Repeat-Test Reproducibility of Initially Positive Serum Galactomannan Results in a Tertiary-Care University Hospital: A 7-Year Single-Center Experience

Repeat-Test Reproducibility of Initially Positive Serum Galactomannan Results in a Tertiary-Care University Hospital: A 7-Year Single-Center Experience | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Repeat-Test Reproducibility of Initially Positive Serum Galactomannan Results in a Tertiary-Care University Hospital: A 7-Year Single-Center Experience Demet Timur, Mehmet Tekinsoy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9057396/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Purpose Serum galactomannan (GM) testing is widely used as a mycological marker in the diagnostic work-up of invasive aspergillosis (IA), but results near the assay cut-off may vary on repeat measurement. This study evaluated how often initially positive serum GM results remained positive when the same specimen was retested and how GM optical density index (ODI) values changed between the first and second tests. We also examined whether GM positivity and retest confirmation differed by sex, requesting service, underlying diagnoses, and initial GM-ODI ranges. Methods We retrospectively analyzed 27,014 serum samples from 3,121 patients requested by pediatric and adult services over seven years. Samples with an initial GM-ODI ≥ 0.5 and no known prior GM positivity were retested using a new aliquot of the same specimen within 72 hours. Results Of 27,014 serum samples, 1,918 were initially GM-positive, and 1,361 were retested within 72 hours. On repeat testing, 883/1,361 (65%) converted to negative, whereas 478/1,361 (35%) remained positive. GM-ODI values were significantly lower on retesting (p < 0.001). Final GM positivity did not differ by sex (p = 0.066) but was higher in pediatric than adult service requests (5.8% vs 3.4%, p < 0.001). Among final GM-positive specimens, the distribution of underlying diagnoses was associated with requesting service (p < 0.001). Retest positivity increased with higher initial GM-ODI ranges (p < 0.001) and varied across disease groups. Conclusion Repeat testing of initially GM-positive serum samples within 72 hours frequently resulted in reversion to negative. Confirmation was more likely at higher initial GM-ODI ranges. These findings indicate substantial short-term variability in serum GM results in routine practice and support cautious interpretation of low-positive results. Galactomannan invasive aspergillosis Platelia Aspergillus Ag reproducibility serum biomarker Figures Figure 1 Introduction Aspergillus species are ubiquitous environmental molds and can cause invasive aspergillosis (IA), particularly in patients with impaired host defenses. The risk is highest in individuals with hematological malignancies, prolonged or profound neutropenia, and in recipients of hematopoietic stem cell or solid organ transplants receiving intensive immunosuppression [ 1 – 3 ]. Galactomannan (GM) is a major Aspergillus cell wall polysaccharide released during active fungal growth and is detectable in body fluids, particularly serum and bronchoalveolar lavage (BAL), most commonly by enzyme immunoassays expressed as an optical density index (ODI). Because the lungs are the most common site of IA, GM testing is most often used in the diagnostic work-up of suspected invasive pulmonary aspergillosis (IPA). When invasive sampling is not feasible, serum GM testing is recommended; when BAL can be obtained, BAL GM testing is recommended together with conventional microbiological methods such as microscopy and culture. These approaches are supported by both North American and European guidelines, and GM is also included as a mycological criterion in the updated EORTC/MSGERC definitions of invasive fungal disease. [ 4 – 6 ] In clinical practice and in many contemporary studies, serum and BAL galactomannan are commonly measured with the Platelia Aspergillus Ag assay (Bio-Rad Laboratories, Marnes-la-Coquette, France), a one-stage sandwich enzyme immunoassay performed on microplates and reported as an ODI. [ 7 – 10 ] Although the Platelia Aspergillus Ag assay is widely used, positive results should be interpreted cautiously because the negative predictive value is generally high whereas the positive predictive value may be modest, particularly in settings with low pre-test probability or when results are close to the assay cut-off. This is partly because false-positive signals may arise from assay-, laboratory-, or patient-related factors, including cross-reactivity with other fungi, exposure to certain penicillin-based antibiotics, intake of galactofuranose-containing foods, laboratory contamination, or non-specific assay reactivity. For this reason, the manufacturer recommends retesting all positive patient results using a new aliquot of the same specimen. [ 10 ] In this study, we aimed to evaluate the repeat-test reproducibility of positive results obtained with the Platelia serum GM assay across a large test volume and to assess whether confirmation patterns differed according to patient groups. Our analysis reflects real-world routine laboratory practice and provides a robust assessment of how often initial positivity is confirmed on repeat testing. To our knowledge, this represents one of the largest single-center cohorts examining repeat testing of positive serum GM results with Platelia. Material and Methods A total of 27,014 serum samples from 3,121 patients received between January 2013 and December 2019 at Bursa Uludag University Faculty of Medicine Hospital were retrospectively evaluated for Aspergillus galactomannan (GM). Serum GM testing was performed using the Platelia Aspergillus Ag assay according to the manufacturer’s instructions. Briefly, specimens underwent heat pre-treatment (120°C for 6 minutes), followed by centrifugation, and the supernatant was analyzed by enzyme immunoassay. Optical density (OD) values were converted to an OD index (ODI) relative to the cut-off control (R4), and GM-ODI ≥ 0.5 was considered positive. Each run included the manufacturer-provided negative, cut-off, and positive controls (R3, R4, and R5). An initial positive result was defined as a first-test GM-ODI ≥ 0.5. Specimens with an initial positive result were considered for repeat testing if no documented prior GM positivity was present in the laboratory records. In such cases, a new aliquot of the same specimen was stored at 4°C and retested within 72 hours. A repeat test positive result was defined as a specimen with GM-ODI ≥ 0.5 in both the initial and repeat tests. Specimens were classified as retest-confirmed positive if the repeat GM-ODI remained ≥ 0.5 and as retest-negative if the repeat GM-ODI was < 0.5. Not all initially positive specimens were retested. In routine practice, repeat testing was omitted for some initially positive specimens when prior documented GM positivity and/or concurrent microbiological evidence, including fungal culture positivity, was already available. For the overall laboratory evaluation, a final positive result was defined as either a repeat test-positive specimen or an initial positive specimen that was not retested because prior GM positivity and/or concurrent microbiological evidence had already been documented. All analyses were based on routinely collected laboratory data. Because analyses were performed at the specimen level, multiple specimens from the same patient were not excluded and could contribute multiple observations. Statistical analysis All statistical analyses were performed using SPSS version 27. Continuous variables were presented as medians and interquartile ranges (IQR). The comparison of GM-ODI values between the initial test and retest was performed using the Wilcoxon Signed-Rank Test. Differences in categorical variables were evaluated using the Pearson chi-square test. When multiple comparisons were made, significance levels were adjusted using the Bonferroni correction. The relationship between the increase in ODI levels and retest positivity was examined using the linear-by-linear association test. A two-tailed p-value of < 0.05 was considered statistically significant. Results A total of 27,014 serum specimens from 3,121 patients submitted to our laboratory were retrospectively analyzed. Including repeat testing