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ABSTRACTThe European AspergillusPCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated “in vivo” specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and ß-d-glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and ß-d-glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and ß-d-glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and ß-d-glucan proved optimal (area under the curve [AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests.

ABSTRACTThe efficacy of voriconazole is potentially compromised by considerable pharmacokinetic variability. There are increasing insights into voriconazole concentrations that are safe and effective for treatment of invasive fungal infections. Therapeutic drug monitoring is increasingly advocated. Software to aid in the individualization of dosing would be an extremely useful clinical tool. We developed software to enable the individualization of voriconazole dosing to attain predefined serum concentration targets. The process of individualized voriconazole therapy was based on concepts of Bayesian stochastic adaptive control. Multiple-model dosage design with feedback control was used to calculate dosages that achieved desired concentration targets with maximum precision. The performance of the software program was assessed using the data from 10 recipients of an allogeneic hematopoietic stem cell transplant (HSCT) receiving intravenous (i.v.) voriconazole. The program was able to model the plasma concentrations with a high level of precision, despite the wide range of concentration trajectories and interindividual pharmacokinetic variability. The voriconazole concentrations predicted after the last dosages were largely concordant with those actually measured. Simulations provided an illustration of the way in which the software can be used to adjust dosages of patients falling outside desired concentration targets. This software appears to be an extremely useful tool to further optimize voriconazole therapy and aid in therapeutic drug monitoring. Further prospective studies are now required to define the utility of the controller in daily clinical practice.

Single-nucleotide polymorphisms (SNPs) appear to influence the risk of invasive mould disease (IMD) in immunocompromised patients. This raises the question of whether genetic risk prediction can be used to alter clinical practice. This review focuses on the current status of genetic association studies regarding invasive fungal disease among hematology patients, with an emphasis on IMD.

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

Host-pathogen interactions at epithelial barriers play an important role in health and disease. This also applies to the clinical setting of stem cell transplantation (SCT) in which deregulated sensing of microbes and their cell wall components by pattern recognition receptors (PRRs) can contribute to inflammatory and infectious complications. The role of Candida species herein has recently been rediscovered since a 'loss-of-function' Y238X polymorphism in dectin-1, a C-type lectin receptor recognizing the β-1,3-glucan motif of Candida,resulted in diminished membrane expression and lower cytokine responses upon β-1,3-glucan recognition, and was associated with increased Candida colonization of SCT recipients, rendering them at risk for candidaemia. In addition, Candida colonization was associated with an increased incidence of acute graft-versus-host disease (GvHD), but only in those individuals with the wild-type dectin-1 allele. The Th17 mediated immune responses might provide a common link in these processes, as they have recently been implicated in anti-Candidaimmunity as well as the pathogenesis of GvHD. These new insights suggest that immunogenetics could contribute to a more individualized risk-based strategy for managing SCT recipients, for example concerning antifungal prophylaxis. In addition, modulating host-pathogen interactions by selectively modulating PRR activity could be exploited in SCT to achieve better outcomes.

ABSTRACTPCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European AspergillusPCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the AspergillusPCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as “compliant” or “noncompliant,” according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 µl showed a negative correlation with sensitivity. The efficiency of the AspergillusPCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (=3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 µl.

Voriconazole is a triazole antifungal developed for the treatment of life-threatening fungal infections in immunocompromised patients. The drug, which is available for both oral and intravenous administration, has broad-spectrum activity against pathogenic yeasts, dimorphic fungi, and opportunistic molds. Voriconazole has a nonlinear pharmacokinetic profile with a wide inter- and intraindividual variety. This variability is caused by many factors such as gender, age, genotypic variation, liver dysfunction, the presence of food, and so on. Another important factor influencing voriconazole's pharmacokinetic profile is drug-drug interactions with CYP450 inhibitors as well as inducers. Variability in plasma concentrations, as a result of the previously mentioned aspects, may lead to variability in efficacy or toxicity. Determination of plasma concentrations is indicated in situations to guide dosing and to individualize and improve the treatment options resulting in better therapeutic outcome or fewer side effects. In this article, we review factors influencing voriconazole pharmacokinetic profile, the data supporting exposure-effect and exposure-toxicity relationships, review the gaps in current knowledge, which make broad recommendations for therapeutic drug monitoring difficult for voriconazole, provide the indications in which therapeutic drug monitoring is reasonable based on currently available data (eg, children), and outline the ways in which this problem could be solved. We provide a summary of the problem so that further research can be conducted to address this are of clinical need.

