Johan Maertens

Advances in the serological diagnosis of invasive Aspergillus infections in patients with haematological disorders

A reliable diagnosis of invasive aspergillosis in patients with haematological malignancies is seldom achieved antemortem. Conventional laboratory diagnostic methods are insensitive and time‐consuming, resulting in late diagnosis and treatment and contributing to unacceptably high mortality. As a result, routine antifungal prophylaxis and early empirical treatment have been recommended. However, overtreatment associated with these strategies results in increased toxicity and cost. The use of sensitive and rapid non‐culture‐based diagnostic assays, such as detection of Aspergillus antigens (galactomannan, β‐d‐glucan) or detection of genomic DNA sequences may allow a shift in emphasis from empirical to pre‐emptive therapy, especially when substantiated by suggestive radiological findings. These new tools may be used to confirm a presumed diagnosis of invasive aspergillosis, or, when used to screen high‐risk patients, may identify an infection at the early stage of disease. The excellent negative predictive value of these assays should convince clinicians to withhold antifungal therapy in persistently febrile neutropenic patients with no other signs of fungal infection. On the other hand, consecutive positive results in a high‐risk population should at least trigger a complete diagnostic work‐up. This review will focus on the diagnostic utility as well as on the pitfalls of serial screening for the presence of circulating fungal antigens in haematology patients.

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA.

Risk factors for breakthrough invasive fungal infection during secondary prophylaxis

BACKGROUNDnIntensive chemotherapy with severe neutropenia is associated with invasive fungal infections (IFIs) leading to high mortality rates. During leukaemia induction chemotherapy, IFI often prohibited further curative treatment, thus predisposing for leukaemia relapse. Continuing myelosuppressive chemotherapy after diagnosis of IFI has become feasible with the now expanding arsenal of safe and effective antifungals. Secondary prophylaxis of IFI is widely administered, but reliable data on outcome and risk factors for recurrent IFI during subsequent chemotherapy are not available. This study determines risk factors for recurrent IFI in leukaemia patients.nnnMETHODSnFrom 25 European cancer centres, 166 consecutive patients with acute myelogenous leukaemia (AML) and a recent history of proven or probable pulmonary IFI were included. Patients were followed for recurrence or breakthrough IFI during the subsequent chemotherapy cycle.nnnRESULTSnOf the 166 patients included, 69 (41.6%) were female, the median age was 53 years (range 2-81) the and 3 (1.8%) were <16 years. Recurrent IFI occurred in 26 patients (15.7%). Multiple logistic regressions yielded predisposing factors: duration of neutropenia [per additional day; odds ratio (OR) 1.043, confidence interval (CI) 1.008-1.078], high-dose cytarabine (OR 3.920, CI 1.120-12.706), number of antibiotics (per antibiotic; OR 1.504, CI 1.089-2.086), partial response as outcome of prior IFI (OR 4.037, CI 1.301-12.524) and newly diagnosed AML (OR 3.823, CI 0.953-15.340). Usage of high efficiency particulate air filter appeared protective (OR 0.198, CI 0.036-1.089).nnnCONCLUSIONSnDuration of neutropenia, high-dose cytarabine, prior antibiotic therapy and a partial response to the first IFI therapy were risk factors for recurrent IFI and should be considered in AML patients with prior pulmonary IFI undergoing further chemotherapy.

Mucorales -specific T cells emerge in the course of invasive mucormycosis and may be used as a surrogate diagnostic marker in high-risk patients

Mucorales-specific T cells were investigated in 28 hematologic patients during the course of their treatment. Three developed proven invasive mucormycosis (IM), 17 had infections of known origin but other than IM, and 8 never had fever during the period of observation. Mucorales-specific T cells could be detected only in patients with IM, both at diagnosis and throughout the entire course of the IM, but neither before nor for long after resolution of the infection. Such T cells predominantly produced IL-4, IFN-γ, IL-10, and to a lesser extent IL-17 and belonged to either CD4(+) or CD8(+) subsets. The specific T cells that produced IFN-γ were able to directly induce damage to Mucorales hyphae. None of the 25 patients without IM had Mucorales-specific T cells. Specific T cells contribute to human immune responses against fungi of the order Mucorales and could be evaluated as a surrogate diagnostic marker of IM.

Role of Non-Culture-Based Tests, with an Emphasis on Galactomannan Testing for the Diagnosis of Invasive Aspergillosis

An established diagnosis of invasive aspergillus is seldom achieved premortem. Conventional laboratory diagnostic methods such as culture and microscopy, although very useful when positive, are insensitive and time-consuming, resulting in late diagnosis and treatment and contributing to high mortality rates. As a result, routine antifungal prophylaxis and early empirical treatment have been recommended. The use of sensitive and rapid non-culture-based diagnostic assays for the detection of Aspergillus antigens (using commercially available tests to detect galactomannan and 1, 3 β-D-glucan) or detection of genomic DNA sequences may allow a shift in emphasis from empirical to preemptive therapy, especially when substantiated by suggestive radiological findings. These new tools may be used to confirm a presumed diagnosis of invasive aspergillosis, or, when used to screen high-risk patients, may identify an infection at an early stage of disease. Their excellent negative predictive value should convince clinicians to withhold antifungal therapy in patients with no other signs of fungal disease. On the other hand, consecutive positive results should at least trigger a complete diagnostic workup. This article will review the diagnostic utility as well as the pitfalls of using these non-culture-based tools for diagnosing invasive aspergillosis.

