Galactomannan Detection Research Articles

Performance of the IMMY® sona Aspergillus lateral flow assay for the detection of galactomannan in tracheal aspirate samples from Brazilian patients with COVID-19-associated pulmonary aspergillosis: Cross-sectional and systematic review of literature.

During the COVID-19 pandemic, many patients in intensive care units (ICUs) were affected by invasive fungal infections, including aspergillosis, contributing to a high mortality rate. Diagnosing proven COVID-19-associated pulmonary aspergillosis (CAPA) requires clinical and radiological evaluations, along with laboratory testing of bronchoalveolar lavage samples or lung biopsies. However, these procedures and equipment are often inaccessible in developing countries or regions with limited resources, including Brazil. Consequently, alternative diagnostic methods, such as measuring Aspergillus galactomannan (GM) in tracheal aspirate (TA), have been explored for CAPA diagnosis. Nonetheless, research on the efficacy of TA-based diagnostic tests is limited. This study aimed to assess the performance of the IMMY® Sona Aspergillus lateral flow assay (LFA) for GM detection in TA samples from 60 ICU patients with suspected CAPA at two tertiary hospitals in Campo Grande, Brazil. The ELISA method (Platelia Aspergillus AG, Bio-Rad®) was used to detect Aspergillus GM in TA samples, serving as the microbiological criterion and reference test. Fifteen patients (12.4%) were identified as having possible CAPA. The overall accuracy of LFA was 94%, and the tests demonstrated an agreement of 93.1% (Cohen's kappa of 0.83). Based on our findings, the LFA for Aspergillus GM detection in TA samples exhibited excellent performance, proving to be a valuable diagnostic tool for potential CAPA. In a systematic review, two studies were included, and the meta-analysis revealed pooled estimates provided a sensitivity of 86% (95% CI, 80%-91%) and specificity of 93% (95% CI, 86%-97%). The diagnostic odds ratio (DOR) for identification of Aspergillus using LFA was 103.38 (95% CI, 38.03-281.03). Despite its lower sensitivity compared to our study, the LFA appears to be a promising diagnostic option for CAPA, particularly in suspected cases that have not received antifungal therapy. This enables timely antifungal treatment and could reduce mortality rates in regions where bronchoscopy is unavailable or limited.

Metagenomic next-generation sequencing for the diagnosis of invasive pulmonary aspergillosis in type 2 diabetes mellitus patients

Invasive pulmonary aspergillosis (IPA) in patients with diabetes mellitus has high incidence, especially in Type 2 diabetes mellitus (T2DM). The aim of this study was to evaluate the diagnostic efficacy of metagenomic next-generation sequencing (mNGS) for IPA in patients with T2DM. A total of 66 patients with T2DM were included, including 21 IPA and 45 non-IPA patients, from January 2022 to December 2022. The demographic characteristics, comorbidities, laboratory test results, antibiotic treatment response, and 30-day mortality rate of patients were analyzed. The diagnostic accuracy of mNGS and conventional methods was compared, including sensitivity, specificity, positive predictive value and negative predictive value. The sensitivity and specificity of mNGS were 66.7% and 100.0%, respectively, which were significantly higher than those of fluorescence staining (42.1% and 100%), serum 1,3-β-D-glucan detection (38.1% and 90.9%), serum galactomannan detection (14.3% and 94.9%) and BALF galactomannan detection (47.3% and 70.7%). Although the sensitivity of BALF culture (75.0%) was higher than that of mNGS (66.7%), the turnover time of mNGS was significantly shorter than that of traditional culture (1.6 days vs. 5.0 days). The sensitivity of mNGS combined with BALF culture reached 100.0%. In addition, mNGS has a stronger ability to detect co-pathogens with IPA. 47.6% of T2DM patients with IPA were adjusted the initial antimicrobial therapy according to the mNGS results. This is the first study to focus on the diagnostic performance of mNGS in IPA infection in T2DM patients. MNGS can be used as a supplement to conventional methods for the diagnosis of IPA in patients with T2DM.

Aspergillosis in Critically Ill Patients with and Without COVID-19 in a Tertiary Hospital in Southern Brazil.