ABSTRACTDetection of circulating galactofuranose (galf) antigens, including galactomannan (GM), by the Platelia Aspergillus(PA) enzyme-linked immunosorbent assay (ELISA) is an important tool in the early diagnosis of invasive aspergillosis (IA). We used a modified pretreatment technique (MT) on consecutive negative PA ELISA plasma samples from IA patients in order to improve the detection of the fungal components present. Plasma samples (52) were collected from healthy donors, and 174 plasma samples with a galactomannan index (GMI) below 0.5 were collected from 25 unclassifiable and 23 IA patients. The PA ELISA reactivity of pretreated samples was determined before (conventional technique [CT]) and after (MT) filtration using a Microcon filter with a 50-kDa cutoff (Millipore). For the MT, the sensitivity of the PA ELISA increased from 42.9% (CT) to 78.6% (MT) using a cutoff for the GMI of 1.5 in the probable and proven group, whereas specificity slightly decreased from 98.7% to 96.1% in the control group. The 10-fold concentration step increased the GMI as high as 121-fold. The MT resulted not only in positive reactivity in samples that tested negative with the CT but also in the earlier detection of antigen by 2 to 17 days.

ABSTRACTWe report on the correlation of the outcomes for two cohorts of patients who had been treated for candidemia (126 episodes) or oropharyngeal candidiasis (110 episodes) with various doses of fluconazole and the MIC of fluconazole obtained by using the EUCAST standard for fermentative yeasts. Of 145 episodes caused by an isolate with a fluconazole MIC ≤2 mg/liter, 93.7% (136 of 145) responded to fluconazole treatment. The response for those infected with a strain with a MIC of 4 mg/liter was 66% but reached 100% when the dose was greater than 100 mg/day, whereas the response for those infected with strains with MICs ≥8 mg/liter was only 12%. Hence, a MIC of 2 mg/liter or 4 mg/liter was able to predict successful treatment. A cure rate of 93.9% (140 of 149) was achieved when the dose/MIC ratio was ≥100 but fell to 14.6% (16 of 109) when the ratio was less. The dose/MIC required to achieve a response rate of 50% (the 50% effective concentration) was 43.7 for the cohort of patients with oropharyngeal candidiasis. Classification and regression analysis indicated that a dose/MIC of 35.5 was the threshold for the prediction of cure or failure. However, an increase in exposure above this threshold further increased the probability of cure, and all patients were cured when the dose/MIC exceeded 100. Monte Carlo simulations showed a probability of target attainment of 99% at MICs ≤2 mg/liter and a pharmacodynamic target of a dose/MIC ratio of 100, which was equivalent to an unbound fraction of the fluconazole area under the curve versus the MIC of 79.

BACKGROUND: Pharmacodynamic investigations with antimicrobials define the relationship between the infecting organism and achievable drug concentrations with clinical outcome.OBJECTIVE: To examine this relationship for meropenem in a population of patients who are at high risk of infection-related morbidity and mortality.METHODS: The study was a retrospective analysis of a multicenter, randomized, blinded clinical trial. A population-based predictive model was created using data from adults with febrile neutropenia and the nonparametric modeling program, NPEM. Patient age, body weight, and serum creatinine level were covariates in the model used to predict unbound concentrations for each patient. Pathogen susceptibility was estimated using product literature minimum inhibitory concentrations for effectiveness against 50% of microorganisms (MIC50) for specific organisms. The pharmacodynamic index of percent time above MIC (% T>MIC) was analyzed for its association with clinical outcome.RESULTS: A 2-compartment pharmacokinetic model using patient covariates of body weight and renal function best described the pharmacokinetics of meropenem in febrile neutropenic patients. Sixty patients with confirmed gram-positive or -negative bacteremia were studied. An average of 83% T>MIC was identified for the 42 clinical responders compared with 59% T>MIC for the 18 nonresponders (p = 0.04). An 80% clinical response rate was evident when the % T>MIC for meropenem exceeded 75% of the dosing interval (p = 0.01).CONCLUSIONS: To our knowledge, this is the first published report of a relationship between a pharmacodynamic index and clinical outcome in a febrile neutropenic population. Based on this relationship, dosing with intravenous meropenem 500 mg every 6 hours is predicted to be comparable to the currently recommended 1 g every 8 hours for serious infections. Our model provides further justification for a prospective clinical trial to evaluate a pharmacodynamically targeted meropenem dosing schedule as to its ability to improve clinical outcome in these patients.

