Variability in Galactomannan detection by platelia Aspergillus EIA™ according to the Aspergillus species

Variabilidade na detecção de galactomanana pelo Platelia Aspergillus EIA® de acordo com a espécie de Aspergillus

Abstracts

Here we investigate the extent to which different Aspergillus species release galactomannan (GM) in vitro. Marked variability was observed in GM reactivity between and within Aspergillus species, with A. terreus strains showing the highest GM indexes. The in vivo significance of these findings remains to be determined.

Invasive aspergillosis; Galactomannan; Diagnosis

O estudo objetivou investigar a liberação in vitro de galactomanana (GM) em distintas espécies patogênicas de fungos do gênero Aspergillus. Grande variabilidade foi detectada tanto intra quanto inter espécies, sendo as cepas da espécie A. terreus relacionadas aos maiores índices de GM detectados. O significado in vivo destes achados permanece em aberto, porém merece investigação.

INTRODUCTION

Galactomannan (GM) is a termostable polysaccharide that is a component of the cell wall of a diverse range of fungi, including those belonging to the genus Aspergillus ¹. Since GM is released during fungal hyphal growth, its detection in circulation or in other body fluids allows for an early diagnosis of invasive aspergillosis (IA)⁴. However, when interpreting a positive GM result in a patient with suspected IA, one might consider the variety of factors that can potentially interfere with the test, including antibiotic use, dietary factors and cross-reactivity with non-Aspergillus fungi³,⁹,¹⁰. Although early investigations have suggested that GM levels may vary among Aspergillus species⁷, there is still only very limited data to support this. Here we report that marked inter-species and intra-species variation occurs in the Aspergilli, considering GM release in vitro.

MATERIALS AND METHODS

Exoantigen testing was based on the method described by SWANINK et al. ⁸, in 12 well-characterized Aspergillus strains. All strains were kindly provided by Myconostica (National Aspergillosis Centre, UK). These included A. fumigatus (n = 4) strains Af294, Af10, Af71, and Af13073; A. flavus (n = 2), Afl5, and Afl16883; A. niger (n = 3), An9029, An1015, and An186; A. terreus (n = 2), At10071, and At49; and A. nidulans (n = 1), strain NEQAS UK. In brief, strains were subcultured to obtain pure young cultures in Sabouraud at 25 °C for 48 h (A. fumigatus, A. flavus and A. niger) or 96 h (A. terreus and A. nidulans). One loop of each strain was used to prepare the inoculum in liquid Sabouraud medium, which was adjusted by spectrophotometry (530 nm) to 80-82% T (2x10⁵ to 2.5x10⁶ FC/mL). Strains were incubated at 35 °C for 48-96 h, centrifuged for five min with 2500 rpm and then filtered (Millipore 0.45 µm). Tenfold dilutions were applied successively, and reactivity of the sandwich ELISA to GM detection was determined in duplicate at the 10^-6 dilution, using Platelia Aspergillus EIA kit (BioRad). The Platelia Aspergillus test was performed according to manufacturer's instructions. In short, 100 µL of Platelia treatment solution (4% ethylenediaminetetraacetic acid solution) was added to 300 µL of the adjusted innoculum, homogenized, and heated to 120 °C for six min in a heat block, followed by centrifugation at 10,000g for 10 min. Next, 50 µL of the supernatant and 50 µL of the horseradish peroxidase labeled monoclonal antibody (EBA-2) were incubated in antibody precoated microplates for 90 min at 37 °C. The plates were washed five times and incubated with 200 µL of substrate chromogen reaction solution for 30 ± 5 min in the dark at room temperature. The reaction was stopped with 1.5 N sulfuric acid solution, and the plates were read at an optical density (OD) of 450 nm, with a reference filter of 620/630 nm. Positive, negative, and cut-off controls were incorporated in each assay. GM results were expressed as optical densities (OD) - samples were considered positive when the ratio between the OD observed for the sample and the mean cut-off OD was > 0.5. Strains resulting in negative GM readings were again tested in duplicate at the previous dilution (10^-5).

RESULTS

Median GM indexes (range) at the 10^-6 dilution were: A. terreus, 3.82 (1.30 to > 6.35); A. nidulans, 1.3; A. fumigatus, 0.88 (0.17-1.50); A. niger, 0.28 (0.24-0.79), and A. flavus, 0.16 (0.14-0.18). Considering A. fumigatus as the comparator, GM reactivity for the other Aspergillus species was 434% for A. terreus, 148% for A. nidulans, 32% for A. niger, and 18% for A. flavus. At the 10^-6 dilution 50% of the A. fumigatus strains (Af10 and Af71) were GM negative, as well as the two A. flavus strains and two out of the three A. niger strains tested (An186 and An9029). These were all positive at 10^-5 dilution, with GM indexes varying from 0.51 to 1.02.

