Detection Of Aspergillus Galactomannan Research Articles

Time-resolved fluoroimmunoassay for Aspergillus detection based on anti-galactomannan monoclonal antibody from stable cell line

Invasive Aspergillosis is a high-risk illness with a high death rate in immunocompromised people due to a lack of early detection and timely treatment. Based on immunology study, we achieved an efficient production of anti-galactomannan antibody by Chinese hamster ovary (CHO) cells and applied it to time-resolved fluoroimmunoassay for Aspergillus galactomannan detection. We first introduced dual promoter expression vector into CHO host cells, and then applied a two-step screening strategy to screen the stable cell line by methionine sulfoximine pressurization. After amplification and fermentation, antibody yield reached 4500 mg/L. Then we conjugated the antibodies with fluorescent microspheres to establish a double antibody sandwich time-resolved fluoroimmunoassay, which was compared with the commercial Platelia™ Aspergillus Ag by clinical serum samples. The preformed assay could obtain the results in less than 25 min, with a limit of detection for galactomannan of approximately 1 ng/mL. Clinical results of the two methods showed that the overall percent agreement was 97.7% (95% CI: 96.6%–98.4%) and Cohen's kappa coefficient was 0.94. Overall, the assay is highly consistent with commercial detection, providing a more sensitive and effective method for the rapid diagnosis of invasive aspergillosis.

Detection of Aspergillus Galactomannan in Blood for Early Diagnosis of Pulmonary Aspergillosis in HIV Positive Subjects with and without Tuberculosis in Calabar, Nigeria

This study was aimed at determining the prevalence of pulmonary aspergillosis using Aspergillus galactomannan detection in human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/ AIDS) subjects with and without tuberculosis. The study was a prospective cohort study carried out at the University of Calabar Teaching Hospital and Dr. Lawrence Henshaw Memorial Hospital in Calabar. Subjects were HIV-positive patients with and without tuberculosis. Blood and sputum samples were obtained from 215 subjects who consented to the study. A structured questionnaire was administered to assess demograph¬ic data and medical history. Sputum samples were subjected to direct microscopy, culture, and Ziehl-Nel¬seen test. Aspergillus galactomannan assay and CD4 counts were performed on the blood samples. The results of Aspergillus galactomannan-positive subjects were communicated to the attending Physicians for proper patient management. Aspergillus species were recovered from 35(16.3%) sputum samples. Asper¬gillus fumigatus was the most encountered isolate 18(8.4%). The most common presenting symptoms were cough 35(100%) and fatigue 30(85.7%). Out of the 35 subjects with Aspergillus isolates, 21(60.0%) had TB, 9(25.7%) had no TB and 5(14.3%) had co-morbidity of HIV and TB. All the subjects with aspergillosis had CD4 counts less than 200 cellsμl-1 and there was a statistically significant association between pulmonary aspergillosis and CD4 levels (H=7.02; p = 0.03). Galactomannan assay could be used for early diagnosis of invasive aspergillosis and timely management of patients as it is sensitive, cost-effective, and has a turna¬round time of less than 1 hour compared to culture methods.

Implementation of Lateral Flow Assays for the Diagnosis of Invasive Aspergillosis in European Hospitals: A Survey from Belgium and a Literature Review of Test Performances in Different Patient Populations.

