Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Clinical Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JCM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Mycology

Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients

Juergen Loeffler, Julia Hafner, Carlo Mengoli, Clemens Wirth, Claus Peter Heussel, Claudia Löffler, P. Lewis White, Andrew J. Ullmann, Denise Michel, Verena Wiegering, Matthias Wölfl, Paul Gerhardt Schlegel, Hermann Einsele, Jan Springer, Matthias Eyrich
D. W. Warnock, Editor
Juergen Loeffler
aMedizinische Klinik & Poliklinik II, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Juergen Loeffler
Julia Hafner
bKinderklinik und Poliklinik, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Carlo Mengoli
cDepartment of Histology, Microbiology, and Medical Biotechnology, University of Padua, Padua, Italy
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Clemens Wirth
dDepartment of Radiology, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Claus Peter Heussel
eDepartment of Diagnostic and Interventional Radiology, University Hospital of Heidelberg, Heidelberg, Germany
fDepartment of Diagnostic and Interventional Radiology with Nuclear Medicine, Thoraxklinik at University of Heidelberg, Heidelberg, Germany
gTranslational Lung Research Center Heidelberg (TLRC), Member of the German Center for Lung Research (DZL), Heidelberg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Claudia Löffler
aMedizinische Klinik & Poliklinik II, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
P. Lewis White
hPublic Health Wales Microbiology Cardiff, Cardiff, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andrew J. Ullmann
aMedizinische Klinik & Poliklinik II, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Denise Michel
aMedizinische Klinik & Poliklinik II, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Verena Wiegering
bKinderklinik und Poliklinik, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Matthias Wölfl
bKinderklinik und Poliklinik, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul Gerhardt Schlegel
bKinderklinik und Poliklinik, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hermann Einsele
aMedizinische Klinik & Poliklinik II, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jan Springer
aMedizinische Klinik & Poliklinik II, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Matthias Eyrich
bKinderklinik und Poliklinik, University Medical Center Würzburg, Würzburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
D. W. Warnock
University of Manchester
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JCM.01682-16
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Combined biomarker screening is increasingly used to diagnose invasive aspergillosis (IA) in high-risk patients. In adults, the combination of galactomannan (GM) and fungal DNA detection has proven to be beneficial in the diagnosis of IA. Data in purely pediatric cohorts are scarce. Here, we monitored 39 children shortly before and after allogeneic stem cell transplantation twice weekly by use of a commercial GM enzyme-linked immunosorbent assay (ELISA) and a PCR assay based on amplification of the pan-Aspergillus ITS1/5.8S ribosomal operon. In addition, clinical data were recorded and classification of IA was performed according to the European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria. Among the 39 high-risk children, we identified 4 patients (10.3%) with probable and 2 (5.1%) with possible IA. All patients with probable IA were repeatedly positive for both tests (means of 9.5 and 6.8 positive GM and PCR samples, respectively), whereas both possible IA cases were detected by PCR. The sensitivity and specificity were, respectively, 67% and 89% for GM and 100% and 63% for PCR. Positive and negative predictive values were, respectively, 50% and 100% for GM and 27% and 100% for PCR. For the combined testing approach, both values were 100%. The number of positive samples seemed to be lower in patients undergoing antifungal therapy. Sporadically positive tests occurred in 12% (GM) and 42% (PCR) of unclassified patients. In summary, our data show that combined monitoring for GM and fungal DNA also results in a high diagnostic accuracy in pediatric patients. Future studies have to determine whether combined testing is suitable for early detection of subclinical disease and how antifungal prophylaxis impacts assay performance.

INTRODUCTION

Invasive aspergillosis (IA) is the most significant opportunistic fungal infection in neutropenic adult and pediatric patients following allogeneic hematopoietic stem cell transplantation (alloHSCT) (1). Diagnosis of IA is challenging, as clinical symptoms are often nonspecific and classical diagnosis is poor (2). Methods such as high-resolution computed tomography (CT) scans show only typical signs once the infection is established, and even then specific signs can be transient (2, 3). Serological tests that detect galactomannan (GM) and β-d-glucan have low positive predictive values (PPVs), better used for the exclusion rather than the diagnosis of IA (2, 3). Such limitations have led to the development of PCR-based assays to detect fungal DNA in patient specimens. However, such assays operate at the very limit of detection due to the small amounts of fungal DNA recovered from blood samples and in pediatrics is further compounded by the relatively small blood volumes typically available. Recent studies in adults indicate that combining the PCR-based assays with antigen testing for the diagnosis of IA may be beneficial (4–6). However, few data exist for these combined strategies in high-risk pediatric patients. This article describes a highly standardized diagnostic schedule involving twice weekly systematic screening of high-risk children by GM and PCR assays around alloHSCT. In parallel, a large variety of clinical signs and symptoms and microbiological data were collected. These data were retrospectively compared to those of a recently described adult cohort (7), and the findings are discussed below.

RESULTS

The entire set of samples comprised 543 blood specimens collected from 39 high-risk pediatric patients shortly before or after alloHSCT. Among all patients, 4 cases of probable IA (10%), 2 cases of possible IA (5%), and 33 unclassified patients with no specific radiological signs of IA (85%) were classified according to the current European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria (8). No proven IA was documented.

