Detection Of Invasive Pulmonary Aspergillosis Research Articles

Improved diagnostic markers for invasive pulmonary aspergillosis in COPD patients.

The prevalence of invasive pulmonary aspergillosis (IPA) among patients with chronic obstructive pulmonary disease (COPD) is steadily increasing, leading to high mortality. Although early diagnosis can significantly reduce mortality, the efficacy of current diagnostic methods is limited. Consequently, there is a need for novel approaches for early IPA detection. This retrospective study involved 383 hospitalized COPD patients with GOLD stages III and IV. The IPA group (67 patients) and non-IPA group (316 patients) were identified at the First Affiliated Hospital of Guangzhou Medical University between January 2016 and February 2022. We analyzed common serological indicators in our hospital to identify predictive indicators for the early diagnosis of IPA in COPD patients. The sensitivity and specificity of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), procalcitonin (PCT), lactate dehydrogenase (LDH), and ceruloplasmin (CER) for diagnosing IPA in COPD patients were as follows: CRP (91.2%, 57.7%), ESR (77.5%, 73.0%), PCT (60.5%, 71.4%), LDH (50.0%, 88.8%), and CER (60.7%, 74.3%). Combinations of biomarkers, such as CRP-ESR, CRP-LDH, ESR-LDH, ESR-CER, and LDH-CER, showed promising diagnostic potential, with larger area under the curve (AUC) values for IPA diagnosis in COPD patients. However, no statistically significant difference was observed between the diagnostic efficacy of single biomarkers and combined biomarkers. Notably, compared to those in the unassisted ventilation group, the patients in the assisted ventilation group (including noninvasive ventilation and tracheal intubation/incision-assisted ventilation group) exhibited significantly greater PCT and LDH levels, while the CER significantly decreased (p=0.021). There were no significant differences in biomarker levels between the ICU group and the non-ICU group. CRP (p<0.01), ESR (p=0.028), PCT (p<0.01), and CER (p<0.01) were positively correlated with hospitalization duration, whereas LDH was not correlated with hospitalization duration. Our study highlights the diagnostic potential of CRP, ESR, PCT, LDH, and CER for IPA in COPD patients. CRP and LDH can also initially predict the need for assisted ventilation, while CRP can initially estimate the length of hospitalization. This study represents the first report of the potential of CER for diagnosing IPA, suggesting its significance for further research.

Galactomannan-Antigen Testing from Non-Directed Bronchial Lavage for Rapid Detection of Invasive Pulmonary Aspergillosis in Critically Ill Patients: A Proof-of-Concept Study.

Invasive pulmonary aspergillosis is associated with high mortality. For diagnosis, galactomannan-antigen in serum and bronchoalveolar lavage fluid is recommended, with higher sensitivity in bronchoalveolar lavage fluid. Because of invasiveness, bronchoalveolar lavage might be withheld due to patients' or technical limitations, leading to a delay in diagnosis while early diagnosis is crucial for patient outcome. To address this problem, we performed an analysis of patient characteristics of intubated patients with invasive pulmonary aspergillosis with comparison of galactomannan-antigen testing between non-directed bronchial lavage (NBL) and bronchoalveolar lavage fluid. A total of 32 intubated ICU patients with suspected invasive pulmonary aspergillosis could be identified. Mycological cultures were positive in 37.5% for A. fumigatus. Galactomannan-antigen in NBL (ODI 4.3 ± 2.4) and bronchoalveolar lavage fluid (ODI 3.6 ± 2.2) showed consistent results (p-value 0.697). Galactomannan-antigen testing for detection of invasive pulmonary aspergillosis using deep tracheal secretion showed comparable results to bronchoalveolar lavage fluid. Because of widespread availability in intubated patients, galactomannan-antigen from NBL can be used as a screening parameter in critical risk groups with high pretest probability for invasive aspergillosis to accelerate diagnosis and initiation of treatment. Bronchoalveolar lavage remains the gold standard for diagnosis of invasive aspergillosis to be completed to confirm diagnosis, but results from NBL remove time sensitivity.

