Long-Term Stability at −20°C of Aspergillus Galactomannan in Serum and Bronchoalveolar Lavage Specimens

Wheat, L. Joseph; Nguyen, M. Hong; Alexander, Barbara D.; Denning, David W.; Caliendo, Angela M.; Lyon, G. Marshall; Baden, Lindsey R.; Marty, Francisco M.; Clancy, Cornelius J.; Kirsch, Emily J.; Noth, Pamela; Witt, John R.; Sugrue, Michele W.; Wingard, John R.

doi:10.1128/jcm.03500-13

Cited by 16 publications

(16 citation statements)

References 14 publications

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“…Galactomannan (GM) is a circulating polysaccharide component of Aspergillus species that is released into the bloodstream by growing hyphae and germinating conidia and can be detected by using a commercial ELISA (Bio-Rad Laboratories, Marnes-laCoquette, France) (13)(14)(15). Although the performance of serum GM testing seems to be promising only in neutropenic patients, who usually develop angioinvasive forms of IPA (1,16), bronchoalveolar lavage (BAL) fluid (BALF) GM seems the method of choice in nonneutropenic patients who tend to develop airway-invasive forms (17)(18)(19).…”

mentioning

confidence: 99%

Novel Tests for Diagnosis of Invasive Aspergillosis in Patients with Underlying Respiratory Diseases

Prattes

Flick²,

Prüller

et al. 2014

Am J Respir Crit Care Med

117

114

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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-b-D-glucan, and Aspergillus-specific lateralflow 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. 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 b-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).

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mentioning

confidence: 99%

Novel Tests for Diagnosis of Invasive Aspergillosis in Patients with Underlying Respiratory Diseases

Prattes

Flick²,

Prüller

et al. 2014

Am J Respir Crit Care Med

117

114

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“…However, the observed reduction in equivocal results could also be due to an effect of sample storage at Ϫ20°C prior to testing, which was introduced for all samples not being tested on the day of receipt in our center around the time of DS2 automation. The low reproducibility of GM-positive results after storage at Ϫ20°C has been highlighted in two studies (10,19); however, the longterm stability of galactomannan in serum at Ϫ20°C has been demonstrated to be up to 5 years (20). This suggests that freezing may lead to a reduction in false positivity but not affect genuinely positive samples.…”

Section: Discussionmentioning

confidence: 94%

“…Prior to implementation in clinical practice, the DS2 system was evaluated using control serum samples and pooled sera spiked with increasing volumes (10,20,30,40, and 50 l) of a galactomannan positive control (supplied by Bio-Rad). Samples were processed on the day of spiking, manually and on two identical DS2 platforms, to allow analysis of interplatform reliability.…”

Section: Methodsmentioning

confidence: 99%

Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System

Gorton

White

Bagkeris³

et al. 2015

J Clin Microbiol

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T he Bio-Rad Platelia Aspergillus galactomannan antigen sandwich enzyme immunoassay (GM-EIA) is widely used as a screening method for prospective surveillance of invasive aspergillosis (IA) in patients at high risk of disease. The assay is well established, as reflected by its recommendation in the European Organization for Research and Treatment of Cancer (EORTC) consensus criteria for defining invasive fungal disease (IFD) (1). Despite this fact, the diagnostic performance of the GM-EIA is variable, with meta-analyses showing combined sensitivities ranging from 0.71 to 0.78 and specificities ranging from 0.81 to 0.89 (2, 3). The false positivity experienced using the GM-EIA has been associated with antimicrobial treatment (e.g., piperacillin-tazobactam), other invasive fungal diseases (e.g., fusariosis), and even ingestion of an ice-pop (4-7). Consequently, the GM-EIA should not be used as a stand-alone diagnostic test but is an important component of the diagnostic strategy for managing IA (1).Although initially the GM-EIA reproducibility was reported to be excellent between laboratories (8), recent reports documented a lack of reproducibility for repeat testing of positive samples (9, 10). In particular, samples with an optical density index (ODI) at or around the positivity threshold (Х0.5) of the assay were regularly found to be negative on repeat testing (8, 11). The storage conditions of specimens appear to have an impact on reproducibility, with a significant decline in the sample ODI reported after storage at Ϫ80°C (11). An IA diagnosis also appears to be important, with nonreproducibility observed more frequently for retesting of false-positive samples from patients without IA (11,12). A significant correlation between the serum albumin concentration and the difference in ODI value on retesting was also recently reported. A larger reduction in indices was observed on retesting sera with increasing albumin concentrations (11).While storage and disease status have been shown to affect the reproducibility of GM-EIA, other factors, such as human error, environmental contamination at the point of testing, and variability in local testing conditions between laboratories, may also have impacts on the assay's performance and reproducibility. The GM-EIA is performed manually by most laboratories and, consequently, is susceptible to fluctuations in the environmental tem-