The impact of invasive pulmonary aspergillosis (IPA) on non-neutropenic critically ill patients in intensive care units (ICU) has been demonstrated in recent decades. Furthermore, after the start of the COVID-19 pandemic, COVID-19 associated with pulmonary aspergillosis (CAPA) has become a major concern in ICUs. However, epidemiological data from different regions are scarce. We evaluated the prevalence and clinical-epidemiological data of IPA in patients with COVID-19 requiring mechanical ventilation (MV) in the ICU ("severe COVID-19") and non-COVID ICU patients in MV of a tertiary hospital in the southern region of Brazil. Eighty-seven patients admitted between June 2020 and August 2022 were included; 31 with severe COVID-19. For the diagnosis of IPA or CAPA, algorithms including host factors and mycological criteria (positive culture for Aspergillus spp., immunoassay for galactomannan detection, and/or qPCR) were utilized. The overall incidence of IPA and CAPA in our ICU was 73 cases/1000 ICU hospitalizations. Aspergillosis occurred in 13% (4/31) of the COVID-19 patients, and in 16% (9/56) of the critically ill patients without COVID-19, with mortality rates of 75% (3/4) and 67% (6/9), respectively. Our results highlight the need for physicians enrolled in ICU care to be aware of aspergillosis and for more access of the patients to sensitive and robust diagnostic tests by biomarkers detection.

Performance of the galactomannan test for the diagnosis of invasive pulmonary aspergillosis using non-invasive proximal airway samples

To diagnose invasive pulmonary aspergillosis (IPA), galactomannan (GM) detection in serum or bronchoalveolar lavage fluid (BALF) is widely used. However, the utility of proximal airway GM test (from induced sputum or tracheal aspirate) has not been well elucidated. In this retrospective cohort study, we evaluated the diagnostic performance of proximal airway GM in diagnosis of IPA including COVID-19 associated pulmonary aspergillosis (CAPA). Between January 2022 and January 2023, patients who had been tested for GM with clinical suspicion or for surveillance from any specimen (serum, induced sputum, tracheal aspirate, and BALF) were screened. IPA was diagnosed using EORTC/MSGERC criteria, and CAPA was diagnosed following the 2020 ECMM/ISHAM consensus criteria. Of 624 patients with GM results, 70 met the criteria for proven/probable IPA and 427 had no IPA. The others included possible IPA and chronic form of aspergillosis. The sensitivities and specificities of serum, proximal airway, and BALF GM for proven/probable IPA versus no IPA were 78.9% and 70.6%, 93.1% and 78.7%, and 78.6% and 91.0%, respectively. Areas under the receiver operating characteristic curve (AUCs) were 0.742 for serum GM, 0.935 for proximal airway GM, and 0.849 for BALF GM (serum GM vs proximal airway GM, p=0.014; proximal airway GM vs BALF GM, p=0.334; serum GM vs BALF GM, p=0.286). This study demonstrates that the performance of GM test from non-invasive proximal airway samples is comparable or even better than those from serum and distal airway sample (BALF).

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.

Comparison of diagnostic techniques in patients with COVID-19-associated pulmonary aspergillosis (CAPA) in a Venezuelan population

Background: Invasive aspergillosis related to COVID-19 or CAPA is defined by the association of a group of histological, mycological, radiological and clinical criteria that establish this disease as a secondary infection of high morbidity without timely diagnosis. Clinical and histological methods, due to the non-specific characterization of the disease, have been supported by techniques based on microbiological methods, such as culture and direct examination, with a sensitivity close to 50%, allowing the identification of biomarkers such as the GM of the pathogen, being the immunological techniques of greater sensitivity and specificity, the use of radial immunodiffusion and ELISA with antigens secreted by Aspergillus spp. are recommended. The objective of this article was to compare three different serological diagnostic methods used in the mycology laboratory to diagnose CAPA in a Venezuelan population. Methods: Based on a longitudinal study, 77 patient samples diagnosed as positive for SARS-CoV-2 by RT-PCR were analyzed, of which 24 CAPA BAL samples were subsequently evaluated by conventional mycological diagnostic methods (direct examination and culture) and corresponding CAPA serum samples by three serological methods: DID, ELISA with Aspergillus pool antigen, and the Galactomannan detection with Lateral flow (GM- LFA) essay. Results: By microscopy, fungal structures were observed in 18/24 patients and 16/24 were positive for GM-LFA. Regarding the DID assays, precipitating bands were obtained in 21/24 patients with 86% sensitivity; and 97% specificity. To use ELISA technique, shows that 16/24 sera from patients with aspergillosis were positive, which represents 66% sensitivity and 97% specificity. As for GM- LFA, all sera were tested and 16/24 positive sera were obtained with the commercial kit, representing 66% sensitivity and 97% specificity. Conclusions: Finally, when comparing the three tests studied, all techniques were found to be highly sensitive and have excellent specificity, recommending that the diagnosis of CAPA and IPA be based on a combination of diagnostic assays.