Diagnostics of Pulmonary Aspergillosis in Critically Ill Hematological and Non-Hematological Patients in the Intensive Care Unit By Combined Use of Galactomannan, 1-3-Beta-D-Glucan, Aspergillus Specific PCR and Conventional Culture in Concurrent Blood and Bronchoalveolar Lavage Samples — Results of a Multicenter Prospective Pilot Study

Invasive pulmonary aspergillosis (IPA) is a life-threatening infectious complication mainly affecting immunocompromised hematological patients. Therapeutic success requires early and precise diagnostics. Few data are available on the performance of current diagnostic tests in patients admitted to the intensive care unit (ICU) as the majority of diagnostic studies is conducted in hematological patients and results cannot readily be transferred to patients in the ICU setting.In a prospective multicenter study, we evaluated concurrent bronchoalveolar lavage (BAL) samples and concurrent blood samples from 44 patients admitted to the ICU who required mechanical ventilation and therefore underwent a diagnostic work-up including chest CT scan and BAL (NCT01695499). The subsequent diagnostic work-up comprised of conventional methods for direct detection of Aspergillus spp. as well as biomarkers such as galactomannan (GM) and 1-3-beta-D-glucan (BDG) and an Aspergillus specific nested polymerase chain reaction (PCR).14 patients (32%) suffered from a hematological/hematooncological disease, of which 7 had received allogeneic stem cell transplantation. 6 patients (14%) had a solid tumor requiring chemotherapy. The remaining 24 patients (54%) had no hematological or oncological disease. 11 patients were neutropenic with ANC <1.0/nl (10 belonged to the hematooncological group). According to AspICU classification (Blot et al., Am J Respir Crit Care Med 2012) 9 patients (20%) had putative IPA (3 patients with hematological malignancy). Other invasive fungal diseases (IFD) besides IPA were observed: positive growth of Rhizopus microsporus in BAL was seen in one patient. Furthermore, two cases of disseminated candidiasis with positive growth in blood culture (C.albicans, C.krusei) and one case with positive growth in ascites (C.albicans). Additionally, two cases of Pneumocystis jirovecii pneumonia (PcP) were diagnosed.Diagnostic performance of each single test and of test combinations was analyzed. GM and PCR showed superior performance in BAL (sensitivity/specificity of 56%/94% (GM) and 44%/94% (PCR) compared to 33%/97% and 11%/94% in serum). Despite a better sensitivity of 89%, BDG showed a specificity of only 31% in BAL and 26% in serum, reducing the diagnostic odds ratio (DOR) to 3.7 and 2.8, respectively. For hematological patients the mean GM optical density index (ODI) in BAL was 0.97 and in serum 0.58 compared to 0.59 and 0.16 in non-hematological patients. The strength of agreement between GM in BAL and GM in serum as calculated by Cohen's kappa intertest coherence analysis was 0.62 [0.29-0.95], which is considered to be good. BDG levels and performance of the PCR assay did not differ between hematological and non-hematological patients. The combined use of biomarkers in BAL showed a diagnostic performance of sensitivity/specificity/PPV/NPV and DOR of 89%/31%/25%/92% and 3.67. Testing of GM and BDG solely in serum resulted in a combined test performance of 89%/26%/ 24%/ 90% and 2.77. GM (BAL) combined with BDG (serum) showed high joint sensitivity of 100% with specificity of 25% (DOR 6.81). Using PCR instead of GM dropped sensitivity to 89% (specificity 23%, DOR 2.37). Diagnostic performance of combined GM and PCR testing in BAL showed sensitivity/specificity/PPV/NPV and DOR 67%/89%/60%/91% and 15.0. Cohen's kappa for PCR (BAL) vs. GM (BAL) was 0.49 [0.145 to 0.840], which is considered to be moderate. If a diagnostic algorithm defines BDG (serum) as an initial test that is, in case of a positive result, followed by a confirming test combination from BAL with strong or even exclusive specificity for Aspergillus spp. (GM, PCR and culture) a diagnostic performance is achieved with sensitivity/specificity/positive predictive value/negative predictive value of 88%/97%/88%/97% and DOR >100.This study intended as a pilot study underscores the concept of combined testing in serum and BAL for optimized detection of IPA and provides valuable data for the emerging cohort of critically ill patients, both hematological and non-hematological. Further studies with hematological ICU patients in larger cohorts are planned. DisclosuresBoch:Pfizer research grant (WS 2274964): Research Funding. Buchheidt:Pfizer research grant (WS 2274964): Research Funding.

