratracheally inoculated at day 0 (d0) and then followed for 60 days. P. carinii DNA was detected in lungs until d29 in two separate experiments and thereafter remained undetectable. A transient air excretion of Pneumocystis DNA was observed between d14 and d22 in the first experiment and between d9 and d19 in the second experiment; it was related to the peak of infection in lungs. IgM and IgG anti-P. carinii antibody increase preceded clearance of P. carinii in the lungs and cessation of airborne excretion. In rats receiving a second challenge 3 months after the first inoculation, Pneumocystis was only detected at a low level in the lungs of 2 of 3 rats at d2 post challenge and was never detected in air samples. Anti-Pneumocystis antibody determinations showed a typical secondary IgG antibody response. This study provides the first direct evidence that immunocompetent hosts can excrete Pneumocystis following a primary acquired infection. Lung infection was apparently controlled by the immune response since fungal burdens decreased to become undetectable as specific antibodies reached high titres in serum. This immune response was apparently protective against reinfection 3 months later.

Bayesian development of a dose-response model for Aspergillus fumigatus and invasive aspergillosis.

Invasive aspergillosis (IA) is a major cause of mortality in immunocompromized hosts, most often consecutive to the inhalation of spores of Aspergillus. However, the relationship between Aspergillus concentration in the air and probability of IA is not quantitatively known. In this study, this relationship was examined in a murine model of IA. Immunosuppressed Balb/c mice were exposed for 60 minutes at day 0 to an aerosol of A. fumigatus spores (Af293 strain). At day 10, IA was assessed in mice by quantitative culture of the lungs and galactomannan dosage. Fifteen separate nebulizations with varying spore concentrations were performed. Rates of IA ranged from 0% to 100% according to spore concentrations. The dose-response relationship between probability of infection and spore exposure was approximated using the exponential model and the more flexible beta-Poisson model. Prior distributions of the parameters of the models were proposed then updated with data in a Bayesian framework. Both models yielded close median dose-responses of the posterior distributions for the main parameter of the model, but with different dispersions, either when the exposure dose was the concentration in the nebulized suspension or was the estimated quantity of spores inhaled by a mouse during the experiment. The median quantity of inhaled spores that infected 50% of mice was estimated at 1.8 × 10(4) and 3.2 × 10(4) viable spores in the exponential and beta-Poisson models, respectively. This study provides dose-response parameters for quantitative assessment of the relationship between airborne exposure to the reference A. fumigatus strain and probability of IA in immunocompromized hosts.

Efficacy of liposomal amphotericin B for prophylaxis of acute or reactivation models of invasive pulmonary aspergillosis.

The efficacy of antifungal prophylaxis for prevention of invasive aspergillosis (IA) may depend on whether IA results from recent inhalation of spores or reactivation of latent colonisation. Compare the efficacy of liposomal amphotericin B (LAmB) for prophylaxis in acute and reactivation models of IA. In the acute model, mice immunosuppressed from day 0 were challenged at day 3 with an aerosol of Aspergillus fumigatus. LAmB (15 mg kg(-1) ) was administered at day 0 or at challenge. In the reactivation model, naïve mice exposed to A. fumigatus remained untreated until clearance of spores from the lungs, then immunosuppressed to induce reactivation. A single LAmB dose was administered at start of immunosuppression. In the acute model, a single administration of LAmB at start of immunosuppression was not effective, but an additional administration resulted in a significant decrease in lung fungal burden (P < 0.05 vs. controls). A significant prophylactic efficacy was observed when LAmB was administered once at challenge (P < 0.01). In the reactivation model, a single LAmB administration at start of immunosuppression significantly reduced both reactivation rate and fungal burden vs. controls (P < 0.01). Our results show that the conditions under which IA develop and timing of administration of LAmB were determinant variables for prophylactic efficacy.

Prevalence of opportunistic intestinal parasitic infections among HIV-infected patients with low CD4 cells counts in France in the combination antiretroviral therapy era.