Biomarker assay performance.As outlined in Table 1, both GM and PCR were positive in 4/4 patients with probable IA, generating a sensitivity of 100%. In these four patients with probable IA, 38/74 (51% [95% confidence interval {CI}, 40 to 62%]) and 27/77 (35% [95% CI, 25 to 46%]) samples proved to be GM and PCR positive, respectively. In 2 patients with possible IA, PCR was positive in 2/22 samples, whereas no positive GM ELISA occurred in these cases. In the unclassified patients, 4/33 and 13/33 patients showed either a positive GM or a positive PCR result, respectively, representing 9/437 (2.1% [95% CI, 1.1 to 3.9%]) and 17/441 (3.9% [95% CI, 2.4 to 6.1%]) samples positive by GM and PCR, respectively. No unclassified patients were positive by both PCR and GM, generating a combined specificity of 100%. The PPVs were 50% for GM ELISA and 27% for PCR; the negative predictive values (NPVs) were 100% for GM and 100% for PCR testing. When the GM ELISA and PCR were considered a combined test system, the PPV and NPV values were both 100% for the detection of probable IA. One patient with a pulmonary aspergilloma never had any positive GM or PCR test result.

View this table:
  • View inline
  • View popup
TABLE 1

Number of positive GM or PCR assays with respective EORTC/MSG classification of patientsa

If cases of both probable and possible IA were considered positive for the calculation of the clinical performance of the assay, the sensitivity and NPV for the GM test were diminished to 67% and 94%, respectively. While sensitivity and NPV for the combined test system were reduced to 67% and 94% as well, the PPV for the PCR assay increased to 35%.

Investigating the effect of antifungal therapy on biomarker assay performance.The effect of using Aspergillus-active prophylaxis or empirical therapy on biomarker assay positivity testing in unclassified patients was investigated. As shown in Table 1, during the use of prophylaxis or empirical therapy, there was a trend (P = 0.0519) toward a reduced rate of positive test results (14% [95% CI, 6 to 32%] compared to 60% [95% CI, 23 to 88%] positive samples in the untreated unclassified population). Interestingly, 8 of 28 (29% [95% CI, 15 to 41] of these treated patients had positive test results outside their treatment or prophylaxis period, resulting in a doubling in the percentage of positive test results compared to that during the prophylaxis/treatment period (29% versus 14%, respectively). This lower incidence of positive biomarkers during antifungal prophylaxis or empirical therapy might indicate a positive clinical effect of antifungal therapy, reducing the release of mold into the blood circulation. The times from initiation of screening to biomarker positivity in patients with probable disease were comparable, with 49 ± 39 days versus 46 ± 40 days (mean ± standard deviation [SD]) for GM versus PCR testing, respectively.

Associations between CT, PCR, and GM positivity.Sixty-seven CT scans were available from 33 patients; in 6/33 patients, CT scans displayed lesions compatible with IA (Fig. 1). PCR was positive in 6/6 patients (sensitivity, 100%), compared to 10 PCR-positive patients in 27 CT-negative patients (specificity, 63%). The Pearson chi-square test of independence applied to the association between a positive CT scan and a positive PCR result suggested a positive correlation between the two tests (chi-square [1 degree of freedom] = 7.7917, P = 0.005).

FIG 1
  • Open in new tab
  • Download powerpoint
FIG 1

Venn diagram illustrating the relationship between the CT-based detection of aspergillosis (CT), the change from a negative to a positive PCR result (asp) in a given patient, and a positive GM assay result (gm). Out of 16 positive Aspergillus PCR events, 6 were truly positive and 10 were false positive (PPV, 37.5%). All positive CT scans were detected by Aspergillus PCR (sensitivity, 100%). Out of 7 positive GM events, 4 were truly positive and 3 showed a false-positive GM assay result (PPV, 57.1%). The GM assay failed to detect 2 out of 6 positive CT scans (sensitivity, 66.7%).

The GM ELISA was positive in 4/6 CT-positive patients (sensitivity, 67%) and in 3/27 CT-negative patients (specificity = 89%). The same chi-square test of independence between a positive CT scan and a positive GM ELISA was significant (chi-square [1 degree of freedom] = 9.0659, P = 0.003). Using the GM index as the classifying variable and the Aspergillus PCR assay as the reference variable, receiver operating characteristic (ROC) analysis revealed that the area under curve (AUC) was 0.7335 (95% CI, 0.64233 to 0.82475), with a standard error of 0.0465. The maximal Youden index, J, was 49.9%, corresponding to a GM index of 0.4 (Fig. 2), demonstrating that a GM index of 0.4 was the best cutoff point to predict when the Aspergillus PCR assay will be positive.

FIG 2
  • Open in new tab
  • Download powerpoint
FIG 2

ROC curve analysis demonstrating the capability of the GM ELISA index predicting a positive Aspergillus PCR assay. The Youden index, J (red line), suggests a GM cut-point of 0.4 as the best for a discriminative purpose. This corresponds to a sensitivity of 55% and a specificity of 95%.

