Holger Hebart

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.

Risk factors for treatment failures in patients receiving PCR-based preemptive therapy for CMV infection

PCR-based preemptive therapy with ganciclovir has been shown to reduce the incidence of CMV disease after BMT. Failures of this treatment strategy are CMV disease and secondary non-viral infections. Eighty-six consecutive patients at high risk for CMV disease who received PCR-based preemptive therapy with ganciclovir were assessed for treatment failures and possible risk factors. Ganciclovir was initiated in 57 of 86 patients (66%). Only 28 of 86 (32%) patients received 4 or more weeks of ganciclovir. Recurrence of CMV infection after successful treatment was more frequent among recipients of a BMT from an unrelated compared to a sibling donor (P = 0.004). three (3.5%) patients developed non-fatal early onset cmv disease and seven of 68 (10.3 %) late onset cmv disease (>100 days post transplant). Risk factors for late onset CMV disease were cGVHD (P = 0.0017) and duration of prior antiviral therapy >4 weeks (P = 0.0073). The incidence of secondary non-viral infections was 28% with the duration of antiviral treatment being a significant risk factor for secondary bacterial (P = 0.0045) and invasive fungal infections (P = 0.006). Thus, PCR-based preemptive treatment with ganciclovir reduces early onset CMV disease, but the duration of antiviral therapy prior to day +100 is a significant risk factor for late onset CMV disease as well as secondary non-viral infections. Bone Marrow Transplantation (2000) 25, 757–763.

Early Detection of Aspergillus Infection after Allogeneic Stem Cell Transplantation by Polymerase Chain Reaction Screening

Invasive aspergillosis (IA) has become a major cause of mortality in patients after allogeneic stem cell transplantation. To assess the potential of prospective polymerase chain reaction (PCR) screening for early diagnosis of IA, 84 recipients of an allogeneic stem cell transplant were analyzed with the investigators blinded to clinical and microbiologic data. Of 1193 blood samples analyzed, 169 (14.2%) were positive by PCR. In patients with newly diagnosed IA (n=7), PCR positivity preceded the first clinical signs by a median of 2 days (range, 1-23 days) and preceded clinical diagnosis of IA by a median of 9 days (range, 2-34 days). Pretransplantation IA (relative risk [RR], 2.37), acute graft-versus-host disease (RR, 2.75), and corticosteroid treatment (RR, 6.5) were associated with PCR positivity. The PCR assay revealed a sensitivity of 100% (95% confidence interval [CI], 48%-100%) and a specificity of 65% (95% CI, 53%-75%). None of the PCR-negative patients developed IA during the study period. Thus, prospective PCR screening allows for identification of patients at high risk for subsequent onset of IA.

Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome

Following extensive propagation in fibroblasts, human cytomegalovirus (HCMV) loses tropism for a number of otherwise natural host cells, in particular, endothelial cells. In this study, the hypothesis was tested that loss of endothelial tropism is associated with the appearance of genomic variants. Initial quantitative focus expansion assays on endothelial monolayers demonstrated that, while the laboratory strains AD169 and Towne failed to form detectable foci, 29 out of 30 recent clinical HCMV isolates had the potential to expand in endothelial cell culture. By long-term adaptation in fibroblast cultures, nonendotheliotropic strains could be selected from clinical HCMV isolates, while long-term endothelial-adapted strains of the same isolates retained both fibroblast tropism and endothelial tropism. Such differentially adapted isolate pairs always displayed genomic differences in restriction fragment length analyses. Coinfection of endothelial cells by two nonendotheliotropic HCMV strains yielded an endotheliotropic recombinant HCMV variant combining portions of the genomes of both parental viruses. When DNA purified from various isolates was transfected into fibroblasts, progeny virus retained the specific tropism of parental virus from which the DNA was isolated. These findings demonstrate that endothelial tropism is an inherent property of most clinical HCMV isolates and is determined by the viral genome. Although the specific determinants of HCMV cell tropism are still unknown, this study provides the first evidence for a genetic contribution.

Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus.

