The contribution of galactomannan detection in the diagnosis of invasive aspergillosis in bone marrow transplant recipients

Until recently, accurate microbiological diagnosis of invasive aspergillosis (IA) was seldom established in HSCT recipients. Blood samples are rarely positive for Aspergillus species, the reliability of the cultures depends of the specimen (if taken from a normally sterile site or not) and biopsy samples require invasive procedures, rarely recommended in patients with severe thrombocytopenia. Implementing the international consensus defining the microbiological criteria for the diagnosis of Aspergillus infection, we retrospectively evaluated the role of serum galactomannan (GM) detection by EIA to diagnose IA among HSCT patients with proven invasive fungal infection (IFI) and the impact of serum storage in GM concentrations. The EIA assay allowed categorizing as “probable” 5 of the 10 cases of “possible” aspergillosis (50%). Considering a lower cut-off level for the reaction (1.0), 80% of the cases could be categorized as “probable” aspergillosis. Positive or undetermined results were detected one to 4 months before the diagnosis of IA in eight of the 11 patients (72.7%) with proven IFI. Retesting the stored samples after a second storage for four years, we could observe lower reactivity in 20% of the samples. The detection of galactomannan by the EIA test represents a major advance in the diagnosis of IA in HSCT recipients at high risk of IA. A better understanding of the kinetics of the GM in different clinical situations is necessary to maximize the benefit of the test in Aspergillus surveillance.     

Detection of a Serum Siderophore by LC-MS/MS as a Potential Biomarker of Invasive Aspergillosis

Invasive aspergillosis (IA) is a life-threatening systemic mycosis caused primarily by Aspergillus fumigatus. Early diagnosis of IA is based, in part, on an immunoassay for circulating fungal cell wall carbohydrate, galactomannan (GM). However, a wide range of sensitivity and specificity rates have been reported for the GM test across various patient populations. To obtain iron in vivo, A. fumigatus secretes the siderophore, N,N'',N"-triacetylfusarinine C (TAFC) and we hypothesize that TAFC may represent a possible biomarker for early detection of IA. We developed an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for TAFC analysis from serum, and measured TAFC in serum samples collected from patients at risk for IA. The method showed lower and upper limits of quantitation (LOQ) of 5 ng/ml and 750 ng/ml, respectively, and complete TAFC recovery from spiked serum. As proof of concept, we evaluated 76 serum samples from 58 patients with suspected IA that were investigated for the presence of GM. Fourteen serum samples obtained from 11 patients diagnosed with probable or proven IA were also analyzed for the presence of TAFC. Control sera (n = 16) were analyzed to establish a TAFC cut-off value (≥6 ng/ml). Of the 36 GM-positive samples (≥0.5 GM index) from suspected IA patients, TAFC was considered positive in 25 (69%). TAFC was also found in 28 additional GM-negative samples. TAFC was detected in 4 of the 14 samples (28%) from patients with proven/probable aspergillosis. Log-transformed TAFC and GM values from patients with proven/probable IA, healthy individuals and SLE patients showed a significant correlation with a Pearson r value of 0.77. In summary, we have developed a method for the detection of TAFC in serum that revealed this fungal product in the sera of patients at risk for invasive aspergillosis. A prospective study is warranted to determine whether this method provides improved early detection of IA.     

Correlation between Circulating Fungal Biomarkers and Clinical Outcome in Invasive Aspergillosis

