Dot Immunobinding Assay for the Rapid Serodetection of Scedosporium/Lomentospora in Cystic Fibrosis Patients

Round 1

Reviewer 1 Report

The authors present a very interesting and relevant manuscript on the diagnosis of Scedosporium/Lomentospora species in cystic fibrosis patients. However, the reviewer would like to obtain more details about the DIA assay as well as to offer the authors the possibility to explore better some points, which were detailed below.

 

Abstract

-        The sentence “This fact is particularly worrying in patients with Cystic Fibrosis (CF), where these fungi are the second most common filamentous fungi isolated, because it can worsen the prognosis and increase the risk of infection.” should be clarified. What do the authors mean by the increased risk of infection?

-        What is the fungal antigen used in the DIA?

 

Introduction

-        The introduction will gain more visibility and attractivity with the addition of more details on similar diagnosis tests to the proposed in the present study. Another relevant curiosity is about the nature of the fungal antigen used in the DIA assay proposed to detect Scedosporium/Lomentospora infections.

 

Methods

-        Change “non-fumigatus spp” to “non-fumigatus Aspergillus species”.

-        Why did the authors decide to use the Whole-Cell Protein Extract from S. boydii instead of other Scedosporium/Lomentospora species? And why do the authors choose to use Whole-Cell Protein Extract from the fungus instead of another antigen? If the reviewer is not wrong, S. apiospermum is the main Scedosporium species recovered from infections in cystic fibrosis patients. In this sense, why did the authors not use this species? Please, the reviewer strongly suggests to the authors to explain all these issues.

-        Another issue is about the nature of the Whole-Cell Protein Extract, the authors elucidated the protein content of this extract? Are there only proteins? Glycoproteins are absent from that fungal extract?

-        The Whole-Cell Protein Extract from S. boydii was obtained after growth in which culture medium? It is well-known that the culture medium directly influences the synthesis of surface-located antigens. In this context, it will be very interesting to culture S. boydii in a medium mimicking the lungs of cystic fibrosis patients in order to induce a more reliable synthesis of fungal surface-located (glyco)proteins. Culturing the fungal cells in a cystic fibrosis medium, it will generate more specific antigens related to the infections of Scedosporium species in this specific environment.

General

-        Using the present diagnosis test is it possible to detect the Scedosporium/Lomentospora infection causing other clinical manifestations, like mycetoma?

Author Response

REVIEWER 1

The authors present a very interesting and relevant manuscript on the diagnosis of Scedosporium/Lomentospora species in cystic fibrosis patients. However, the reviewer would like to obtain more details about the DIA assay as well as to offer the authors the possibility to explore better some points, which were detailed below. (In all cases, the pages and lines indicated refer to the final version of the manuscript without marked changes).

Abstract

The sentence “This fact is particularly worrying in patients with Cystic Fibrosis (CF), where these fungi are the second most common filamentous fungi isolated, because it can worsen the prognosis and increase the risk of infection.” should be clarified. What do the authors mean by the increased risk of infection?

• The sentence has been changed in page 1, lines 23-25, to: “This fact is particularly worrying in patients with Cystic Fibrosis (CF), where these fungi are the second most common filamentous fungi isolated, because a poor and delayed diagnosis can worsen the prognosis of the disease.”

What is the fungal antigen used in the DIA?

• The fungal antigen has been detailed in page 1, lines 27-28, as follows: “A crude protein extract from conidia and hyphae of Scedosporium boydii was employed as fungal antigen.”

 

Introduction

The introduction will gain more visibility and attractivity with the addition of more details on similar diagnosis tests to the proposed in the present study. Another relevant curiosity is about the nature of the fungal antigen used in the DIA assay proposed to detect Scedosporium/Lomentospora infections.

• The following paragraph concerning the currently available POC tests for Aspergillus has been added (Page 2; lines 58-65): “Among them, there are three main rapid tests commercially available based on Lateral Flow Devices (LFDs), “Aspergillus Galactomannan Lateral Flow Assay”, “Aspergillus-specific Lateral Flow Device Test” and “Aspergillus Proximity Ligation Antigen Assay”. These assays are developed to detect cell-wall antigens, indicative of fungal presence, which under a given infectious symptomatology is decisive for the diagnosis of infection [3]. They can be performed on-demand, require minimal hands-on time, technical skills and equipment to operate, and provide rapid results (minutes), but unfortunately they are not available for Scedosporium/Lomentospora.”