Diagnosis of invasive aspergillosis is based on a combination of criteria, of which the detection of Aspergillus galactomannan (GM) often is decisive. To date, the most commonly used method to determine GM is an enzyme-linked immune assay (EIA). But since a few years lateral flow assays (LFAs) were introduced, providing the possibility for rapid single sample testing. More and more LFAs are entering the market, but, although often being equated, all use their own antibodies, procedures and interpretation criteria. A recent European survey revealed that about 24-33% of laboratories implemented a lateral flow assay on-site. We conducted a survey at 81 Belgian hospital laboratories regarding the implementation of LFAs in their centre. In addition, we performed an extensive review of all publicly available studies on the performance of lateral flow assays to diagnose invasive aspergillosis. Response rate to the survey was 69%. Of the 56 responding hospital laboratories, 6 (11%) used an LFA. The Soña Aspergillus galactomannan LFA (IMMY, Norman, Oklahoma, USA) was used in 4/6 centres, while two centres used the QuicGM (Dynamiker, Tianjin, China) and one centre used the FungiXpert Aspergillus Galactomannan Detection K-set LFA (Genobio [Era Biology Technology], Tianjin, China). One centre used 2 distinct LFAs. In 3/6 centres, the sample is sent to another lab for confirmation with GM-EIA when the LFA result is positive and in 2/6 when the LFA results is negative. In one centre, a confirmatory GM-EIA is always performed in house. In three centres the LFA result is used as a complete substitute for GM-EIA. Available LFA performance studies are very diverse and results vary in function of the study population and type of LFA. Apart from the IMMY and OLM LFA, only very limited performance data are available. From two out of three LFAs used in Belgium, no clinical performance studies are published in literature. A large variety of LFAs are used in Belgian Hospitals, some of which no clinical validation studies are published. These results do likely have implications for other parts of Europe and for the rest of the world as well. Due to the variable performance of LFA tests and the limited validation data available, each laboratory must check the available performance information of the specific test considered for implementation. In addition, laboratories should perform an implementation verification study.

P460 The Aspergillus Lateral Flow Assay for the diagnosis of invasive Aspergillosis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesInvasive Aspergillosis (IA) is a life-threatening opportunistic mycosis that occurs in some people with a compromised immune system. The detection of Aspergillus galactomannan (GM) rapidly gained widespread acceptance as part of the diagnostic workup of a patient suspected of IA. Recently, a Novel QuicGM™ Aspergillus Galactomannan Ag Lateral Flow Assay (LFA) from Dynamiker Biotechnology (Tianjin) Co., Ltd. was released as a screening test of IA. It is a fluorescent immunochromatographic cassette test which using a monoclonal antibody against GM and Europium nanoparticles (Eu NP) (Fig. 1). It is a semi-quantitative test which was easy to operate, rapid with portable detection devices, and can be widely accepted by clinical and primary medical (Fig. 2). We conducted a comparative study between the Dynamiker QuicGMTM Aspergillus Galactomannan Ag (LFA) and the Bio-Rad Platelia™.MethodsA total of 66 serum samples and 54 BALF samples were collected, 15 serum samples from the College of American Pathologists (CAP) certificated lab were also included in this study. The samples were tested in parallel using the Bio-Rad Platelia™ Aspergillus Ag (ELISA) and Dynamiker QuicGM™ Aspergillus Galactomannan Ag (LFA).ResultsThe total coincidence rate of the two assays was 98.48% (Kappa value was 0.97 >0.75) and 90.74% (Kappa value was 0.81 >0.75) in 66 serum samples and 54 BALF samples respectively.ConclusionsThe performance of the Dynamiker QuicGM™Aspergillus Galactomannan Ag (LFA) and Bio-Rad Platelia™ Aspergillus Ag (ELISA) were highly consistent, and the performance of Dynamiker Aspergillus Galactomannan kit were all in line with the statement of the manufacturer. The results are accurate and reliable, and it could be used as an aid for the early rapid screening test of IA.

Performance of Two Commercial Assays for the Detection of Serum Aspergillus Galactomannan in Non-Neutropenic Patients.

Besides the relevance of aspergillosis in neutropenic patients, this mycosis has gained significance among non-neutropenic patients in last years. The detection of Aspergillus galactomannan has been used for aspergillosis diagnosis and follow-up in neutropenic patients. This study evaluated the applicability of two commercial tests for galactomannan detection in non-neutropenic patients with different clinical forms of aspergillosis. Serum samples from patients with chronic pulmonary aspergillosis, aspergilloma, invasive aspergillosis, and COVID-19 associated pulmonary aspergillosis were evaluated using the IMMY sōna AGM lateral flow assay and the Bio-Rad Platelia sandwich ELISA. Serum specimens from patients with tuberculosis, histoplasmosis, paracoccidioidomycosis, and from healthy individuals were used as controls. The Bio-Rad Platelia sandwich ELISA presented greater sensitivity, whereas the IMMY sōna AGM lateral flow assay presented greater specificity. The accuracies of the tests were similar, as demonstrated by a receiver operator characteristic analysis. Moreover, the best cut-off values determined by this analysis were closer to that recommended by both manufacturers for neutropenic patients. The galactomannan indexes determined by different methodologies were strongly related, and a substantial agreement was observed between results. Both tests can be used in non-neutropenic patients with the cut-off values defined by the manufacturers. Histoplasma cross-reactions may occur in areas where histoplasmosis is endemic.