Antibody-guided in vivo imaging of Aspergillus fumigatus lung infections during antifungal azole treatment

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immunocompromised humans, caused by the opportunistic fungal pathogen Aspergillus fumigatus. Inadequacies in current diagnostic procedures mean that early diagnosis of the disease, critical to patient survival, remains a major clinical challenge, and is leading to the empiric use of antifungal drugs and emergence of azole resistance. A non-invasive procedure that allows both unambiguous detection of IPA and its response to azole treatment is therefore needed. Here, we show that a humanised Aspergillus-specific monoclonal antibody, dual labelled with a radionuclide and fluorophore, can be used in immunoPET/MRI in vivo in a neutropenic mouse model and 3D light sheet fluorescence microscopy ex vivo in the infected mouse lungs to quantify early A. fumigatus lung infections and to monitor the efficacy of azole therapy. Our antibody-guided approach reveals that early drug intervention is critical to prevent complete invasion of the lungs by the fungus, and demonstrates the power of molecular imaging as a non-invasive procedure for tracking IPA in vivo.

Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology.

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus is a life-threatening lung disease of immunocompromised patients. Diagnosis currently relies on non-specific chest CT, culture of the fungus from invasive lung biopsy, and detection of the cell wall carbohydrate galactomannan (GM) in serum or in BAL fluids recovered during invasive bronchoscopy. Urine provides an ideal bodily fluid for the non-invasive detection of pathogen biomarkers, with current urine-based immunodiagnostics for IPA focused on GM. Surrogate protein biomarkers might serve to improve disease detection. Here, we report the development of a monoclonal antibody (mAb), PD7, which is specific to A. fumigatus and related species in the section Fumigati, and which binds to its 18 kDa ribotoxin Asp f I. Using PD7, we show that the protein is secreted during hyphal development, and so represents an ideal candidate for detecting invasive growth. We have developed a lateral-flow device (Afu-LFD®) incorporating the mAb which has a limit of detection of ~15 ng Asp f I/mL urine. Preliminary evidence of the test’s diagnostic potential is demonstrated with urine from a patient with acute lymphoid leukaemia with probable IPA. The Afu-LFD® therefore provides a potential novel opportunity for non-invasive urine-based detection of IPA caused by A. fumigatus.

Advances in the In Vivo Molecular Imaging of Invasive Aspergillosis.

Invasive pulmonary aspergillosis (IPA) is a life-threatening infection of immunocompromised patients with Aspergillus fumigatus, a ubiquitous environmental mould. While there are numerous functioning antifungal therapies, their high cost, substantial side effects and fear of overt resistance development preclude permanent prophylactic medication of risk-patients. Hence, a fast and definitive diagnosis of IPA is desirable, to quickly identify those patients that really require aggressive antimycotic treatment and to follow the course of the therapeutic intervention. However, despite decades of research into this issue, such a diagnostic procedure is still not available. Here, we discuss the array of currently available methods for IPA detection and their limits. We then show that molecular imaging using positron emission tomography (PET) combined with morphological computed tomography or magnetic imaging is highly promising to become a future non-invasive approach for IPA diagnosis and therapy monitoring, albeit still requiring thorough validation and relying on further acceptance and dissemination of the approach. Thereby, our approach using the A. fumigatus-specific humanized monoclonal antibody hJF5 labelled with 64Cu as PET-tracer has proven highly effective in pre-clinical models and hence bears high potential for human application.

Detection of Invasive Pulmonary Aspergillosis in COVID-19 with Nondirected BAL

Detection of Invasive Pulmonary Aspergillosis in COVID-19 with Nondirected BAL

Detection of invasive pulmonary aspergillosis in critically ill patients by combined use of conventional culture, galactomannan, 1-3-beta-D-glucan and Aspergillus specific nested polymerase chain reaction in a prospective pilot study

