Human Toxocariasis in Portugal—An Overview of a Neglected Zoonosis over the Last Decade (2010–2020)

Round 1

Reviewer 1 Report

The subject is interesting, but it lacks methodology and the results are incomplete. For these reasons it cannot be accepted.

Below are some errors

Introduction

Lines 39-42.

It's confusing. They do not separate the mechanisms of infection by egg or larvae.

Line 45

Humans are paratenic hosts.

Methods

The authors conducted a study from 2010 to 2020

29 medical institutions sent the serum to a reference laboratory.

They made:

a) ELISA with TES (Excretory-secretory antigen from second-stage larvae of T. canis.

b) Immunoprecipitation with TES.

c) Counterimmunoelectrophoresis against TES.

d) Counterimmunoelectrophoresis against Ascaris suum.

They used a commercial kit (ELISA- IgG) in doubtful results (ELISA - and CIE +)

The authors failed to say:

-If the protocol was accepted in all the institutions involved

-There is no letter of informed consent. The authors say it does not apply. However, taking a peripheral blood sample requires a letter of consent and assent.

The authors did not say: how did they storage the sera for 10 years?

When did they perform the techniques?

The TES of larvae was from one batch or several batches over the course of 10 years?

The authors did not say how they make the ELISAs and the CIE with the TES, the description is missing.

Results

The authors found 152 positive sera from 846 samples.

They did not say how many sera were positive with ELISA and immunoprecipitation using TES

They did not say how many sera were positive for Ascaris suum

Figure 2.

They show the result of 772 sera, 74 are missing. They had 10 cases with unknown age, thus, 64 sera are missing. Where are the sera?

The sum of the percentages is 137.9% (doubtful result)

Figure 3.

The sum of the sera is 837. Where are the missing 9?

They show 148 positives. Where are the missing 4?

Author Response

We acknowledge the evaluation of the manuscript by both Reviewers. Responses to their comments and suggestions can be found below.

Reviewer #1

• The subject is interesting, but it lacks methodology, and the results are incomplete. For these reasons it cannot be accepted. Answer: The authors thank the Reviewer for her/his comments and suggestions. The manuscript was changed accordingly.

• Below are some errors. Introduction Lines 39-42. It's confusing. They do not separate the mechanisms of infection by egg or larvae. Answer: To attend the suggestion passed by Reviewer #1, lines 39-42 were rephrased.

1.3 Line 45: Humans are paratenic hosts. Answer: Text was adapted accordingly.

1.4 Methods: The authors conducted a study from 2010 to 2020, 29 medical institutions sent the serum to a reference laboratory. They made: a) ELISA with TES (Excretory-secretory antigen from second-stage larvae of T. canis. b) Immunoprecipitation with TES. c) Counterimmunoelectrophoresis against TES. d) Counterimmunoelectrophoresis against Ascaris suum. They used a commercial kit (ELISA- IgG) in doubtful results (ELISA - and CIE +) The authors failed to say:

1.4.1. If the protocol was accepted in all the institutions involved. Answer: The authors are grateful to the reviewer for correcting this misunderstood and improving the manuscript. Indeed, the protocol was accepted by all the institutions involved. A sentence stating that was added to the M&M section.

1.4.2 There is no letter of informed consent. The authors say it does not apply. However, taking a peripheral blood sample requires a letter of consent and assent. Answer: The authors are grateful to the reviewer for highlighting this misunderstood. A letter of consent and assent was obtained at the hospital where the patient was attended, and then serum samples were sent to reference laboratories, as the Portuguese Institute of Hygiene and Tropical Medicine.

1.4.3 The authors did not say: how did they storage the sera for 10 years? Answer: Serum samples were tested exactly at the time they were received and discharged after laboratory results and not stored.

1.4.4 When did they perform the techniques? Answer: Samples were tested exactly at the time they were received once it’s a required for the patient’s diagnosis and, if confirmed, to start a specific treatment. Thereby, these samples started to be tested in 2010 and finished at the end of 2020.

1.4.5 The TES of larvae was from one batch or several batches over the course of 10 years? Answer: The TES of larvae were from several batches over the decade, which were tested and optimized before its application. This information was added to the M&M section.

1.4.6 The authors did not say how they make the ELISAs and the CIE with the TES, the description is missing. Answer: To attend the suggestion passed by Reviewer #1 a full description of the ELISAs and the CIE was added to the M&M section.

1.5.1 Results: The authors found 152 positive sera from 846 samples. They did not say how many sera were positive with ELISA and immunoprecipitation using TES. Answer: The authors are grateful to the reviewer for this comment. In fact, the results of the re-test with the commercial kit ELISA-IgG® were not included in the initial manuscript. For that reason, a whole new statistical analysis was performed, and those results are now presented:

Out of the 846 ELISA tested serum samples, 159 were positive (18.80%), 678 negative (80.1%) and 9 (1.1%) remained inconclusive, after re-testing with the commercial kit ELISA-IgG®. By CIE against A. suum crude adult worm’s antigen, 73 samples were positive (8.6%) and 773 samples were negative (91.4%). By CIE against TES, 30 samples were positive (3.5%) and 816 samples were negative (96.5%). This information was added right in the beginning of the result section.

1.5.2 They did not say how many sera were positive for Ascaris suum. Answer: By CIE against A. suum crude adult worm’s antigen, 73 samples were positive (8.6%) and 773 samples were negative (91.4%). This information was added in the results section.

1.6 Figure 2. They show the result of 772 sera, 74 are missing. They had 10 cases with unknown age, thus, 64 sera are missing. Where are the sera? Answer: Yes, that’s correct. Overall, age was unknown in 74 cases, of which 10 were positives.