of initially positive specimens, a total of 28,375 GM assay runs were performed. On the initial test, 1,918 serum specimens were GM-positive, of which 1,361 underwent repeat testing within 72 hours. Among these 1,361 initially positive specimens, 883 (64.9%) became negative on repeat testing, whereas 478 (35.1%) remained positive. The median GM-ODI values were 0.83 (IQR, 0.61–1.52) in the initial test and 0.29 (IQR, 0.17–0.75) on repeat testing. In paired analysis of the retested specimens, GM-ODI values were significantly lower on repeat testing than on the initial test (Wilcoxon signed-rank test, p < 0.001). No significant association was found between sex and final GM status (Table 1 ). By contrast, final GM positivity was observed more often in serum specimens requested by pediatric services than in those requested by adult services (5.8% vs 3.4%, p < 0.001). This finding indicates that the proportion of final GM-positive specimens differed according to requesting service. Table 1 Association of final GM status with sex and requesting service among serum specimens. Variable Positive n (%) Negative n (%) Total n p-value Female 375 (3.6) 10,151 (96.4) 10,526 0.066 Male 660 (4.0) 15,828 (96.0) 16,488 Pediatric services 295 (5.8) 4,789 (94.2) 5,084 < 0.001 Adult services 740 (3.4) 21,190 (96.6) 21,930 Counts represent unique serum specimens. Positive results reflect the final GM classification used in the analysis: specimens were classified as final positive if they were repeat test positive (GM-ODI ≥ 0.5 in both the initial and repeat tests) or if they were initially positive but not retested because prior documented GM positivity and/or concurrent microbiological evidence, including fungal culture positivity, was already available. All other specimens were classified as negative. P values were calculated using the Pearson chi-square test. Abbreviations: GM, galactomannan; ODI, optical density index. We next examined whether the underlying diagnoses of final GM-positive serum specimens varied according to requesting service (Table 2 ). The overall distribution was associated with pediatric versus adult service requests; however, this finding was driven only by selected diagnostic categories. After Bonferroni correction, renal diseases were more common among adult-service requests, whereas solid organ tumors were more common among pediatric-service requests (p < 0.005 for both). The remaining diagnostic categories showed no significant differences between services. Table 2 Distribution of underlying diagnoses by requesting service among final GM-positive serum specimens. Diagnosis Pediatric services Adult services p-value n (%) n (%) Other diseases 5 (16.1) 28 (83.9) 0.110 Hematological diseases 18 (27.7) 47 (72.3) 0.841 Hematological malignancies 231 (27.6) 606 (72.4) 0,162 Renal diseases 1 (3.3) 29 (96.7) 0.002 Solid organ tumors 40 (57.1) 30 (42.9) < 0.001 This table shows the distribution of underlying diagnoses among final GM-positive serum specimens according to requesting service (pediatric vs adult). Final GM-positive specimens were defined as specimens that were repeat test positive (GM-ODI ≥ 0.5 in both the initial and repeat tests) or initially positive specimens that were not retested because prior documented GM positivity and/or concurrent microbiological evidence, including fungal culture positivity, was already available. Percentages are shown within each diagnostic category. Bonferroni-adjusted p values are presented for post hoc comparisons. Abbreviations: GM, galactomannan. The distributions of initial and repeat GM-ODI values are shown in Fig. 1 . When repeat-test positivity was analyzed according to initial ODI category, repeat positivity was lowest in the 0.5–0.99 range and was generally higher in the higher ODI categories (Table 3 ), with a significant overall trend across categories (linear-by-linear association test, p < 0.001). Finally, repeat-test positivity rates also varied across underlying disease groups, as summarized in Table 4 . Table 3 Repeat-test confirmation of initially positive serum GM results by the initial GM-ODI range Cut-off ODI Retest positive n (%) Retest negative n (%) Total (First test positive) n (%) 0.5–0.99 224 (27.7) 584 (72.3) 808 (100) 1.0-1.49 86 (41,7) 120 (58.3) 206 (100) 1.5–1.99 27 (39.7) 41 (60.3) 68 (100) ≥ 2.0 141 (50.5) 138 (49.5) 279 (100) This table summarizes repeat-test outcomes for specimens with an initially positive serum GM result (GM-ODI ≥ 0.5), stratified by the initial GM-ODI range. Repeat positivity was defined as a repeat GM-ODI ≥ 0.5 using a new aliquot of the same specimen. A significant overall trend toward higher repeat-test positivity across increasing initial GM-ODI categories was observed (linear-by-linear association test, p < 0.001). Abbreviations: GM, galactomannan; ODI, optical density index. Table 4 Repeat-test confirmation rates of initially positive serum GM results according to underlying disease group. Disease Group First test positive (n) Retest positive (n) Retest negative (n) Retest positivity (%) AML 399 113 286 28 ALL 378 154 224 41 CLL 14 9 5 64 CML 4 1 3 25 AA 29 10 19 35 MDS 16 10 6 63 NHL HL MM CKD Others 146 23 135 70 147 57 5 32 22 65 89 18 103 48 82 39 22 24 31 44 This table presents the number of initially positive specimens, repeat-positive specimens, repeat-negative specimens, and the repeat-test positivity rate according to underlying disease group. Repeat-test positivity rates differed significantly across disease categories (p < 0.001). The repeat-test positivity rate was calculated as the number of repeat-positive results divided by the number of initially positive results in each disease category. Because analyses were performed at the specimen level, repeated specimens from the same patient were not excluded. Others includes disease categories not listed separately. A bbreviations: GM, galactomannan; ODI, optical density index; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; AA, aplastic anemia; MDS, myelodysplastic syndrome; NHL, non-Hodgkin lymphoma; HL, Hodgkin lymphoma; MM, multiple myeloma; CKD, chronic kidney disease. Discussion Serum GM testing is widely used as a mycological marker in the diagnostic work-up of suspected invasive pulmonary aspergillosis, but interpreting results near the assay cut-off remains challenging. In this large single-center cohort, repeat testing of initially positive serum samples frequently yielded discordant findings when the same specimen was retested within 72 hours, and confirmation became more likely as the initial GM-ODI increased. Together, these results indicate substantial variability in serum GM measurements in routine practice, particularly for low-positive values. Several mechanisms can underlie either false-positive GM results or discordant repeat results after an initially positive finding, and these two phenomena should be distinguished. False-positive GM results may occur when exogenous GM-containing sources or analytical interferences generate a true signal in the absence of invasive aspergillosis; in such situations, positivity may persist on repeat testing of the same specimen. Examples include contamination or exposure from dextrose-containing fluids, which has been associated with clusters of false-positive serum GM results, and penicillin-based antibiotics such as piperacillin–tazobactam or amoxicillin–clavulanate, which have historically been linked to false positivity [ 11 – 13 ]. Dietary or enteral products, including oral/enteral nutritional supplements, have been implicated as potential sources of false-positive GM reactivity, particularly in patients with mucositis or impaired intestinal barrier integrity [ 13 , 14 ]. Beyond these exogenous sources, galactomannan is not entirely specific to Aspergillus , and cross-reactivity has been reported with other fungi, including Fusarium spp. and Histoplasma capsulatum , which may further contribute to non-specific GM reactivity in selected settings [ 4 , 13 ]. Environmental contamination during specimen handling is theoretically possible, but available evidence indicates that only heavy conidial contamination is sufficient to alter GM readings, making this an unlikely explanation under routine laboratory conditions. [ 15 ] By contrast, discordant repeat results, defined as loss of positivity on