In patients with probable IA, a mean of 9.5 GM samples (range, 6 to 14 samples; mean optical density [OD], 3.1) and 6.8 PCR samples (range, 3 to 14 samples) showed positive results.

Next, binary and continuous covariates were analyzed using a time-to-event approach, running the nonparametric log rank method. Time-event variables included the day of binary Aspergillus PCR status change, as first conversion from negativity to positivity, and the day of first GM conversion to a positive index. We revealed that liposomal amphotericin B use reduced the risk of a positive Aspergillus biomarker test result, whereas CT positivity was associated with a significant increase of risk (P = 0.042). Using the GM conversion to positivity as the endpoint, CT positivity was associated with a markedly increased risk for IA (P = 0.0375). Four covariates (use of corticosteroids, other herpesviruses, caspofungin, CT positivity) were significantly associated with an increased risk for IA.

Longitudinal investigations.For all 4 patients with probable IA, a longitudinally detailed analysis was performed. Figure 3 shows as examples the characteristic time courses for 2/4 patients (see Fig. S1 in the supplemental material for data for the 2 other patients).

FIG 3
  • Open in new tab
  • Download powerpoint
FIG 3

Representative medical histories of two patients suffering from probable IA with different courses and outcomes. Graphs display the course of C-reactive protein (CrP, red) and neutrophils counts (blue) over time, together with the results of the GM/PCR assays, presence of clinical symptoms, CT scans (bars above the graph), and antifungal treatment (bars below the graph). For CT scans and GM/PCR assays, red indicates a positive test result and blue indicates a negative test result. (A) A 12-year-old boy who received an alloHSCT due to a refractory AML. He developed de novo IA during aplasia but recovered. Finally, he died from further disease progression but without clinical signs of IA. (B) A 15-year-old boy with refractory ALL who was referred to our center for ALL immunotherapy. Despite intensified antifungal treatment, his preexisting IA further progressed, from which he ultimately died. During treatment, all GM/PCR tests remained continuously positive with increasing values (GM index in the final three samples is above the upper limit of 8).

Patient 1 (Fig. 3A), a 12-year-old boy with refractory acute myeloid leukemia (AML) who had received a matched unrelated donor transplant, was given prophylactic caspofungin due to prolonged neutropenia. During aplasia after alloHSCT, he developed fever, cough, tachycardia, and elevated C-reactive protein (CRP) levels and oxygen demand. CT scans revealed a new 1.4-cm nodule in the left upper lobe, compatible with pulmonary IA. After neutrophil regeneration and intensified antifungal treatment, he cleared his IA within 24 days, as confirmed by repeat CT. Interestingly, GM samples (1 test) and PCR samples (3 tests) became positive again, later during the reconvalescence, without clinical symptoms but in association with neutropenia.

Patient 2 (Fig. 3B), a 15-year-old boy who suffered from refractory acute lymphocytic leukemia (ALL), was referred to our center to receive bispecific antibody treatment and subsequent alloHSCT. The referring institution had already suspected a pulmonary IA, which was confirmed by double-positive GM and PCR tests as well as radiologically by the presence of diffuse nodules up to 2.3 cm in diameter in both lungs. Although antifungal treatment was further intensified, he progressed clinically and showed 14 double-positive samples in one series with increasing GM indices and DNA load in the real-time PCR assay. This patient ultimately died from IA, prior to alloHSCT.

DISCUSSION

The aim of this study was to determine whether intense (twice weekly) and standardized GM ELISA and PCR screening could increase the diagnostic accuracy for the detection of IA in high-risk pediatric patients. For children, the number of existing studies on this matter is scarce, patient populations are often heterogeneous, and currently no methodological standards similar to the recommendations of the European Aspergillus PCR Initiative recommendations (EAPCRI) exist. As discussed recently by Lehrnbecher et al., there are only 19 studies reporting on GM ELISA testing and 11 studies reporting on PCR testing in pediatric cancer or HSCT patients (9). In addition, disease pathology and the physiology of children differ from those of adults (e.g., children-specific diseases, immunology, metabolism, body weight, and blood volume). In contrast to findings in adults, the typical signs of IA (halo and air crescent signs) are rarely seen in young children and radiographic findings are often unspecific (10). In addition, the nutrition of babies and children as well as the maturation state of the mucosa might be responsible for significantly higher rates of false-positive GM ELISAs (11). Similarly, the antibiotics piperacillin-taxobactam and amoxicillin-clavulanate have been reported to yield false-positive results in GM assays, although recently this finding has been questioned (12) (13). Recently, Buchheidt et al. presented a review focusing on studies of PCR-based assays to diagnose IA in immunocompromised pediatric patients (14). The authors concluded that the value of PCR in pediatric patients has not yet been defined and that a large variety of assay protocols exist. These previous studies relied on nested-PCR assays (15, 16), did not include any cases of probable IA (16, 17), and used extremely small blood volumes (17, 18). In addition, study sizes were very small (17) and sampling frequencies were low (e.g., once per week [16]). Nevertheless, authors found high sensitivities of up to 96% (19) and/or high NPVs of up to 100% (17, 19). Here, we used an established PCR assay which has been validated in an adult cohort upfront. Our data from close longitudinal follow-up combined with a detailed clinical history clearly show that PCR assays are also of diagnostic value in pediatric patients. As expected, on the single-test level, PCR was more sensitive than GM testing, whereas specificity was superior for GM testing. It should be kept in mind that by the EORTC/MSG definitions, all patients with probable IA must be positive for a mycological criterion, which in this study was GM, whereas all patients with possible IA are subsequently negative. This incorporation bias may lead to an overestimation of performance measures for GM and in parallel might negatively influence the performance measures of PCR.