Intestinal mucositis is an important cause of cancer treatment‐related morbidity and mortality, carrying a serious economic burden. Currently, objective parameters are lacking that would enable the monitoring of gut damage in routine clinical practice, thus hindering the development of clinical studies designed to investigate potential new strategies aimed at reducing or preventing this side effect. The authors investigated the characteristics of serum citrulline concentration compared with sugar permeability tests with respect to its use as a marker for cancer treatment‐induced small bowel injury.In this prospective study, 10 patients with hematologic malignancies who were receiving myeloablative therapy had gut toxicity assessed with sugar permeability tests. Serum citrulline concentrations also were determined using archival serum samples. The association between both parameters and their respective characteristics were analyzed and compared with data from the literature.Sensitivity and specificity were better for the citrulline assay compared with sugar permeability tests. Maximum gut damage assessed with the citrulline assay was observed 1–2 weeks earlier compared with the sugar permeability test. Similarly, citrulline indicated recovery of gut damage at 3 weeks after transplantation, whereas most sugar permeability tests remained abnormal.The simplicity of the method, the low costs, and the lack of drawbacks to the method make the citrulline assay the first choice for measuring and monitoring treatment‐related gut damage and provides an objective parameter for cancer treatment‐related gut toxicity. Cancer 2005. © 2004 American Cancer Society.

A frequent complication of anticancer treatment, oral and gastrointestinal (GI) mucositis, threatens the effectiveness of therapy because it leads to dose reductions, increases healthcare costs, and impairs patients' quality of life. The Multinational Association of Supportive Care in Cancer and the International Society for Oral Oncology assembled an international multidisciplinary panel of experts to create clinical practice guidelines for the prevention, evaluation, and treatment of mucositis. The panelists examined medical literature published from January 1966 through May 2002, presented their findings at two separate conferences, and then created a writing committee that produced two articles: the current study and another that codifies the clinical implications of the panel's findings in practice guidelines. New evidence supports the view that oral mucositis is a complex process involving all the tissues and cellular elements of the mucosa. Other findings suggest that some aspects of mucositis risk may be determined genetically. GI proapoptotic and antiapoptotic gene levels change along the GI tract, perhaps explaining differences in the frequency with which mucositis occurs at different sites. Studies of mucositis incidence in clinical trials by quality and using meta-analysis techniques produced estimates of incidence that are presented herein for what to our knowledge may be a broader range of cancers than ever presented before. Understanding the pathobiology of mucositis, its incidence, and scoring are essential for progress in research and care directed at this common side-effect of anticancer therapies. Cancer 2004;100(9 Suppl):1995–2025. © 2004 American Cancer Society.

ABSTRACTThe susceptibilities of 13 clinical isolates of Scedosporium apiospermumand 55 clinical isolates of S. prolificansto new and conventional drugs belonging to three different classes of antifungal agents, the azoles (miconazole, itraconazole, voriconazole, UR-9825, posaconazole), the polyenes (amphotericin B, nystatin and liposomal nystatin), and allylamines (terbinafine), were studied by use of proposed standard M38-P of NCCLS. Low growth-inhibitory antifungal activities were found in vitro for most of the drugs tested against S. prolificansisolates, with the MICs at which 90% of isolates are inhibited (MIC90s) being >8 μg/ml; the MIC90s of voriconazole and UR-9825, however, were 4 μg/ml. S. apiospermumisolates were more susceptible in vitro, with the highest activity exhibited by voriconazole (MIC90s, 0.5 μg/ml), followed by miconazole (MIC90s, 1 μg/ml), UR-9825 and posaconazole (MIC90s, 2 μg/ml), and itraconazole (MIC90s, 4 μg/ml). The MICs of terbinafine, amphotericin B, and the two formulations of nystatin (for which no statistically significant differences in antifungal activities were found for the two species) for S. apiospermumisolates were high. Cross-resistance was observed among all the azoles except posaconazole and among all the polyenes except the lipid formulation. A distribution analysis was performed with the MICs of each drug and for each species. Bimodal and skewed MIC distributions were obtained, and cutoffs indicating the borders of different MIC subpopulations of the distributions were determined on the basis of the normal plot technique. These cutoffs were in many cases reproducible between 48 and 72 h.