Invasive pulmonary aspergillosis (IPA) is an emerging and life-threatening infectious disease in patients admitted to the intensive care unit (ICU). Most diagnostic studies are conducted in hematological patients and results cannot readily be transferred to ICU patients lacking classical host factors.In a multicenter, prospective clinical trial including 44 ICU patients, hematological (n = 14) and non-hematological patients (n = 30), concurrent serum and bronchoalveolar lavage (BAL) samples were analyzed by conventional culture, galactomannan (GM), 1-3-beta-D-glucan (BDG) as well as an Aspergillus specific nested polymerase chain reaction (PCR). Nine patients (20%) had putative IPA according to AspICU classification.GM and PCR showed superior performance in BAL with sensitivity/specificity of 56%/94% and 44%/94% compared to 33%/97% and 11%/94% in serum. Despite better sensitivity of 89%, BDG showed poor specificity of only 31% (BAL) and 26% (serum). Combination of GM and PCR (BAL) with BDG (serum) resulted in 100% sensitivity, but also reduced specificity to 23%. Whereas mean GM levels were significantly higher in hematological patients BDG and PCR did not differ between hematological and non-hematological patients.Under present clinical conditions test combinations integrating both BAL and blood samples are advantageous. BDG might best serve as possible indicator for ruling out IPA. Trial registrationClinicalTrials.gov Identifier: NCT01695499. First posted: September 28, 2012, last update posted: May 8, 2017.

Serial assessment of pulmonary lesion volume by computed tomography allows survival prediction in invasive pulmonary aspergillosis.

Serial chest CT is the standard of care to establish treatment success in invasive pulmonary aspergillosis (IPA). Data are lacking how response should be defined. Digital CT images from a clinical trial on treatment of IPA were re-evaluated and compared with available biomarkers. Total volume of pneumonia was added up after manual measurement of each lesion, followed by statistical analysis. One-hundred and ninety CT scans and 309 follow-up datasets from 40 patients were available for analysis. Thirty-one were neutropenic. Baseline galactomannan (OR 4.06, 95%CI: 1.08-15.31) and lesion volume (OR 3.14, 95%CI: 0.73-13.52) were predictive of death. Lesion volume at d7 and trend between d7 and d14 were strong predictors of death (OR 20.01, 95%CI: 1.42-282.00 and OR 15.97, 95%CI: 1.62-157.32) and treatment being rated as unsuccessful (OR 4.75, 95%CI: 0.94-24.05 and OR 40.69, 95%CI: 2.55-649.03), which was confirmed by a Cox proportional hazards model using time-dependent covariates. Any increase in CT lesion volume between day 7 and day 14 was a sensitive marker of a lethal outcome (>50%), supporting a CT rescan each one and 2weeks after initial detection of IPA. The predictive value exceeded all other biomarkers. Further CT follow-up after response at day 14 was of low additional value. • CT evaluation offers good prediction of outcome for invasive pulmonary aspergillosis. • Predictive capability exceeds galactomannan, blood counts, and lesion count. • Any progression between day 7 and day 14 constitutes a high-risk scenario.

Towards Translational ImmunoPET/MR Imaging of Invasive Pulmonary Aspergillosis: The Humanised Monoclonal Antibody JF5 Detects Aspergillus Lung Infections In Vivo.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy or bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus. Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [64Cu]DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [64Cu]NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [64Cu]NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant β1,5-galactofuranose (Galf) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [64Cu]NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting.

Comparison of Serum Galactomannan and 1,3-Beta-d-Glucan Determination for Early Detection of Invasive Pulmonary Aspergillosis in Critically Ill Patients with Hematological Malignancies and Septic Shock

Invasive pulmonary aspergillosis (IPA) is an important cause of morbidity/mortality in critically ill patients with hematological malignancies. New diagnosis strategies include the noninvasive biomarkers 1,3-beta-D-glucan (BDG) and serum galactomannan (GM). For early detection of IPA, we compared BDG Fungitell assay with GM Platelia assay. Twenty-two out of 30 patients (74%) had elevated BDG levels (mean 306pg/ml) beyond the cutoff of 80pg/ml. GM levels were elevated in only 3 patients (10%) over the ODI cutoff of >0.5. Following the BDG/GM and microbiological findings, 10 (34%) cases were classified as probable IPA and 12 (40%) as possible IPA. Eight (26%) were classified as no IPA. An overall sensitivity of 90% (95% CI 86-96%) and specificity of 85% (95% CI 79-86%) was found for the BDG Fungitell assay in IPA. In contrast, an overall sensitivity of 30% (95% CI 26-38%) and specificity of 98% (95% CI 94-100%) was found for the GM Platelia assay. A false-negative rate of 70% for probable IPA and 85% for probable/possible IPA was detected for GM. The false-negative rate for BDG was 0% in cases of probable IPA and 45% in cases of possible cases. BDG is a sensitive marker for patients' surveillance at risk of IPA. In patients with hematological malignancies and septic shock, early diagnosis of IPA might be significantly improved by BDG compared to GM, also considering that BDG has the advantage of detecting fungal diseases other than IPA.