For both PCR and GM assays, a positive CT scan was associated with a higher risk for test conversion. PCR conversion was negatively associated with the use of liposomal amphotericin B, which is in line with the lower rate of PCR positivity when antifungal prophylaxis or therapy is used (Table 1). As reported before (20), both tests were unable to detect noninvasive forms of IA.

The number of unclassified patients with at least one positive PCR specimen in this study of children was higher than expected. However, this could not be attributed to the prophylactic or empirical use of Aspergillus-active agents, since the majority of positive test results occurred in patients without prophylaxis or outside the treatment or prophylaxis period. This observation deviates from our former cohort analysis of 213 adult hematological patients (7). There, we showed that PCR screening is of limited value in patients receiving mold prophylaxis, with 72% (53/73 patients) receiving antifungal prophylaxis generating a potentially false-positive PCR result, compared to only 14% in this pediatric cohort. The reason for these conflicting data are unknown. However, we hypothesize that in pediatric patients with mold prophylaxis, subclinical Aspergillus infections might be less frequent than those in adults and, in consequence, fewer positive PCR tests occur in children receiving mold prophylaxis (4/28 patients) (Table 1). This again argues for more controlled trials on the role of Aspergillus-active prophylaxis in allogeneic transplant patients.

Furthermore, we demonstrated that in adult patients suffering from probable IA, only means of 2.1 and 1.9 samples were positive for GM and PCR, respectively (7), compared to the 9.5 and 6.8 positive samples per affected patient in the pediatric cohort described herein. While the mean OD value of all positive GM tests in pediatric patients with probable IA was 3.1, the corresponding OD value in adults was only 1.5 (7). The reason for this observation is not entirely clear; however, it may well be that in a clinically overt IA, fungal components are more concentrated in pediatric blood samples, since total body blood volumes of children can be up to 10 times smaller than those in adults, while the blood volumes sampled for GM and PCR assays did not markedly differ between the two cohorts.

A closer look at the individual clinical courses of patients provides new insights but also raises several questions. First, positivity of both tests significantly suggests IA, and the level of the GM index or the PCR quantification cycle (Cq) values may eventually serve as a surrogate marker of the clinical course. However, it remains unclear whether single positive tests in cases of possible or unclassifiable IA actually represent false-positive results or whether they truly indicate fungal cell components in the bloodstream without any disease pathology. Furthermore, due to the relatively low number of probable IA cases, we were not able to determine whether the combined testing approach also allows a preemptive detection of IA, before clinical symptoms become apparent. It is worth noting that all high-risk patients in our cohort received antifungal prophylaxis, including the 4 patients developing probable IA, independent of their neutrophil counts. This prophylaxis included posaconazole (10 mg/kg of body weight), caspofungin (50 mg/m2 of body surface area), or liposomal amphotericin B (1 mg/kg of body weight). The impact of antifungal prophylaxis on diagnostic test systems as well as on disease development in the pediatric setting remains elusive and requires further investigations focused on this question.

Despite antifungal prophylaxis, 4 patients developed probable IA, and in parallel, several others frequently had single positive GM or PCR assay results. Although our PCR assay covers a broad range of Aspergillus species, we could not isolate the responsible mold. Thus, we do not have any data on antibiotic resistance in these cases. However, this circumstance also points to the need for more conclusive clinical data and a global consensus on antifungal prophylaxis and coordinated stewardship programs to promote the consistent and appropriate use of antifungal drugs, especially in neonates and children (21).

In summary, we were able to show that in pediatric patients, a combined PCR and GM screening method yielded greater diagnostic accuracy than single testing, providing 100% specificity/PPV, and detected 4 out of 4 patients with probable IA. A single positive test result occurrs frequently without indicating overt disease, while dual testing plays a decisive role in the diagnosis and management of IA and allows the identification of pediatric patients with IA. The role of antifungal prophylaxis and its interaction with diagnostic test systems in pediatric patients remains to be further evaluated in larger multicenter trials.

MATERIALS AND METHODS

Patients.Since 2012, all patients from the University Children's Hospital Würzburg were screened twice weekly by GM ELISA and PCR-based diagnostic assays. In July 2015, all patients to date (n = 39) were selected for retrospective data analysis. All patients (24 males, median age of 9.5 years [range, 4 to 21 years]; 15 females, median age of 10 years [3 to 19 years]) had alloHSCT. Patients were diagnosed with the following underlying diseases: acute lymphoblastic leukemia (n = 22), myelodysplastic syndrome (n = 5), acute myeloid leukemia (n = 3), and other diseases, including chronic myeloid leukemia, Hodgkin lymphoma, Diamond-Blackfan anemia, thalassemia, neuroblastoma, Fanconi anemia, adrenoleukodystrophy, severe aplastic anemia, and Omenn syndrome (n = 1 each).

In this cohort, 85% of the patients received one or two mold-active antifungal drugs as prophylaxis or empirical therapy prior to commencing or during diagnostic testing (among them, 97%, 13%, 13%, and 17% received liposomal amphotericin B, voriconazole, posaconazole, and caspofungin, respectively). The durations of antifungal prophylaxis and empirical therapy were 27 ± 28 days, 35 ± 33 days, and 99 ± 84 days (mean ± SD) in patients with unclassified, possible, and probable disease, respectively.

In total, 543 blood samples (mean, 13.9 samples per patient; range, 6 to 33 samples) were collected. Sampling started in the preparative phase for alloHSCT (median, 14 days [range, 0 to 74 days] before transplantation) and continued for all patients until day +100 after alloHSCT or, if before day +100, until the patient died.

Diagnostic molecular assays.Samples were tested as part of routine diagnostic care involving concomitant GM ELISA (Platelia; Bio-Rad, Munich, Germany) and by PCR testing. We used an index of ≥0.5 to define a positive GM ELISA. A single positive test was required to define a positive GM ELISA. For the PCR, DNA was extracted from a 1-ml cell-free blood fraction using the QIAamp UltraSens virus kit (Qiagen, Hilden, Germany) (22). The protocol used was compliant with the European Aspergillus PCR Initiative recommendations (EAPCRI) for serum to guarantee the highest diagnostic standards (23). The study was designed and performed on test serum samples. If occasionally serum samples were not available, plasma specimens were used instead to continue consecutive sampling for all patients. Real-time PCR, involving an internal control, was performed as previously described (7). Clinical signs and microbiological data were recorded for each individual patient, with IA defined according to revised European Organization for the Research and Treatment of Cancer/Mycoses Study Group ((EORTC/MSG) criteria (8). Data were compared with those for a previously published adult hematology cohort that was screened by Aspergillus-specific biomarkers (7).

CT technology and reading process.Sixty-seven CT scans were performed as part of the clinical management. CT scans of the thorax were acquired as low-dose examinations during inspiration with institutional age- and weight-adapted parameters (80 to 120 kV, 30 to 50 mA, Care Dose 4D). All scans were performed on a 64-slice state-of-the-art scanner (Siemens Sensation 64; Siemens Healthcare, Erlangen, Germany). The scans were reconstructed iteratively in thin slices (1 mm). In addition to the original analysis, two independent experts who were blind to clinical and mycological information retrospectively reviewed the CT images for confirmation. The definition of Aspergillus-specific lung lesions was based on published EORTC/MSG criteria (8).

Covariate analysis.Individual patient-specific covariates were recorded, including gender, neutropenia (neutrophil count of <500/μl for >10 days), corticosteroids (0.3 mg/kg of body weight for more than 21 days), T-cell-immunosuppressive medication (cyclosporine, tacrolimus, or mycophenolate mofetil [MMF] at any dose), cytomegalovirus (DNA detection in plasma), diagnosis of other herpesviridae (herpes simplex virus 1 or 2, human herpesvirus 6, Epstein-Barr virus, varicella-zoster virus) or adenoviridae, detection of respiratory viruses (e.g., influenza virus, parainfluenza virus, coronavirus, or rhinovirus), use of itraconazole, fluconazole, liposomal amphotericin B, voriconazole, posaconazole, or caspofungin (all in therapeutic dosages), CT evidence of IA, fever (>38.5°C, or >38°C for >4 h), cough, tachycardia, oxygen demand, and tachypnea. In addition, the continuous covariates of body weight (kg), days of neutropenia (neutrophil count of <500/μl for >10 days), days of corticosteroid use (0.3 mg/kg body weight), age, GM index, numbers of CD3+, CD4+, CD8+, CD16/CD56+, and CD19+ cells per μl of blood, and numbers of leukocytes, monocytes, neutrophils, and lymphocytes per μl of blood were recorded.

Statistics.A frequency analysis test was performed: any patient with at least one positive CT scan was considered CT positive, whereas any patient with no positivity in CT scans was considered CT negative. The same scheme was performed for Aspergillus PCR and GM assays. Then, the association between CT positivity, Aspergillus positivity, and GM positivity was explored using the Pearson chi-square test of independence.

The relationship between the Aspergillus PCR assay result (as a binary reference variable) and the GM index (as a continuous classifying variable) was explored by means of the ROC curve method for all observations reporting both assays.

Moreover, a nonparametric time-to-event (log rank) analysis was performed. Two main event variables were identified and used to perform two corresponding approaches to time-to-event analysis: (i) the day of the binary Aspergillus PCR status change from negative to positive, and (ii) the day of the first conversion of the serum GM assay to a positive result (galactomannan assay index [GMI] ≥ 0.5). The effect of each binary and continuous covariate was estimated singly for both outcomes.

Statistical methods were performed with Stata 14.1 (StataCorp, College Station, TX, USA) and R version 3.2.3 (R Foundation, Vienna, Austria).

ACKNOWLEDGMENTS

This work was supported by Pfizer Pharma GmbH, Berlin, Germany (grant no. IIR-WI-187426).

C. P. Heussel has received lecture and consultation fees from Schering-Plough, Pfizer, Basilea, Boehringer Ingelheim, Novartis, Roche, Astellas, Gilead, Merck Sharp & Dohme, Lilly, Intermune, Fresenius, Olympus, Essex, AstraZeneca, Bracco, MEDA Pharma, Intermune, Chiesi, Siemens, Covidien, Pierre Fabre, Bayer, and Grifols, as well as research funding from Siemens, Pfizer, Boehringer Ingelheim, and MeVis. The other authors report no conflicts of interest.

FOOTNOTES

    • Received 11 August 2016.
    • Returned for modification 1 September 2016.
    • Accepted 12 October 2016.
    • Accepted manuscript posted online 19 October 2016.
  • Supplemental material for this article may be found at https://doi.org/10.1128/JCM.01682-16 .

  • Copyright © 2016 American Society for Microbiology.

All Rights Reserved .

REFERENCES

  1. 1.↵
    1. Neofytos D,
    2. Horn D,
    3. Anaissie E,
    4. Steinbach W,
    5. Olyaei A,
    6. Fishman J,
    7. Pfaller M,
    8. Chang C,
    9. Webster K,
    10. Marr K
    . 2009. Epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of Multicenter Prospective Antifungal Therapy (PATH) Alliance registry. Clin Infect Dis48:265–273. doi:10.1086/595846.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    1. Hope WW,
    2. Denning DW
    . 2004. Invasive aspergillosis: current and future challenges in diagnosis and therapy. Clin Microbiol Infect10:2–4. doi:10.1111/j.1469-0691.2004.00809.x.
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    1. Barton RC
    . 2013. Laboratory diagnosis of invasive aspergillosis: from diagnosis to prediction of outcome. Scientifica (Cairo)2013:459405. doi:10.1155/2013/459405.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Rogers TR,
    2. Morton CO,
    3. Springer J,
    4. Conneally E,
    5. Heinz W,
    6. Kenny C,
    7. Frost S,
    8. Einsele H,
    9. Loeffler J
    . 2013. Combined real-time PCR and galactomannan surveillance improves diagnosis of invasive aspergillosis in high risk patients with haematological malignancies. Br J Haematol161:517–524. doi:10.1111/bjh.12285.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Aguado JM,
    2. Vazquez L,
    3. Fernandez-Ruiz M,
    4. Villaescusa T,
    5. Ruiz-Camps I,
    6. Barba P,
    7. Silva JT,
    8. Batlle M,
    9. Solano C,
    10. Gallardo D,
    11. Heras I,
    12. Polo M,
    13. Varela R,
    14. Vallejo C,
    15. Olave T,
    16. Lopez-Jimenez J,
    17. Rovira M,
    18. Parody R,
    19. Cuenca-Estrella M
    , for the PCRAGA Study Group, Spanish Stem Cell Transplantation Group, Study Group of Medical Mycology of the Spanish Society of Clinical Microbiology and Infectious Diseases, Spanish Network for Research in Infectious Diseases. 2015. Serum galactomannan versus a combination of galactomannan and polymerase chain reaction-based Aspergillus DNA detection for early therapy of invasive aspergillosis in high-risk hematological patients: a randomized controlled trial. Clin Infect Dis60:405–414. doi:10.1093/cid/ciu833.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Morrissey CO,
    2. Chen SCA,
    3. Sorrell TC,
    4. Milliken S,
    5. Bardy PG,
    6. Bradstock KF,
    7. Szer J,
    8. Halliday CL,
    9. Gilroy NM,
    10. Moore J,
    11. Schwarer AP,
    12. Guy S,
    13. Bajel A,
    14. Tramontana AR,
    15. Spelman T,
    16. Slavin MA
    , Australasian Leukaemia Lymphoma Group and the Australia and New Zealand Mycology Interest Group. 2013. Galactomannan and PCR versus culture and histology for directing use of antifungal treatment for invasive aspergillosis in high-risk haematology patients: a randomised controlled trial. Lancet Infect Dis13:519–528. doi:10.1016/S1473-3099(13)70076-8.
    OpenUrlCrossRefPubMedWeb of Science
  7. 7.↵
    1. Springer J,
    2. Lackner M,
    3. Nachbaur D,
    4. Girschikofsky M,
    5. Risslegger B,
    6. Mutschlechner W,
    7. Fritz J,
    8. Heinz WJ,
    9. Einsele H,
    10. Ullmann AJ,
    11. Loffler J,
    12. Lass-Florl C
    . 2016. Prospective multicentre PCR-based Aspergillus DNA screening in high-risk patients with and without primary antifungal mould prophylaxis. Clin Microbiol Infect22:80–86. doi:10.1016/j.cmi.2015.09.009.
    OpenUrlCrossRef
  8. 8.↵