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.

Microbiological diagnosis of pulmonary invasive aspergillosis in critically ill patients with severe SARS-CoV-2 pneumonia: a bronchoalveolar study

BackgroundDiagnosing COVID-19-associated pulmonary aspergillosis (CAPA) can be challenging since radiological and clinical criteria in the critically ill patient are nonspecific. Microbiological diagnostic support is therefore crucial. The aim of this study was to document the incidence of aspergillosis using bronchoalveolar lavage (BAL) as the diagnostic method and to determine the performance of the current mycological diagnostic tests most widely used for the diagnosis of CAPA, together with evaluation of the Asp lateral flow device (LFD).MethodsProspective cohort study conducted between March 2020 and June 2022. Inclusion criteria were critically ill patients admitted to the ICU with SARS-CoV-2 pneumonia requiring invasive mechanical ventilation. Diagnostic bronchoscopy and BAL were performed at the beginning of invasive mechanical ventilation. The sensitivity, specificity, positive and negative predictive value (PPV and NPV), positive and negative likelihood ratio (LR + and LR-) of BAL culture, direct examination with calcofluor white stain, ELISA (Platelia) and LFD (AspLFD) for detection of galactomannan (GM) were evaluated. Aspergillus-qPCR was applied when discrepancies between diagnostic tests arose.ResultsOf the 244 critically ill patients with SARS-CoV-2 pneumonia admitted to the ICU, the majority (n = 200, 82%) required invasive mechanical ventilation. Diagnostic bronchoscopic procedures were performed in 160 patients (80%), who were enrolled in this study. The incidence of CAPA was 18.7% (n = 30). LFD-GM demonstrated a sensitivity of 84%, specificity of 99%, PPV 94%, NPV 97%, LR(+) of 84, and LR(-) of 0.16. At GM-ELISA indices of ≥ 0.5 and ≥ 1.0, sensitivity was 92% and 79%, specificity was 95% and 99%, PPV 76% and 91%, NPV 99% and 96%, LR(+) 18 and 79, and LR(-) 0.08 and 0.21, respectively. The optimal cut-off index from the ROC curve was 0.48, with sensitivity of 95% and specificity of 95%.ConclusionsUsing a diagnostic strategy based on bronchoscopy and BAL, we documented a high incidence of pulmonary aspergillosis in patients with severe SARS-CoV-2 pneumonia. Asp-LFD showed moderate sensitivity and excellent specificity, with a high PPV, and could be used for rapid diagnosis of patients with suspected CAPA.

False-positive Aspergillus galactomannan immunoassay in the glucose component of total parenteral nutrition products.

In October 2022, we experienced a significant increase in samples showing high galactomannan (GM) indexes ranging from 6.22 to 10.58, as determined by the Platelia Aspergillus antigen immunoassay, also known as the GM test. After reviewing the medical records of nine GM antigenemia cases that did not show evidence of invasive aspergillosis, we found that these patients had received total parenteral nutrition (TPN) products from the same manufacturer, whose supplier of the glucose component had recently changed. The TPN products supplied by the specific manufacturer in October were subjected to a GM assay. The glucose component of the products from three different lot numbers exhibited strong positive results in the GM assay. Microbiological investigations through fungal culture and PCR on the TPN products were negative. The present study demonstrated that the glucose component of the TPN products contained a high level of GM antigen, which caused false-positive GM test results. The source of GM in the glucose component was glucoamylase, which was produced from Aspergillus niger to obtain glucose monohydrate from starch. Investigation of three commercially available glucoamylase products exhibited positive GM and 1,3-β-D-glucan tests with various titers positive up to 1:1,000 dilutions, while fungal cultures were all negative. Quality assurance measures of TPN products to prevent GM contamination should be emphasized during the manufacturing process to avoid unnecessary additional diagnostic procedures and overtreatment of invasive aspergillosis due to false-positive GM tests.IMPORTANCEThis manuscript describes an occurrence of false-positive GM tests in patients receiving TPN products from a manufacturer who had recently changed the supplier of the glucose component. We describe the clinical presentation of nine false-positive cases and the results of serologic and microbiological investigations of the TPN products suspected of contamination with GM. Attempts to detect GM in parenteral nutrition products were made since the detection of GM in sodium gluconate-containing solutions in 2007, but none of them identified the source of elevated GM indexes in TPN products. However, the present study demonstrated that the glucose component of the TPN products contained a high level of GM antigen, which caused false-positive GM assay results. The source of GM was glucoamylase, which was derived from A. niger in the manufacturing process. Physicians and clinical microbiology laboratories should be aware of this issue to improve interpretation and patient care.

