Weight loss and abnormal lung inflammation in mice chronically exposed to secondary organic aerosols.

Secondary organic aerosols (SOAs) have emerged recently as a major component of fine particulate matter. Cell culture studies revealed a role for SOAs in cell oxidative stress, toxicity and inflammation and only a few studies investigated short-term SOA exposure in animal models. Here, mice were chronically exposed to naphthalene-derived SOAs for one and two months. Weight monitoring indicated a marked mass loss, especially in females, following chronic exposure to SOAs. Significantly, a cytokine antibody microarray approach revealed SOA-induced abnormal lung inflammation similar to that seen in cigarette smoke-induced chronic obstructive pulmonary disease (COPD). This in vivo study testifies to the pathogenic role of sub-chronic SOA exposure on human health.

A 10-year retrospective analysis of Toxoplasma gondii qPCR screening in allogeneic hematopoietic stem cell transplantation recipients.

Weekly blood Toxoplasma gondii DNA screening using real-time quantitative polymerase chain reaction (qPCR) has been implemented in all allogeneic hematopoietic stem cell transplantation (alloHSCT) recipients at our hospital. We retrospectively analyzed the consequences of a positive blood qPCR in the management of Toxoplasma infection (TI) and disease (TD).From 2011 to 2020, 52 (4.13%) of 1 257 alloHSCT recipients had at least one positive qPCR, 45 (3.5%) with TI and seven (0.56%) with TD (central nervous system involvement). Forty-four patients were qPCR-positive before day 100, 30 without and 14 with anti-Toxoplasma prophylaxis. Twenty-five of them (56.8%) started or continued prophylactic dosage treatment: all became qPCR-negative, including 20 (80%) receiving only prophylactic dosage treatment. Twenty-four of them (54.5%) received non-prophylactic dosage treatment: qPCR became negative in 22/24 (91.7%), while TI contributed to death in two cases. Six of the eight patients diagnosed after D100 had breakthrough TI or TD. No death was attributable to TI or TD. qPCR kinetics available for 24 patients increased until anti-Toxoplasma treatment began, then decreased with all treatment regimens.Clinical follow-up and qPCR monitoring with quantification of the parasitic load appears a reasonable strategy to avoid TD and to use minimal effective dosage of anti-Toxoplasma treatments.

A cell impedance-based real-time in vitro assay to assess the toxicity of amphotericin B formulations.

Aerosolized liposomal amphotericin B (L-AmB) has been investigated as prophylaxis against invasive aspergillosis. However, the clinical results are controversial and some trials suggest that toxicity could be a limitation for wider use. Our aim was to assess the dynamics of cell toxicity induced in a human alveolar epithelial cell line (A549) after exposure to L-AmB (50 to 400µg/ml) or amphotericin B deoxycholate (D-AmB; 50 to 200µg/ml) by monitoring real-time A549 cell viability using an impedance-based technology. Results were expressed as cell index values integrating cell adhesion, proliferation, and survival. In parallel, the gene expression of proinflammatory cytokines was quantified at 6 and 24h after drug addition by real-time RT-PCR on cell lysates. No sustained reduction of cell indexes was observed with L-AmB or empty liposomes, even at 400µg/ml. Only the highest concentration tested of L-AmB (400µg/ml) yielded transient significant 6-fold and 4-fold induction of TNF-α and IL-8 mRNAs, respectively. In contrast, D-AmB induced a decrease in cell indexes and only the 50µg/ml concentration of D-AmB was followed by cell recovery, higher concentrations leading to cell death. Significant 4-fold, 7-fold and 3-fold inductions of TNF-α, IL-8 and IL-33 mRNAs were also observed at 6h with 50µg/ml of D-AmB. In conclusion, continuous cell impedance measurement showed no toxicity on overall cellular behavior although a slight proinflammatory cytokine expression is possible after L-AmB challenge. Real-time kinetics of cell impedance is an interesting tool for initial screening of cell toxicity.

Polymorphism study of Cryptosporidium hominis gp60 subtypes circulating in Tunisia.

