Performance of Legiolert Test vs. ISO 11731 to Confirm Legionella pneumophila Contamination in Potable Water Samples

Round 1

Reviewer 1 Report

The paper makes a valuable contribution to those considering using using the IDEXX Legiolert system for monitoring potable water. This contribution would be enhanced with more detail on they type of water analyzed in the project and a brief comment in the results and discussion as to whether Legiolert performed differently or better with one type of water quality matrix (high/low iron, high/low TOC etc) or one type of water (low TOC groundwater, high TOC surface water etc) versus others.

Overall the paper is clear but the second two paragraphs in the Results section reporting the positive Legiolert samples are confusing. "10 mL water samples showed that 34/123 (27.6%) samples were positive", "100 mL water samples detected 37/123 (30%) samples were positive", "28 or 33 positive samples from the 100 mL and 10 mL tests, respectively". Perhaps a graphic would clarify this.

Line 4 of the introduction (ending with the word amoebae) needs this citation: Rowbotham TJ. 1980. Preliminary report on the pathogenicity of Le-gionella pneumophila for freshwater and soil amoebae. J Clin Pathol33:1179–1183.

There are just a few grammatical/typographical revisions needed:

"where can it "be found"  should be: where it can be found

"equal performance than traditional plate" should be: equal performance to

Table 10. mL and   should be Table 1: 10 mL and....

I have no objection to IDEXX funding this work. No one else funds these types of studies.

Author Response

Dear Reviewer,

thank you for your attention and for the time you have spent for revising our manuscript.

Please find below the responses to your revision.

The manuscript has been English editing by a native English-speaking colleague.

Reviewer 1

Answer/comment The paper makes a valuable contribution to those considering using the IDEXX Legiolert system for monitoring potable water. This contribution would be enhanced with more detail on the type of water analyzed in the project and a brief comment in the results and discussion as to whether Legiolert performed differently or better with one type of water quality matrix (high/low iron, high/low TOC etc) or one type of water (low TOC groundwater, high TOC surface water etc) versus others. Response: It would have been very interesting to have this information, unfortunately it was not included in the study design and therefore we cannot provide it.

Answer/comment Overall, the paper is clear but the second two paragraphs in the Results section reporting the positive Legiolert samples are confusing. "10 mL water samples showed that 34/123 (27.6%) samples were positive", "100 mL water samples detected 37/123 (30%) samples were positive", "28 or 33 positive samples from the 100 mL and 10 mL tests, respectively". Perhaps a graphic would clarify this. Response: Results Lines 97-100. The discrepancy observed in the numbers (for Legiolert 100, initially 34 and then 28 or Legiolert 10 initially 37 and then 33) is due to the restrictions imposed by the “ISO 17994, 2014: Requirements for the comparison of the relative recovery of microorganisms by two quantitative methods”, for which, as indicated in the text, eight samples exceeded the MPN count by Legiolert (Too numerous to count, TNTC) ), therefore they were excluded from calculation.

Answer/comment Line 43 of the introduction (ending with the word amoebae) needs this citation: Rowbotham TJ. 1980. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol33:1179–1183. Response: Introduction, Line 43:The reference has been added.

Answer/comment/correction. There are just a few grammatical/typographical revisions needed:

"where can it "be found"  should be: where it can be found

"equal performance than traditional plate" should be: equal performance to

Table 10. mL and   should be Table 1: 10 mL and.... Response: Introduction, Lines 42 and 77. Corrections have been done.

Reviewer 2 Report

This is a good ms comparing two test to detect Legionella in water. Just minor suggestion which is to discuss further the following statement and include solutions or weakness:

"One limitation of the Legiolert method is that it is designed to detect only Legionella pneumophila,
while other species remain undetectable. Legionella pneumophila is the most common species
responsible for LD cases in Europe and, for this reason, in a few regions, such as France, Belgium,
province of Quebec, Canada, it has been decided to monitor only Legionella pneumophila, while in
other countries there is still a great debate on this matter."

Author Response

Dear Reviewer,

Thank you for your useful revision and for taking the time to review our manuscript. The manuscript has undergone
English revision by a native English speaker.

Please find below the responses to your comments.

Answer/comment This is a good ms comparing two test to detect Legionella in water. Just minor suggestion which is to discuss further the following statement and include solutions or weakness “One limitation of the Legiolert method is that it is designed to detect only Legionella pneumophila, while other species remain undetectable. Legionella pneumophila is the most common species responsible for LD cases in Europe and, for this reason, in a few regions, such as France, Belgium, province of Quebec, Canada, it has been decided to monitor only Legionella pneumophila, while in other countries there is still a great debate on this matter”.

Response:Discussion, Lines  158-180.The following sentences have been added in the Discussion:

Three fundamental aspects could be in favor of monitoring exclusively for  Legionella pneumophila: ​​ the first is risk -  L. pneumophila  is the species almost always cited in clinical cases and outbreaks and is the species most commonly in the environment; the second is that laboratories may save time, human resources and money, and they can employ those saved resources to  analyze additional samples or locations instead of identifying other Legionella species which represent a much lower health risk; the third is that routinely monitoring only for the most pathogenic species of a bacteria is already established practice. For example,  Pseudomonas aeruginosa is routinely monitored rather than all species of Pseudomonas,  .