ABSTRACTA colorimetric assay for antifungal susceptibility testing ofAspergillusspecies (Aspergillus fumigatus,Aspergillus flavus,Aspergillus terreus,Aspergillus nidulans, and Aspergillus ustus) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 µg of XTT/ml with 25 µM menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 102CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.

To investigate whether or not the considerable regional differences in prescribing further modifications to initial empirical regimen were due to differences in the patient populations or were the result of other factors we re-analysed the data from a large multicentre study of monotherapy for the empirical treatment of febrile, neutropenic patients. We matched 151 episodes treated in 14 centres in Europe with the same number treated in 17 centres in North America for age > 46 years, gender, acute leukaemia, antibacterial prophylaxis, intravascular catheter use, receipt of ceftazidime or piperacillin plus tobramycin which were the regimens in the original study, the presence of bacteraemia at the onset of fever and the occurrence of a focus of infection within the first 3 days of empirical therapy. The initial regimen was in fact unchanged and successful after 72h for 74% of episodes treated in European centres and 45% of those treated in North American centres (p<.0001) and 38% and 21% of episodes respectively were managed successfully without any modification of therapy (p=.0044). Amphotericin and acyclovir were given in 35% and 11% of episodes treated in European centres compared with 53% and 25% of episodes treated in North American centres (p=.0053 and.0029). Moreover, empirical therapy was changed after a mean 4.8 days (95% Cl± 0.8) in North American centres but after 7.1 days (95%CI±1.0) in European centres (p=.0004) and there were 2.3±0.3 changes to therapy compared with 1.5±0.3 changes respectively (p=.0002). However there was no significant difference in survival (97% in and 96%), in the number of episodes complicated by a subsequent infection (10% and 11%), in the incidence of other adverse event (11% and 10%) nor in the proportion of episodes in which fever persisted longer than 5 days (53% and 48%) in North American centres and European centres respectively. The duration of fever (8.4±1.4 days and 7.1 ±1.2 days), antimicrobial therapy (19.8±1.9 days and 16.3±1.5 days) and neutropenia <0.5×109/L (19.9±2.9 days and 16.8±2.1 days) were also similar for North American centres and European centres respectively. These results suggest a genuine regional difference in medical practice does, in fact, exist.

Patterns of bacterial infection have remained fairly static with Gram-positive bacteria predominating, although this varies between institutions and between patient populations. Similarly, antimicrobial resistance rates differ widely and reflect antibiotic use and exposure. Hence, each institution should devote microbiological resources to maintain surveillance and employ vigilance so that antimicrobial treatment protocols can be adapted to meet any new challenges. Most changes are being seen in the diagnosis of fungal infections, particularly aspergillosis, and the way in which it is being incorporated into treatment strategies.

ABSTRACTThe susceptibility of 30 clinical isolates belonging to six different species of filamentous fungi (Aspergillus fumigatus, Aspergillus flavus, Scedosporium prolificans, Scedosporium apiospermum, Fusarium solani, and Fusarium oxysporum) was tested against six antifungal drugs (miconazole, voriconazole, itraconazole, UR9825, terbinafine, and amphotericin B) with the microdilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (M38-P). The MICs were compared with the MICs obtained by a colorimetric method measuring the reduction of the dye 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan by viable fungi. The levels of agreement between the two methods were 96 and 92% for MIC-0 (clear wells) and MIC-1 (75% growth reduction), respectively. The levels of agreement were always higher for Aspergillusspp. (97% ± 2.5%), followed byScedosporiumspp. (87% ± 10.3%) and Fusariumspp. (78% ± 7.8%). The NCCLS method was more reproducible than the MTT method: 98 versus 95% for MIC-0 and 97 versus 90% for MIC-1. However, the percentage of hyphal growth as determined visually by the NCCLS method showed several discrepancies when they were compared with the percentages of MTT reduction. A new simplified assay that incorporates the dye MTT with the initial inoculum and in which the fungi are incubated with the dye for 48 h or more was developed, showing comparable levels of agreement and reproducibility with the other two methods. Furthermore, the new assay was easier to perform and more sensitive than the MTT method.