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.

Risk factors and outcomes of fungal superinfections in patients with severe COVID-19: an observational study from Pisa academic hospital.

Superinfections acquired during the hospital course represent common complications in COVID-19 patients. Several studies reported an increasing incidence of COVID-19 associated pulmonary aspergillosis (CAPA) and candidaemia. The aim of this study is to describe fungal superinfections in a large cohort of hospitalized patients with COVID-19 and identify factors independently associated with the risk of fungal superinfections. Observational study including patients with COVID-19 admitted to the tertiary-care, University Hospital of Pisa, Italy from April 2020 to May 2021. Patients with pneumonia and laboratory confirmed SARS-CoV-2 infection with a RT-PCR test on a nasopharyngeal swab, were eligible for the study. Patients who died within 24 hours from admission and those with missing data were excluded. Data about fungal superinfections were collected. To identify factors independently associated with the development of fungal superinfections, a multivariate regression analysis was performed. Among 983 patients with COVID-19, 52 (5.3%) fungal superinfections were detected. Fungal superinfections included: 24/52 (46%) CAPA, 27/52 (51.9%) episodes of candidaemia and 1 case of pulmonary pneumocystosis in a haematological patient. All patients with CAPA were cared for in intensive care unit (ICU). The majority of patients received liposomal amphotericin B as antifungal treatment (83.3%). In-hospital mortality was 41.7%. Among 27 episodes of candidaemia, 16 (59.3%) occurred in ICU while 11 (40.7%) in medical wards. In-hospital mortality was 14.8%. Overall, patients with fungal superinfections had a median age of 73 (IQRs 59-77) years and a median length of ICU stay of 40 (17-50) days. In-hospital mortality among all patients with superinfections was 28.8%. On multivariable analysis, ICU stay (OR 17.63, 95% CI 8.3-37.41, p<0.001), high-dose steroids (OR 13.48, 95% CI 6.68-27.26, p<0.001), and diabetes mellitus (OR 2.14, 95% CI 1.09-4.17, p=0.026) were factors independently associated with the risk of developing a fungal superinfection. Fungal superinfections may complicate the hospital course of COVID-19 patients, especially of those admitted to ICU. Surveillance with detection of galactomannan on bronchoalveolar lavage in patients with clinical deterioration should be performed. A rational use of steroids is essential to avoid the risk of developing a fungal superinfection.

Evaluation of the EUROIMMUN Aspergillus antigen immunoenzyme assay in serum and bronchoalveolar lavage fluid samples

IntroductionThe most widely used marker for the diagnosis of invasive aspergillosis (IA) is the detection of galactomannan by ELISA. This study describes the evaluation of the results obtained by Euroimmun Aspergillus antigen ELISA (EIA-GM-E) in serum samples and bronchoalveolar lavage fluid (BAL) from patients at risk of IA, and compares these results with those obtained by Bio-Rad Galactomannan EIA (EIA-GM-BR). MethodsAnonymous retrospective case–control comparative study in 64 serum samples and 28 BAL from 51 patients. ResultsOverall agreement of the results of the two assays was observed in 72 of 92 samples (78.3%). The sensitivity of EIA-GM-BR and EIA-GM-E in serum samples was 88.9% and 43.2%, respectively, and 100% and 88.9% for BAL. The specificity of EIA-GM-BR and EIA-GM-E in serum samples was 91.9% for both assays, and 68.4% and 84.2% in BAL. There were no statistically significant differences in the results of both assays. ConclusionsBoth methods show good results for the discrimination of patients with IA when BAL is tested, or serum in case of EIA-GM-BR.

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan.

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

Blood Mucorales PCR to track down Aspergillus and Mucorales co-infections in at-risk hematology patients: A case-control study.