Bone Suppression Increases the Visibility of Invasive Pulmonary Aspergillosis in Chest Radiographs

ObjectiveChest radiographs (CXR) are an important diagnostic tool for the detection of invasive pulmonary aspergillosis (IPA) in critically ill patients, but their diagnostic value is limited by a poor sensitivity. By using advanced image processing, the aim of this study was to increase the value of chest radiographs in the diagnostic work up of neutropenic patients who are suspected of IPA.MethodsThe frontal CXRs of 105 suspected cases of IPA were collected from four institutions. Radiographs could contain single or multiple sites of infection. CT was used as reference standard. Five radiologists and two residents participated in an observer study for the detection of IPA on CXRs with and without bone suppressed images (ClearRead BSI 3.2; Riverain Technologies). The evaluation was performed separately for the right and left lung, resulting in 78 diseased cases (or lungs) and 132 normal cases (or lungs). For each image, observers scored the likelihood of focal infectious lesions being present on a continuous scale (0–100). The area under the receiver operating characteristics curve (AUC) served as the performance measure. Sensitivity and specificity were calculated by considering only the lungs with a suspiciousness score of greater than 50 to be positive.ResultsThe average AUC for only CXRs was 0.815. Performance significantly increased, to 0.853, when evaluation was aided with BSI (p = 0.01). Sensitivity increased from 49% to 66% with BSI, while specificity decreased from 95% to 90%.ConclusionThe detection of IPA in CXRs can be improved when their evaluation is aided by bone suppressed images. BSI improved the sensitivity of the CXR examination, outweighing a small loss in specificity.

Electronic Nose Technology for Detection of Invasive Pulmonary Aspergillosis in Prolonged Chemotherapy-Induced Neutropenia: a Proof-of-Principle Study

Although the high mortality rate of pulmonary invasive aspergillosis (IA) in patients with prolonged chemotherapy-induced neutropenia (PCIN) can be reduced by timely diagnosis, a diagnostic test that reliably detects IA at an early stage is lacking. We hypothesized that an electronic nose (eNose) could fulfill this need. An eNose can discriminate various lung diseases through the analysis of exhaled volatile organic compounds (VOCs). An eNose is cheap and noninvasive and yields results within minutes. In a single-center prospective cohort study, we included patients who were treated with chemotherapy expected to result in PCIN. Based on standardized indications, a full diagnostic workup was performed to confirm invasive aspergillosis or to rule it out. Patients with no aspergillosis were considered controls, and patients with probable or proven aspergillosis were considered index cases. Exhaled breath was examined with a Cyranose 320 (Smith Detections, Pasadena, CA). The resulting data were analyzed using principal component reduction. The primary endpoint was cross-validated diagnostic accuracy, defined as the percentage of patients correctly classified using the leave-one-out method. Accuracy was validated by 100,000 random classifications. We included 46 subjects who underwent 16 diagnostic workups, resulting in 6 cases and 5 controls. The cross-validated accuracy of the eNose in diagnosing IA was 90.9% (P = 0.022; sensitivity, 100%; specificity, 83.3%). Receiver operating characteristic analysis showed an area under the curve of 0.93. These preliminary data indicate that PCIN patients with IA have a distinct exhaled VOC profile that can be detected with eNose technology. The diagnostic accuracy of the eNose for invasive aspergillosis warrants validation.

Interlaboratory and interstudy reproducibility of a novel lateral-flow device and influence of antifungal therapy on detection of invasive pulmonary aspergillosis.

