Invasive fungal infections are a leading cause of death in immunocompromised patients (13
species are responsible for many of these infection-related deaths, with a mortality rate of approximately 60 to 90% depending on the immunity and underlying disease of the host (4
). Poor outcomes are, in part, associated with delayed diagnosis and inadequate or inappropriate early antimicrobial therapies (18
In the past, the diagnosis of invasive aspergillosis (IA) relied solely on clinical, radiological, and histopathologic examination. Unfortunately, these methods lack both sensitivity and specificity, particularly in the early stages of infection, when the diagnosis is often missed. The Platelia Aspergillus
enzyme immunoassay (EIA) (Bio-Rad) detects circulating galactomannan (GM), a major component of the Aspergillus
cell wall. It is a double-sandwich EIA that uses a rat monoclonal antibody (EB-A2) to capture the β-1,5-galactofuranoside side chain of the GM molecule. In multiple studies, the GM EIA has been shown to be a useful diagnostic test for IA in neutropenic patients with cancer and recipients of hematopoietic stem cell transplants (5
); however, estimates of sensitivity and specificity vary, ranging from 50 to 92.6% and 82 to 99.6%, respectively (5
The GM EIA results are interpreted as an optical density (OD) index, which is the ratio of the sample OD divided by the mean OD of two threshold controls. When the GM EIA was originally marketed in Europe, the recommended cutoff to define a positive test was an index of ≥1.5. Since then, multiple studies have shown that sensitivity can be increased with minimal loss of specificity by lowering the cutoff to the 0.5 to 0.7 range (3
). The U.S. Food and Drug Administration (FDA) cleared the test using an index cutoff of 0.5; another methodological change with FDA approval is the recommendation that the test be repeated on the same specimen to confirm positivity with an index cutoff of 0.5.
The goal of this study was to examine the reproducibility of the Platelia GM EIA, focusing on samples with low-level GM indices (<1.5). A multilaboratory study was conducted to evaluate intra-assay and interassay reproducibility using multiple kit lots tested on multiple days.
Confidence in the reproducibility of the Platelia GM EIA is vital for the interpretation of results in the clinical setting, where the assay may be used not only to facilitate the early diagnosis of IA but also to monitor the course of infection. This study was performed to evaluate the reproducibility of test results, focusing on samples that have low-negative (<0.5) and low-positive (≥0.5) indices. The results of this study suggest that the assay demonstrates excellent reproducibility between laboratories and between days; however, some variability in results appeared to be associated with the use of different kit lots between runs. Analysis of the “real-time” performance of repeat testing of patient sera also demonstrates some lack of reproducibility when a sample with a GM index from 0.5 to 0.7 is tested in a different run. These results suggest that some caution may be needed when interpreting a single test with an index value between 0.5 and 0.7 as positive.
Test results were generally reproducible between laboratories and between days, even for samples with low index values. However, we did find significant variation between results generated in different runs using different kit lots, at all index values, with particularly high CV%s at low index values. The within-lot/run CV% was also high for these index values. Unfortunately, because of our study design, we were not able to distinguish between interlot and interrun variability. As we did not find a large variance between days, the variability is likely to reflect differences in kit lots. Lot variables that may be different include properties of the monoclonal antibody and, perhaps more likely, OD values of the threshold controls. The relatively high total CV% (19.5%) for the threshold controls, almost all of which represented between-site and between-lot/run variability, supports this hypothesis (Table 1
). These results add to the findings of Verweij et al., who demonstrated significant between-run variability using the same kit lot (17
). Measurement of serum GM levels over time to monitor the course of aspergillosis is not an FDA-approved indication for this assay; however, there is evidence that favorable outcomes are associated with decreasing GMIs and that poor outcomes are associated with increasing GMIs (8
). The interlot/interrun variation seen in this study may be particularly relevant for clinicians when comparing patients' GMIs over time using different kit lots.
With the exception of sample B and the threshold controls, between-laboratory results were reproducible, although one laboratory (site 3) did generate more variability than sites 1 and 2. EIAs are multistepped tests prone to variation and error. Explanations for between-site variability include different equipment and calibration and factors associated with between-run variability: mainly, methodological inconsistencies including the timing of each step, reagent temperature, pipetting errors, and the effectiveness of the washing step. In our reproducibility study, we sought to minimize these factors by ensuring proficiency of the operator before the study was started.
As shown in Table 1
, the variance components appeared to increase as the index value increased. With the exception of between-lot/run variability, the increase in variance was not significant. As variance is a function of the mean value, this increase in variance is expected. Indeed, when the variance component is standardized by the mean index value to create the CV%, the highest CV% is seen at the lower index values.
In the clinical study, 10.2% of initially positive tests were not confirmed on subsequent testing. Lack of confirmation was almost exclusively seen when the initial GM index was between 0.5 and 0.7. The majority (85%) of the patients from whom these specimens were drawn did not have IA according to published criteria. A weakly positive GMI may represent one of the following: low fungal burden, cross-reactivity with other serum antigens, gut translocation of galactomannan from dietary sources, and specimen contamination with environmental Aspergillus
species. Recently, we have demonstrated that concomitant administration of antifungal agents is associated with lower GMIs and decreased sensitivity of the assay (11
). One of 61 (1.6%) tests with high initial GM indices (≥1.5) was not positive in the confirmity run, suggesting that laboratory contamination was not a large issue during the course of the study. The finding of variability in the low-positive range may reflect between-run variability, to which the clinical laboratory is particularly susceptible, given that different operators perform different test runs. Unfortunately, we do not know how many different kit lots were used to test the samples and the impact of such on the data.
The optimal cutoff for defining a positive result continues to be a subject of investigation and discussion. The current FDA recommendation is to use a cutoff index of 0.5, to confirm an initial positive test by repeating the assay on the same specimen, and to report the result as positive only if the subsequent index is also ≥0.5. In a recent paper, Maertens et al. demonstrated improved sensitivity (96.5%) and specificity (98.6%) when a dynamic cutoff of two tests with GM indices of ≥0.5 is employed, compared with sensitivity and specificity results when a static cutoff of a single test with a GM index of ≥1.5 and ≥0.5 is used (82.7% and 100%, and 96.5% and 85.1%, respectively) (6
). Mindful of the lowered cutoff (0.5), we designed this study to investigate reproducibility at low levels of GM positivity, where the lack of reproducibility might have the greatest impact on diagnostic and treatment decisions. Our data suggest that between-lot/run and within-lot/run variability have an impact on the reproducibly of tests with index values around the low-positive range, and analysis of “real-time” results from the clinical microbiology laboratory confirmed some lack of reproducibility for tests having initial low-positive results (0.5 to 0.7). These results highlight the need for repeat testing of the same sample prior to reporting a positive test result.
There are some study limitations that need to be considered when analyzing our results. First, one site tested samples A and G three and five additional times, respectively, and sample B three fewer times than required by the study protocol; as each sample was tested approximately 81 times, we believe that the impact of this deviation on the final results is negligible. The serum samples used to prepare the panels had been frozen prior to use in this study. Although freezing does not alter the stability of GM (C. Bentsen, personal communication), it might impact the stability of other cross-reactive antigens.
In conclusion, the data from our reproducibility study demonstrate excellent reproducibility of the Platelia GM assay at low levels of GM positivity between laboratories and days. Some variability in results was seen with the use of different kit lots between runs and within runs, particularly at low index values. In the clinical study, the majority of results that were not reproducibly positive had low initial GM positivity with indices between 0.5 and 0.7. These findings indicate good overall reproducibility of the Platelia GM EIA; however, caution should be exercised when making clinical decisions based on changes in a patient's serum GM level when different kit lots are used or when interpreting single samples with indices between 0.5 and 0.7.