 

 

 

Methods

Change “non-fumigatus spp” to “non-fumigatus Aspergillus species”.

• This has been changed.

 

Why did the authors decide to use the Whole-Cell Protein Extract from S. boydii instead of other Scedosporium/Lomentospora species? And why do the authors choose to use Whole-Cell Protein Extract from the fungus instead of another antigen? If the reviewer is not wrong, S. apiospermum is the main Scedosporium species recovered from infections in cystic fibrosis patients. In this sense, why did the authors not use this species? Please, the reviewer strongly suggests to the authors to explain all these issues.

• Regarding Spain, there are no studies conducted in CF patients discriminating between species within these genera, however in France and in Germany, S. boydii is the most prevalent species. In order to clarify this aspect a new section “2.3.1. Selection of the Fungal Species and Obtaining of the Antigenic Extract” has been included in page 4 lines 141-157 with the following information describing the reason for the selection of the species as well as the protein extraction process:

To select the fungal species for antigen extraction, the origin of the CF serum samples (Spain and Germany) was considered. The latest epidemiological study in CF patients in Germany [5] reports Scedosporium boydii as the most isolated Scedosporium/Lomentospora species. Regarding Spain, there are no studies conducted in CF patients discriminating between species within these genera, but in the nearby country France, S. boydii is also the most prevalent species [6]. Moreover, a crude antigenic extract of this fungus was previously analysed by our research group and demonstrated to be useful discriminating by ELISA Scedosporium/Lomentospora positive CF patients [7]. Therefore, a Whole-Cell Protein (WCP) extract of S. boydii conidia and hyphae was obtained following the protocol optimized by our research group. Briefly, the fungus was grown for 24 h at 37°C and 120 rpm in Potato Dextrose Broth (PDB) (Condalab, Spain). Fungal growth was recovered by filtration, washed with Phosphate Buffered Saline pH 7.4 (PBS), resuspended in PBS supplemented with 1%(v/v) b-mercaptoethanol and 1%(v/v) ampholytes pH 3-10 (GE Healthcare, Germany) and disrupted by bead-beating for 20 min at 30Hz using theMillMix20 (Tethnica, Slovenia). Cell debris was discarded by centrifugation, and the resulting WCP suspension was sonicated and stored at -80°C.

 

Another issue is about the nature of the Whole-Cell Protein Extract, the authors elucidated the protein content of this extract? Are there only proteins? Glycoproteins are absent from that fungal extract?

• The Whole-Cell Protein Extract is a crude extract obtained from conidia and hyphae of boydii where all the proteins are recovered, including glycoproteins. No additional steps have been followed to discard glycoproteins. The procedure is described in previous articles published by our research group (Martin-Souto et al. Front Cell Infect Microbiol., 2020) included in the text, and in the page 4; lines 149-157 of the manuscript.

The Whole-Cell Protein Extract from S. boydii was obtained after growth in which culture medium? It is well-known that the culture medium directly influences the synthesis of surface-located antigens. In this context, it will be very interesting to culture S. boydii in a medium mimicking the lungs of cystic fibrosis patients in order to induce a more reliable synthesis of fungal surface-located (glyco)proteins. Culturing the fungal cells in a cystic fibrosis medium, it will generate more specific antigens related to the infections of Scedosporium species in this specific environment.

• It is a very interesting suggestion the use of a culture medium mimicking the CF lung mucus that we are considering for our future studies.

 

General

Using the present diagnosis test is it possible to detect the Scedosporium/Lomentospora infection causing other clinical manifestations, like mycetoma?

• The diagnostic utility of the WCP has been assessed only with CF patients sera with apparently no other clinical manifestations. Therefore, we don’t know if DIA would be suitable for these malignancies. We can suppose it will be able to detect IgG produced in other malignances too, but the suitability should be studied.

 

 

Reviewer 2 Report

This article is of great scientific interest and is highly applicable in clinical practice. The only aspect that needs to be modified or further developed is to delimit the technique's specificity, as the terms Scedosporium and Scedosporium/Lomentospora are used interchangeably in different paragraphs. It would have been interesting to include samples from CF patients from Australia in the analyses, where the most prevalent species in this country is S. aurantiacum.

The procedures for species identification could be improved; e.g. indicating the fragment of Tubulin sequenced.

Other modifications/suggestions are included in the ms.

Comments for author File: Comments.pdf

Author Response

REVIEWER 2

General

This article is of great scientific interest and is highly applicable in clinical practice. The only aspect that needs to be modified or further developed is to delimit the technique's specificity, as the terms Scedosporium and Scedosporium/Lomentospora are used interchangeably in different paragraphs.

• Since CF samples of patients with Scedosporium or Lomentospora have been considered within the group of Positive Culture, and no distinction between species has been performed to carry out the different analyses. Therefore the term Scedosporium has been replaced by Scedosporium/Lomentospora in the manuscript.

It would have been interesting to include samples from CF patients from Australia in the analyses, where the most prevalent species in this country is S. aurantiacum.

• We totally agree with the reviewer. It would be very interesting to study the humoral response of Australian CF samples. In fact, in the discussion we suggest to perform a larger-scale worldwide multicentric study, which we would like to carry out the following years.

Procedures for species identification could be improved; e.g. indicating the fragment of Tubulin sequenced.

• AUTHORS:      The fragment of Tubulin sequenced has been indicated, lines 94 and 101: “… BT2, exon 2-4 …”

 

Methods

Page 3, line 115: and that they have a positive result for culture

• Not only a positive culture result. The requirement to sample inclusion in this study was to have a sputum-culture result (positive/negative) performed the same day of the blood extraction. We have modified the sentence to clarify this point (Page 3 lines 108-111): “The inclusion criteria for the initial selection of serum samples were that they derived from CF patients, and that they have data for the culture of respiratory secretions obtained on the same day of serum extraction as well as availability of other concomitant clinical data.”

Why did you use Scedosporium boydii to obtain Whole-Cell Protein Extract and not the most prevalent species in this type of infection, Scedosporium apiospermum?

• Regarding Spain, there are no studies conducted in CF patients discriminating between species within these genera, however in France and in Germany, S. boydii is the most prevalent species. In order to clarify this aspect a new section “2.3.1. Selection of the Fungal Species and Obtaining of the Antigenic Extract” has been included in page 4 lines 141-157 with the following information describing the reason for the selection of the species as well as the protein extraction process:

“To select the fungal species for antigen extraction, the origin of the CF serum samples (Spain and Germany) was considered. The latest epidemiological study in CF patients in Germany [5] reports Scedosporium boydii as the most isolated Scedosporium/Lomentospora species. Regarding Spain, there are no studies conducted in CF patients discriminating between species within these genera, but in the nearby country France, S. boydii is also the most prevalent species [6]. Moreover, a crude antigenic extract of this fungus was previously analysed by our research group and demonstrated to be useful discriminating by ELISA Scedosporium/Lomentospora positive CF patients [7]. Therefore, a Whole-Cell Protein (WCP) extract of S. boydii conidia and hyphae was obtained following the protocol optimized by our research group. Briefly, the fungus was grown for 24 h at 37°C and 120 rpm in Potato Dextrose Broth (PDB) (Condalab, Spain). Fungal growth was recovered by filtration, washed with Phosphate Buffered Saline pH 7.4 (PBS), resuspended in PBS supplemented with 1%(v/v) b-mercaptoethanol and 1%(v/v) ampholytes pH 3-10 (GE Healthcare, Germany) and disrupted by bead-beating for 20 min at 30Hz using theMillMix20 (Tethnica, Slovenia). Cell debris was discarded by centrifugation, and the resulting WCP suspension was sonicated and stored at -80°C.”

Highlighted nl

• “nl” has been changed to “nL”

 

Results

Changes in Figure 4 “P. ellipsoidea” and “Scedosporium/Lomentospora”

• Figure 4 has been changed.

 

In results text, the referee has included the follow comment: “The first time you use a specific name, you should refer to it in complete form”.

• This has been checked in the manuscript. All complete name of microorganism species was used in the Methods section 2.2 (the first time they are mentioned).

 

Round 2

Reviewer 1 Report

The manuscript improves after revision and it is acceptable for publication in the Journal of Fungi in this current form.