repeat testing of the same specimen, are more plausibly explained by analytical imprecision and operational or preanalytical factors that can shift values around the assay cut-off, particularly for low-positive indices. Potential contributors include between-run or between-lot variability, small differences in multistep assay workflow (such as timing, pipetting, or washing), and storage- or matrix-related changes that may reduce GM signal on retesting. Such variability has been documented for the Platelia Aspergillus Ag assay, where reproducibility is lower near the cut-off and operational factors within the workflow may generate non-reproducible positives that revert on repeat measurement [ 16 – 18 ]. In our cohort, this interpretation is supported by the substantial repeat-test discordance observed: 64.9% of initially positive samples became negative on repeat testing, and confirmation rates increased with higher initial GM-ODI categories. This pattern was particularly evident across initial ODI categories, with the lowest repeat-test confirmation observed in samples with initial values of 0.5–0.99, whereas confirmation was generally higher in the higher GM-ODI categories. This is consistent with the broader understanding that results near the manufacturer-recommended threshold of 0.5 are more prone to non-confirmation, while higher ODI values are generally associated with greater specificity and a higher likelihood of clinically meaningful positivity. In line with this, updated consensus definitions adopted a serum cutoff of 1.0 to increase diagnostic likelihood compared with 0.5, and even higher thresholds, such as 1.5, have been argued to further improve specificity and positive predictive value, albeit at the cost of lower sensitivity. [ 4 , 16 , 19 ] Beyond the assay-level pattern, the clinical context in which GM testing was requested also appeared to influence positivity rates. In our cohort, overall GM positivity was higher in pediatric service requests than in adult service requests, and no significant difference was observed by sex. This may reflect more targeted test ordering and a higher pre-test probability in pediatric settings rather than a purely biological difference in assay behavior. Such an interpretation is plausible because invasive aspergillosis remains an important concern in immunocompromised pediatric populations, particularly in children receiving chemotherapy, hematopoietic stem cell transplantation, or intensive immunosuppression [ 20 ]. This interpretation is further supported by differences in the distribution of underlying diagnoses among GM-positive cases. Renal diseases were more frequent among requests from adult services, whereas solid organ tumors were more frequent among requests from pediatric services (Table 2 ). However, these subgroup findings should be interpreted cautiously, as some diagnostic categories included relatively small numbers of cases. Repeat-test positivity also varied across the major hematologic subgroups, although these patterns should be viewed as exploratory rather than definitive. In our cohort, retest positivity was higher than the overall average in ALL and NHL, but lower in AML and multiple myeloma. At least part of this variation may reflect differences in service-level case mix rather than disease biology alone, since ALL is predominantly a pediatric malignancy, whereas AML becomes more common with increasing age, NHL is overall more frequent in adults despite recognized pediatric forms, and multiple myeloma is predominantly a disease of older adults. In addition, because repeated specimens from the same patient were not excluded from the analysis, disease groups with more frequent serial sampling may have been overrepresented, potentially influencing subgroup-specific repeat-confirmation rates. Accordingly, the observed differences across disease categories may reflect a combination of case-mix differences, sampling frequency, and disease-specific clinical or biological factors rather than solely intrinsic assay behavior. The low repeat-test positivity observed in our cohort with multiple myeloma is consistent with prior reports suggesting that GM reactivity in this setting may be prone to non-specific positivity, although this subgroup finding should be interpreted cautiously [ 21 ]. This study has several limitations. First, it was a single-center retrospective analysis based on routinely generated laboratory data. Second, patients were not classified according to EORTC/MSGERC criteria as having possible, probable, or proven invasive aspergillosis; therefore, false positivity could not be definitively distinguished from non-confirmation related to analytical or preanalytical variability. Third, analyses were performed at the specimen level, and repeated specimens from the same patient were not excluded, which may have influenced subgroup-level comparisons and the apparent distribution of positivity across diagnostic categories. Finally, not all initially positive specimens underwent repeat testing, as retesting was sometimes omitted when prior positivity and/or concurrent microbiological evidence had already been documented, potentially introducing selection bias. Conclusion Repeat testing of initially positive serum galactomannan results frequently yielded discordant findings in routine practice, particularly for low-positive values near the assay cut-off. Repeat-test confirmation was more likely at higher initial GM-ODI levels and varied across underlying disease groups. These findings indicate substantial short-term variability in serum GM measurements and support cautious interpretation of low-positive results, especially when clinical correlation is limited. In routine laboratory workflows, repeat assessment of initially positive samples may improve confidence in result interpretation. Declarations Competing Interests The authors declare that they have no financial or non-financial interests that are directly or indirectly related to the work submitted for publication. Ethical Approval Ethical approval was obtained from the Bursa City Hospital Clinical Research Ethics Committee (approval date: 04 September 2024; decision no: 2024-14/7). Consent to Participate Given the retrospective nature of the study and the use of routinely collected laboratory data, the institutional review board waived the requirement for informed consent. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Author Contributions The study was conceived and designed by Demet Timur. Material preparation, data collection, and data curation were performed by Demet Timur. Formal analysis was performed by Demet Timur and Mehmet Tekinsoy. The investigation was carried out by Demet Timur and Mehmet Tekinsoy. The first draft of the manuscript was written by Demet Timur and Mehmet Tekinsoy, and both authors contributed to the review and editing of subsequent versions. Supervision was provided by Demet Timur. Both authors read and approved the final manuscript. Acknowledgements The authors thank the mycology laboratory staff of Bursa Uludag University Faculty of Medicine Hospital for their support in routine GM testing and laboratory record maintenance. They also thank Dr. Ahmet Timur for statistical support and Prof. Beyza Ener for her guidance during the study. Funding This research received no external funding. References Douglas AP, et al. Consensus guidelines for the diagnosis and management of invasive aspergillosis, 2021. Intern Med J. 2021;51(Suppl 7):143–76. Lass-Florl C. How to make a fast diagnosis in invasive aspergillosis. Med Mycol. 2019;57(Supplement2):S155–60. Susianti H, et al. Diagnostic value of serum human Galactomannan aspergillus antigen and 1,3-beta-D-glucan in immunocompromised patient suspected fungal infection. J Clin Lab Anal. 2021;35(6):e23806. Mercier T, et al. Defining Galactomannan Positivity in the Updated EORTC/MSGERC Consensus Definitions of Invasive Fungal Diseases. Clin Infect Dis. 2021;72(Suppl 2):S89–94. Donnelly JP, et al. Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin Infect Dis. 2020;71(6):1367–76. Ullmann AJ, et al. Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect. 2018;24(Suppl 1):e1–38. Lamberink H, et al. Multicenter validation of a galactomannan chemiluminescence immunoassay for the diagnosis of pulmonary aspergillosis on serum of patients with hematological disease. J Clin Microbiol. 