Empiric therapy is necessary for febrile, neutropenic patients in order to minimise morbidity and mortality. Certain agents are now available for monotherapy which offcrcomparable success to combinations of either an aminoglycoside with a β-lactam or two β-lactams. However, no regimen offers complete treatment under all circumstances in all patients. It is also apparent that febrile, neutropenic patients comprise a more heterogeneous group than just those with bacteraemia, clinically apparent infection and unexplained fever. Localized infections occur in just under a third of cases at the onset of fever and a similar number will develop during the course of fever. Mortality is higher in infections that are accompanied by bacteraemia and also those that develop subsequently, especially when related to the lung. The aetiological agent also differs with each type of infection as does the duration of fever and symptoms. Consequently modifications are required more often. The length of treatment may also differ. Therefore, during the first 3-4 days of empiric therapy, every effort should be made to identify incipient localized infections in addition to detecting bacteraemia. Changes in therapy can then be based on objective grounds rather than continued fever offering more patients individual treatment than is possible when relying only on the temperature chart.

Three hundred episodes of neutropenia were reviewed for the occurrence of potential sites of infection. Ninety sites (30 per cent) were identified at the onset of fever independent of initial bacteraemia which was encountered in 104 episodes (35%) and predominantly involved Gram-positive cocci. A further 90 sites were recorded involving mainly the lower respiratory tract (58%) and skin and soft tissue (18%). These changes evolved significantly later (mean of 5.1 and 4.3 days respectively) than did other foci which mainly presented at the onset of fever (p < 0.01). However the infectious aetiology was established in only 54 cases overall with fungi being responsible for 25 of 45 cases of lower respiratory tract infections with a known microbiological aetiology. The mortality associated with initial bacteraemia, focus at onset and unexplained fever was 11-14% while that associated with the development of a subsequent focus was 28% with lung infiltrates carrying the worst prognosis. Therefore rather than being seen as a final solution for possible infectious complications, empiric therapy provides an opportunity for daily review of the patient thereby increasing the likelihood of both explaining initial fever and diagnosing subsequent infection.

In recent years there has been some progress in diagnosing invasive aspergillosis at an early stage of disease. Polymerase chain reaction (PCR) assays are more sensitive than culture for detecting Aspergillus sp in bronchoalveolar lavage (BAD fluid specimens but diagnostic reliability may be diminished by false-positive reactions when patients are colonized with Aspergillus conidia or by laboratory contamination. Aspergillus antigens can be detected with a rapid, commercially-available, latex-agglutination (LA) test which employs the monoclonal antibody EB-A2 raised against Aspergillus galactomannan. The sensitivity of this test was found to be 38–95, and positive test results were obtained only during advanced stages of disease. Recently, a sandwich ELISA has been developed which employs the same monoclonal antibody but was almost 10 times more sensitive than the LA test. This ELISA test yielded a sensitivity of 90 when a series of serum samples was employed. Furthermore, Aspergillus galactomannan was detected in the serum at an earlier stage of infection than that detected by the LA test. The development of sensitive diagnostic methods such as these offers an opportunity for optimizing diagnosis and improving the management of invasive aspergillosis. By allowing selective targeting of patients for antifungal therapy to pre-empt invasive aspergillosis, the outlook for patients at risk should be improved in terms of both morbidity and mortality.

We studied the efficacy and safety of itraconazole for the prevention of fungal infection in neutropenic patients given cytotoxic chemotherapy for hematologic malignancies. Patients were randomly allocated to receive either itraconazole (200 mg bd) or placebo in addition to oral amphotericin B until the patient either developed fungal infection or had completed an-tileukemic treatment. Forty six patients (83 neutropenic episodes) treated with itraconazole and 46 placebo treated patients (84 neutropenic episodes) were evaluable. No specific toxicity was noted. Nine fungal infections developed in the itraconazole group, of which four were histologically or microbiologically proven and 15 in the patients given placebo (eight proven) (p > 0.12). All these patients received IV amphotericin B. The incidence of Candida albicans infections tended to be lower in the itraconazole group, but overall, there was no measurable improvement in the prevention of fungal infections and mortality by itraconazole.

New and unusual pathogens continue to cause infection in the immunocompromised patient, although common organisms still predominante. Protozoal infections are usually lethal, but infrequent, posing a dilemma as to who should receive prophylaxis and when. Infections caused by both common and rare bacteria, however, can be treated successfully in the vast majority of cases, making the need for prophylaxis less compelling. Viral infections are usually self-limiting, although those due to cytomegalovirus are sufficiently life threatening to make pre-emptive or early therapy necessary. By making the most of the limited diagnostic measures at our disposal and the finite number of antifungal agents available (the efficacy of which might be imporved by giving granlocyte-macrphage colony stimulating factor adjunctively), this approach may prove the most effective in managing deep-seated fungal infections involving the mould.

