Juergen Loeffler

Resistance to fluconazole and cross‐resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol Δ5,6‐desaturation

Fluconazole resistance occurs in >10% of cases of candidosis during the late stages of AIDS. We show here in two clinical isolates that resistance was caused by defective sterol Δ5,6‐desaturation. This altered the type of sterol accumulating under fluconazole treatment from 14α‐methylergosta‐8,24(28)‐dien‐3β,6α‐diol to 14α‐methylfecosterol which is capable of supporting growth. A consequence of this mechanism of azole resistance is that an absence of ergosterol causes cross‐resistance to the other major antifungal agent available, amphotericin B. The results also show that growth arrest after fluconazole treatment of C. albicans in clinical conditions is caused by 14α‐methylergosta‐8,24(28)‐dien‐3β,6α‐diol accumulation.

Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4

Invasive aspergillosis (IA) is a leading cause of mortality in haematological patients. Appropriate activation of the innate immune system is crucial for the successful clearance of IA. Therefore, we studied the Aspergillus fumigatus‐mediated activation of human granulocytes and monocyte‐derived immature dendritic cells (DCs), as well as murine bone marrow‐derived DCs (BMDCs) from wild type, toll‐like receptor (TLR)4‐deficient, TLR2 knockout, and TLR2/TLR4 double deficient mice. Aspergillus fumigatus antigens induced the activation and maturation of immature DCs as characterized by CD83 expression, upregulation of major histocompatibility complex and co‐stimulatory molecules. Moreover, fungal antigens enhanced the phagocytosis and production of interleukin (IL)‐8 in granulocytes. The release of IL‐12 by BMDCs in response to A. fumigatus antigens was dependent on the expression of TLR2, whereas the release of IL‐6 was dependent on the expression of functional TLR4 molecules. The protein precipitate of A. fumigatus supernatant provided strong stimulation of DCs and granulocytes, indicating that a factor secreted by A. fumigatus might activate innate immune cells. In conclusion, A. fumigatus antigens induced the activation of DCs and granulocytes. Our results indicated that this activation was mediated via TLR2 and TLR4. Future studies are needed to assess the clinical impact of these findings in patients at high risk for IA.

Identification of rare Candida species and other yeasts by polymerase chain reaction and slot blot hybridization

Invasive candidiasis has become a major cause of morbidity and mortality in immunocompromised hosts. Here we describe a fast and reliable DNA extraction and PCR amplification method in combination with a slot blot hybridization assay. A genus-specific probe was designed that allowed to detect DNA from a broad range of Candida species and 3 other yeasts. In addition, species-specific oligonucleotides for emerging Candida and other yeast species allowed to identify DNA extracted from Candida lusitaniae, Candida humicola, Candida kefyr, Candida inconspicua, Candida solani, Malassezia furfur and Trichosporon cutaneum. A sensitivity of at least 10(1) CFU, corresponding to 100 fg of fungal DNA, was documented for all species-specific probes and the common Candida probe. In addition, the 18S rRNA genes of 7 yeast species (C. humicola, C. kefyr, C. solani, C. inconspicua, C. norvegensis, C. utilis and M. furfur) were completely sequenced. The sequencing primers described bind to highly conserved primer binding sites. Therefore, these primers would allow rapid cycle sequence of additional ribosomal genes throughout the whole kingdom of fungi.

A CTL epitope from human cytomegalovirus IE1 defined by combining prediction of HLA binding and proteasomal processing is the target of dominant immune responses in patients after allogeneic stem cell transplantation

OBJECTIVE AND METHODS In an attempt to define HCMV IE1-derived, HLA-A(*)0201-restricted epitopes, an advanced computer-based epitope prediction combining HLA binding and proteasomal cleavages in silico was performed. RESULTS This prediction algorithm clearly confirmed VLEETSVML to be the most likely CTL epitope. By tetramer staining, HCMV pp65 NLVPMVATV-specific CD8(+) T cells were detectable in 18/24 HCMV seropositive HLA-A(*)0201-expressing individuals (median frequency 0.58%; range 0.1%-4.7%), and IE1 VLEETSVML-specific CD8(+) T cells in 5/24 (median frequency 2.1%; range 0.1%-4.3%), respectively (p<0.01). Also in recipients of an allogeneic SCT, VLEETSVML- and NLVPMVATV-specific CD8(+) T cells were detectable in comparable frequencies, but again the number of patients with detectable pp65-specific CD8(+) T cells was higher (p=0.014). In 4/15 individuals, all demonstrating IE1 VLEETSVML-specific CD8(+) T cells prior to peptide stimulation, VLEETSVML-specific T cell lines (purity of 42.6%-98.6% of all CD3(+)/CD8(+) T cells) were successfully generated after 2-4 weeks of culture using the IFN-gamma secretion assay. CONCLUSION In conclusion, this novel prediction strategy efficiently predicted an immunodominant viral T-cell epitope.

Standardization of Aspergillus PCR diagnosis.

Blennow and Mattsson1 recently concluded in a reply to Meije et al.2 that standardization of Aspergillus PCR diagnosis is needed. We agree with this, which is why we set up a Working Group of the International Society of Human and Animal Mycology, known as the European Aspergillus PCR initiative (EAPCRI) (http://www.eapcri.eu). This group has forged ahead, developing the necessary framework for establishing standards.3

Quantification of δRec-ψJα Signal Joint T-Cell Receptor Excision Circle DNA in Patients after Autologous and Allogeneic Stem Cell Transplantation

Myeloablative chemotherapy followed by stem cell transplantation is often associated with a prolonged and substantial, potentially detrimental period of T-cell immunodeficiency (1). The depletion of T-cells by intensive chemotherapy may lead to an increased number of viral and fungal infections. For a complete reconstitution of immunity, the generation of de novo T-cells in the thymus with a broad T-cell receptor repertoire is essential (2). However, the thymus is gradually replaced by adipose tissue and its activity is age-dependent (3). Additional factors that influence the thymic activity are graft-versus-host disease and direct damage from chemo- and radiotherapy. Measuring thymopoietic capacity only on the basis of phenotyping naive T-cells is of limited value as CD45RA+ T-cells may immediately convert into memory T-cells, may proliferate antigen-independently or may persist most of their life span. As an alternative approach to monitor thymic activity, the frequency of T-cell receptor excision circles among peripheral blood cells can be determined (4).

Quantification and Speciation of Fungal DNA in Clinical Specimens Using the LightCycler Instrument

Invasive fungal infections have become a major cause of morbidity and mortality in immunocompromised patients such as bone marrow and solid organ transplant recipients, patients receiving intense chemotherapy, AIDS patients, patients with cystic fibrosis, neonates, and severe burn patients [1]. Invasive aspergillosis has become a leading cause of death, mainly among hematology patients. The incidence is estimated to be 25% and the mortality is up to 90% in patients with acute leukemia [2]. Early diagnosis is essential for appropriate and successful antifungal therapy. Conventional tests for the detection of Aspergillus spp., such as blood culture and serology, have limited sensitivity and specificity. Diagnostic assays based on in vitro amplification and speciation of fungal DNA show promising sensitivity and specificity, indicating potential for the early diagnosis of invasive fungal infections [3–5]. However, published assays require 6–8 h for fungal DNA extraction, followed by a minimum of 9 h for DNA amplification and amplicon detection. Post-PCR analysis is mainly based on qualitative or semi-quantitative methods such as gel electrophoresis or hybridization by Southern blot or PCR-ELISA techniques.