BACKGROUND: The use of combination antiretroviral therapy (cART) has dramatically reduced the prevalence of opportunistic infections, however data on the prevalence of intestinal parasitic infections in HIV-infected patients with low CD4 cell counts in the cART era are scarce. METHODS: We performed a prospective cohort study among HIV-infected patients with CD4 cell counts <100/mm(3) seen at a university hospital in Paris. Medical records were reviewed and stool samples were obtained for macroscopic examination and detection of parasites including cryptosporidia and microsporidia, whether or not the patient had diarrhea. Stool cultures were performed for patients with diarrhea. Factors associated with the detection of parasites were then identified. RESULTS: Stools samples from 143 consecutive patients were analyzed. Patients were mostly men (76%), and the median patient age was 41 years. The median CD4 cell count was 32/mm(3), and 59% were receiving cART. Diarrhea was present in 85 patients (59%), 19 of whom (22%) had intestinal parasites detected in stools. Three patients with diarrhea were diagnosed with Salmonella typhimurium, Campylobacter coli, and Clostridium difficile infections. Among the 58 patients without diarrhea, parasitic intestinal pathogens were still identified in six (10%). The overall prevalence of intestinal parasites was 17%, with cryptosporidia (n=8), microsporidia (n=6), and Giardia duodenalis (n=5) being the most frequent pathogens. Patients with intestinal parasites had diarrhea more often (76% vs. 56%, p=0.025) and were more often at US Centers for Disease Control and Prevention (CDC) clinical stage C (84% vs. 69%, p=0.024) than patients without parasites. CONCLUSIONS: The prevalence of intestinal parasitic infections remains significant in HIV-infected patients with low CD4 counts in the cART era. A systematic search for parasitic pathogens including microsporidia, cryptosporidia, and G. duodenalis should be performed even in the absence of diarrhea.

The strategy for the diagnosis of invasive pulmonary aspergillosis should depend on both the underlying condition and the leukocyte count of patients with hematologic malignancies.

The identification of the causative organism in invasive pulmonary aspergillosis (IPA) is recommended. We investigated whether a mycologic diagnostic strategy could be optimized based on patient characteristics. Fifty-five patients were enrolled in a prospective study. The presence of Aspergillus in respiratory samples occurred more frequently in non-acute leukemia (AL) patients than in AL patients (P = .0003), and in patients with leukocyte counts more than 100/mm(3) (P = .002). In a logistic regression model, these 2 factors appeared to be independent, with an adjusted odds ratio of 7.14 (95% confidence interval, 1.40-36.5) for non-AL patients and an adjusted odds ratio of 6.97 (95% confidence interval, 1.33-36.5) for patients with leukocyte counts more than 100/mm(3). A positive mycologic result was also more frequent among patients with lung CT scan signs of airway-invasive disease than among other patients (P = .043). Airway-invasive signs were more frequent among non-AL patients (P = .049), whereas angioinvasive disease was more frequent among both AL patients (P = .01) and patients with leukocyte counts less than 100/mm(3) (P = .001). A concomitant pulmonary infection was identified more frequently among non-AL patients (P = .005 vs allogeneic hematopoietic stem cell transplant and P = .048 vs others). Our results suggest that different strategies for diagnosing IPA should be considered based on the underlying condition.

Prospective evaluation of clinical and biological markers to predict the outcome of invasive pulmonary aspergillosis in hematological patients.

Early evaluation of treatment efficacy in invasive aspergillosis (IA), a leading cause of morbidity and mortality in hematological patients, remains a challenge. We conducted a prospective study to evaluate the performance of different markers in predicting the outcome of patients with IA. Both clinical and biological criteria were assessed 7, 14, 21, and 45 days after inclusion in the study, and mortality was assessed at day 60. The association between baseline data and their evolution and the day 45 response to treatment was analyzed. A total of 57 patients (4 with proven, 44 with probable, and 9 with possible aspergillosis according to the revised EORTC/MSG [European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group] definitions) were included. At day 45, 30 patients (53%) were determined to be responders, 25 (44%) were nonresponders, and 2 were not able to be evaluated. Twenty patients died within the 60 days of follow-up. We found that a poor day 45 outcome was associated with patients who had high baseline serum galactomannan (GM) antigen levels and those receiving steroids at the time of IA. A consistently negative serum GM index was associated with a good outcome, and the day 14 clinical evaluation was predictive of the day 45 outcome. No association was found between Aspergillus antibodies or DNA detection and patients' outcome. We conclude that the GM index value at diagnosis of IA, GM index kinetics, and clinical evaluation at day 14 are good markers for predicting the outcome of patients with IA and should be taken into account for adapting antifungal treatment.

Sequential air-liquid exposure of human respiratory cells to chemical and biological pollutants.