Invasive Pulmonary Aspergillosis.

Invasive pulmonary aspergillosis (IPA) remains difficult to diagnose and to treat. Most common risk factors are prolonged neutropenia, hematopoietic stem cell or solid organ transplantation, inherited or acquired immunodeficiency, administration of steroids or other immunosuppressive agents including monoclonal antibodies and new small molecules used for cancer therapy. Critically ill patients are also at high risk of IPA. Clinical signs are unspecific. Early computed tomography (CT)-scan identifies the two main aspects, angioinvasive and airway invasive aspergillosis. Although CT-scan findings are not fully specific they usually allow early initiation of therapy before mycological confirmation of the diagnosis. Role of 18F-fludeoxyglucose positron emission tomography with computed tomography (18F-FDG PET/CT) is discussed. Confirmation is based on microscopy and culture of respiratory samples, histopathology in case of biopsy, and importantly by detection of Aspergillus galactomannan using an immunoassay in serum and bronchoalveolar lavage fluid. Deoxyribonucleic acid detection by polymerase chain reaction is now standardized and increases the diagnosis yield. Two point of care tests detecting an Aspergillus glycoprotein using a lateral flow assay are also available. Mycological results allow classification into proven (irrespective of underlying condition), probable or possible (for cancer and severely immunosuppressed patients) or putative (for critically ill patients) IPA. New antifungal agents have been developed over the last 2 decades: new azoles (voriconazole, posaconazole, isavuconazole), lipid formulations of amphotericin B (liposomal amphotericin B, amphotericin B lipid complex), echinocandins (caspofungin, micafungin, anidulafungin). Results of main trials assessing these agents in monotherapy or in combination are presented as well as the recommendations for their use according to international guidelines. New agents are under development.

Aspergillus galactomannan detection: Trichoderma as a cause of positive results.

Aspergillus galactomannan immunoassay is a main diagnostic and monitoring tool in medical mycology. However, the specificity of the method can be skewered by the presence of several other fungi. Trying to diagnose a possible fungal infection of the lower respiratory tract in a haematology patient, it appeared that the fungus Trichoderma longibrachiatum is an additional probable cause of positive galactomannan results. Although, that Trichoderma is a rare but emerging pathogen in immunocompromised patients, the above information could be a caution point in the clinical evaluation of diagnostic results.

Aspergillus galactomannan detection in exhaled breath condensate compared to bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in immunocompromised patients

ObjectivesExhaled breath condensate (EBC) is a noninvasive means of sampling the airways that has shown significant promise in the diagnosis of many disorders. There have been no reports of its usefulness in the detection of galactomannan (GM), a component of the cell wall of Aspergillus. The suitability of EBC for the detection of GM for the diagnosis of invasive aspergillosis (IA) using the Platelia Aspergillus enzyme-linked immunosorbent assay was investigated. MethodsProspective, cross-sectional study of lung transplant recipient and haemotologic malignancy patients at a university centre. EBC samples were compared to concomitant bronchoalveolar lavage (BAL) samples among lung transplant recipients and healthy controls. EBC was collected over 10 minutes using a refrigerated condenser according to the European Respiratory Society/American Thoracic Society recommendations, with the BAL performed immediately thereafter. ResultsA total of 476 EBC specimens with 444 matched BAL specimens collected from lung transplant recipients (n = 197) or haemotologic malignancy patients (n = 133) were examined. Both diluted and untreated EBC optical density (OD) values (0.0830, interquartile range (IQR) 0.0680–0.1040; and 0.1130, IQR 0.0940–0.1383), respectively, from all patients regardless of clinical syndrome were significantly higher than OD values in healthy control EBCs (0.0508, IQR 0.0597–0.0652; p < 0.0001). However, the OD index values did not correlate with the diagnosis of IA (44 samples were associated with IA). Furthermore, no significant correlation was found between EBC GM and the matched BAL specimen. ConclusionsGM is detectable in EBC; however, no correlation between OD index values and IA was noted in lung transplant recipients.

