Prospective comparison of the diagnostic potential of real-time PCR, double-sandwich enzyme-linked immunosorbent assay for galactomannan, and a (1-->3)-beta-D-glucan test in weekly screening for invasive aspergillosis in patients with hematological disorders

Abstract

The establishment of an optimal noninvasive method for diagnosing invasive aspergillosis (IA) is needed to improve the management of this life-threatening infection in patients with hematological disorders, and a number of noninvasive tests for IA that target different fungal components, including galactomannan, (1-->3)-beta-d-glucan (BDG), and Aspergillus DNA, have been developed. In this study, we prospectively evaluated the diagnostic potential of three noninvasive tests for IA that were used in a weekly screening strategy: the double-sandwich enzyme-linked immunosorbent assay (ELISA) for galactomannan (Platelia Aspergillus), a real-time PCR assay for Aspergillus DNA (GeniQ-Asper), and an assay for BDG (beta-glucan Wako). We analyzed 149 consecutive treatment episodes in 96 patients with hematological disorders who were at high risk for IA and diagnosed 9 proven IA cases, 2 probable IA cases, and 13 possible invasive fugal infections. In a receiver-operating characteristic (ROC) analysis, the area under the ROC curve was greatest for ELISA, using two consecutive positive results (0.97; P = 0.036 for ELISA versus PCR, P = 0.055 for ELISA versus BDG). Based on the ROC curve, the cutoff for the ELISA could be reduced to an optical density index (O.D.I.) of 0.6. With the use of this cutoff for ELISA and cutoffs for PCR and BDG that give a comparable level of specificity, the sensitivity/specificity/positive predictive value/negative predictive value of the ELISA and the PCR and BDG tests were 1.00/0.93/0.55/1.00, 0.55/0.93/0.40/0.96, and 0.55/0.93/0.40/0.96, respectively. In conclusion, among these weekly screening tests for IA, the double-sandwich ELISA test was the most sensitive at predicting the diagnosis of IA in high-risk patients with hematological disorders, using a reduced cutoff of 0.6 O.D.I.

FIG. 1.

(A to C) ROC curves of the GM (A), PCR (B), and BDG (C) tests for screening for IA. Both methods I and II were used. The ROC curves obtained by estimate A/B are shown in red, and those obtained by estimate C are shown in blue. The ROC curves obtained by method II are indicated by solid lines, and those obtained by method I are indicated by dotted lines. (D) Combination of ROC curves of the GM test (method II) and those of the PCR and BDG tests (method I).

FIG. 2.

Number of days from when GM assays become positive to the onset of treatment, using a threshold of 0.6 O.D.I. by method II (solid arrowheads) or 1.0 O.D.I. by method II (open arrowheads), or positive findings on CT. Open triangles indicate the date of positive culture, and solid triangles indicate when the histopathological diagnosis was made (Px, biopsy; Ax, autopsy). The values in parentheses indicate the number of days after the onset of treatment. For example, for episode 11, CT showed specific findings 50 days after the onset of treatment and the GM assay become positive 2 days before treatment. Episode numbers correspond to those in Table 2. Episodes whose GM assays did not reach the threshold are not shown. For episodes 2 and 9, a CT scan was not performed, and for episodes 7, 8, 10, 17, 19, 21, and 22, the CT findings were nonspecific and could not be used for decision-making. Each treatment was started at the discretion of the physician, taking into account various prices of clinical information, including CT findings and the results of GM assays. For Episode 8, IA was not suspected and no antifungal agent was administered. Therefore, the date of death was used instead of the date of treatment onset.

FIG. 3.

Number of days before death that each test gave positive results. Solid triangles indicate the date when GM became positive, using a threshold of 0.6 O.D.I. by method II; open triangles indicate the date when PCR exceeded a cutoff value of 40 copies/ml; and shaded triangles indicate the date when the BDG test exceeded a cutoff value of 11 ng/ml, by method I. In episode 2, PCR never exceeded the cutoff value. Episode numbers correspond to those in Table 2.