Although indoor air has wide ranging effects on human health, the effects of environmental, chemical, and biological pollutants on the respiratory system are not fully understood. In order to clarify the health effects of airborne pollutant exposure, it would appear that toxicological evidence is needed to complement epidemiological observations to support by providing biological plausibility. The aim of this study is to manage air-liquid successive exposures to different pollutants such as a chemical pollutant (formaldehyde--FA), and a biological contaminant (Aspergillus fumigatus--Asp) using our in vitro model. Human alveolar cells (A549) were exposed at the air-liquid interface in an exposure module, firstly to an environmental level of FA (50 µg/m³) (or air) for 30 min, and 14 h later to Asp (7×108 spores/m³) (or air) for 30 min. After 10 h post-incubation, cellular viability was assessed. Inflammation biomarkers (IL-8, MCP-1) were assayed by ELISA and by RT-PCR. Whatever the conditions, no cytotoxic effect was observed. FA followed by air exposure did not induce modification of production and expression of cytokines, confirming results with a unique FA exposure. Air followed by Asp exposure tended to induce IL-8 expression whereas IL-8 production tended to increase after FA and Asp exposure compared to FA and air exposure. The reaction of cells to sequential exposure to FA and Asp was moderate. These results show the feasibility of our model for sequential exposures to different types of environmental pollutants, allowing using it for preliminary assessment of cellular activity modification induced by airborne contaminants.

Dynamics of Pneumocystis carinii air shedding during experimental pneumocystosis.

To better understand the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected rats was quantified by means of a real-time polymerase chain reaction assay, in parallel with the kinetics of P. carinii loads in their lungs. P. carinii DNA was detected in the air 1 week after infection and increased until 4-5 weeks after infection before stabilizing. A significant correlation was shown between lung burdens and the corresponding airborne levels, suggesting the possibility of estimating the fungal lung involvement through quantification of Pneumocystis in the exhaled air.

Accumulation and transport of microbial-size particles in a pressure protected model burn unit: CFD simulations and experimental evidence.

BACKGROUND: Controlling airborne contamination is of major importance in burn units because of the high susceptibility of burned patients to infections and the unique environmental conditions that can accentuate the infection risk. In particular the required elevated temperatures in the patient room can create thermal convection flows which can transport airborne contaminates throughout the unit. In order to estimate this risk and optimize the design of an intensive care room intended to host severely burned patients, we have relied on a computational fluid dynamic methodology (CFD). METHODS: The study was carried out in 4 steps: i) patient room design, ii) CFD simulations of patient room design to model air flows throughout the patient room, adjacent anterooms and the corridor, iii) construction of a prototype room and subsequent experimental studies to characterize its performance iv) qualitative comparison of the tendencies between CFD prediction and experimental results. The Electricité De France (EDF) open-source software Code_Saturne® (http://www.code-saturne.org) was used and CFD simulations were conducted with an hexahedral mesh containing about 300 000 computational cells. The computational domain included the treatment room and two anterooms including equipment, staff and patient. Experiments with inert aerosol particles followed by time-resolved particle counting were conducted in the prototype room for comparison with the CFD observations. RESULTS: We found that thermal convection can create contaminated zones near the ceiling of the room, which can subsequently lead to contaminate transfer in adjacent rooms. Experimental confirmation of these phenomena agreed well with CFD predictions and showed that particles greater than one micron (i.e. bacterial or fungal spore sizes) can be influenced by these thermally induced flows. When the temperature difference between rooms was 7°C, a significant contamination transfer was observed to enter into the positive pressure room when the access door was opened, while 2°C had little effect. Based on these findings the constructed burn unit was outfitted with supplemental air exhaust ducts over the doors to compensate for the thermal convective flows. CONCLUSIONS: CFD simulations proved to be a particularly useful tool for the design and optimization of a burn unit treatment room. Our results, which have been confirmed qualitatively by experimental investigation, stressed that airborne transfer of microbial size particles via thermal convection flows are able to bypass the protective overpressure in the patient room, which can represent a potential risk of cross contamination between rooms in protected environments.

Quantification and spread of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis pneumonia.

BACKGROUND: Airborne transmission of Pneumocystis has been demonstrated in animal models and is highly probable in humans. However, information concerning burdens of Pneumocystis jirovecii (human-derived Pneumocystis) in exhaled air from infected patients is lacking. Our objective is to evaluate P. jirovecii air diffusion in patients with Pneumocystis pneumonia. METHODS: Patients admitted with Pneumocystis pneumonia were prospectively enrolled from 9 January 2008 to 21 July 2009. Air samples (1.5 m(3)) were collected on liquid medium with a commercial sampler at 1-, 3-, 5-, and 8-m distances from patients' heads. Air control samples were collected away from Pneumocystis pneumonia patient wards and outdoors. Samples were examined for P. jirovecii detection and quantification using a real-time polymerase chain reaction assay targeting the mitochondrial large subunit ribosomal RNA gene. RESULTS: Forty patients were diagnosed as having Pneumocystis pneumonia. Air sampling was performed in the environment for 19 of them. At a 1-m distance from patients' heads, P. jirovecii DNA was detected in 15 (79.8%) of 19 patients, with fungal burdens ranging from 7.5 X 10³ to 4.5 X 106 gene copies/m(3). These levels decreased with distance from the patients (P < .002). Nevertheless, 4 (33.3%) of the 12 samples taken at 8 m, in the corridor adjacent to their room, were still positive. Forty control samples were collected and remained negative. CONCLUSION: This study provides the first quantitative data on the spread of P. jirovecii in exhaled air from infected patients. It sustains the risk of P. jirovecii direct transmission in close contact with patients with Pneumocystis pneumonia and leads the way for initiating a quantitative risk assessment for airborne transmission of P. jirovecii.