(40) - Aspergillus Galactomannan Detection in Exhaled Breath Condensate for the Diagnosis of Invasive Aspergillosis in Lung Transplant Recipients

(40) - Aspergillus Galactomannan Detection in Exhaled Breath Condensate for the Diagnosis of Invasive Aspergillosis in Lung Transplant Recipients

Role of galactomannan assay in bronchoalveolar lavage fluid in the diagnosis of invasive aspergillosis in high risk patients with hematological disorders

Introduction: Invasive aspergillosis (IA) is the most frequent invasive fungal infection occurring in high risk patients with haematological disorders and also an important cause of morbidity and mortality in them. Lungs comprise the primary site of IA in the vast majority of cases, and the most frequently occurring form of IA. Early diagnosis followed by early initiation of therapy improves outcome. The current culture based diagnostic methods are insensitive and slow. Detection of Aspergillus galactomannan (GM) in serum using the platelia Aspergillus enzyme immunoassay (GM EIA) represents sensitive, non-culture-based tool for the early diagnosis of IA. Recent studies have shown that during IPA, GM is released earlier and in higher concentration in the BAL fluid than in serum. Thus, an early and accurate diagnosis of IPA can be reached by detecting antigen in BAL samples. Materials and Methods: Thirty consecutive high risk patients with haematological disorders satisfying the inclusion and exclusion criteria according to EORTC-MSG diagnostic criteria and undergoing indoor treatment were recruited between November 2013 and December 2014 into the study. In all the patients, bronchoalveolar lavage (BAL) fluid GM and serum GM assays were assayed. Results: The diagnostic accuracy of the two tests was measured by calculating the sensitivity, specificity, predictive values and receiver operating characteristic curve (ROC). The optimal BAL GM index for diagnosing probable and possible IPA was >1.00 OD, resulting in a sensitivity and specificity of 93.75% and 78.57% respectively. As per ROC curve, when the optimal BAL GM index for the diagnosis of probable and possible invasive pulmonary aspergillosis (IPA) was OD >0.5, it resulted in sensitivity and specificity of 50% and 71.43% respectively. BAL GM was thus found to have a significantly higher sensitivity as compared to serum GM. Conclusion: In all high-risk patients with haematological disorders where the clinical and radiological findings favour the possibility of invasive pulmonary aspergillosis, conducting a GM assay in BAL fluid sample is far more rewarding than carrying out the traditional serum GM assay. BAL GM has a high diagnostic accuracy when correlated with the EORTC/MSG diagnostic criteria, and this high diagnostic yield is useful in obviating the unnecessary widespread use and abuse of empirical antifungal therapy in patients with IPA.

High negative predictive value diagnostic strategies for the reevaluation of early antifungal treatment: A multicenter prospective trial in patients at risk for invasive fungal infections

Early antifungal therapeutic strategies are proposed during invasive fungal infection (IFI), but antifungal stewardship programs should institute a systematic reevaluation of prescriptions, particularly in the context of empirical treatment. Here, we aimed to evaluate the performances and particularly the negative predictive value (NPV) of diagnostic strategies, including a whole blood panfungal quantitative PCR assay (PF-qPCR) in a high risk population for IFI. The first step was to standardize and optimize a new PF-qPCR targeting ITS2 region. Then, this method was evaluated in a multicenter prospective study including 313 patients with suspected IFI for whom an early antifungal treatment was prescribed. All patients enrolled at day 0 of their treatment benefited from serum Aspergillus galactomannan (GM) antigen detection twice a week, weekly PF-qPCR assay, and when indicated and feasible, CT-scan and mycological sampling. In total, 125 of 313 patients were diagnosed with IFI: 68 invasive aspergillosis (eight proven, 48 probable and 12 possible), one fusariosis, 47 candidemia, three disseminated candidiasis and six cryptococcosis. Globally, the sensitivity of the PF-qPCR assay was only 40%, but the specificity, PPV and NPV were 96%, 88% and 69%, respectively. In the population of patients at high risk for invasive aspergillosis who also benefited from Aspergillus GM detection, the sensitivity and the NPV of the combined detection reached to 78% and 84%, respectively. Even higher NPV were obtained when combining negative PF-qPCR and CT scan (95%) as well as negative GM and CT scan (93%), thus allowing to rationalize and re-evaluate the prescription of empirical treatment in such highly selected population.

