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.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Respiratory specimens and the diagnostic accuracy of Aspergillus lateral flow assays (LFA-IMMY™): real-life data from a multicentre study.

OBJECTIVES: Proper diagnosis of invasive aspergillosis is challenging because conventional methods lack sensitivity and are complicated by time-consuming incubation processes. To meet the requirement for early diagnosis the new Aspergillus-specific point-of-care test LFA-IMMY™ was evaluated with respect to the ability to accurately detect Aspergillus in bronchoalveolar fluids and sputa, and to clarify the potential of cross-reactivity with other fungal pathogens. METHODS: Respiratory specimens (n = 398) from non-selected patients (n = 390) underwent either fungal microscopy, culture or both before Aspergillus lateral flow assay (LFA-IMMY) testing. RESULTS: For Aspergillus culture- and microscopy-positive samples, sensitivity (48/52) and specificity (44/48) were 92% (95% CI 8.0%-9.7%) and 91% (95% CI 7.9%-9.7%), respectively; cross-reactivity was documented with non-Aspergillus pathogens. CONCLUSION: LFA-IMMY is a reliable diagnostic tool for the detection of Aspergillus in respiratory samples.

The performance of galactomannan in combination with 1,3-ß-D-glucan or aspergillus-lateral flow device for the diagnosis of invasive aspergillosis: Evidences from 13 studies.

Galactomannan (GM), 1,3-ß-D-glucan (BDG) and aspergillus-lateral flow device (LFD) are recognized as diagnostic tools for invasive aspergillosis (IA). The combined performance of these assays, however, is inconsistent in various studies. We undertook a meta-analysis of 13 studies involving 1513 patients to evaluate the utility of GM in combination with BDG or LFD for diagnosing IA. The pooled SEN, SPE, PLR, NLR and diagnostic odds ratio (DOR) were calculated and constructed to summarize the overall combined performance. Combining both positive results of GM and BDG assays leaded to the pooled SEN 0.49 (95%CI 0.27-0.72), SPE 0.98 (95%CI 0.94-1.00), PLR 31.68 (95%CI 5.36-187.37), NLR 0.52 (95%CI 0.32-0.84) and DOR 61.23 (95%CI 6.96-538.90). Comparing with GM and BDG assays, both positive results of GM and LFD leaded to high SEN, similar SPE, low PLR and NLR. At least one positive result of GM or LFD conferred great SEN 0.93 and low NLR 0.08. Both positive results of GM and BDG or LFD assay were in favor of confirming the existence of IA. And both negative results of GM and LFD were beneficial to rule out IA. Further studies with sufficient sample size should focus on the diagnostic performance and cost-effectiveness of these combined tests in clinical setting.

Lateral Flow Assays for the Diagnosis of Invasive Aspergillosis: Current Status.

PURPOSE OF REVIEW: Diagnosis during early stages of invasive aspergillosis (IA) and targeted antifungal treatment has the potential to improve survival significantly. Despite advances in the diagnostic arsenal, invasive mold infections remain difficult to diagnose-especially at early stages before typical radiological signs develop. Varying availability and time-to-results are important limitations of current approved biomarkers and molecular assays for diagnosis of IA. Here, we will give an update on the Aspergillus-specific lateral-flow device (LFD) test. We further review promising findings on feasibility of point-of-care (POC) detection of urinary excreted fungal galactomannan-like antigens. RECENT FINDINGS: POC LFD assays for detection of Aspergillus antigens are currently in development. The Aspergillus-specific LFD test, which is based on the JF5 antibody (Ab), detects an extracellular glycoprotein antigen secreted during active growth of Aspergillus spp. The test has shown promising results in various studies. In addition, a monoclonal Ab476-based LFD for POC detection of urinary excreted fungal galactomannan-like antigens has been developed but needs further validation. SUMMARY: Important advances have been made in the development of LFD assays for IA. Most promising is the Aspergillus-specific LFD test; commercial availability is still pending, however. The search for reliable POC tests for other molds, including mucorales, continues.

Serological Approaches.

The diagnosis of invasive fungal diseases (IFD) based on clinical, radiological, and conventional microbiological findings is not reliable and is often delayed. Non-culture-based methods with higher sensitivity and specificity may reduce diagnostic time and result in decreased IFD morbidity and mortality. These methods are now increasingly used to manage patients at risk of IFD. Among available biomarkers, fungal antigens have been investigated as an aid to early diagnosis and are predominantly used as screening tests to prompt antifungal treatment mainly in patients with hematological malignancies. The revised version of the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) consensus definitions includes some of these biological markers (galactomannan, 1,3-beta-D-glucan, cryptococcus antigen).