Objective means are needed to predict and assess clinical response in patients treated for invasive aspergillosis (IA). We examined whether early changes in serum galactomannan (GM) and/or β-D-glucan (BDG) can predict clinical outcomes. Patients with proven or probable IA were prospectively enrolled, and serial GM and BDG levels and GM optical density indices (GMI) were calculated twice weekly for 6 weeks following initiation of standard-of-care antifungal therapy. Changes in these biomarkers during the first 2 and 6 weeks of treatment were analyzed for associations with clinical response and survival at weeks 6 and 12. Among 47 patients with IA, 53.2% (25/47) and 65.9% (27/41) had clinical response by weeks 6 and 12, respectively. Changes in biomarkers during the first 2 weeks were associated with clinical response at 6 weeks (GMI, P = 0.03) and 12 weeks (GM+BDG composite, P = 0.05; GM, P = 0.04; GMI, P = 0.02). Changes in biomarkers during the first 6 weeks were also associated with clinical response at 6 weeks (GM, P = 0.05; GMI, P = 0.03) and 12 weeks (BDG+GM, P = 0.02; GM, P = 0.02; GMI, P = 0.01). Overall survival rates at 6 weeks and 12 weeks were 87.2% (41/47) and 79.1% (34/43), respectively. Decreasing biomarkers in the first 2 weeks were associated with survival at 6 weeks (BDG+GM, P = 0.03; BDG, P = 0.01; GM, P = 0.03) and at 12 weeks (BDG+GM, P = 0.01; BDG, P = 0.03; GM, P = 0.01; GMI, P = 0.007). Similar correlations occurred for biomarkers measured over 6 weeks. Patients with negative baseline GMI and/or persistently negative GMI during the first 2 weeks were more likely to have CR and survival. These results suggest that changes of biomarkers may be informative to predict and/or assess response to therapy and survival in patients treated for IA.     

Local retrospective analysis of galactomannan cut-off values in bronchoalveolar lavage fluids for diagnosis of invasive aspergillosis

Galactomannan antigen (GM) testing has been used for decades to screen immunocompromised patients for invasive aspergillosis (IA). Recent publications suggested that using a higher cut-off value than 0.5 in bronchoalveolar lavage fluid (BALF) could be more discriminant for hematology patients. We retrospectively analyzed the values of GM in BALF over 7 years (from 2010 to 2016). Performance indicators of the GM in BALF, according to three different cut-off values (0.5, 0.8, 1.5), were calculated using Stata 14.1. IA classification for hematology patients was based on European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria, as defined in 2008. A number of 716 GM were performed on BALF from 2010 to 2016 (597 patients) and 66 were positive (>?0.5). Among these 597 patients, 27 IA were diagnosed, 13 with a positive GM in BALF, 9 with a negative GM in BALF, and 5 unclassified IA (ICU patients). The analysis of performance indicators, based on our local data, did not demonstrate any significant difference using a higher cut-off value of GM in BALF. This result may be explained by the local recruitment of patients and by pre-analytic variations during BALF realization.     

Diagnostic Performance of an Aspergillus-Specific Nested PCR Assay in Cerebrospinal Fluid Samples of Immunocompromised Patients for Detection of Central Nervous System Aspergillosis

Central nervous system (CNS) invasive aspergillosis (IA) is a fatal complication in immunocompromised patients. Confirming the diagnosis is rarely accomplished as invasive procedures are impaired by neutropenia and low platelet count. Cerebrospinal fluid (CSF) cultures or galactomannan (GM) regularly yield negative results thus suggesting the need for improving diagnostic procedures. Therefore the performance of an established Aspergillus-specific nested polymerase chain reaction assay (PCR) in CSF samples of immunocompromised patients with suspicion of CNS IA was evaluated. We identified 113 CSF samples from 55 immunocompromised patients for whom CNS aspergillosis was suspected. Of these patients 8/55 were identified as having proven/probable CNS IA while the remaining 47 patients were classified as having either possible (n = 22) or no CNS IA (n = 25). PCR positivity in CSF was observed for 8/8 proven/probable, in 4/22 possible CNS IA patients and in 2/25 NoIA patients yielding sensitivity and specificity values of 1.0 (95% CI 0.68–1) and 0.93 (95% CI 0.77–0.98) and a positive likelihood ratio of 14 and negative likelihood ratio of 0.0, respectively, thus resulting in a diagnostic odds ratio of ∞. The retrospective analysis of CSF samples from patients with suspected CNS IA yielded a high sensitivity of the nested PCR assay. PCR testing of CSF samples is recommended for patients for whom CNS IA is suspected, especially for those whose clinical condition does not allow invasive procedures as a positive PCR result makes the presence of CNS IA in that patient population highly likely.     