Cryptosporidium spp. are a major cause of gastrointestinal diseases in humans worldwide. While a single subtype of Cryptosporidium hominis has been shown to be responsible for several large outbreaks related to water contamination in developed countries, little is known about the epidemiology of C. hominis in developing countries. This study reports the first genetic characterization of C. hominis at the subtype level in several human populations in Tunisia using the gp60 gene. Eighteen isolates were identified as C. hominis by a restriction fragment length polymorphism (RFLP) analysis. The prevalence of this species in different human populations ranges from 1.53% to 13.04% with a high prevalence being reported in immunocompromised children (13.04%) followed by patients with malignent myeloma (5.5%) and HIV-infected patients (4.59%). The gp60 analysis on C. hominis isolates, performed in 14 cases, showed the presence of a single subtype family: "Ia". Different subtypes were identified within this family (A11G1R1, A12R3, A23G1R1, A26G1R1, A27G1R1, A28G1R1). The IaA26G1R1 subtype was the most dominant subtype described in this area (50%). Despite the high genetic diversity of Cryptosporidium spp, a low heterogeneity at the subtype level was observed within C. hominis circulating in Tunisia. This distribution is an indicator for intensive and stable anthroponotic cryptosporidiosis in this region. Besides, the presence of a unique genotype in 5 HIV-infected patients attending the same hospital ward suggests the possible occurrence of hospital-acquired infection and underlines the need to implement preventive measures to avoid nosocomial transmission.

Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells.

Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of aflatoxin B1 (AFB1) and gliotoxin, two mycotoxins secreted by Aspergillus molds, on the biological activity of the human corneal epithelial (HCE) cells. After 24, 48, and 72 h of exposure, cellular viability and inflammatory response were assessed. Both endpoint cell viability colorimetric assays and continuous cell impedance measurements, providing noninvasive real-time assessment of the effect on cells, were performed. Cytokine gene expression and interleukin-8 release were quantified. Gliotoxin appeared more cytotoxic than AFB1 but, at the same time, led to a lower increase of the inflammatory response reflecting its immunosuppressive properties. Real-time cell impedance measurement showed a distinct profile of cytotoxicity for both mycotoxins. HCE cells appeared to be a well-suited in vitro model to study ocular surface reactivity following biological contaminant exposure. Low, but persistent inflammation, caused by environmental factors, such as fungal toxins, leads to irritation and sensitization, and could be responsible for allergic manifestations which, in turn, could lead to mucosal hyper-reactivity.

Encephalitozoon hellem in a patient with CD4+ T-cell prolymphocytic leukemia: case report and genomic identification.

Five cases of microsporidioses among leukemic patients, 4 in myeloid-leukemic patients and 1 in a chronic lymphocytic leukemia, have been described until now. We report a case of microsporidiosis and the genomic identification of Encephalitozoon hellem in a patient with CD4(+) T-cell prolymphocytic leukemia.

Molecular diagnosis of toxoplasmosis in immunocompromised patients: a 3-year multicenter retrospective study.

Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of Toxoplasma gondii DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (n = 337 samples), cerebrospinal fluid (n = 101 samples), and aqueous humor (n = 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV(+); the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (P < 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.

Utility of adding Pneumocystis jirovecii DNA detection in nasopharyngeal aspirates in immunocompromised adult patients with febrile pneumonia.

Detection of viral and bacterial DNA in nasopharyngeal aspirates (NPAs) is now a routine practice in emergency cases of febrile pneumonia. We investigated whether Pneumocystis jirovecii DNA could also be detected in these cases by conducting retrospective screening of 324 consecutive NPAs from 324 adult patients (198 or 61% were immunocompromised) admitted with suspected pulmonary infections during the 2012 influenza epidemic season, using a real-time quantitative polymerase chain reaction (PCR) assay (PjqPCR), which targets the P. jirovecii mitochondrial large subunit ribosomal RNA gene. These NPAs had already been tested for 22 respiratory pathogens (18 viruses and 4 bacteria), but we found that 16 NPAs (4.9%) were PjqPCR-positive, making P. jirovecii the fourth most prevalent of the 23 microorganisms in the screen. Eleven of the 16 PjqPCR-positive patients were immunocompromised, and five had underlying pulmonary conditions. Nine NPAs were also positive for another respiratory pathogen. Six had PjqPCR-positive induced sputa less than 3 days after the NPA procedure, and five were diagnosed with pneumocystis pneumonia (four with chronic lymphoproliferative disorders and one AIDS patient). In all six available pairs quantification of P. jirovecii DNA showed fewer copies in NPA than in induced sputum and three PjqPCR-negative NPAs corresponded to PjqPCR-positive bronchoalveolar lavage fluids, underscoring the fact that a negative PjqPCR screen does not exclude a diagnosis of pneumocystosis. Including P. jirovecii DNA detection to the panel of microorganisms included in screening tests used for febrile pneumonia may encourage additional investigations or support use of anti-pneumocystis pneumonia prophylaxis in immunocompromised patients.

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.

Epidemiology, clinical, immune, and molecular profiles of microsporidiosis and cryptosporidiosis among HIV/AIDS patients.

BACKGROUND: The objective of this study was to determine the prevalence of intestinal parasites, with special emphasis on microsporidia and Cryptosporidium, as well as their association with human immunodeficiency virus (HIV) symptoms, risk factors, and other digestive parasites. We also wish to determine the molecular biology definitions of the species and genotypes of microsporidia and Cryptosporidium in HIV patients. METHODS: In this cross-sectional study, carried out in Kinshasa, Democratic Republic of the Congo, stool samples were collected from 242 HIV patients (87 men and 155 women) with referred symptoms and risk factors for opportunistic intestinal parasites. The analysis of feces specimen were performed using Ziehl-Neelsen stainings, real-time polymerase chain reaction (PCR), immunofluorescence indirect monoclonal antibody, nested PCR-restriction fragment length polymorphism, and PCR amplification and sequencing. Odds ratio (OR) and 95% confidence intervals were used to quantify the risk. RESULTS: Of the 242 HIV patients, 7.8%, 0.4%, 5.4%, 0.4%, 2%, 10.6%, and 2.8% had Enterocytozoon bieneusi, Encephalitozoon intestinalis, Cryptosporidium spp., Isospora belli, pathogenic intestinal protozoa, nonpathogenic intestinal protozoa, and helminths, respectively. We found five genotypes of E. bieneusi: two older, NIA1 and D, and three new, KIN1, KIN2, and KIN3. Only 0.4% and 1.6% had Cryptosporidium parvum and Cryptosporidium hominis, respectively. Of the patients, 36.4%, 34.3%, 31%, and 39% had asthenia, diarrhea, a CD4 count of <100 cells/mm(3), and no antiretroviral therapy (ART), respectively. The majority of those with opportunistic intestinal parasites and C. hominis, and all with C. parvum and new E. bieneusi genotypes, had diarrhea, low CD4+ counts of <100 cells/mm(3), and no ART. There was a significant association between Entamoeba coli, Kaposi sarcoma, herpes zoster, chronic diarrhea, and asthenia, and the presence of 28 cases with opportunistic intestinal parasites. Rural areas, public toilets, and exposure to farm pigs were the univariate risk factors present in the 28 cases with opportunistic intestinal parasites. In logistic regression analysis, a CD4 count of <100 cells/mm(3) (OR = 4.60; 95% CI 1.70-12.20; P = 0.002), no ART (OR = 5.00; 95% CI 1.90-13.20; P < 0.001), and exposure to surface water (OR = 2.90; 95% CI 1.01-8.40; P = 0.048) were identified as the significant and independent determinants for the presence of opportunistic intestinal parasites. CONCLUSION: E. bieneusi and Cryptosporidium are becoming more prevalent in Kinshasa, Congo. Based on the findings, we recommend epidemiology surveillance and prevention by means of hygiene, the emphasis of sensitive PCR methods, and treating opportunistic intestinal parasites that may be acquired through fecal-oral transmission, surface water, normal immunity, rural area-based person-person and animal-human infection, and transmission of HIV. Therapy, including ART and treatment with fumagillin, is needed.

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.

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.

Polymerase chain reaction for diagnosing pneumocystis pneumonia in non-HIV immunocompromised patients with pulmonary infiltrates.

RATIONALE: Pneumocystis jiroveci polymerase chain reaction (PCR) has higher sensitivity than conventional stains but cannot distinguish colonization from infection. METHODS: We compared P jiroveci PCR and conventional stains in HIV-uninfected immunocompromised patients. RESULTS: Among the 448 patients, 296 (66%) patients had hematologic malignancies; 72 (16.1%), bone marrow transplants; 44 (9.8%), solid tumors; 21 (4.7%), renal transplants; and 15 (3.4%) were taking immunosuppressants for systemic diseases. Diagnostic strategy consisted of BAL in 351 patients and induced sputum (IS) in 97 patients. Conventional pneumocystic pneumonia (PCP) stain was positive in 39 (8.7%) patients, including 34 with positive PCR. In addition, PCR was positive in 32 patients, including 21 with complete follow-up, of whom 14 were diagnosed with probable or definitive PCP (a 36% increase). PCR was 87.2% sensitive and 92.2% specific; positive and negative predictive values were 51.5% and 98.7%, respectively. Sensitivity and negative predictive value were 100% on IS. CONCLUSIONS: In HIV-uninfected immunocompromised patients with acute pulmonary infiltrates, P jiroveci PCR correlates with clinical evidence of PCP. A negative PCR allows withdrawing anti-PCP therapy.

Invasive pulmonary infection due to Trichoderma longibrachiatum mimicking invasive Aspergillosis in a neutropenic patient successfully treated with voriconazole combined with caspofungin.

Trichoderma longibrachiatum, a filamentous fungus, was recently described as an emerging pathogen in immunocompromised patients. Here, we report the first case, to our knowledge, of isolated invasive lung infection by T. longibrachiatum in a patient with hematologic malignancy. The infection mimicked invasive pulmonary aspergillosis and was successfully treated with a combination of voriconazole and caspofungin.

Clinical picture of Pneumocystis jiroveci pneumonia in cancer patients.

BACKGROUND: Pneumocystis pneumonia (PCP) is common in patients with HIV infection but may also occur in patients with other causes of immunodeficiency, including hematologic and solid malignancies. METHODS: To better describe the clinical picture of PCP as to maintain a high level of suspicion in adequate cases, we studied 56 cancer patients with PCP and compared them to 56 cancer patients with bacterial pneumonia. RESULTS: Among 56 PCP patients, 44 patients (78.6%) had hematologic malignancies (18 recipients of bone marrow transplantation) and 12 patients had solid tumors. The time since diagnosis was 24 months (range, 4 to 49 months). All patients with solid tumors and 20 patients (45.4%) with hematologic malignancies were receiving steroids. Only six patients were receiving PCP prophylaxis. The main symptoms were fever (85.7%), dyspnea (78.6%), and cough (57.1%). Time from symptom onset was 7 days (range, 3 to 14 days). PCP presented as severe pneumonia (Pao(2), 58 mm Hg [range, 50 to 70 mm Hg]) with bilateral interstitial infiltrates (80.4%) and bilateral ground-glass attenuation (89.3%) by CT. Of the 24 ICU patients (42.9%), 16 patients (19.6%) required mechanical ventilation. Eleven patients (19.6%) died. Compared to 56 patients with bacterial pneumonia, PCP patients were more likely to have non-Hodgkin lymphoma and be receiving long-term steroids; they had longer times since diagnosis, longer symptom duration, higher frequencies of fever and of diffuse lung disease (diffuse crackles, bilateral infiltrates, and hypoxemia), higher frequency of ground-glass opacities, and lower frequency of pleural involvement. CONCLUSIONS: PCP presents as subacute, febrile, hypoxemic, and diffuse pulmonary involvement in patients with solid tumors or hematologic malignancies receiving long-term steroids.

Inhibitory activity of human immunodeficiency virus aspartyl protease inhibitors against Encephalitozoon intestinalis evaluated by cell culture-quantitative PCR assay.

Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis. A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis-encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis.

Comparison of PCR-enzyme-linked immunosorbent assay and real-time PCR assay for diagnosis of an unusual case of cerebral toxoplasmosis in a stem cell transplant recipient.

A PCR-enzyme-linked immunosorbent assay and a real-time PCR assay were compared for diagnosis and follow-up of cerebral toxoplasmosis in a stem cell transplant recipient. The sensitivity of detection was similar for both assays but was higher when the assays were performed on buffy coat rather than on whole blood or serum.