2025;63(2):e0105324. Montesinos I et al. Diagnostic Value of Serum Biomarkers for Invasive Aspergillosis in Haematologic Patients. J Fungi (Basel), 2024. 10(9). Albert E, et al. Comparative performance of the Platelia Aspergillus Antigen and Aspergillus Galactomannan antigen Virclia Monotest immunoassays in serum and lower respiratory tract specimens: a real-life experience. Microbiol Spectr. 2024;12(8):e0391023. Laboratories B-R. PLATELIA™ ASPERGILLUS Ag-Instructions for Use (package insert) . 2013 [cited 2026 22.02.2026]; 62794:[Instructions for Use]. Available from: https://commerce.bio-rad.com/webroot/web/pdf/inserts/CDG/en/62794_881115_EN.pdf Lee R et al. Dextrose-containing fluids causing false-positive serum galactomannan: a case-control study and interrupted time series analysis. Clin Microbiol Infect, 2024. 30(5): p. 682 e1-682 e4. Gerlinger MP, et al. False positive galactomannan Platelia due to piperacillin-tazobactam. Med Mal Infect. 2012;42(1):10–4. Garg P, et al. Evaluating the causes associated with false positive Galactomannan assay in suspected cases of respiratory fungal infections. J Family Med Prim Care. 2025;14(2):736–42. Shin DW, et al. False-positive results of galactomannan assays in patients administered glucose-containing solutions. Sci Rep. 2024;14(1):2552. Cabana AL, et al. Can Aspergillus fumigatus conidia cause false-positive results in the galactomannan enzyme immunoassay test? Rev Soc Bras Med Trop. 2018;51(3):387–9. Upton A, et al. Reproducibility of low galactomannan enzyme immunoassay index values tested in multiple laboratories. J Clin Microbiol. 2005;43(9):4796–800. Guigue N, et al. Importance of operational factors in the reproducibility of Aspergillus galactomannan enzyme immune assay. PLoS ONE. 2015;10(4):e0124044. Kimpton G, White PL, Barnes RA. The effect of sample storage on the performance and reproducibility of the galactomannan EIA test. Med Mycol. 2014;52(6):618–26. Leeflang MM, et al. Galactomannan detection for invasive aspergillosis in immunocompromised patients. Cochrane Database Syst Rev. 2015;2015(12):CD007394. Wehbe S et al. Pediatric vs. Adult Invasive Aspergillosis in Cancer and Hematopoietic Transplant Patients: Insights from a Matched Cohort at a Tertiary Cancer Center. J Fungi (Basel), 2025. 11(11). Abe Y, et al. Higher frequency of false-positive serum galactomannan tests among older subjects and the association with elevated serum immunoglobulin G levels. Mycoses. 2019;62(9):773–9. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 15 May, 2026 Reviews received at journal 13 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviews received at journal 26 Apr, 2026 Reviewers agreed at journal 13 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor assigned by journal 09 Mar, 2026 Submission checks completed at journal 09 Mar, 2026 First submitted to journal 07 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9057396","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625465514,"identity":"cff427be-af1b-49a7-8f70-aac8cbffd906","order_by":0,"name":"Demet Timur","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDCCAwwGDAlAml8CzJWQIUaLYQNIi+QMBsYGoBYe4rSAaIMbYC0MhLXw3T68/cHDnMP5xrebjz+6UWPBw8B++OgGfFokz6UVNiRuO2y57c6xxOacY0CH8aSl3cCnxeAMjyFIi4HZjRzD5hw2oBYJHjPitBjPAGn5R4oWAwmgltw2IrRInmErnJG4Ld1A4kZa4uzcPgkeNkJ+4TvDvOHjz23WBvwzkg98zvlWJ8fPfvgYXi2YgI005aNgFIyCUTAKsAEAdq9LczzD1KQAAAAASUVORK5CYII=","orcid":"","institution":"Bursa Uludag University Faculty of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Demet","middleName":"","lastName":"Timur","suffix":""},{"id":625465515,"identity":"b9775cfe-49cf-4800-b3a8-8ff6994fbd7c","order_by":1,"name":"Mehmet Tekinsoy","email":"","orcid":"","institution":"Bursa Uludag University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Tekinsoy","suffix":""}],"badges":[],"createdAt":"2026-03-07 09:53:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9057396/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9057396/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107355941,"identity":"cc6ff177-8571-4ab9-95ec-8834cc350424","added_by":"auto","created_at":"2026-04-20 16:55:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117059,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of initial and repeat serum GM-ODI values among specimens retested within 72 hours.\u003c/p\u003e\n\u003cp\u003eThis figure shows GM-ODI values from the initial and repeat tests for specimens retested using a new aliquot of the same specimen within 72 hours. Overall, repeat GM-ODI values were lower than initial values.\u003c/p\u003e\n\u003cp\u003eAbbreviations: GM, galactomannan; ODI, optical density index.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9057396/v1/fb0e04d8e0e6cb19f31ea69d.png"},{"id":107356013,"identity":"20c78ad4-fedb-4639-875e-c2c911d95da1","added_by":"auto","created_at":"2026-04-20 16:55:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":473692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9057396/v1/a5bb2610-3c12-4498-b0a2-9a29fae3d5f4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Repeat-Test Reproducibility of Initially Positive Serum Galactomannan Results in a Tertiary-Care University Hospital: A 7-Year Single-Center Experience","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAspergillus species are ubiquitous environmental molds and can cause invasive aspergillosis (IA), particularly in patients with impaired host defenses. The risk is highest in individuals with hematological malignancies, prolonged or profound neutropenia, and in recipients of hematopoietic stem cell or solid organ transplants receiving intensive immunosuppression [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Galactomannan (GM) is a major \u003cem\u003eAspergillus\u003c/em\u003e cell wall polysaccharide released during active fungal growth and is detectable in body fluids, particularly serum and bronchoalveolar lavage (BAL), most commonly by enzyme immunoassays expressed as an optical density index (ODI). Because the lungs are the most common site of IA, GM testing is most often used in the diagnostic work-up of suspected invasive pulmonary aspergillosis (IPA). When invasive sampling is not feasible, serum GM testing is recommended; when BAL can be obtained, BAL GM testing is recommended together with conventional microbiological methods such as microscopy and culture. These approaches are supported by both North American and European guidelines, and GM is also included as a mycological criterion in the updated EORTC/MSGERC definitions of invasive fungal disease. [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn clinical practice and in many contemporary studies, serum and BAL galactomannan are commonly measured with the Platelia Aspergillus Ag assay (Bio-Rad Laboratories, Marnes-la-Coquette, France), a one-stage sandwich enzyme immunoassay performed on microplates and reported as an ODI. [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Although the Platelia Aspergillus Ag assay is widely used, positive results should be interpreted cautiously because the negative predictive value is generally high whereas the positive predictive value may be modest, particularly in settings with low pre-test probability or when results are close to the assay cut-off. This is partly because false-positive signals may arise from assay-, laboratory-, or patient-related factors, including cross-reactivity with other fungi, exposure to certain penicillin-based antibiotics, intake of galactofuranose-containing foods, laboratory contamination, or non-specific assay reactivity. For this reason, the manufacturer recommends retesting all positive patient results using a new aliquot of the same specimen. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn this study, we aimed to evaluate the repeat-test reproducibility of positive results obtained with the Platelia serum GM assay across a large test volume and to assess whether confirmation patterns differed according to patient groups. Our analysis reflects real-world routine laboratory practice and provides a robust assessment of how often initial positivity is confirmed on repeat testing. To our knowledge, this represents one of the largest single-center cohorts examining repeat testing of positive serum GM results with Platelia.