    1. De Pauw B,
    2. Walsh TJ,
    3. Donnelly JP,
    4. Stevens DA,
    5. Edwards JE,
    6. Calandra T,
    7. Pappas PG,
    8. Maertens J,
    9. Lortholary O,
    10. Kauffman CA,
    11. Denning DW,
    12. Patterson TF,
    13. Maschmeyer G,
    14. Bille J,
    15. Dismukes WE,
    16. Herbrecht R,
    17. Hope WW,
    18. Kibbler CC,
    19. Kullberg BJ,
    20. Marr KA,
    21. Munoz P,
    22. Odds FC,
    23. Perfect JR,
    24. Restrepo A,
    25. Ruhnke M,
    26. Segal BH,
    27. Sobel JD,
    28. Sorrell TC,
    29. Viscoli C,
    30. Wingard JR,
    31. Zaoutis T,
    32. Bennett JE
    . 2008. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis46:1813–1821. doi:10.1086/588660.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Lehrnbecher T,
    2. Robinson PD,
    3. Fisher BT,
    4. Castagnola E,
    5. Groll AH,
    6. Steinbach WJ,
    7. Zaoutis TE,
    8. Negeri ZF,
    9. Beyene J,
    10. Phillips B,
    11. Sung L
    . 27August2016. Galactomannan, beta-d-glucan, and polymerase chain reaction-based assays for the diagnosis of invasive fungal disease in pediatric cancer and hematopoietic stem cell transplantation: a systematic review and meta-analysis. Clin Infect Dis. Epub ahead of print.
  10. 10.↵
    1. Groll AH,
    2. Castagnola E,
    3. Cesaro S,
    4. Dalle JH,
    5. Engelhard D,
    6. Hope W,
    7. Roilides E,
    8. Styczynski J,
    9. Warris A,
    10. Lehrnbecher T
    , Fourth European Conference on Infections in Leukaemia, Infectious Diseases Working Party of the European Group for Blood Marrow Transplantation (EBMT-IDWP), Infectious Diseases Group of the European Organisation for Research and Treatment of Cancer (EORTC-IDG), International Immunocompromised Host Society (ICHS), European Leukaemia Net (ELN). 2014. Fourth European Conference on Infections in Leukaemia (ECIL-4): guidelines for diagnosis, prevention, and treatment of invasive fungal diseases in paediatric patients with cancer or allogeneic haemopoietic stem-cell transplantation. Lancet Oncol15:e327–e340. doi:10.1016/S1470-2045(14)70017-8.
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    1. Mennink-Kersten MA,
    2. Donnelly JP,
    3. Verweij PE
    . 2004. Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis4:349–357. doi:10.1016/S1473-3099(04)01045-X.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    1. Zandijk E,
    2. Mewis A,
    3. Magerman K,
    4. Cartuyvels R
    . 2008. False-positive results by the platelia Aspergillus galactomannan antigen test for patients treated with amoxicillin-clavulanate. Clin Vaccine Immunol15:1132–1133. doi:10.1128/CVI.00022-08.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Vergidis P,
    2. Razonable RR,
    3. Wheat LJ,
    4. Estes L,
    5. Caliendo AM,
    6. Baden LR,
    7. Wingard JR,
    8. Baddley J,
    9. Assi M,
    10. Norris S,
    11. Chandrasekar P,
    12. Shields R,
    13. Nguyen H,
    14. Freifeld A,
    15. Kohler R,
    16. Kleiman M,
    17. Walsh TJ,
    18. Hage CA
    . 2014. Reduction in false-positive Aspergillus serum galactomannan enzyme immunoassay results associated with use of piperacillin-tazobactam in the United States. J Clin Microbiol52:2199–2201. doi:10.1128/JCM.00285-14.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Buchheidt D,
    2. Reinwald M,
    3. Spiess B,
    4. Boch T,
    5. Hofmann WK,
    6. Groll AH,
    7. Lehrnbecher T
    , Working Group “Infections in Hematology and Oncology,” German Paul-Ehrlich-Society. 2016. Biomarker-based diagnostic work-up of invasive pulmonary aspergillosis in immunocompromised paediatric patients—is Aspergillus PCR appropriate?Mycoses59:67–74. doi:10.1111/myc.12443.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Bialek R,
    2. Moshous D,
    3. Casanova JL,
    4. Blanche S,
    5. Hennequin C
    . 2002. Aspergillus antigen and PCR assays in bone marrow transplanted children. Eur J Med Res7:177–180.
    OpenUrlPubMed
  16. 16.↵
    1. Reinwald M,
    2. Konietzka CA,
    3. Kolve H,
    4. Uhlenbrock S,
    5. Ahlke E,
    6. Hummel M,
    7. Spiess B,
    8. Hofmann WK,
    9. Buchheidt D,
    10. Groll AH
    . 2014. Assessment of Aspergillus-specific PCR as a screening method for invasive aspergillosis in paediatric cancer patients and allogeneic haematopoietic stem cell recipients with suspected infections. Mycoses57:537–543. doi:10.1111/myc.12192.
    OpenUrlCrossRefPubMed
  17. 17.↵
    1. Mandhaniya S,
    2. Iqbal S,
    3. Sharawat SK,