Serum Mucorales PCR can precede the final diagnosis of invasive mucormycosis by several days or weeks and could therefore be useful as a non-invasive screening tool. We assessed the performance of a commercial Mucorales PCR assay (MucorGenius®, PathoNostics, Maastricht, The Netherlands) on prospectively collected banked sera from hematology patients at risk for invasive mould infections. We evaluated if there is an underestimated incidence of missed Mucorales co-infections in patients with invasive aspergillosis (IA). We tested Mucorales PCR on the sera of all patients with a diagnosis of at least possible IA (EORTC-MSGERC consensus criteria) before the start of any antifungal therapy, and in a control group of similar high-risk hematology patients without IA (in a 1:4 ratio). When a positive Mucorales PCR was observed, at least 5 serum samples taken before and after the positive one were selected. Mucorales PCR was performed in 46 diagnostic serum samples of cases and in 184 controls. Serum Mucorales PCR was positive in 4 cases of IA (8.7%; 12.9% of probable cases) and in 1 control case (0.5%) (p=0.0061, OR=17.43 (1.90-159.96). Post-mortem cultures of the positive control became positive for Rhizopus arrhizus. Mortality of IA cases with and without a positive Mucorales PCR was not significantly different. Only in the PCR positive control case, serial serum samples before and after the diagnostic sample were also positive. It is not entirely clear what a positive Mucorales PCR in these cases implies since the 4 Mucorales PCR positive cases were treated with antifungals with activity against Mucorales. In addition, PCR was positive only once. This study does not provide enough evidence to implement Mucorales PCR screening. However, our findings emphasize once more the importance of considering the possibility of dual mould infections, even in patients with a positive galactomannan detection.

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.

P436 A study to compare detection of Aspergillus galactomannan in patients with clinically suspected invasive Aspergillosis using the Lateral Flow Assay (LFA) in comparison with Enzyme Linked Immunosorbent Assay (ELISA) and identification of new risk factors as per BM-AspICU criteria

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesTo compareperformance characteristics of lateral flow assay (Aspergillus galactomannan LFA, Immy, USA) in comparison with ELISA (Platelia™ Aspergillus antigen, Bio-Rad, France) for detection of galactomannan antigen in blood and BAL samples from suspected cases of Invasive Aspergillosis (IA)turn-around time for reporting and the cost per test for both the methodsTo identify new host factors for categories of IA as per BM-AspICU (Hamam et al. Ann. Intensive Care, 2021) guidelinesMethodsA total of 50 consecutive leftover samples (30 blood and 20 BAL) received for testing by Galactomannan assay by ELISA method were included. LFA and ELISA were performed as per manufacturers’ instructions. Results were recorded using the Digital Reader provided which gives readings as Positive ≥0.5 and <0.5 Negative. Additionally, results were read visually by two different people and graded as 0 (negative), 1+ (very faint band), 2+ (faint band), and 3+ (strong band). The values obtained by ELISA were reported as Galactomannan Index. Results of LFA and ELISA were compared. Patient profiles were studied to identify the possible and probable cases of IA as per BM-AspICU guidelines.ResultsOverall, 92% samples (n = 46) were positive with ELISA. Results of LFA and ELISA were comparable in 68% samples (n = 34). For all except two samples, visual readings of LFA (5 with 3+, 12 with 2 + and 19 with 1 + readings) correlated with digital readings. Sensitivity of LFA was 73.9% (95% CI). Applying BM-AspICU criteria, 28% (n = 14) were categorized as probable, 64% (n = 32) as possible cases of IA and 8% (n = 4) as NO IA. A total of 12 patients had classical host factors, 42% (n = 21) had other risk factors and we were able to identify 16% (n = 8) cases with new risk factors which were CNS involvement/brain injury/hemorrhage on mechanical ventilation. Clinical features were present in all of these 58% (n = 29) cases whereas radiological imaging was done in only 25 cases out of which 28% (n = 14) were positive. BM-AspICU criteria require ≥2 mycological criteria, but only two of the BAL samples were culture positive for Aspergillus species. These criteria could not be strictly applied to most patients in our study since invasive procedures such as BAL was not done, instead, blood was sent for galactomannan detection.The average turn-around time was 3-7 days for ELISA since the test was run twice a week whereas it was 5-24 h for the LFA. The pre-processing of samples and time required to perform the procedure for both the assays are similar. The cost per test for ELISA was Rs.1675/- as against LFA for Rs.750/-.ConclusionLFA for galactomannan detection is an easier to perform, cost-effective method for diagnosis of IA as compared with ELISA. LFA overcomes the difficulty of insufficient volume of calibrators and controls provided with the ELISA kit. Useful especially for patients admitted in ICU where reports can be issued on the same day instead of batch testing. Identification of new risk factors and inclusion of the same under guidelines is essential to suspect and send samples for testing for early initiation of treatment.

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.