Fever and infection remain the most frequent complications arising during neutropenia. Many strategies and schemes have been proposed for prevention, but as yet, there is no consensus on the optimum scheme, or which patient would gain the most benefit. A majority of fevers remain unexplained, underscoring the limitations of present diagnostic approaches. New insights into sepsis and its manifestations, perhaps combined with C-reactive protein, may help to better discriminate febrile episodes. Blood cultures and other diagnostic tests provide information only after onset of fever, thus initial treatment is empiric. While combination therapy is regarded by many as being the standard, evidence continues to accumulate that shows monotherapy to be a viable alternative. It is also clear that many patients will require modification of treatment during neutropenia, but this should be guided by daily clinical review and the most recent diagnostic information.

33 consecutive allogeneic bone marrow transplant recipients who were likely to develop streptococcal bacteraemia were treated for 5 days with clindamycin (900 mg i.v. t.d.s.) and ceftazidime (2 g t.d.s.) for the initial management of fever associated with severe oral mucositis. Bacteraemia due to ‘viridans’ streptococci was encountered in 23 cases (70) as mucositis progressed to peak severity and occurred a day before fever in 8 cases. At the end of treatment with clindamycin only 2 patients had defervesced although the streptococci were successfully eradicated. C-reactive protein (CRP) levels continued to rise in 18 cases and declined by more than 10 in only 7 cases. Severe oromucositis rather than infection appeared to induce an acute phase response with fever suggesting bacteraemia due to ‘viridans’ streptococci to have been a consequence of mucosal damage. Indeed, oromucositis was the only primary focus of inflammation in 22 patients and only after its resolution did both fever and CRP levels diminish. By then, patients had also begun to recover from granulocytopenia. These data indicate that rather than including a specific antimicrobial like clindamycin in an empirical regimen, it would be more beneficial to evolve strategies that minimise mucosal damage in this patient population.

ABSTRACTTwelve laboratories evaluated candidate material for an AspergillusDNA calibrator. The DNA material was quantified using limiting-dilution analysis; the mean concentration was determined to be 1.73 × 1010units/ml. The calibrator can be used to standardize aspergillosis diagnostic assays which detect and/or quantify nucleic acid.

ABSTRACTA standardized protocol for extracting DNA from Aspergillus fumigatushas been proposed by the European AspergillusPCR Initiative (EAPCRI). Using meta-regression analysis, the EAPCRI showed certain stages of the process to be critical to providing a satisfactory analytical sensitivity. The study investigated each step of the EAPCRI protocol by elimination and monitored the influence on AspergillusPCR performance.

ABSTRACTThe European Committee on Antimicrobial Susceptibility Testing (EUCAST) Subcommittee on Antifungal Susceptibility Testing recently published a standard for determining the susceptibility of fermentative yeasts to antifungals. From the beginning, the EUCAST and its North American counterpart, the CLSI, decided to work together in order to establish common standards. As part of this exercise, the susceptibility of a set of 475 yeast isolates was tested by both standards. The intraclass correlation coefficient and the equations defining the linear regression between both methods were estimated. Both methods produced very similar results, with an intraclass correlation coefficient of 0.954 (0.945 to 0.962), although linear regression analysis shows that the EUCAST standard resulted in slightly lower MICs. There were only eight isolates showing at least four twofold dilution MIC differences between both standards. After 24 h of incubation, the MICs obtained by the CLSI method were equivalent to those obtained by the EUCAST standard. In summary, both methods produce very similar MICs, indicating that methodology does not pose any obstacle to obtaining uniform standards for antifungal susceptibility testing of yeasts.

ABSTRACTThe fluconazole MIC distributions for Candida glabratafrom testing 34 different clinical isolates and performing 51 tests on a single isolate mirrored each other. Since what is perceived as biological variation in isolates without resistance mechanisms is mainly methodological variation, breakpoints which divide this distribution not only lack a sound biological basis but also result in poor reproducibility of susceptibility characterization. This makes 2, 4, 8, and possibly 16 μg/ml unsuitable breakpoints for C. glabrataand fluconazole.