Aspergillus galactomannan detection in comparison to a real-time PCR assay in serum samples from a high-risk group of patients.

Invasive aspergillosis (IA) is a severe infection with a 70% mortality rate. Aspergillus fumigatus is responsible for over 90% of those infections.The diagnosis of invasive aspergillosis is based on clinical sample culture and detection of fungal hyphae in histopathological examination. Additional tests may include the detection of the galactomannan antigen and of fungal genetic material in serum and bronchoalveolar washings. The present study was to assess the use of these two rapid tests in the diagnosis of invasive aspergillosis: serological one – to detect the galactomannan antigen (ELISA assay), and real-time PCR, and to establish a possible correlation between these two methods.

A Bad Bug Takes on a New Role as a Cause of Ventilator-associated Pneumonia

Despite use of preventive strategies and significant advances in supportive care and antimicrobial therapy, ventilator-associated pneumonia (VAP) remains an important cause of morbidity and mortality (1). Although bacteria cause most VAPs, invasive pulmonary aspergillosis (IPA) is extremely uncommon, except when classical risk factors for aspergillosis (hematologic cancer, neutropenia, transplantation) are present. In this issue of the Journal (pp. 27‐34), Meersseman and colleagues (2) report their findings using detection of Aspergillus galactomannan (GM) (cell wall constituent released during tissue invasion) in bronchoalveolar lavage (BAL) fluid (BAL-GM) for diagnosing VAP caused by Aspergillus spp. Upon diagnosis of pneumonia, 110 adults at risk for IPA underwent BAL for culture and GM detection, serum GM, and chest computed tomography (CT). Only 22% of patients were neutropenic and 67% had underlying diseases other than hematologic cancer, including cirrhosis and chronic obstructive pulmonary disease (COPD). Twenty-six patients (24%) developed histopathologically proven IPA. The diagnostic sensitivity and specificity of BAL-GM was 88 and 87%, respectively, compared with 42% sensitivity for serum GM. Serum GM and BAL culture remained negative in 11 patients (42%) in whom BAL-GM was positive. Chest CT was of limited diagnostic value. The findings of this prospective study, backed by an impressive 95% autopsy rate, underscore the increasingly important role that Aspergillus species play as a cause of VAP in apparently immunocompetent intensive care unit (ICU) patients

Well-Characterized Monoclonal Antibodies against Cell Wall Antigen of Aspergillus Species Improve Immunoassay Specificity and Sensitivity

The diagnosis of invasive aspergillosis (IA) based on the detection of Aspergillus galactomannan (GM) is complicated by the presence of cross-reactive GM epitopes in patient specimens. We have developed a novel and specific Aspergillus antigen-capture enzyme-linked immunosorbent assay (ELISA) by the selection of two well-characterized monoclonal antibodies from 17 candidate antibodies. The epitopes recognized by the monoclonal antibodies were present on the cell walls of the hyphae and the conidia of Aspergillus species, which were circulating or excreted as immunodominant antigens during the acute phase of IA established in the animal models. The detection of experimental Aspergillus-mediated antigenemia was suitably sensitive, and the sensitivity was comparable to that of a commercial GM detection ELISA kit (the Platelia Aspergillus assay). Moreover, the specificity of this assay was 100% when it was used to test 382 serum specimens and 120 urine specimens from healthy individuals. Cross-reactivity with other common opportunistic fungi, such as Penicillium and Candida species, and with purified GM protein derived from Aspergillus was not evident. Therefore, the chemical nature of the epitopes captured in this assay is most likely not associated with the GM structure, indicating that this newly developed Aspergillus antigen-capture ELISA is a promising tool for the diagnosis of IA without the risk of the false-positive results that are problematic with current GM antigen assays.