Treatment outcome of invasive mould disease after sequential exposure to azoles and liposomal amphotericin B.

OBJECTIVESnTo analyse the potential antagonism between azoles, which inhibit ergosterol synthesis, and polyenes, which bind directly to ergosterol in cell membranes, in patients receiving sequential azole-polyene treatment.nnnMETHODSnIn an earlier randomized, double blind study of liposomal amphotericin as initial therapy for invasive filamentous fungal infection (IFFI), a 3 mg/kg/day dose had a favourable overall response rate of 50% and 12 week survival rate of 72%. No improved outcome was seen with 10 mg/kg/day for the first 14 days. The study population was further analysed for the effect of prior azole exposure on treatment responses to liposomal amphotericin B. The protocol allowed prior treatment with azoles for prophylaxis or empirical therapy, and for up to 4 days for the confirmed IFFI before starting liposomal amphotericin B. Outcomes were compared for subsets of patients based on receipt of any azole and receipt of voriconazole during the 30 day screening period prior to study treatment.nnnRESULTSnOf 201 patients with data review board-confirmed IFFI, 116 (57.7%) received prior azoles and 36 (17.9%) received prior voriconazole. Favourable responses were achieved in 57 (49.1%) patients with prior azole exposure, in 39 (45.9%) without prior azole and in 13 (36.1%) with prior voriconazole. Numbers of patients alive at 12 weeks were 74 (63.8%) with any prior azole, 56 (65.9%) without prior azole and 26 (72.2%) after prior voriconazole. No differences were statistically significant.nnnCONCLUSIONSnPrior treatment with any azole or specifically with voriconazole did not seem to impact on overall response or survival in patients treated with liposomal amphotericin B for confirmed IFFI.

Economic Comparison of an Empirical Versus Diagnostic-Driven Strategy for Treating Invasive Fungal Disease in Immunocompromised Patients:

PURPOSEnPatients with persistent or recurrent neutropenic fevers at risk of invasive fungal disease (IFD) are treated empirically with antifungal therapy (AFT). Early treatment using a diagnostic-driven (DD) strategy may reduce clinical and economic burdens. We compared costs and outcomes of both strategies from a UK perspective.nnnMETHODSnAn empirical strategy with conventional amphotericin B deoxycholate (C-AmB), liposomal amphotericin B (L-AmB), or caspofungin was compared with a DD strategy (initiated based on positive ELISA results for galactomannan antigen) and/or positive results for Aspergillus species on polymerase chain reaction assay) using C-AmB, voriconazole, or L-AmB in a decision-analytic model. Rates of IFD incidence, overall mortality, and IFD-related mortality in adults expected to be neutropenic for ≥10xa0days were obtained. The empirical strategy was assumed to identify 30% of IFD and targeted AFT to improve survival by a hazard ratio of 0.589. AFT-specific adverse events were obtained from a summary of product characteristics. Resource use was obtained, and costs were estimated by using standard UK costing sources. All costs are presented in 2012 British pounds sterling.nnnFINDINGSnTotal costs were 32% lower for the DD strategy (£1561.29) versus the empirical strategy (£2301.93) due to a reduced incidence of adverse events and decreased use of AFT. Administration of AFT was reduced by 41% (DD strategy, 74 of 1000; empirical strategy, 125 of 1000), with similar survival rates.nnnIMPLICATIONSnThis study suggests that a DD strategy is likely to be cost-saving versus empirical treatment for immunocompromised patients with persistent or recurrent neutropenic fevers.

Pharmacodynamics of Isavuconazole for Invasive Mold Disease: Role of Galactomannan for Real-Time Monitoring of Therapeutic Response

Summary An analysis of serial galactomannan index (GMI) in patients with invasive aspergillosis from a phase 3 clinical trial demonstrated that increases in GMI in the first week of therapy significantly increase the likelihood of death and unsuccessful response.

Mucorales-Specific T Cells in Patients with Hematologic Malignancies

Background Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients. Methods and Findings By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD): 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD. Conclusions Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

European guidelines for primary antifungal prophylaxis in adult haematology patients: summary of the updated recommendations from the European Conference on Infections in Leukaemia

The European Conference on Infections in Leukaemia (ECIL) updated its guidelines on antifungal prophylaxis for adults using the grading system of IDSA. The guidelines were extended to provide recommendations for other haematological diseases besides AML and recipients of an allogeneic haematopoietic stem cell transplantation (HSCT). Posaconazole remains the drug of choice when the incidence of invasive mould diseases exceeds 8%. For patients undergoing remission-induction chemotherapy for AML and myelodysplastic syndrome (MDS), fluconazole can still offer an alternative provided it forms part of an integrated care strategy that includes screening with biomarkers and imaging. Similarly, aerosolized liposomal amphotericin B combined with fluconazole can be considered for patients at high risk of invasive mould diseases but other formulations of the polyene are discouraged. Fluconazole is still recommended as primary prophylaxis for patients at low risk of invasive mould diseases during the pre-engraftment phase of allogeneic HSCT whereas only a moderate recommendation could be made for itraconazole, posaconazole and voriconazole for patients at high risk. Posaconazole is strongly recommended for preventing invasive mould disease post-engraftment but only when graft-versus-host disease (GvHD) was accompanied by other risk factors such as its severity, use of an alternative donor or when unresponsive to standard corticosteroid therapy. The need for primary prophylaxis for other patient groups was less clear and should be defined by the estimated risk of invasive fungal disease (IFD).