Cornely: Gilead Sciences: Consultancy, Research Funding, Speakers Bureau. Off Label Use: Liposomal Amphotericin B (AmBisome®) is a broad spectrum antifungal agent indicated for the treatment of severe systemic and/or deep mycoses, empiric treatment of presumed fungal infections in febrile neutropenic patients and treatment of visceral leishmaniasis. In this study the efficacy, safety and tolerability of prophylactic liposomal amphotericin B (AmBisome®) was explored for the prevention of invasive fungal infections in subjects receiving remission-induction chemotherapy for Acute Lymphoblastic Leukaemia (the AmBiGuard trial).. Leguay:Gilead Sciences: Research Funding. Maertens:Gilead Sciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Castagnola:Gilead Sciences: Research Funding. Anagnostopoulos:Gilead Sciences: Research Funding. Allione:Gilead Sciences: Research Funding. Heussel:Gilead Sciences: Consultancy, Speakers Bureau. Donnelly:Astellas, Gilead, Merck, Pfizer: Consultancy, Honoraria, Research Funding. Agrawal:Gilead, Pfizer, Astellas, MSD, Bio-Rad: Honoraria, Research Funding. Garner:Gilead Sciences: Employment, Equity Ownership. Simcock:Gilead Sciences: Employment, Equity Ownership. Hawkins:Gilead Sciences: Employment, Equity Ownership. Goekbuget:Gilead Sciences: Consultancy, Honoraria.

Background:Adult patients undergoing remission-induction (RI) chemotherapy for newly diagnosed acute lymphoblastic leukaemia (ALL) are at high risk of invasive fungal disease (IFD), particularly invasive aspergillosis. The prevalence of IFD in this population has not been widely studied. There is currently no approved standard for antifungal prophylaxis and it is recommended by the European Working Group for Adult ALL that azole antifungal agents be avoided because of drug interactions with vincristine, a standard component of induction chemotherapy regimens.

Background: Infection remains a leading cause of morbidity and mortality in hematopoetic stem cell transplant (HSCT) recipients. The objective of the present study was to identify the relationship between C-reactive protein (CRP), serum citrulline as an indicator of gut mucosal barrier injury, and bacteremia in patients undergoing autologous peripheral blood (PB)HSCT for multiple myeloma. Methods: 29 consecutive patients given high dose melphalan as conditioning therapy for a PBHSCT according to a national protocol from May 2004 to May 2005 were included in the analysis. Blood cultures were obtained at least twice weekly during three weeks of hospital admission; bacteremia was defined by any positive blood culture. SPSS for Windows version 12 was used to perform statistical calculations. CRP values were transformed to log10 whereas the actual citrulline values were used. Results: The mean age of the patients was 55 (range 33–65) years and the mean length of hospital stay was 23 (17–34) days. Blood cultures of 19 patients yielded bacterial growth. For the overall study population a specific pattern of citrulline levels that was not correlated with the occurrence of bacteremia was observed. At baseline the levels (mean ± SD) amounted to 25.4 ± 6.2 micromol/L, after 1 week 14.1 ± 4.7 micromol/L, after 2 weeks 7.1 ± 3.5 micromol/L and after 3 weeks 9.3 ± 5.2 micromol/L. The corresponding CRP levels showed a high SD pointing at a patient-specific level. The respective values (mean ± SD) were: 5 ± 3 mg/L at baseline, 8 ± 10 mg/L lst week, 95 ± 86 mg/L 2nd week and 47 ± 83 mg/L 3rd week. A strong correlation (p<0.01) was found between positive blood cultures and low citrulline (7.6 ± 3.8 micromol/L); for CRP such a relation was not established. Conclusion: The occurrence of bacteremia coincides with low serum citrulline levels, but not with CRP levels. Extending our observation might be of help to establish a lower limit of citrulline levels that is indicative of an imminent bacteremia.

Background:

