Evaluation of the Double Agar Gel Immunodiffusion Test and of the Enzyme-Linked Immunosorbent Assay in the Diagnosis and Follow-Up of Patients with Chronic Pulmonary Aspergillosis

Abstract

The diagnosis of chronic pulmonary aspergillosis (CPA) depends on the radiologic image and the identification of specific antibodies. The present study aimed to evaluate accuracy parameters of enzyme-linked immunosorbent assay (ELISA) and of the determination of serum galactomannan level in the diagnosis of patients with CPA, comparing these results with the double agar gel immunodiffusion (DID) test. In addition, the prevalence of cross-reactivity and the serological progression after treatment were evaluated by comparing DID and ELISA. Six study groups were formed: G1: 22 patients with CPA, 17 of whom had Aspergillus fungus ball, one chronic cavitary pulmonary aspergillosis (CCPA) and four chronic fibrosing pulmonary aspergillosis (CFPA); G2: 28 patients with pulmonary tuberculosis (TB); G3: 23 patients with histoplasmosis (HST); G4: 50 patients with paracoccidioidomycosis (PCM); G5: 20 patients with cryptococcosis (CRC); and G6: 200 healthy controls. Serum antibodies were measured by DID and ELISA, with two antigen preparations—Aspergillus fumigatus (DID1, ELISA1) and a pool of A. fumigatus, A. flavus and A. niger antigens (DID2, ELISA2). The Platélia Aspergillus Enzyme Immunoassay (EIA) kit was used to measure galactomannan. The cut-off points of ELISA were determined for each antigen preparation and for the 95% and 99% confidence intervals. Despite the low sensitivity, DID was the technique of choice due to its specificity, positive and negative predictive values and positive likelihood ratio–especially with the antigen pool and due to the low frequency of cross-reactivity. ELISA1 and a 0.090 cut-off showed high sensitivity, specificity and negative predictive value, but a high frequency of cross-reactivity with CRC. The best degree of agreement was observed between ELISA1 and ELISA2. The detection of serum galactomannan showed high sensitivity, comparable to ELISA2. The immunodiffusion test showed an excellent relationship with the progression after treatment, which made it the reaction of choice for patient follow-up.

Accuracy of serological tests for diagnosis of chronic pulmonary aspergillosis: a systematic review and meta-analysis

“…The characteristics of the included studies are presented in S1Table. The earliest study was published in 1983 [22] and the five most current articles were published in 2015 [23], 2016 [24, 25, 26] and 2018 [27]. Nine studies took place in five countries: Japan [25, 28], Brazil [23], United Kingdom [24, 29], France [26, 30, 31] and India [27], but in 4 articles, the study countries were not reported [22, 32, 33, 34].…”

“…Nine articles presented DID as the reference test [22, 23, 25, 27, 28, 30, 32, 33, 34]; an article presented two reference tests, DID and CIE [34] and four studies presented only CIE as the reference test [24, 26, 29, 31].…”

“…When we performed data extraction, some important differences were observed and deserve to be highlighted. Seven articles performed in-house ELISA tests [22, 23, 28, 30, 32, 33, 34] and six articles described their studies with commercial tests [24, 25, 26, 27, 29, 31]. Different Aspergillus antigens and cut-off points were used in the in-house ELISA tests; the articles that used commercial tests also used several types of antigens and cutoff points included by authors beyond those established by the manufacturer and are described in S1 Table.…”

“…During the extraction of ELISA antigen concentration data, five studies with in-house tests presented concentrations varying from 0.1 mcg to 250 mcg per well [22, 23, 30, 33, 34]; in two articles these concentrations were not reported [28, 32].…”

“…In the in-house tests, we still find other differences, such as ELISA secondary antibody dilution, with concentrations ranging from 1: 100 to 1: 300 when they were described [22, 23, 33, 34]; in three articles these dilutions were not reported [28, 30, 32]. When we evaluated the cut-off for ELISA, several descriptions were found with titers ranging from 1: 100 to 1: 800; we also found values in OD (optic density), au / mL, in percentage and in absorbance, and there was no comparable value in in-house tests [22, 23, 33, 34]; in three articles, the cut-off was not described [28, 30, 32]. For the ELISA substrate, TMB (3,3′,5,5′-Tetramethylbenzidine) was found in two articles [22, 23], also pNPP (Alkaline Phosphatase Yellow) [33, 34] and OPD (o-Phenylenediamine) [30]; in two articles the substrate was not reported [28, 32].…”