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eA total of 27,014 serum samples from 3,121 patients received between January 2013 and December 2019 at Bursa Uludag University Faculty of Medicine Hospital were retrospectively evaluated for Aspergillus galactomannan (GM). Serum GM testing was performed using the Platelia Aspergillus Ag assay according to the manufacturer\u0026rsquo;s instructions. Briefly, specimens underwent heat pre-treatment (120\u0026deg;C for 6 minutes), followed by centrifugation, and the supernatant was analyzed by enzyme immunoassay. Optical density (OD) values were converted to an OD index (ODI) relative to the cut-off control (R4), and GM-ODI\u0026thinsp;\u0026ge;\u0026thinsp;0.5 was considered positive. Each run included the manufacturer-provided negative, cut-off, and positive controls (R3, R4, and R5).\u003c/p\u003e \u003cp\u003eAn initial positive result was defined as a first-test GM-ODI\u0026thinsp;\u0026ge;\u0026thinsp;0.5. Specimens with an initial positive result were considered for repeat testing if no documented prior GM positivity was present in the laboratory records. In such cases, a new aliquot of the same specimen was stored at 4\u0026deg;C and retested within 72 hours. A repeat test positive result was defined as a specimen with GM-ODI\u0026thinsp;\u0026ge;\u0026thinsp;0.5 in both the initial and repeat tests. Specimens were classified as retest-confirmed positive if the repeat GM-ODI remained\u0026thinsp;\u0026ge;\u0026thinsp;0.5 and as retest-negative if the repeat GM-ODI was \u0026lt;\u0026thinsp;0.5.\u003c/p\u003e \u003cp\u003eNot all initially positive specimens were retested. In routine practice, repeat testing was omitted for some initially positive specimens when prior documented GM positivity and/or concurrent microbiological evidence, including fungal culture positivity, was already available. For the overall laboratory evaluation, a final positive result was defined as either a repeat test-positive specimen or an initial positive specimen that was not retested because prior GM positivity and/or concurrent microbiological evidence had already been documented.\u003c/p\u003e \u003cp\u003eAll analyses were based on routinely collected laboratory data. Because analyses were performed at the specimen level, multiple specimens from the same patient were not excluded and could contribute multiple observations.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using SPSS version 27. Continuous variables were presented as medians and interquartile ranges (IQR). The comparison of GM-ODI values between the initial test and retest was performed using the Wilcoxon Signed-Rank Test. Differences in categorical variables were evaluated using the Pearson chi-square test. When multiple comparisons were made, significance levels were adjusted using the Bonferroni correction. The relationship between the increase in ODI levels and retest positivity was examined using the linear-by-linear association test. A two-tailed p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 27,014 serum specimens from 3,121 patients submitted to our laboratory were retrospectively analyzed. Including repeat testing of initially positive specimens, a total of 28,375 GM assay runs were performed. On the initial test, 1,918 serum specimens were GM-positive, of which 1,361 underwent repeat testing within 72 hours. Among these 1,361 initially positive specimens, 883 (64.9%) became negative on repeat testing, whereas 478 (35.1%) remained positive. The median GM-ODI values were 0.83 (IQR, 0.61\u0026ndash;1.52) in the initial test and 0.29 (IQR, 0.17\u0026ndash;0.75) on repeat testing. In paired analysis of the retested specimens, GM-ODI values were significantly lower on repeat testing than on the initial test (Wilcoxon signed-rank test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eNo significant association was found between sex and final GM status (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By contrast, final GM positivity was observed more often in serum specimens requested by pediatric services than in those requested by adult services (5.8% vs 3.4%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This finding indicates that the proportion of final GM-positive specimens differed according to requesting service.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociation of final GM status with sex and requesting service among serum specimens.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal n\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e375 (3.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10,151 (96.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10,526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e660 (4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15,828 (96.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16,488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePediatric services\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e295 (5.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4,789 (94.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5,084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdult services\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e740 (3.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21,190 (96.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21,930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eCounts represent unique serum specimens. Positive results reflect the final GM classification used in the analysis: specimens were classified as final positive if they were repeat test positive (GM-ODI\u0026thinsp;\u0026ge;\u0026thinsp;0.5 in both the initial and repeat tests) or if they were initially positive but not retested because prior documented GM positivity and/or concurrent microbiological evidence, including fungal culture positivity, was already available. All other specimens were classified as negative. P values were calculated using the Pearson chi-square test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e GM, galactomannan; ODI, optical density index.\u003c/p\u003e\u003cp\u003eWe next examined whether the underlying diagnoses of final GM-positive serum specimens varied according to requesting service (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The overall distribution was associated with pediatric versus adult service requests; however, this finding was driven only by selected diagnostic categories. After Bonferroni correction, renal diseases were more common among adult-service requests, whereas solid organ tumors were more common among pediatric-service requests (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005 for both). The remaining diagnostic categories showed no significant differences between services.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of underlying diagnoses by requesting service among final GM-positive serum specimens.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDiagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePediatric services\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdult services\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther diseases\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(16.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(83.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematological diseases\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(27.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(72.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.841\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematological malignancies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(27.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(72.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal diseases\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(3.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(96.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolid organ tumors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e(57.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e(42.