The Performance of Real-Time PCR, Galactomannan, and Fungal Culture in the Diagnosis of Invasive Aspergillosis in Ventilated Patients with Chronic Obstructive Pulmonary Disease (COPD)

Emerging reports have associated chronic pulmonary obstructive disease (COPD) with invasive aspergillosis (IA), particularly in patients treated with mechanical ventilation and/or corticosteroids. This is a multicentre study in which COPD patients demonstrating a new lung infiltrate while being mechanically ventilated were prospectively evaluated for the presence of IA. From the 47 patients studied, Aspergillus fumigatus was recovered in culture in two patients (4.2%). While serum galactomannan (GM) was negative for 94% of patients, GM levels in respiratory samples were >0.5, >1.0 and >1.5 for 74.5, 40.5, and 21.3% of patients, respectively. PCR was positive for 10 patients in the study but did not differentiate Aspergillus colonization from infection. The combination of PCR and GM in respiratory samples may be an interesting alternative to diagnose IA in COPD patients.     

The Role of Biomarkers for Diagnosis of and Therapeutic Decisions Related to Invasive Aspergillosis in Children

Invasive aspergillosis (IA) is a feared opportunistic infection for many immuncompromised children resulting in significant morbidity and mortality. Timely diagnosis based on traditional testing modalities and clinical evaluation has proven to be challenging. Assays designed to measure IA biomarkers such as galactomannan (GM) and beta-D-glucan (BG) represent a potential significant improvement in the ability to diagnose pediatric IA. However, as with any new diagnostic tool, clinicians need to be aware of the operating characteristics of these assays in order to apply them in the appropriate settings. Published adult and pediatric data regarding GM and BG testing are reviewed and guidance regarding the appropriate utility of these assays in children is offered.     

Diagnosis of aspergillosis: Role of proteomics

The expansion of the antifungal armamentarium and the implementation of imaging techniques and new nonculture-based fungal diagnostics (NCBFDs) have improved the survival of patients with invasive asper-gillosis (IA). However, mortality rates still remain high, possibly influenced by several pitfalls, affecting NCBFDs and reducing the window of opportunity for earlier treatment. A large body of in vitro and in vivo studies has demonstrated that several fungal proteic components are strongly immunogenic, and both the adaptive immunity and the innate branch are heavily involved in the recognition and clearance of fungal pathogens, resulting, on occasion, in a useful tool for the treatment of IA. By evaluating these studies, this review considers the possibility of exploiting either components of the innate or adaptive immunity to support the rapid and early diagnosis of IA.     

Detection of Urinary Excreted Fungal Galactomannan-like Antigens for Diagnosis of Invasive Aspergillosis

Mortality associated with invasive aspergillosis (IA) remains high, partly because of delayed diagnosis. Detection of microbial exoantigens, released in serum and other body fluids during infection, may help timely diagnosis. In course of IA, Aspergillus galactomannan (GM), a well established polysaccharide biomarker, is released in body fluids including urine. Urine is an abundant, safely collected specimen, well-suited for point-of-care (POC) testing, which could play an increasing role in screening for early disease. Our main objective was to demonstrate GM antigenuria as a clinically relevant biological phenomenon in IA and establish proof-of-concept that it could be translated to POC diagnosis. Utilizing a novel IgM monoclonal antibody (MAb476) that recognizes GM-like antigens from Aspergillus and other molds, we demonstrated antigenuria in an experimental animal IA model (guinea pig), as well as in human patients. In addition, we investigated the chemical nature of the urinary excreted antigen in human samples, characterized antigen detection in urine by immunoassays, described a putative assay inhibitor in urine, and indicated means of alleviation of the inhibition. We also designed and used a lateral flow immunochromatographic assay to detect urinary excreted antigen in a limited number of IA patient urine samples. In this study, we establish that POC diagnosis of IA based on urinary GM detection is feasible. Prospective studies will be necessary to establish the performance characteristics of an optimized device and define its optimal clinical use.     