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“…; University of Florida, Gainesville, Florida, USA h W e thank Dr. Johnson and coauthors for reemphasizing in their letter (1) some of the limitations of our study of the stability of galactomannan in frozen specimens (2), namely, the small sample size, lack of clinical information, and potential for confounding factors in pooled specimens. We also acknowledge that our findings differ from those of several other studies, including their own.…”

mentioning

confidence: 99%

Reply to “Insufficient Demonstration of Long-Term Stability of Aspergillus Galactomannan”

Wheat¹,

Alexander

Denning

et al. 2014

J Clin Microbiol

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h W e thank Dr. Johnson and coauthors for reemphasizing in their letter (1) some of the limitations of our study of the stability of galactomannan in frozen specimens (2), namely, the small sample size, lack of clinical information, and potential for confounding factors in pooled specimens. We also acknowledge that our findings differ from those of several other studies, including their own.While clinical information is desirable, the point of our study was the stability of galactomannan in specimens that had been stored frozen. As the assay uses monoclonal antibodies that are specific for galactomannan and as false-positive results are caused by galactomannan present in other fungi or components produced by Penicillium or Aspergillus or present in contaminated medications or electrolyte solutions, the lack of clinical information is unlikely to influence observations about galactomannan stability.Greater variability was observed in the retrospective study of frozen specimens that had initially been tested 5 years earlier than in the prospective study, which used pools created at the time of initial testing. Greater variability in archived specimens is understandable given the multiple factors affecting variability: assay-toassay, kit-to-kit, and operator-to-operator variation. Furthermore, and very importantly, the assay was not developed for quantification and does not use a standard curve for calculation of galactomannan concentration. That said, the agreement in the retrospective study was good. Samples (10 of the 30 tested) that showed a Ͼ20% decline in their galactomannan level and galactomannan index (GMI) are listed in Table 1. Only one of the 10 specimens was negative (had an index of less than 0.5) with repeat testing, and 5 of the 10 had relatively low initial GMIs (0.83 to 2.48), in which greater variability is expected. Only 2 specimens showed a major decline, both of which were BAL fluid specimens (GMIs, 2.58 to 0.61 [76%] and 7.10 to 2.78 [62%]).The prospective study of pooled specimens was conducted to control for the variables affecting the retrospective study. Pooled specimens were used because of the insufficient volume of individual specimens to study four prospective time points. No reduction in GMI was observed over the 11 months for any of the pools. In fact, the GMIs increased: 21.4% of slightly positive serum samples, 2% of highly positive serum samples, 13% of slightly positive BAL fluid samples, and 29% of highly positive BAL fluid samples.In conclusion, our findings support the stability of galactomannan in specimens that have been stored frozen and the use of frozen specimens in evaluating new diagnostic tests based on galactomannan detection. Retrospective studies are unlikely to resolve this controversy. A statistically powered prospective study testing specimens from individual patients at 0 and 11 months, by the same operator using the same kit lot, might settle the controversy.

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Long-Term Stability at −20°C of Aspergillus Galactomannan in Serum and Bronchoalveolar Lavage Specimens

Cited by 16 publications

References 14 publications

Novel Tests for Diagnosis of Invasive Aspergillosis in Patients with Underlying Respiratory Diseases

Novel Tests for Diagnosis of Invasive Aspergillosis in Patients with Underlying Respiratory Diseases

Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System

Reply to “Insufficient Demonstration of Long-Term Stability of Aspergillus Galactomannan”

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