Monitoring and prophylaxis

Invasive deep mycoses following bone marrow and solid-organ transplantation remain a major cause of morbidity and mortality. Species of Candida and Aspergillus account for more than 80% of these mycoses. Because these infections are often difficult to diagnose and treat successfully, antifungal prophylaxis is recommended in high-risk patients. Fluconazole is useful in patients who are at risk of invasive candidiasis, including bone marrow transplants, liver and pancreatic transplants. Although invasive aspergillosis is frequent in patients with bone marrow, lung and heart transplantation, no established methods have been available for its prophylaxis. Recently, efforts to improve the efficiency of diagnostic tests have been directed toward the detection of fungal components or metabolites. The requirements for clinical use (monitoring) are as follows: capability of early diagnosis, quantitative measurement, and easy sampling and simple assay procedure. The detection of plasma (1-3)-beta-D-glucan (BDG), a characteristic cell wall component of almost all fungi, is widely used in Japan. Twenty-seven episodes of fungemia were observed in our hematology ward and all were positive with BDG. Positive results were observed before the documentation of fungemia in 14 patients (51.9%). Although the positive rate of BDG also was 100% in 17 patients with invasive aspergillosis, it rose slightly at an early stage of the disease in 13 patients (76.5%). The determination of plasma BDG appears useful in the monitoring of deep fungal infection, but its usefulness for early diagnosis remains to be determined. The utility of detection of Aspergillus galactomannan by ELISA and fungal DNA by polymerase chain reaction are also discussed.

Antigen detection in the diagnosis and management of a patient with probable cerebral aspergillosis treated with voriconazole

This report describes the diagnosis and management of a 16-year-old boy who developed neurological signs and symptoms suggestive of cerebral aspergillosis following a haploidentical bone marrow transplant. A new sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Aspergillus galactomannan circulating antigens (Platelia Aspergillus, Sanofi Diagnostic Pasteur, France) was used on serum and cerebrospinal fluid to obtain a presumptive diagnosis and to monitor the course of the disease. Having failed conventional therapy with amphotericin B, the patient received compassionate treatment with voriconazole for a period of 37 days. High levels of voriconazole were observed in both serum and cerebrospinal fluid (CSF), with a trend toward accumulation. After 7 days a marked improvement in the patient's neurological symptoms was noted, and ELISA data indicated a corresponding decrease in Aspergillus galactomannan levels in both serum and CSF. Voriconazole was well tolerated, with only transient increases in ALT/AST recorded during therapy. Although the patient survived the acute Aspergillus infection, he subsequently died of an unrelated infection.

Detection of antigen in sera of patients with invasive aspergillosis: intra- and interlaboratory reproducibility. The Dutch Interuniversity Working Party for Invasive Mycoses.

The intra- and interlaboratory reproducibilities of a commercial sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Aspergillus galactomannan in serum (Platelia Aspergillus; Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) were evaluated in six laboratories of university hospitals. Twenty serum samples were obtained from 12 neutropenic patients including 6 with invasive aspergillosis. These samples were blinded and sent to each center together with eight blinded ELISA-negative serum samples spiked with known concentrations of galactomannan. The centers were provided with ELISA microtiter plates from a single batch and a detailed protocol. Ten clinical samples showed ELISA reactivity, while 10 samples were ELISA negative. The mean coefficient of variation (CV) of the optical density values was 4.24% within a single assay and 25.6% between runs. The interassay CV of the ratios for the serum samples tested was 18.6%. Analysis of ordinal interpretation of the ELISA result (i.e., negative, gray zone, or positive) showed excellent reproducibility. Recalculation of the cutoff values for positive and negative samples suggested that the cutoff level recommended by the manufacturer could be lowered from 1.0 to 0.8 for negative samples and from 1.5 to 1.0 for positive samples. The intra- and interlaboratory reproducibilities were excellent when the ELISA results were interpreted as ordinal data, but considerable variation in optical density values and, to a lesser extent, in the ratios for the serum samples tested, was observed between runs. High assay variability was also found for serum samples spiked with known concentrations of galactomannan. Therefore, antigen titers in serum samples from a single patient, measured in different runs, should be compared with caution.