The susceptibility of monocyte-derived immature dendritic cells (DC) to infection by various strains of human cytomegalovirus (HCMV) was analysed. Immature DC were generated by incubation of peripheral blood monocytes with interleukin-4 and granulocyte-macrophage colony-stimulating factor for 7 days and were characterized by a CD1a+/CD40+/CD80+/CD86+/HLA-DR+/CD14- phenotype. Viral antigen expression and production of infectious progeny virus were analysed in infected immature DC cultures. Immature DC were 80-90 % susceptible to HCMV strains that had been propagated in endothelial cell culture, whereas the infection rate was negligible with fibroblast-adapted HCMV strains. Immature DC infection resulted in expression of viral immediate early, early and late genes. Productive infection was proven by the detection of infectious virus in single-step growth curves and in infectious centre assays. It is concluded that HCMV might interfere with the host immune reaction by permissive, lytic infection of immature DC.

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.

Screening for CMV-specific T cell proliferation to identify patients at risk of developing late onset CMV disease

Thirty patients undergoing allogeneic BMT were screened post-transplant together with their marrow donors for CMV-specific T cell proliferation and the occurrence of CMV disease. Twenty-one of these patients received a marrow transplant from an HLA-matched sibling donor, and nine from an HLA-matched unrelated donor. All these patients were either CMV seropositive and/or had received a transplant from a CMV-seropositive donor. Patients were monitored for CMV-viraemia until day +100 post-BMT by PCR and virus culture, and thereafter by virus culture only when clinically indicated. The proliferative T cell response was investigated at regular monthly intervals beginning on day +30. A proliferative response to HCMV (median, day +123) was documented in these patients between day +37 and +730 post-BMT. None of the patients with a documented CMV-specific T cell proliferation on day 120 (n = 17) developed CMV disease in the later post-transplant period, but of the patients lacking CMV- specific proliferation (n = 13), 30.8% developed CMV disease after day 120. Thus, patients lacking a CMV- specific T-helper cell response might benefit from sensitive screening for CMV infection and pre-emptive therapy after day +100.

Detection of PCR-amplified fungal DNA by using a PCR-ELISA system.

In order to speed up and standardize polymerase chain reaction (PCR)-based detection of medically important fungi in clinical samples we established a combination of commercially available kits for DNA extraction, PCR amplification and detection of the amplicons. The PCR plate assay proved to be as sensitive and specific as our previously published assay (5 cfu ml(-1) blood). Moreover, in a selected group of patients, all patients with proven and probable invasive fungal infection were found to be PCR-positive. Thus the PCR plate assay was found to be a sensitive, technically simplified and standardized method with potential for adaptation to automation.

Nucleic Acid Sequence-Based Amplification of Aspergillus RNA in Blood Samples

ABSTRACT Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of Aspergillus RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of theAspergillus 18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevantAspergillus species, including Aspergillus fumigatus. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection ofAspergillus RNA with potential for routine diagnosis, including the possibility to test the viability of cells.

A prospective randomized controlled trial comparing PCR-based and empirical treatment with liposomal amphotericin B in patients after allo-SCT.

We compared the efficacy and safety of empirical plus PCR-based vs empirical liposomal amphotericin B treatment after Allo-SCT. Allo-SCT recipients were randomized to receive either PCR-based preemptive therapy (group A; n=198) or empirical antifungal therapy (group B; n=211) with liposomal amphotericin B. In group A, therapy was started after one positive PCR result or after 120 h of febrile neutropenia refractory to broad-spectrum antibacterial therapy. In group B, liposomal amphotericin B was started after 120 h of refractory febrile neutropenia. Demographic and clinical characteristics were well balanced. A total of 112 (57.1%) patients in group A and 76 (36.7%) patients in group B received antifungal therapy (P<0.0001). Twelve patients in group A and 16 patients in group B developed proven invasive fungal infection (IFI). Survival curves showed better survival until day 30 when close PCR monitoring was performed (mortality 1.5 vs 6.3%; P=0.015), but there was no difference at day 100. At day 100, no difference was observed in the incidence of IFI (primary end point) and survival between the two arms. Further studies are required to assess the benefit of using PCR in patients after SCT.