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eThis table shows the distribution of underlying diagnoses among final GM-positive serum specimens according to requesting service (pediatric vs adult). Final GM-positive specimens were defined as specimens that were repeat test positive (GM-ODI\u0026thinsp;\u0026ge;\u0026thinsp;0.5 in both the initial and repeat tests) or initially positive specimens that were not retested because prior documented GM positivity and/or concurrent microbiological evidence, including fungal culture positivity, was already available. Percentages are shown within each diagnostic category. Bonferroni-adjusted p values are presented for post hoc comparisons.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e\u003cstrong\u003eAbbreviations:\u0026nbsp;\u003c/strong\u003eGM, galactomannan.\u003c/p\u003e\u003cp\u003eThe distributions of initial and repeat GM-ODI values are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. When repeat-test positivity was analyzed according to initial ODI category, repeat positivity was lowest in the 0.5\u0026ndash;0.99 range and was generally higher in the higher ODI categories (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), with a significant overall trend across categories (linear-by-linear association test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Finally, repeat-test positivity rates also varied across underlying disease groups, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRepeat-test confirmation of initially positive serum GM results by the initial GM-ODI range\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCut-off ODI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRetest\u003c/p\u003e \u003cp\u003epositive n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRetest\u003c/p\u003e \u003cp\u003enegative n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal (First test positive) n (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5\u0026ndash;0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e224 (27.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e584 (72.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e808 (100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.0-1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86 (41,7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120 (58.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e206 (100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.5\u0026ndash;1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 (39.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41 (60.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68 (100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e141 (50.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e138 (49.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e279 (100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eThis table summarizes repeat-test outcomes for specimens with an initially positive serum GM result (GM-ODI\u0026thinsp;\u0026ge;\u0026thinsp;0.5), stratified by the initial GM-ODI range. Repeat positivity was defined as a repeat GM-ODI\u0026thinsp;\u0026ge;\u0026thinsp;0.5 using a new aliquot of the same specimen. A significant overall trend toward higher repeat-test positivity across increasing initial GM-ODI categories was observed (linear-by-linear association test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e GM, galactomannan; ODI, optical density index.\u003c/p\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRepeat-test confirmation rates of initially positive serum GM results according to underlying disease group.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisease\u003c/p\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirst test\u003c/p\u003e \u003cp\u003epositive (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRetest\u003c/p\u003e \u003cp\u003epositive (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRetest\u003c/p\u003e \u003cp\u003enegative (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRetest\u003c/p\u003e \u003cp\u003epositivity (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAML\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCML\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHL\u003c/p\u003e \u003cp\u003eHL\u003c/p\u003e \u003cp\u003eMM\u003c/p\u003e \u003cp\u003eCKD\u003c/p\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e146\u003c/p\u003e \u003cp\u003e23\u003c/p\u003e \u003cp\u003e135\u003c/p\u003e \u003cp\u003e70\u003c/p\u003e \u003cp\u003e147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57\u003c/p\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e32\u003c/p\u003e \u003cp\u003e22\u003c/p\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e89\u003c/p\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e103\u003c/p\u003e \u003cp\u003e48\u003c/p\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39\u003c/p\u003e \u003cp\u003e22\u003c/p\u003e \u003cp\u003e24\u003c/p\u003e \u003cp\u003e31\u003c/p\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eThis table presents the number of initially positive specimens, repeat-positive specimens, repeat-negative specimens, and the repeat-test positivity rate according to underlying disease group. Repeat-test positivity rates differed significantly across disease categories (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The repeat-test positivity rate was calculated as the number of repeat-positive results divided by the number of initially positive results in each disease category. Because analyses were performed at the specimen level, repeated specimens from the same patient were not excluded. Others includes disease categories not listed separately.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003ebbreviations:\u003c/strong\u003e GM, galactomannan; ODI, optical density index; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; AA, aplastic anemia; MDS, myelodysplastic syndrome; NHL, non-Hodgkin lymphoma; HL, Hodgkin lymphoma; MM, multiple myeloma; CKD, chronic kidney disease.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSerum GM testing is widely used as a mycological marker in the diagnostic work-up of suspected invasive pulmonary aspergillosis, but interpreting results near the assay cut-off remains challenging. In this large single-center cohort, repeat testing of initially positive serum samples frequently yielded discordant findings when the same specimen was retested within 72 hours, and confirmation became more likely as the initial GM-ODI increased. Together, these results indicate substantial variability in serum GM measurements in routine practice, particularly for low-positive values.\u003c/p\u003e \u003cp\u003eSeveral mechanisms can underlie either false-positive GM results or discordant repeat results after an initially positive finding, and these two phenomena should be distinguished. False-positive GM results may occur when exogenous GM-containing sources or analytical interferences generate a true signal in the absence of invasive aspergillosis; in such situations, positivity may persist on repeat testing of the same specimen. Examples include contamination or exposure from dextrose-containing fluids, which has been associated with clusters of false-positive serum GM results, and penicillin-based antibiotics such as piperacillin\u0026ndash;tazobactam or amoxicillin\u0026ndash;clavulanate, which have historically been linked to false positivity [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Dietary or enteral products, including oral/enteral nutritional supplements, have been implicated as potential sources of false-positive GM reactivity, particularly in patients with mucositis or impaired intestinal barrier integrity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Beyond these exogenous sources, galactomannan is not entirely specific to \u003cem\u003eAspergillus\u003c/em\u003e, and cross-reactivity has been reported with other fungi, including \u003cem\u003eFusarium\u003c/em\u003e spp. and \u003cem\u003eHistoplasma capsulatum\u003c/em\u003e, which may further contribute to non-specific GM reactivity in selected settings [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Environmental contamination during specimen handling is theoretically possible, but available evidence indicates that only heavy conidial contamination is sufficient to alter GM readings, making this an unlikely explanation under routine laboratory conditions. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eBy contrast, discordant repeat results, defined as loss of positivity on repeat testing of the same specimen, are more plausibly explained by analytical imprecision and operational or preanalytical factors that can shift values around the assay cut-off, particularly for low-positive indices. Potential contributors include between-run or between-lot variability, small differences in multistep assay workflow (such as timing, pipetting, or washing), and storage- or matrix-related changes that may reduce GM signal on retesting. Such variability has been documented for the Platelia Aspergillus Ag assay, where reproducibility is lower near the cut-off and operational factors within the workflow may generate non-reproducible positives that revert on repeat measurement [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In our cohort, this interpretation is supported by the substantial repeat-test discordance observed: 64.9% of initially positive samples became negative on repeat testing, and confirmation rates increased with higher initial GM-ODI categories. This pattern was particularly evident across initial ODI categories, with the lowest repeat-test confirmation observed in samples with initial values of 0.5\u0026ndash;0.99, whereas confirmation was generally higher in the higher GM-ODI categories. This is consistent with the broader understanding that results near the manufacturer-recommended threshold of 0.5 are more prone to non-confirmation, while higher ODI values are generally associated with greater specificity and a higher likelihood of clinically meaningful positivity. In line with this, updated consensus definitions adopted a serum cutoff of 1.0 to increase diagnostic likelihood compared with 0.5, and even higher thresholds, such as 1.5, have been argued to further improve specificity and positive predictive value, albeit at the cost of lower sensitivity. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eBeyond the assay-level pattern, the clinical context in which GM testing was requested also appeared to influence positivity rates. In our cohort, overall GM positivity was higher in pediatric service requests than in adult service requests, and no significant difference was observed by sex. This may reflect more targeted test ordering and a higher pre-test probability in pediatric settings rather than a purely biological difference in assay behavior. Such an interpretation is plausible because invasive aspergillosis remains an important concern in immunocompromised pediatric populations, particularly in children receiving chemotherapy, hematopoietic stem cell transplantation, or intensive immunosuppression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This interpretation is further supported by differences in the distribution of underlying diagnoses among GM-positive cases. Renal diseases were more frequent among requests from adult services, whereas solid organ tumors were more frequent among requests from pediatric services (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, these subgroup findings should be interpreted cautiously, as some diagnostic categories included relatively small numbers of cases.\u003c/p\u003e \u003cp\u003eRepeat-test positivity also varied across the major hematologic subgroups, although these patterns should be viewed as exploratory rather than definitive. In our cohort, retest positivity was higher than the overall average in ALL and NHL, but lower in AML and multiple myeloma. At least part of this variation may reflect differences in service-level case mix rather than disease biology alone, since ALL is predominantly a pediatric malignancy, whereas AML becomes more common with increasing age, NHL is overall more frequent in adults despite recognized pediatric forms, and multiple myeloma is predominantly a disease of older adults. In addition, because repeated specimens from the same patient were not excluded from the analysis, disease groups with more frequent serial sampling may have been overrepresented, potentially influencing subgroup-specific repeat-confirmation rates. Accordingly, the observed differences across disease categories may reflect a combination of case-mix differences, sampling frequency, and disease-specific clinical or biological factors rather than solely intrinsic assay behavior. The low repeat-test positivity observed in our cohort with multiple myeloma is consistent with prior reports suggesting that GM reactivity in this setting may be prone to non-specific positivity, although this subgroup finding should be interpreted cautiously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, it was a single-center retrospective analysis based on routinely generated laboratory data. Second, patients were not classified according to EORTC/MSGERC criteria as having possible, probable, or proven invasive aspergillosis; therefore, false positivity could not be definitively distinguished from non-confirmation related to analytical or preanalytical variability. Third, analyses were performed at the specimen level, and repeated specimens from the same patient were not excluded, which may have influenced subgroup-level comparisons and the apparent distribution of positivity across diagnostic categories. Finally, not all initially positive specimens underwent repeat testing, as retesting was sometimes omitted when prior positivity and/or concurrent microbiological evidence had already been documented, potentially introducing selection bias.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eRepeat testing of initially positive serum galactomannan results frequently yielded discordant findings in routine practice, particularly for low-positive values near the assay cut-off. Repeat-test confirmation was more likely at higher initial GM-ODI levels and varied across underlying disease groups. These findings indicate substantial short-term variability in serum GM measurements and support cautious interpretation of low-positive results, especially when clinical correlation is limited. In routine laboratory workflows, repeat assessment of initially positive samples may improve confidence in result interpretation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or non-financial interests that are directly or indirectly related to the work submitted for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from the Bursa City Hospital Clinical Research Ethics Committee (approval date: 04 September 2024; decision no: 2024-14/7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the retrospective nature of the study and the use of routinely collected laboratory data, the institutional review board waived the requirement for informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conceived and designed by Demet Timur. Material preparation, data collection, and data curation were performed by Demet Timur. Formal analysis was performed by Demet Timur and Mehmet Tekinsoy. The investigation was carried out by Demet Timur and Mehmet Tekinsoy. The first draft of the manuscript was written by Demet Timur and Mehmet Tekinsoy, and both authors contributed to the review and editing of subsequent versions. Supervision was provided by Demet Timur. Both authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the mycology laboratory staff of Bursa Uludag University Faculty of Medicine Hospital for their support in routine GM testing and laboratory record maintenance. They also thank Dr. Ahmet Timur for statistical support and Prof. Beyza Ener for her guidance during the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDouglas AP, et al. Consensus guidelines for the diagnosis and management of invasive aspergillosis, 2021. Intern Med J. 2021;51(Suppl 7):143\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLass-Florl C. How to make a fast diagnosis in invasive aspergillosis. Med Mycol. 2019;57(Supplement2):S155\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSusianti H, et al. Diagnostic value of serum human Galactomannan aspergillus antigen and 1,3-beta-D-glucan in immunocompromised patient suspected fungal infection. J Clin Lab Anal. 2021;35(6):e23806.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMercier T, et al. Defining Galactomannan Positivity in the Updated EORTC/MSGERC Consensus Definitions of Invasive Fungal Diseases. Clin Infect Dis. 2021;72(Suppl 2):S89\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonnelly JP, et al. Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin Infect Dis. 2020;71(6):1367\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUllmann AJ, et al. Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect. 2018;24(Suppl 1):e1\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLamberink H, et al. Multicenter validation of a galactomannan chemiluminescence immunoassay for the diagnosis of pulmonary aspergillosis on serum of patients with hematological disease. J Clin Microbiol. 2025;63(2):e0105324.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontesinos I et al. Diagnostic Value of Serum Biomarkers for Invasive Aspergillosis in Haematologic Patients. J Fungi (Basel), 2024. 10(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbert E, et al. Comparative performance of the Platelia Aspergillus Antigen and Aspergillus Galactomannan antigen Virclia Monotest immunoassays in serum and lower respiratory tract specimens: a real-life experience. Microbiol Spectr. 2024;12(8):e0391023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaboratories B-R. \u003cem\u003ePLATELIA\u0026trade; ASPERGILLUS Ag-Instructions for Use (package insert)\u003c/em\u003e. 2013 [cited 2026 22.02.2026]; 62794:[Instructions for Use]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://commerce.bio-rad.com/webroot/web/pdf/inserts/CDG/en/62794_881115_EN.pdf\u003c/span\u003e\u003cspan address=\"https://commerce.bio-rad.com/webroot/web/pdf/inserts/CDG/en/62794_881115_EN.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee R et al. Dextrose-containing fluids causing false-positive serum galactomannan: a case-control study and interrupted time series analysis. Clin Microbiol Infect, 2024. 30(5): p. 682 e1-682 e4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerlinger MP, et al. False positive galactomannan Platelia due to piperacillin-tazobactam. Med Mal Infect. 2012;42(1):10\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarg P, et al. Evaluating the causes associated with false positive Galactomannan assay in suspected cases of respiratory fungal infections. J Family Med Prim Care. 2025;14(2):736\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin DW, et al. False-positive results of galactomannan assays in patients administered glucose-containing solutions. Sci Rep. 2024;14(1):2552.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabana AL, et al. Can Aspergillus fumigatus conidia cause false-positive results in the galactomannan enzyme immunoassay test? Rev Soc Bras Med Trop. 2018;51(3):387\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUpton A, et al. Reproducibility of low galactomannan enzyme immunoassay index values tested in multiple laboratories. J Clin Microbiol. 2005;43(9):4796\u0026ndash;800.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuigue N, et al. Importance of operational factors in the reproducibility of Aspergillus galactomannan enzyme immune assay. PLoS ONE. 2015;10(4):e0124044.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKimpton G, White PL, Barnes RA. The effect of sample storage on the performance and reproducibility of the galactomannan EIA test. Med Mycol. 2014;52(6):618\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeeflang MM, et al. Galactomannan detection for invasive aspergillosis in immunocompromised patients. Cochrane Database Syst Rev. 2015;2015(12):CD007394.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWehbe S et al. Pediatric vs. Adult Invasive Aspergillosis in Cancer and Hematopoietic Transplant Patients: Insights from a Matched Cohort at a Tertiary Cancer Center. J Fungi (Basel), 2025. 11(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbe Y, et al. Higher frequency of false-positive serum galactomannan tests among older subjects and the association with elevated serum immunoglobulin G levels. Mycoses. 2019;62(9):773\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Galactomannan, invasive aspergillosis, Platelia Aspergillus Ag, reproducibility, serum biomarker","lastPublishedDoi":"10.21203/rs.3.rs-9057396/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9057396/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eSerum galactomannan (GM) testing is widely used as a mycological marker in the diagnostic work-up of invasive aspergillosis (IA), but results near the assay cut-off may vary on repeat measurement. This study evaluated how often initially positive serum GM results remained positive when the same specimen was retested and how GM optical density index (ODI) values changed between the first and second tests. We also examined whether GM positivity and retest confirmation differed by sex, requesting service, underlying diagnoses, and initial GM-ODI ranges.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe retrospectively analyzed 27,014 serum samples from 3,121 patients requested by pediatric and adult services over seven years. Samples with an initial GM-ODI\u0026thinsp;\u0026ge;\u0026thinsp;0.5 and no known prior GM positivity were retested using a new aliquot of the same specimen within 72 hours.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOf 27,014 serum samples, 1,918 were initially GM-positive, and 1,361 were retested within 72 hours. On repeat testing, 883/1,361 (65%) converted to negative, whereas 478/1,361 (35%) remained positive. GM-ODI values were significantly lower on retesting (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Final GM positivity did not differ by sex (p\u0026thinsp;=\u0026thinsp;0.066) but was higher in pediatric than adult service requests (5.8% vs 3.4%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Among final GM-positive specimens, the distribution of underlying diagnoses was associated with requesting service (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Retest positivity increased with higher initial GM-ODI ranges (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and varied across disease groups.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eRepeat testing of initially GM-positive serum samples within 72 hours frequently resulted in reversion to negative. Confirmation was more likely at higher initial GM-ODI ranges. These findings indicate substantial short-term variability in serum GM results in routine practice and support cautious interpretation of low-positive results.\u003c/p\u003e","manuscriptTitle":"Repeat-Test Reproducibility of Initially Positive Serum Galactomannan Results in a Tertiary-Care University Hospital: A 7-Year Single-Center Experience","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-20 16:54:45","doi":"10.21203/rs.3.rs-9057396/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-15T04:47:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T22:21:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223202806993969099077130877107314559002","date":"2026-05-05T01:36:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3158079209832309690370000258001568352","date":"2026-05-04T17:09:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T11:40:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328721033568130575032918298789808948215","date":"2026-04-13T05:19:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-13T04:59:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-09T06:08:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-09T06:08:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-03-07T09:46:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9e513cf3-91af-4994-aa12-83356fe9fc26","owner":[],"postedDate":"April 20th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-15T04:47:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T22:21:56+00:00","index":86,"fulltext":""},{"type":"reviewerAgreed","content":"223202806993969099077130877107314559002","date":"2026-05-05T01:36:22+00:00","index":83,"fulltext":""},{"type":"reviewerAgreed","content":"3158079209832309690370000258001568352","date":"2026-05-04T17:09:01+00:00","index":82,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T04:54:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-20 16:54:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9057396","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9057396","identity":"rs-9057396","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}