Interest in lateral-flow devices (LFDs) as potential point-of-care assays for the diagnosis of infectious diseases has increased. Our objective was to evaluate the interlaboratory and interstudy reproducibility and the effects of antifungal therapy on an LFD developed for invasive pulmonary aspergillosis (IPA) detection. An established neutropenic guinea pig model of IPA caused by Aspergillus fumigatus was used. At predetermined time points (1 h and 3, 5, and 7 days postinoculation), blood and bronchoalveolar lavage (BAL) fluid were collected from infected and uninfected animals. In a separate experiment, guinea pigs were treated with posaconazole (10 mg/kg of body weight orally [p.o.] twice a day [BID]), voriconazole (10 mg/kg p.o. BID), liposomal amphotericin B (10 mg/kg intraperitoneally [i.p.] once a day [QD]), or caspofungin (2 mg/kg i.p. QD), and samples were collected on days 7 and 11. Each laboratory independently evaluated the IgG monoclonal antibody-based LFD. Galactomannan and (1 → 3)-β-D-glucan were also measured using commercially available kits. Good interlaboratory agreement was observed with the LFD, as the results for 97% (32/33) of the serum and 78.8% (26/33) of the BAL fluid samples from infected animals were in agreement. Good interstudy agreement was also observed. The serum sensitivity of each surrogate-marker assay was reduced in animals treated with antifungals. In contrast, these markers remained elevated within the BAL fluids of treated animals, which was consistent with the fungal burden and histopathology results. These results demonstrate that the LFD assay is reproducible between different laboratories and studies. However, the sensitivity of this assay and other markers of IPA may be reduced with serum in the presence of antifungal therapy.

Comparison of Sensitivity of Low-Dose CT Scan and Serum Galactomannan in Patients with Hematologic Malignancies and Positive Bronchoalveolar Lavage for Invasive Pulmonary Aspergillosis

Abstract 1490▪▪This icon denotes a clinically relevant abstract Background:Invasive pulmonary aspergillosis (IPA) is associated with considerable morbidity and mortality in patients with acute leukemia and those undergoing hematopoietic stem cell transplantation (HSCT). Timely diagnosis of IPA is crucial, but difficult. Strategies for early detection of IPA include serum galactomannan (GM), bronchoalveolar lavage (BAL) GM and characteristic radiologic abnormalities on CT scan; however, the relative sensitivity of these diagnostic tests is unclear. We sought to determine the utility of serum GM and characteristic CT radiologic abnormalities in diagnosing IPA in patients with hematologic malignancies who had a positive BAL for Aspergillus. Methods:We performed a single center retrospective cohort study from 2010 to 2012 to determine the sensitivity serum GM (OD > 0.5) and low dose CT scan in patients with hematologic malignancies. All positive BAL GM samples and Aspergillus isolates from BAL fluid cultures from patients with acute leukemia and those undergoing HSCT were reviewed; all patients had abnormal chest CT scans consistent with pulmonary infection. IPA was classified as proven, probable or possible according to EORTC/MSG criteria. Recent low dose CT scans correlating to the date of the positive BAL GM or Aspergillus isolation were reviewed and graded: halo signs, cavities, crescents and nodules were deemed to be consistent with IPA, while other changes (e.g. ground-glass changes, consolidation) were considered non-specific. Results:A total of 49 BAL samples were included; of these, 31 were considered probable IPA and 18 possible IPA (on the basis of non-specific radiologic findings). Most patients had either no prior or 1–2 days of mold-active antifungal agents. There were 43 cases with positive BAL GM and 11 cases of positive Aspergillus BAL isolates; 5 patients had both. Of the positive BAL GM cases, 27 (63%) were associated with radiologic findings consistent with IPA. The remaining cases were associated with non-specific radiologic findings. Of the patients with Aspergillus isolates on BAL, 55% (6/11) had radiologic features consistent with IPA, while the remaining cases had non-specific radiologic findings.We next evaluated the sensitivity of serum GM. Of 34 patients with BAL GM positivity who had concomitant serum GM testing, 4 (12%) had positive serum GM. Of 8 patients with Aspergillus species isolated on BAL who had serum GM performed, only 1 (12.5%) had positive serum GM. In contrast, 5/8 patients (63%) with Aspergillus isolates on BAL had a positive BAL GM. The combined sensitivity of serum GM was 12% (5/42). Conclusions:Although the majority of patients with positive BAL for Aspergillus (+GM and/or isolates) had characteristic radiologic findings on CT scan, the absence of such findings did not exclude this diagnosis, as over one third had only non-specific radiologic findings. Serum GM had very low sensitivity in this population and should not be used in isolation to diagnose IPA. Disclosures:No relevant conflicts of interest to declare.