Table 1
Marked variations are observed in galactomannan reactivity when different Aspergillus species and strains are tested in vitro. All experiments were performed in duplicate at the 10^-6 dilution using Platelia Aspergillus EIA kit (Bio-Rad). Optical densities represent mean values

Aspergillus strain	Galactomannan optical density
A. fumigatus (Af 294)	1.50
A. fumigatus (Af 10)	0.33
A. fumigatus (Af 71)	0.17
A. fumigatus (Af 13073)	1.43
A. flavus (Afl 5)	0.18
A. flavus (Afl 16883)	0.14
A. niger (An 9029)	0.28
A. niger (An 1015)	0.79
A. niger (An 186)	0.24
A. terreus (At 10071)	1.30
A. terreus (At 49)	> 6.35
A. nidulans (NEQAS UK)	1.30

DISCUSSION

This study showed that marked variations occurred in GM release among Aspergillus species, with the highest GM indexes being observed for A. terreus. On the other hand, A. niger and A. flavus showed less reactivity in the GM ELISA test, in comparison to A. fumigatus. Previous studies had already suggested that in vitro GM release may vary according to the Aspergillus species being tested³,⁸. However, varied results have been observed among studies. For instance, two previous investigations found that A. niger isolates produced more GM than A. fumigatus ⁷,⁸. Even though limited GM release by A. flavus was documented in our study as well as in the study by MENNINK-KERSTEN et al. ⁷ (which was presented in abstract form only), SWANINK et al. ⁸ showed that GM reactivity was 7% higher in A. flavus, in comparison to A. fumigatus. Common to all studies was the observation that A. terreus produces more GM than A. fumigatus ⁷,⁸.

One explanation for the lower amount of in vitro GM release in A. niger and A. flavus strains, in comparison to A. fumigatus, is the fact that A. fumigatus clearly demonstrates a higher germination rate at 37 °C², a temperature that was similar to the incubation temperature used in our experiment (35 °C). However, considering that both A. terreus and A. nidulans showed higher GM release in comparison to A. fumigatus, although they had been submitted to a higher period of incubation (96 h versus 48 h), these two species grew slower than A. fumigatus at 37 °C², and therefore germination rate by itself does not seem to explain the observed difference in GM indexes. Another potential explanation for such variability in GM levels is related to the quantity of β-D-galactofuranosidase produced by different Aspergillus strains. By degrading GM epitopes, the enzyme may interfere with the immunoassay by blocking the antigen-antibody complex formation. The demonstration that A. fumigatus produces 2-20% of the β-D-galactofuranosidase produced by A. niger ⁶ suggests that A. fumigatus could react more avidly in the GM EIA test than A. niger, as found in the current study. The influence of the β-D-galactofuranosidase on GM levels is not completely clear, since similar enzyme activity has been observed for patients with IA caused by A. fumigatus in spite of serum GM levels⁵. The impact of other variables, such as medium pH, glucose concentration and development fungal stage could not be addressed in the present study since all strains were submitted to the same test condition.

If the results of our study are confirmed in vivo, then differences in serum GM levels observed among patients with IA may also be related to the Aspergillus species causing the infection. As a result, caution would be required when diagnosing IA based on the use of a universal cut-off for serum GM detection (i.e., 0.5). In fact, a clinical study involving haematological patients showed higher sensitivity of the Platelia Aspergillus EIA test in the diagnosis of IA when the infection was caused by Aspergillus species other than A. fumigatus (49% vs. 13%, in comparison to A. fumigatus, respectively)⁵. One interesting finding of our study was the observation that variability in GM levels also occurred within each of the Aspergillus species evaluated. For instance, the magnitude of such differences was as high as ninefold, and this could potentially explain the difference in results obtained among studies.

In conclusion, this study showed that marked variations occurred in GM levels among distinct Aspergillus species, as well as among different strains belonging to the same species. It remains to be determined whether such differences could affect the determination of GM concentrations in vivo.

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Publication Dates

• Publication in this collection
  May-Jun 2013

History

• Received
  10 July 2012
• Accepted
  3 Oct 2012