hLF1-11, comprising the N-terminal 11 AA of the human lactoferrin protein, has previously been shown to have a broad antimicrobial spectrum. The efficacy towards several strains of bacteria and fungi was demonstrated in in vitro and in vivo models. At this moment, hLF1-11 is in clinical development for use as broad spectrum anti-fungal and anti-bacterial prophylaxis in hematopoetic stem cell transplantation (HSCT) patients. There is data to support that the hLF1-11 mechanism of action is not restricted to direct killing of the pathogen, but that the indirect, immune modulatory and immune activating properties are important for hLF1-11 efficacy in vivo at low doses. In earlier studies, efficacy of hLF1-11 was shown in neutropenic animals systemically infected with Candida albicans. In the present studies antimicrobial efficacy and safety of hLF1-11 was tested in animal models that resemble the immune compromised state of HSCT patients shortly after transplantation. In cyclophosphamid and cyclosporin treated animals, hLF1-11 significantly reduced intra muscular infection with methicillin resistant Staphylococcus aureus (MRSA) when injected 24 hours after infection. In the same model hLF1-11 showed prophylactic properties in that it was effective even when administered as a single injection 24 hours before local infection with MRSA. In order to assess drug-drug interactions of hLF1-11 with common antibiotics, hLF1-11 was co administered with either vancomycin or ciprofloxacin in the local MRSA infection. Data from these models show that hLF1-11 is effective in combination with standard antibiotic and immunosuppressive care in a HSCT setting. Moreover, the hLF1-11 peptide does not inhibit antibiotic activity of currently used antibiotics. Besides these preclinical safety data, clinical safety was evaluated in a single and multiple ascending dose study in human volunteers. During these studies pharmacokinetic and pharmacodynamic parameters were measured and no drug related adverse effects were observed. Now, preparations are made to evaluate safety and efficacy of hLF1-11 in autologous and allogenic HSCT patients as anti-fungal and anti-bacterial prophylaxis. In conclusion, hLF1-11 is a promising peptide to prevent and treat opportunistic infections in HSCT patients and other immune suppressed patient populations.

hLF1-11, comprising the N-terminal 11 AA of the human lactoferrin protein, has previously been shown to have a broad antimicrobial spectrum. The efficacy towards several strains of bacteria and fungi was demonstrated in in vitroand in vivomodels. At this moment, hLF1-11 is in clinical development for use as broad spectrum anti-fungal and anti-bacterial prophylaxis in hematopoetic stem cell transplantation (HSCT) patients. There is data to support that the hLF1-11 mechanism of action is not restricted to direct killing of the pathogen, but that the indirect, immune modulatory and immune activating properties are important for hLF1-11 efficacy in vivoat low doses. In earlier studies, efficacy of hLF1-11 was shown in neutropenic animals systemically infected with Candida albicans. In the present studies antimicrobial efficacy and safety of hLF1-11 was tested in animal models that resemble the immune compromised state of HSCT patients shortly after transplantation. In cyclophosphamid and cyclosporin treated animals, hLF1-11 significantly reduced intra muscular infection with methicillin resistant Staphylococcus aureus(MRSA) when injected 24 hours after infection. In the same model hLF1-11 showed prophylactic properties in that it was effective even when administered as a single injection 24 hours before local infection with MRSA. In order to assess drug-drug interactions of hLF1-11 with common antibiotics, hLF1-11 was co administered with either vancomycin or ciprofloxacin in the local MRSA infection. Data from these models show that hLF1-11 is effective in combination with standard antibiotic and immunosuppressive care in a HSCT setting. Moreover, the hLF1-11 peptide does not inhibit antibiotic activity of currently used antibiotics. Besides these preclinical safety data, clinical safety was evaluated in a single and multiple ascending dose study in human volunteers. During these studies pharmacokinetic and pharmacodynamic parameters were measured and no drug related adverse effects were observed. Now, preparations are made to evaluate safety and efficacy of hLF1-11 in autologous and allogenic HSCT patients as anti-fungal and anti-bacterial prophylaxis. In conclusion, hLF1-11 is a promising peptide to prevent and treat opportunistic infections in HSCT patients and other immune suppressed patient populations.

We devised a diagnostic approach based on screening plasma for an Aspergillus antigen with use of a sandwich enzyme-linked immunosorbent assay (ELISA), thoracic computed tomographic scanning, and radionuclide imaging for managing patients at risk for invasive aspergillosis. We used a decision analytic model to compare this alternative strategy with the conventional strategy, which relies only on the presence of clinical symptoms, persistent fever, and chest roentgenographic findings. Use of the alternative strategy reduced the number of patients who would receive antifungal treatment empirically, but this strategy was more expensive. The specificity of the sandwich ELISA had a significant impact on cost, but the sensitivity did not. A 13% prevalence of infection resulted in equal costs for both strategies. As much as 43.3% of the patients treated empirically could be given liposomal amphotericin B (L-AmB) before the conventional strategy became the most expensive. The costs of the alternative strategy were less than those of the conventional strategy when >5.3% of all patients, irrespective of strategy, were treated with L-AmB.