Aspergillus-PCR in bronchoalveolar lavage for detection of invasive pulmonary aspergillosis in immunocompromised patients

BackgroundInvasive fungal disease (IFD) is a frequent and serious infectious complication in immunocompromised patients. Culture and cytology in bronchoalveolar lavage (BAL) have a high specificity but low sensitivity for the diagnosis of IFD as assessed by histology. Molecular methods are expected to allow a rapid diagnosis of IFD with a high sensitivity. We evaluated the diagnostic accuracy of conventional nested PCR in the bronchoalveolar fluid to diagnose IFD in severely immunocompromised patients.MethodsConsecutive immunosuppressed patients undergoing bronchoscopy for suspected pulmonary infection in a tertiary care hospital were included. Patients were classified as having “proven”, “probable”, “possible”, and “no” IFD based on definitions of the European Organization for Research and Treatment of Cancer and National Institute of Allergy and Infectious Diseases (EORTC/NIAID) and on clinical grounds. Conventional nested PCR for aspergillus fumigatus, flavus, niger, glaucus, terreus and tomarrii were applied to 2.5 ml bronchoalveolar fluid.ResultsA total of 191 patients were included. Mean age was 51 y, 61% were male. There were 129 patients with hematological conditions, 26 solid organ transplant recipients, 24 auto-immune disorders, and 12 HIV. According to the EORTC/NIAID classification, there were 53 patients with potential IFD: 3 (2%) had proven, 8 (4%) probable, 42 (22%) possible and 138 (72%) no IFD. A total of 111 (58%) of the patients - 10 (90.9%) proven or probable IFD, 32 (76.2%) possible IFD and 69 (50%) “no” IFD) were on anti-fungal therapy at the time of bronchoscopy. Conventional nested PCR for Aspergillus was positive in 55 cases (28.8%). According to these results, sensitivity, specificity, PPV and NPV for “proven” IFD was 0%, 71%, 0%, 98%, respectively and “probable” IFD was 36%, 72%, 7%, 95%, respectively. In 53 (28%) cases there was a strong clinical suspicion of IFD in the chest-x-ray and/or chest-CT irrespective of the EORTC/NIAID classification. However, from those, only 15 (28%) had a positive conventional nested PCR.ConclusionIn our experience, conventional nested Aspergillus PCR in the BAL seems to be of limited usefulness for detection of invasive fungal disease in immunocompromised patients due to the limited sensitivity and specificity of the method.

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 →3)-β-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 → 3)-β-D-glucan assays7.Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria. Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained. An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 →3)-β-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5. Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 → 3)-β-D-glucan assays7. Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

What should be the optimal cut-off of serum 1,3-β-d-glucan for the detection of invasive pulmonary aspergillosis in patients with haematological malignancies?

The detection of 1,3-β-d-glucan (BDG), a cell wall component of several medically important fungi, is a promising tool for the diagnosis of invasive pulmonary aspergillosis. The aim of this study was to evaluate the diagnostic accuracy of the BDG test in invasive pulmonary aspergillosis (IPA) by focusing on the optimal cut-off value. The records of the Infection Control Committee were reviewed to identify patients with haematological malignancies and stem cell transplantation who had at least 1 BDG (Fungitell kit) measurement during the period January 2008 through April 2011. The European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) criteria (independent of BDG results) were used to categorize the patients with IPA. Patients with possible IPA were not included in the study. A total of 128 patients (50 with proven or probable IPA) were included in the study. At the manufacturer's recommended cut-off value of 80 pg/ml, the sensitivity of BDG was 66% (95% CI 51.2-78.7), specificity 75.6% (95% CI 64.6-84.5), positive predictive value (PPV) 63.4%, and negative predictive value (NPV) 77.6%. A receiver operating characteristic (ROC) curve was constructed to define the optimum serum BDG cut-off for the diagnosis of IPA. At a cut-off value of 181 pg/ml, the sensitivity was 52% (95% CI 37.4-66.3), specificity 94.8% (95% CI 87.4-98.6), PPV 86.7%, and NPV 75.5%. Although higher cut-off levels increased the specificity of the BDG test, sensitivity decreased to an unacceptable level; the commercially recommended cut-off value appears to be appropriate for screening purposes.

Molecular Detection and Species-Specific Identification of Medically Important Aspergillus Species by Real-Time PCR in Experimental Invasive Pulmonary Aspergillosis

Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients.