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ABSTRACTAzole resistance in Aspergillus fumigatusis increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevant Aspergillusspecies, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatusstrains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of =1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergilluswas <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillusspecies and 14 A. fumigatussamples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillusspecies directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

Toll-like receptor (TLR)-based signaling pathways in the host may be modulated by pathogens during the course of infection. We describe a novel immunomodulatory mechanism in which Aspergillus fumigatus conidia induce attenuation of TLR2- and TLR4-mediated interleukin (IL)-6 and IL-1β proinflammatory responses in human mononuclear cells with suppression of IL-1β mRNA transcription. Background TLR2 and TLR4 mRNA transcription was not influenced. A. fumigatus conidia induced TLR2 internalization and uptake into the phagosome with a resultant decrease in surface receptor expression. A. fumigatus hyphae, on the other hand, selectively downregulated the TLR4-mediated response. These novel immunosuppressive effects may facilitate the invasiveness of A. fumigatus.

The different manifestations of Candida albicansinfection are dictated by an underlying defect in the immune response of the host. Protective immunity to disseminated candidiasis, the manifestation of C. albicansinfection discussed in this review, has traditionally been ascribed to innate immunity with emphasis on the role of granulocytes. Lately, however, immunological studies have learned that host defence against disseminated candidiasis is based on a complex interplay between innate and cell-mediated immunity. Despite the availability of new antifungal agents, mortality associated with disseminated C. albicansinfection remains high. Immunotherapy that augments host defence is an important strategic option in the battle against disseminated candidiasis. Here, the authors review the chronological events in the pathogenesis of disseminated candidiasis that aid in predicting the impact of existing immunotherapy and the development of future immunomodulating strategies.

ABSTRACTThe aim of the present study was to assess the influence of immunomodulation of host defense with recombinant murine granulocyte colony-stimulating factor (rmG-CSF) on intra-abdominal abscesses caused by Candida albicans. Mice received prophylaxis or therapy with 1 μg of rmG-CSF/day in the presence or absence of antifungal treatment consisting of amphotericin B (0.75 mg/kg of body weight/day) or fluconazole (50 mg/kg/day). The number of CandidaCFU in abscesses was significantly reduced (P< 0.05) in mice receiving rmG-CSF prophylaxis (day −1 or day −1 through 2) compared with controls on day 8 of infection. Administration of rmG-CSF therapy alone (for 5 days starting on day 4 of infection) had no influence on the number of CandidaCFU in abscesses. Amphotericin B treatment was significantly more effective than fluconazole treatment (3.41 log CFU/abscesses; 95% confidence interval [CI], 3.17 log CFU/abscesses; 3.65 versus 3.90 log CFU/abscesses; 95% CI, 3.66 log CFU/abscesses, 4.16 log CFU/abscesses; P< 0.05). Therapeutic administration of rmG-CSF in conjunction with an antifungal agent showed a tendency towards a further reduction of CandidaCFU in abscesses than antifungal treatment only. In conclusion, in this experimental model of intra-abdominal Candidaabscesses, rmG-CSF administration did not have a detrimental influence on the course of infection. Amphotericin B treatment was most effective, and additional rmG-CSF therapy did not antagonize the effect of antifungal treatment. In contrast, addition of rmG-CSF therapy to antifungal treatment might further enhance the beneficial effect of the antifungal agent.

Neutrophils (PMNs) constitute the main mechanism of host defense against acute invasive and disseminated candidiasis. Recent studies have demonstrated that tumor necrosis factor‐α (TNFα), interleukin‐6 (IL‐6) and granulocyte colony‐stimulating factor (G‐CSF) play an important role in the recruitment of PMNs at the site of invasive Candidainfection. In the absence of either TNFα or IL‐6, the course of experimental disseminated candidiasis is more severe, due to defective PMN recruitment. Treatment of mice with recombinant G‐CSF (rG‐CSF) leads to a significantly reduced mortality during disseminated candidiasis. The outgrowth of Candida albicansfrom the organs of rG‐CSF‐treated mice is significantly decreased. Treatment with the combination of rG‐CSF and fluconazole has an additive effect on the reduction of fungal load in the organs. In subacute or chronic disseminated Candidainfection, rG‐CSF is less effective, indicating that neutrophil recruitment and activation are crucial in acute, life‐threatening candidiasis, whereas other host defense mechanisms control the outcome of less overwhelming invasive Candidainfection.

ABSTRACTHost chitinases, chitotriosidase and acidic mammalian chitinase (AMCase), improved the antifungal activity of caspofungin (CAS) against Aspergillus fumigatus in vitro. These chitinases are not constitutively expressed in the lung. Here, we investigated whether chitosan derivatives were able to induce chitinase activity in the lungs of neutropenic rats and, if so, whether these chitinases were able to prolong survival of rats with invasive pulmonary aspergillosis (IPA) or of rats with IPA and treated with CAS. An oligosaccharide-lactate chitosan (OLC) derivative was instilled in the left lung of neutropenic rats to induce chitotriosidase and AMCase activities. Rats instilled with OLC or with phosphate-buffered saline (PBS) were subsequently infected with A. fumigatusand then treated with suboptimal doses of CAS. Survival, histopathology, and galactomannan indexes were determined. Instillation of OLC resulted in chitotriosidase and AMCase activities. However, instillation of OLC did not prolong rat survival when rats were subsequently challenged with A. fumigatus. In 5 of 7 rats instilled with OLC, the fungal foci in the lungs were smaller than those in rats instilled with PBS. Instillation of OLC did not significantly enhance the survival of neutropenic rats challenged with A. fumigatusand treated with a suboptimal dosage of CAS. Chitotriosidase and AMCase activities can be induced with OLC, but the presence of active chitinases in the lung did not prevent the development of IPA or significantly enhance the therapeutic outcome of CAS treatment.