At the same time, it is well known that other Legionella species are pathogenic to humans although they represent a fraction of infections, with the exception of Legionella longbeachae, which is found in soil, rather than water and mostly in Australia and New Zealand, but is beginning to be isolated also in Europe (2.5% of isolated species in 2018, ECDC, unpublished data). Other known and unknown species of Legionella represent only 3.3% of notified cases (ECDC 2018, unpublished data). It should be noted that the identification of species other than L. pneumophila suffers from extensive use of urinary antigen, that detects exclusively Legionella pneumophila serogroup 1, and from the medium used for the isolation of Legionella , which has historically been optimized for Legionella pneumophila. Therefore, many cases caused by other species might not be detected even by culture for this reason. Until a suitable medium for growing other Legionella species is developed, a routine PCR test in diagnosing human specimens, capable of distinguishing between Legionella pneumophila and other species, should be adopted in order to identify the real burden of Legionnaires’ disease, as already demonstrated in a few countries (31-35). The results of these studies will be able to confirm the real incidence of infections caused by other Legionella species and consequently to address the choices on what should be the monitoring focus in the environment.

Reviewer 3 Report

Interesting study examining an alternative to the standard Legionella detection method.

Abstract

The abstract needs a bit more detail

Could you please add information regarding the number and type of samples tested – number or positive and number of negative using the different methods and the t-test results

Introduction

In the last paragraph you mention the volumes used for the Legiolert – it would be good to include the volumes used for the ISO method

Materials and methods

-nursery home – I don’t know what this is – do you mean aged-care facility?

Need to include a bit more information of sampling and handling of samples – how were they transported – how long before analysis – was this all done in the same lab?

Results

From a public health perspective, you need to make it clearer that there were 3 samples that were negative using the Legiolert that were positive using the ISO. I think in the table with the statistical analysis it would also be good to include the number of positive and negative results for each to address this.

Also, I have assumed the positive matched and there were no occasions where the legiolert was positive and the ISO negative but a statement to clarify this would be good

Discussion

legionella – needs to be italisiced and have a capital letter

Reference

Formatting need to be fixed

Some information missing

Author Response

Dear Reviewer,

Thank you for your useful revision and for taking the time to review our manuscript. The manuscript has undergone
English revision by a native English speaker.

Please find below the responses to your comments.

Answer/comment The abstract needs a bit more detail. Could you please add information regarding the number and type of samples tested – number or positive and number of negative using the different methods and the t-test results. Response: Abstract Lines 26-32. The information required have been added to the abstract, highlighted below  in red.

Abstract Detection and enumeration of Legionella in water samples is of great importance for risk assessment analysis. Plate culture is the gold standard but has several well-known criticisms that induced researchers to develop alternative methods. The purpose of this study was to compare Legionella counts obtained by analysis of potable water samples between plate culture and the IDEXX liquid culture Legiolert*methods.  Legionella plate culture, according to the ISO 11731:1998, was performed using 1L volumes of water. Legiolert was performed using both the 10mL and 100 mL Legiolert protocols. Overall, 123 potable water samples were analysed. Thirty-seven (30%) of them, positive for L. pneumophila, serogroups 1 or 2-14 by plate culture, were used for Legiolert protocols comparison. The Legiolert 10 mL test detected 34 positive samples (27.6%) and the Legiolert 100mL test detected 37 positive samples, 27.6% and 30% respectively, of the total samples analysed. No significant difference was found between either the Legiolert 10mL and Legiolert 100mL vs the plate culture (p=0.9 and p=0.3, respectively) or between the Legiolert 10mL and Legiolert 100mL tests (p=0.83). This study confirms the reliability of the IDEXX Legiolert test for Legionella pneumophila detection and enumeration already shown in similar studies. Like plate culture, the Legiolert assay is also suitable to obtain isolates for typing purposes relevant for epidemiological investigation. 

Answer/comment Introduction In the last paragraph you mention the volumes used for the Legiolert – it would be good to include the volumes used for the ISO method. Response: Introduction Line 82. The volume used for the ISO method has been added.

Answer/comment Materials and methods -nursery home – I don’t know what this is – do you mean aged-care facility? Need to include a bit more information of sampling and handling of samples – how were they transported – how long before analysis – was this all done in the same lab? Responses: Materials and methods Lines 211-212, The name “nursery home” was replaced by “elderly health care facility”. Materials and methods Lines 216-218:More information was added of sampling and handling of samples: Water samples were collected according to the protocol contained in the Italian guidelines for Legionella (37) and stored at 5±3°C until they were delivered (within 24 hours) at the Italian reference laboratory for Legionella, where all samples were analysed.

Answer/comment Results From a public health perspective, you need to make it clearer that there were 3 samples that were negative using the Legiolert that were positive using the ISO. I think in the table with the statistical analysis it would also be good to include the number of positive and negative results for each to address this. Also, I have assumed the positive matched and there were no occasions where the Legiolert was positive and the ISO negative but a statement to clarify this would be good. Response: Discussion Lines 187-196 and 200-202. A few comments about these data have already been, done. Anyway these results are difficult to explain and unfortunately we couldn't repeat those samples in order to verify the at least once the data.