Disseminated infection with a new genovar of Encephalitozoon cuniculi in a renal transplant recipient.

Disseminated microsporidiosis is a life-threatening opportunistic infection. Here, we report about a previously undescribed genovar of Encephalitozoon cuniculi causing disseminated infection in a non-HIV-infected renal transplant recipient. Disseminated microsporidiosis must be considered in the differential diagnosis of chronic fever in renal allograft recipients, even those without urinary symptoms.

Identification of cryptosporidium species infecting humans in Tunisia.

Prevalence and species distribution of Cryptosporidium spp. were determined among 633 immunocompetent children less than five years of age and 75 patients hospitalized for immunodeficiency who lived in northern Tunisia. Microscopy was used for initial screening to detect positive samples and a nested polymerase chain reaction and restriction fragment length polymorphism analysis was used to determine the species. Cryptosporidium spp. was identified in 2.7% of cases (19 stool samples), and there was a significant difference between samples collected from immunocompromised patients and those collected from healthy children (10.7% versus 1.7%). Prevalence was also significantly higher in diarrheal specimens than in formed specimens (6.3% versus 1.6%). Cryptosporidium hominis and C. parvum were responsible for most Cryptosporidium spp. infections (78.9%). Cryptosporidium hominis was more prevalent in children from urban areas than in those from rural areas, and C. parvum was found with similar prevalence rates in the two populations. Cryptosporidium meleagridis was identified in four children on farms.

Restoration of Toxoplasma gondii-specific immune responses in patients with AIDS starting HAART.

OBJECTIVE: To study the kinetics and identify factors associated with Toxoplasma-specific immune responses in patients with AIDS starting antiretroviral therapy. METHODS: A prospective study of 38 HIV-infected patients seropositive for Toxoplasma who started antiretroviral therapy with CD4 T cells less than 200 cells/microl. T-cell and B-cell phenotypes, anti-Toxoplasma antibodies titers, Th-1 and Th-2 cytokine production and lymphocyte proliferative responses (LPRs) to Toxoplasma were assessed over 12 months. RESULTS: Median CD4 cell count increased from 122 cells/microl at baseline to 260 cells/microl at 12 months, and the incidence of a positive LPR from 18.4 to 70.5%. A Toxoplasma IgG titer more than 150 IU/ml was the only baseline variable associated with a positive LPR (hazard ratio: 4.6, P = 0.003). Among time-dependent covariates, the number of effector memory (CD45RA-CCR7-) CD4 T cells was associated with a positive LPR (P < 0.02) and the number of terminally differentiated (CD45RA+CCR7-) CD8 T cells was associated with in-vitro production of gamma-IFN (P < 0.008). CONCLUSION: Among patients with low CD4 cell counts, high anti-Toxoplasma IgG titers were associated with LPR to Toxoplasma antigen. After starting antiretroviral therapy, the number of effector memory CD4 T cells and terminally differentiated CD8 T cells were associated with the restoration of Toxoplasma LPR and gamma-IFN production, respectively.

Treatment of parasitic diarrhea in HIV-infected patients.

Parasitic infections responsible for diarrhea have a worldwide distribution, overlapping with AIDS in most countries. Indeed, highly active antiretroviral therapy has markedly reduced the incidence of most parasitic opportunistic infections, but parasite-related diarrhea remains frequent and probably underestimated in developing countries. In this review, we focus on the advances in molecular epidemiology, diagnosis and current treatment of the most prevalent parasitic infections in HIV-infected patients. Most of these parasites are protozoa, whose diagnosis at the laboratory requires some adapted technique and expertise. We highlight the importance of diagnosis and the skill of the laboratory of parasitology, since most parasitic infections responsible for diarrhea in AIDS patients can be treated.