Prevention of invasive aspergillosis in high-risk patients: Universal versus preemptive, targeted treatment

Invasive aspergillosis (IA) is associated with increased morbidity and mortality, and there is a need for better preventative and therapeutic approaches. Successful treatment of documented IA remains difficult, often because of the inability to detect disease at an early stage. An important, recent advance in the management of aspergillosis is the availability of the newer broad-spectrum azoles, voriconazole and posaconazole, which have good activity against Aspergillus spp. In addition, newer diagnostic modalities including Aspergillus galactomannan, β-glucan, and polymerase chain reaction are more readily available. These diagnostic and treatment options have made new strategies possible for the management of IA. Prophylaxis and empirical therapy for high-risk patients have been popular for decades, and now a preemptive, targeted approach to IA management has become more attractive. This article reviews strategies for the prevention and management of IA and compares and contrasts universal prophylaxis to the preemptive, targeted approach for IA in high-risk patients.     

Tratamiento antifúngico individualizado en el paciente crítico con infección fúngica invasora

Invasive candidiasis (IC) is the most common invasive fungal infection (IFI) affecting critically ill patients, followed by invasive pulmonary aspergillosis (IPA). International guidelines provide different recommendations for a first-line antifungal therapy and, in most of them, echinocandins are considered the first-line treatment for IC, and triazoles are so for the treatment of IPA. However, liposomal amphotericin B (L-AmB) is still considered a second-line therapy for both clinical entities. Although in the last decade the management of IFI has improved, several controversies persist. The antifungal drugs currently available may have a suboptimal activity, or be wrongly used in certain IFI involving critically ill patients. The aim of this review is to analyze when to provide individualized antifungal therapy to critically ill patients suffering from IFI, emphasizing the role of L-AmB. Drug-drug interactions, the clinical status, infectious foci (peritoneal candidiasis is discussed), the fungal species involved, and the need of monitoring the concentration of the antifungal drug in the patient are considered.     

21例淋巴瘤合并肺部侵袭性真菌感染的回顾性临床分析

摘要 目的：探讨淋巴瘤合并肺部侵袭性真菌感染( IPFI ) 的高危因素、临床特征和诊疗方案。方法：回顾性分析2019-2020年血液科收治的淋巴瘤合并IPFI患者的临床资料。结果：化疗后出现IPFI的淋巴瘤患者共计21例,总发病率为2.7%,平均年龄60岁,男性占77.8%,其中确诊5例、临床诊断7例、拟诊8例,确诊患者的病原菌为白色念珠菌（80%）和曲霉菌（20%）。肺部CT影像特征不典型,大多表现为弥漫、散在的斑片状絮样密度增高影、结节影,双肺受累多见,在接受一线抗真菌治疗后有18例患者缓解,总有效率为90.4%,其中原发病终末期患者及合并细菌感染患者死亡率高,预后差。结论：老年、男性、原发病控制不佳、高强度或大剂量化疗以及糖皮质激素的使用可能是淋巴瘤IPFI的高危因素,早期临床症状和影像学检查缺乏特异性,常规病原学检出率仍较低,1-3-β-D-葡聚糖试验（G试验）、半乳甘露聚糖试验（GM试验）结合肺部CT检查有助于早期诊断,一线抗真菌药物的使用可显著改善患者预后。     

Dynamic modulation of the glycosphingolipid content in supported lipid bilayers by glycolipid transfer protein

Supported lipid bilayers (SLBs) are popular models of cell membranes. Owing to the importance of glycosphingolipids (GSLs) in modulating structure and function of membranes and membrane proteins, methods to tune the GSL content in SLBs would be desirable. Glycolipid transfer protein (GLTP) can selectively transfer GSLs between membrane compartments. Using the ganglioside GM1 as a model GSL, and two mass-sensitive and label-free characterization techniques—quartz crystal microbalance with dissipation monitoring and ellipsometry—we demonstrate that GLTP is an efficient and robust biochemical tool to dynamically modulate the GSL content of SLBs up to 10 mol % GM1, and to quantitatively control the GSL content in the bulk-facing SLB leaflet. By exploiting what we believe to be a novel tool, we provide evidence that GM1 distributes highly asymmetrically in silica-supported lipid bilayers, with ∼85% of the ganglioside being present in the bulk-facing membrane leaflet. We report also that the pentameric B-subunit of cholera toxin binds with close-to-maximal stoichiometry to GM1 in SLBs over a large range of GM1 concentrations. Furthermore, we quantify the liganding affinity of GLTP for GM1 in an SLB context to be 1.5 μM.     