Evaluation of the diagnostic accuracy of galactomannan from the bronchoalveolar lavage fluid of patients with suspected invasive pulmonary aspergillosis

BackgroundSeveral studies to evaluate the accuracy of galactomannan (GM) in bronchoalveolar lavage fluid (BALF) as a diagnostic tool have been carried out; however, there are still controversies about the optimal cut-off point of BALF GM. AimsThe objective of this study was to determine the diagnostic accuracy and the optimal cut-off point on BALF GM from patients with suspected invasive pulmonary aspergillosis (IPA) in a tertiary care hospital. MethodsA cross-sectional study with 188 patients (≥18 years) that had undergone a bronchoscopy with BAL due to suspected IPA was carried out. IPA was diagnosed according to the EORTC/MSG guidelines. ResultsThe optimal optical density cut-off point for BALF GM was 0.67, with sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 70%, 32.3%, and 100%, respectively. ConclusionsBALF GM detection proved to be a useful supplementary technique in the early diagnosis of IPA in both neutropenic and non-neutropenic patients.

Comparison of the Performance of Two Galactomannan Detection Tests: Platelia Aspergillus Ag and Aspergillus Galactomannan Ag Virclia Monotest.

ABSTRACTThe use of nonculture-based biomarkers such as the determination of galactomannan is sought for the diagnosis of invasive aspergillosis. To investigate the comparative yield of two tests for the detection of galactomannan in patients with or without proven or probable invasive aspergillosis. Overall, 327 samples (327 patients) were analyzed in a retrospective/prospective study performed in 3 hospitals in Madrid, comparing the determination results in serum or bronchoalveolar lavage of two techniques for galactomannan detection, namely, Platelia Aspergillus Ag (Bio-Rad) and Aspergillus galactomannan Ag Virclia Monotest (Vircell S.L.), following the manufacturer’s instructions. Both techniques can automate the process, but the second technique has the advantage of individual processing and assembly of each sample without the need for the additional expense of single-dose strips in controls. In total, 288 of the 327 tests performed showed concordant results between both techniques. The agreement between both methods was к = 0.722, and the correlation between indices was ρ = 0.718. Only 39 samples showed discordant results. In those 39 cases, there were 15 patients with proven or probable invasive aspergillosis criteria. For the samples with clinical criteria as a reference, the areas under the curve of the receiver operating characteristic (ROC) curve were 0.962 for Platelia and 0.968 for VirClia. The VirClia test has been proven to be an alternative for diagnosis due to its friendlier automated format than that of the usual Platelia routine test. The VirClia test also allows individual action and, therefore, a more immediate clinical response.IMPORTANCE Invasive mycoses are increasingly present in immunosuppressed or hospitalized patients with serious illnesses, leading to high rates of morbidity and mortality. Invasive aspergillosis is an infection caused, in a percentage greater than 50%, by the genus Aspergillus. It is vitally important to make an early diagnosis that leads to the application of antifungals in the initial stage of the infection. Therefore, tools are required to help with the early diagnosis of the infection. This comparative study of two enzyme immunoassays is based on the detection of galactomannan antigen in serum and bronchoalveolar lavage samples. A new design based on chemiluminescence and presented in an automated single-dose format is compared to a conventional ELISA technique marketed for years. The results obtained from the prospective and retrospective study indicate a high correlation and degree of agreement between both techniques, as well as in their diagnostic performance.

COVID-19 Associated Pulmonary Aspergillosis: Diagnostic Performance, Fungal Epidemiology and Antifungal Susceptibility.

Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) raises concerns as to whether it contributes to an increased mortality. The incidence of CAPA varies widely within hospitals and countries, partly because of difficulties in obtaining a reliable diagnosis. We implemented a routine screening of respiratory specimens in COVID-19 ICU patients for Aspergillus species using culture and galactomannan (GM) detection from serum and/or bronchoalveolar lavages (BAL). Out of 329 ICU patients treated during March 2020 and April 2021, 23 (7%) suffered from CAPA, 13 of probable, and 10 of possible. In the majority of cases, culture, microscopy, and GM testing were in accordance with CAPA definition. However, we saw that the current definitions underscore to pay attention for fungal microscopy and GM detection in BALs, categorizing definitive CAPA diagnosis based on culture positive samples only. The spectrum of Aspergillus species involved Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus niger, and Aspergillus nidulans. We noticed changes in fungal epidemiology, but antifungal resistance was not an issue in our cohort. The study highlights that the diagnosis and incidence of CAPA is influenced by the application of laboratory-based diagnostic tests. Culture positivity as a single microbiological marker for probable definitions may overestimate CAPA cases and thus may trigger unnecessary antifungal treatment.