    4. Xess I,
    5. Bakhshi S
    . 2012. Diagnosis of invasive fungal infections using real-time PCR assay in paediatric acute leukaemia induction. Mycoses55:372–379. doi:10.1111/j.1439-0507.2011.02157.x.
    OpenUrlCrossRefPubMedWeb of Science
  18. 18.↵
    1. Armenian SH,
    2. Nash KA,
    3. Kapoor N,
    4. Franklin JL,
    5. Gaynon PS,
    6. Ross LA,
    7. Hoffman JA
    . 2009. Prospective monitoring for invasive aspergillosis using galactomannan and polymerase chain reaction in high risk pediatric patients. J Pediatr Hematol Oncol31:920–926. doi:10.1097/MPH.0b013e3181b83e77.
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    1. Landlinger C,
    2. Preuner S,
    3. Baskova L,
    4. van Grotel M,
    5. Hartwig NG,
    6. Dworzak M,
    7. Mann G,
    8. Attarbaschi A,
    9. Kager L,
    10. Peters C,
    11. Matthes-Martin S,
    12. Lawitschka A,
    13. van den Heuvel-Eibrink MM,
    14. Lion T
    . 2010. Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients. Leukemia24:2032–2038. doi:10.1038/leu.2010.209.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Imbert S,
    2. Gauthier L,
    3. Joly I,
    4. Brossas JY,
    5. Uzunov M,
    6. Touafek F,
    7. Brun S,
    8. Mazier D,
    9. Datry A,
    10. Gay F,
    11. Fekkar A
    . 2016. Aspergillus PCR in serum for the diagnosis, follow-up and prognosis of invasive aspergillosis in neutropenic and nonneutropenic patients. Clin Microbiol Infect22:562.e1–8. doi:10.1016/j.cmi.2016.01.027.
    OpenUrlCrossRef
  21. 21.↵
    1. Lestner JM,
    2. Versporten A,
    3. Doerholt K,
    4. Warris A,
    5. Roilides E,
    6. Sharland M,
    7. Bielicki J,
    8. Goossens H
    , ARPEC Project Group. 2015. Systemic antifungal prescribing in neonates and children: outcomes from the Antibiotic Resistance and Prescribing in European Children (ARPEC) Study. Antimicrob Agents Chemother59:782–789. doi:10.1128/AAC.04109-14.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Springer J,
    2. Morton CO,
    3. Perry M,
    4. Heinz WJ,
    5. Paholcsek M,
    6. Alzheimer M,
    7. Rogers TR,
    8. Barnes RA,
    9. Einsele H,
    10. Loeffler J,
    11. White PL
    . 2013. Multicenter comparison of serum and whole-blood specimens for detection of Aspergillus DNA in high-risk hematological patients. J Clin Microbiol51:1445–1450. doi:10.1128/JCM.03322-12.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. White PL,
    2. Mengoli C,
    3. Bretagne S,
    4. Cuenca-Estrella M,
    5. Finnstrom N,
    6. Klingspor L,
    7. Melchers WJ,
    8. McCulloch E,
    9. Barnes RA,
    10. Donnelly JP,
    11. Loeffler J
    . 2011. Evaluation of Aspergillus PCR protocols for testing serum specimens. J Clin Microbiol49:3842–3848. doi:10.1128/JCM.05316-11.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients
Juergen Loeffler, Julia Hafner, Carlo Mengoli, Clemens Wirth, Claus Peter Heussel, Claudia Löffler, P. Lewis White, Andrew J. Ullmann, Denise Michel, Verena Wiegering, Matthias Wölfl, Paul Gerhardt Schlegel, Hermann Einsele, Jan Springer, Matthias Eyrich
Journal of Clinical Microbiology Dec 2016, 55 (1) 101-109; DOI: 10.1128/JCM.01682-16

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Clinical Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients
(Your Name) has forwarded a page to you from Journal of Clinical Microbiology
(Your Name) thought you would be interested in this article in Journal of Clinical Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients
Juergen Loeffler, Julia Hafner, Carlo Mengoli, Clemens Wirth, Claus Peter Heussel, Claudia Löffler, P. Lewis White, Andrew J. Ullmann, Denise Michel, Verena Wiegering, Matthias Wölfl, Paul Gerhardt Schlegel, Hermann Einsele, Jan Springer, Matthias Eyrich
Journal of Clinical Microbiology Dec 2016, 55 (1) 101-109; DOI: 10.1128/JCM.01682-16
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

biomarkers
DNA, Fungal
enzyme-linked immunosorbent assay
invasive pulmonary aspergillosis
Mannans
polymerase chain reaction
Aspergillus fumigatus
PCR
invasive aspergillosis
pediatrics

Related Articles

Cited By...

About

  • About JCM
  • Editor in Chief
  • Board of Editors
  • Editor Conflicts of Interest
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Resources for Clinical Microbiologists
  • Ethics
  • Contact Us

Follow #JClinMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

 

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0095-1137; Online ISSN: 1098-660X