Reproducibility of Positive Results for the Detection of Serum Galactomannan by Platelia™ Aspergillus EIA

Galactomannan (GM) was recently included in consensus guidelines as an indirect mycological criterion for the diagnosis of invasive aspergillosis. Currently, there is an enzyme immunoassay available to detect GM in biological samples, the Platelia? Aspergillus EIA. In this study, the reproducibility of positive results obtained using this assay was evaluated using serum samples from neutropenic patients. A trend toward lower values was observed, and 55 %(27/49) of positive results were negative after retesting. A low reproducibility of positive results for the detection of GM in serum was observed.     

半乳甘露聚糖检测在圈养海洋动物曲霉病早期诊断的应用

<正>曲霉菌是广泛存在于自然界的一种腐生菌。由于曲霉菌孢子较小,在空气中漂浮,一般是通过呼吸道进入机体感染后引起曲霉病,为条件性致病菌(刘正印和王爱夏,2007)。曲霉病在人类、家畜和许多野生物种中都有发现,引起从局部感染到致命以及对吸入孢子的过敏反应。曲霉病会引起海扇珊瑚致命性感染、蜜蜂僵化、鸟类肺部和气囊感染、牛真菌流产和乳腺感染、     

Antifungal Therapy for Invasive Fungal Diseases in Allogeneic Stem Cell Transplant Recipients: An Update

Invasive fungal diseases (IFDs) remain a major cause of morbidity and mortality in allogeneic stem cell transplant (SCT) recipients. While the most common pathogens are Candida spp. and Aspergillus spp., the incidence of infections caused by non-albicans Candida species as well as molds such as Zygomycetes has increased. For many years, amphotericin B deoxycholate (AMB-D) was the only available antifungal for the treatment of IFDs. Within the past decade, there has been a surge of new antifungal agents developed and added to the therapeutic armamentarium. Lipid-based formulations of amphotericin B provide an effective and less nephrotoxic alternative to AMB-D. Voriconazole has now replaced AMB-D as first choice for primary therapy of invasive aspergillosis (IA). Another extended-spectrum triazole, posaconazole, also appears to be a promising agent in the management of zygomycosis, refractory aspergillosis, and for prophylaxis. Members of the newest antifungal class, the echinocandins, are attractive agents in select infections due to their safety profile, and are a more attractive option compared to AMB-D as initial treatment for invasive candidiasis and (based on one study) challenge fluconazole for superiority in management with this mycoses. However, challenges do exist among these newer agents in very high-risk individuals like allogeneic SCT recipients, which may include adverse drug events, drug–drug interactions, variability in oral absorption, and availability of alternative formulations. The addition of newer agents has also stimulated interest in the potential application of combination therapy in serious, life-threatening infections. However, adequate studies are not available for most IFDs; thus, the clinical use of combination therapy is not evidenced based on most cases and preciseness in its use is uncertain. Finally, therapeutic drug monitoring of select antifungals (notably posaconazole and voriconazole) may play an increasing role due to significant interpatient variability in serum concentrations after standard doses.     

Galactomannan-Based and PCR-Based Assays in Bronchoalveolar Lavage to Diagnose Invasive Aspergillosis: Current Status and Future Prospects

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication in patients receiving chemotherapy or undergoing allogeneic haematopoietic stem cell transplantation for acute leukemia. The existing tools to diagnose IPA lack specificity or sensitivity, or both; the search for improved diagnostic tools for IPA has focused on novel serologic and molecular methods. Aspergillus Galactomannan enzyme-linked immunosorbent assay (GM) analyses showed sensitivity rates in serum samples ranging in a wide span; testing GM in bronchoalveolar lavage (BAL) originated from the primary site of the infection seems to be more sensitive in patients with IPA. Other novel diagnostic markers to detect fungal DNA directly in clinical samples, rapidly, early, sensitively and specifically, are provided by polymerase chain reaction (PCR) based assays; higher sensitivity and specificity rates have been observed for BAL samples in IPA, even under antifungal treatment. The clinical place value of a diagnostic approach combining PCR and GM in BAL is unclear.     