Diagnostic accuracy of the Aspergillus-specific bronchoalveolar lavage lateral-flow assay in haematological malignancy patients.

We evaluated the performance of the Aspergillus-specific lateral-flow device (LFD) test for diagnosing invasive pulmonary aspergillosis (IPA) in patients with underlying haematological malignancies. Participating centres were the two Austrian University Hospitals of Graz and Innsbruck. LFD performance was evaluated with 95 bronchoalveolar lavage fluid (BALF) samples from 72 patients collected prospectively in Graz, and with 24 BALF bio bank samples from 23 patients (21 samples with probable IPA) in Innsbruck. Invasive fungal infections were classified according to the revised European Organization of Research and Treatment of Cancer/Mycoses Study Group criteria. Overall, 27 patients (30 samples) had probable IPA, 32 (43 samples) possible and 36 (46 samples) did not fulfil IPA criteria. The vast majority of patients - in particular those with probable IPA - received mould-active treatment before bronchoscopy. Sensitivity, specificity, positive predictive value and negative-predictive-value for probable IPA diagnosis using the BALF-LFD test were 71%, 76%, 35% and 94% for the Graz cohort. Sensitivity of the BALF-LFD test for probable IPA was 57% in Innsbruck bio bank samples. Our results indicate that the BALF-LFD-test provides fast results with moderate sensitivities in patients with underlying haematological malignancies. Similar to other diagnostic tests and biomarkers sensitivity of the test may be influenced by ongoing systemic mould-active treatment.

Non-cultural methods for the diagnosis of invasive fungal disease.

Invasive fungal diseases carry a high mortality risk which can be reduced by early treatment. Diagnosing invasive fungal diseases is challenging, because invasive methods for obtaining histological samples are frequently not feasible in thrombocytopenic immunocompromised patients, while fungal cultures have low sensitivity and a long turn-around time. Non-cultural methods are fundamental for a rapid diagnosis of invasive fungal diseases and they include assays based on the detection of fungal antigens (galactomannan, Aspergillus-lateral flow device, [1,3]-ß-D-glucan, mannan), antibodies, such as anti-mannan, and molecular tests. With the exception of some molecular methods for rare fungi, the non-cultural assays are usually applied to the diagnosis of invasive aspergillosis, invasive candidiasis and pneumocystosis. The performance of a single test or a combination of tests will be discussed, with particular focus on choosing the most appropriate marker(s) for every specific patient population. Reasons for potential false-positive or false-negative results will be discussed.

Novel tests for diagnosis of invasive aspergillosis in patients with underlying respiratory diseases.

RATIONALE: Invasive pulmonary aspergillosis has been increasingly reported in nonneutropenic patients, including those with underlying respiratory diseases. OBJECTIVES: We compared the diagnostic performances of galactomannan, 1,3-ß-D-glucan, and Aspergillus-specific lateral-flow device tests with that of conventional culture by using bronchoalveolar lavage fluid samples from patients with underlying respiratory diseases. METHODS: We analyzed 268 bronchoalveolar lavage samples from 221 patients with underlying respiratory diseases (and without hematologic malignancy or previous solid organ transplantation) that were collected for routine microbiological workup between February 2012 and May 2014 at the University Hospital of Graz, Austria. Invasive pulmonary aspergillosis was defined according to European Organization of Research and Treatment of Cancer/Mycoses Study Group criteria modified for patients with respiratory diseases. MEASUREMENTS AND MAIN RESULTS: Thirty-one patients (14%) had probable or proven, 25 possible, and the remaining 165 patients no invasive pulmonary aspergillosis. Probable/proven aspergillosis was associated with a significantly higher (P = 0.034) 30-day mortality rate of 32%. Sensitivities, specificities, and diagnostic odd ratios differed markedly between galactomannan (cut-off 0.5: optical density index, 0.97, 0.81, 124.4; cut-off 1.0: 0.97, 0.93, 422.1; cut-off 3.0: 0.61, 0.99, 109.8), ß-D-glucan (cut-off 80 pg/ml: 0.90, 0.42, 6.57; cut-off 200 pg/ml: 0.70, 0.61, 3.7), lateral-flow device tests (0.77, 0.92, 41.8), and mycological culture (0.29, 0.97, 14). CONCLUSIONS: Probable or proven invasive pulmonary aspergillosis was diagnosed in 14% of our study population and associated with significantly higher 30-day mortality rates. Although the performance of ß-D-glucan was limited by low specificity and that of mycological culture by low sensitivity, the Aspergillus lateral-flow device seems to be a promising alternative to galactomannan testing, which remains the diagnostic gold standard for aspergillosis. Clinical trial registered with www.clinicaltrials.gov (NCT 02058316).