Meta-Analysis and Cost Comparison of Empirical versus Pre-Emptive Antifungal Strategies in Hematologic Malignancy Patients with High-Risk Febrile Neutropenia

Background

Invasive fungal disease (IFD) causes significant morbidity and mortality in hematologic malignancy patients with high-risk febrile neutropenia (FN). These patients therefore often receive empirical antifungal therapy. Diagnostic test-guided pre-emptive antifungal therapy has been evaluated as an alternative treatment strategy in these patients.

Methods

We conducted an electronic search for literature comparing empirical versus pre-emptive antifungal strategies in FN among adult hematologic malignancy patients. We systematically reviewed 9 studies, including randomized-controlled trials, cohort studies, and feasibility studies. Random and fixed-effect models were used to generate pooled relative risk estimates of IFD detection, IFD-related mortality, overall mortality, and rates and duration of antifungal therapy. Heterogeneity was measured via Cochran’s Q test, I² statistic, and between study τ². Incorporating these parameters and direct costs of drugs and diagnostic testing, we constructed a comparative costing model for the two strategies. We conducted probabilistic sensitivity analysis on pooled estimates and one-way sensitivity analyses on other key parameters with uncertain estimates.

Results

Nine published studies met inclusion criteria. Compared to empirical antifungal therapy, pre-emptive strategies were associated with significantly lower antifungal exposure (RR 0.48, 95% CI 0.27–0.85) and duration without an increase in IFD-related mortality (RR 0.82, 95% CI 0.36–1.87) or overall mortality (RR 0.95, 95% CI 0.46–1.99). The pre-emptive strategy cost $324 less (95% credible interval -$291.88 to $418.65 pre-emptive compared to empirical) than the empirical approach per FN episode. However, the cost difference was influenced by relatively small changes in costs of antifungal therapy and diagnostic testing.

Conclusions

Compared to empirical antifungal therapy, pre-emptive antifungal therapy in patients with high-risk FN may decrease antifungal use without increasing mortality. We demonstrate a state of economic equipoise between empirical and diagnostic-directed pre-emptive antifungal treatment strategies, influenced by small changes in cost of antifungal therapy and diagnostic testing, in the current literature. This work emphasizes the need for optimization of existing fungal diagnostic strategies, development of more efficient diagnostic strategies, and less toxic and more cost-effective antifungals.     

Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA.

The interaction of adenovirus-2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene. Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt-sensitive activities after associating with equal efficiency to both single- and double-stranded DNA; however, a putative C-terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti-serum specific to the EIA carboxy-terminus) showed 10-fold greater affinity to double- versus single-stranded DNA. When examined with Ad2 DNA, the EIA protein had a retention that was at least 2-fold higher compared to calf thymus DNA, suggesting some substrate specificity. It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III. This suggests that the strength of the protein interaction depends on the target DNA sequence. Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA-like protein potentiated the association of the protein to double-stranded calf thymus DNA up to 7-fold. These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co-factor(s) in an indirect fashion.     

Environmental change challenges decision-making during post-market environmental monitoring of transgenic crops

The ability to decide what kind of environmental changes observed during post-market environmental monitoring of genetically modified (GM) crops represent environmental harm is an essential part of most legal frameworks regulating the commercial release of GM crops into the environment. Among others, such decisions are necessary to initiate remedial measures or to sustain claims of redress linked to environmental liability. Given that consensus on criteria to evaluate ‘environmental harm’ has not yet been found, there are a number of challenges for risk managers when interpreting GM crop monitoring data for environmental decision-making. In the present paper, we argue that the challenges in decision-making have four main causes. The first three causes relate to scientific data collection and analysis, which have methodological limits. The forth cause concerns scientific data evaluation, which is controversial among the different stakeholders involved in the debate on potential impacts of GM crops on the environment. This results in controversy how the effects of GM crops should be valued and what constitutes environmental harm. This controversy may influence decision-making about triggering corrective actions by regulators. We analyse all four challenges and propose potential strategies for addressing them. We conclude that environmental monitoring has its limits in reducing uncertainties remaining from the environmental risk assessment prior to market approval. We argue that remaining uncertainties related to adverse environmental effects of GM crops would probably be assessed in a more efficient and rigorous way during pre-market risk assessment. Risk managers should acknowledge the limits of environmental monitoring programmes as a tool for decision-making.