1.7 The sum of the percentages is 137.9% (doubtful result). Answer: Yes, that value corresponded to the percentage of positives per each age group (eg: in the 0-9 years-old age group, there were 56 positives out of the 191, corresponding to 29.3%. Nevertheless, the authors thank the reviewer for correcting this lack of clarity. For that reason, the authors decide to create a whole new graphic and insert the percentage of positives/overall. Now the sum of the percentages is the overall positivity - 18.8%. Additionally, they included a section with the category of “unknown age” to be clearer.  

1.8 Figure 3. The sum of the sera is 837. Where are the missing 9? Answer: Unknown provenance was reported in nine samples, of which four were positive and five were negative. The authors are grateful to the reviewer for this comment. This information was added in the result section as well as in the Figure 3 caption.

1.9 They show 148 positives. Where are the missing 4? Answer: As previously mentioned, unknown provenance was reported in nine samples, of which four were positive and five were negative. The authors are grateful to the reviewer for this comment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Introduction:

The mode of the Toxocara infection is described two times (Line 39-42; 47-51);

Please reduce the amount of details in the introduction part, such as - very detailed description of clinical syndromes of human toxocariasis (Line 54-73);

Material and methods:

The material and methods are described incomprehensibly (the most important information - line 111-121, then you should describe a questionnaire or the way of obtaining information about patients (line 124-128).

Results:

Figure 1 and Figure 2 have two titles (please remove the headlines)

Discussion:

According to the limitations (line 332-342) - I totally agree that the Western blot should be performed in this study, because the prevalence of Toxocara may be incorrect.

Author Response

We acknowledge the evaluation of the manuscript by both Reviewers. Responses to their comments and suggestions can be found below.

2.1 Comments and Suggestions for Authors. Introduction: The mode of the Toxocara infection is described two times (Line 39-42; 47-51). Answer: The authors are grateful to the reviewer for correcting this misunderstood, lines 39-42 and 47-51 were rephrased and put together.

2.2 Please reduce the amount of details in the introduction part, such as - very detailed description of clinical syndromes of human toxocariasis (Line 54-73). Answer: The synthesis suggested passed by Reviewer #2 has been incorporated. The 20-line paragraph has been shortened to 13-line paragraph.

2.3 Material and methods: The material and methods are described incomprehensibly (the most important information - line 111-121, then you should describe a questionnaire or the way of obtaining information about patients (line 124-128). Answer: The authors are grateful to the reviewer for this suggestion. A full description of the process of making the ELISAs and the CIE with the TES was added to the M&M. Additionally, information regarding patient questionnaire was added in detail.

2.4 Results: Figure 1 and Figure 2 have two titles (please remove the headlines). Answer: The authors agree with the reviewer. The headline of each figure was removed.

2.5 Discussion: According to the limitations (line 332-342) - I totally agree that the Western blot should be performed in this study, because the prevalence of Toxocara may be incorrect. Answer: The authors understand the concern of the reviewers as serological cross-reactivity may occur between pathogens and thus would be an advantage to achieve an accurate etiological diagnosis. Indeed, the authors would very much appreciate to have complemented this study with Western blot, although serum was discharged after laboratory results and not stored. Nevertheless, this constraint is discussed in the limitations of our study, line 335-338, where it is clearly stated that “whenever available and economically feasible, serological screenings should be complemented with other methods such as detection of anti-Toxocara antibodies by Western blot using TES antigens, to increase the accuracy of the diagnosis and to establish the implicated species.”

Reviewer 3 Report

This is an interesting study evaluating the positivity of serological tests to Toxocara in Portugal over the last decade (from January 2010 to December 2020). However, some points should be considered or clarified.

Introduction section

L.34-38 Please rephrase. These two sentences are repeated.

L.47-48 “Four recognized human toxocariasis syndromes comprise: visceral larva migrans (VLM), ocular larva migrans (OLM), neural larva migrans (NLM) and covert toxocariasis (CT).”
In fact, there are five main syndromes. You forgot the Common toxocariasis. Please correct.

Material and methods section

Why using two different type of ELISA tests (in-house and commercial) instead of an ELISA technique for screening and a more sensitive and specific Western blot technique for confirmation?

How are these two ELISA tests complementary?

Which Toxocara standardized antigens are used in the commercial kit?

What is the sensitivity and specificity of each kit?

Results section

Do you know the number of patients treated? In whom toxocariasis was considered by clinicians.

Discussion

It would be interesting to specify that a positive serology does not mark an infection but a potential serological scar. There is no tool currently available to distinguish between the two.

Author Response

Author's Reply to the Review Report (Reviewer 3)

Comments and Suggestions for Authors: This is an interesting study evaluating the positivity of serological tests to Toxocara in Portugal over the last decade (from January 2010 to December 2020). However, some points should be considered or clarified.

3.1 Introduction section: L.34-38 Please rephrase. These two sentences are repeated.

Authors: The authors thank the reviewer. The two sentences were rephrased accordingly.

3.2 L.47-48 “Four recognized human toxocariasis syndromes comprise: visceral larva migrans (VLM), ocular larva migrans (OLM), neural larva migrans (NLM) and covert toxocariasis (CT).” In fact, there are five main syndromes. You forgot the Common toxocariasis. Please correct.

Authors: The authors thank the reviewer for correcting this misunderstood and improving the manuscript. The following sentence was added to the text: “CT, representing “common toxocariasis” in adults and “covert toxocariasis” in children is similar to VLM but far more common and with mild manifestations”.

3.3 Material and methods section: Why using two different type of ELISA tests (in-house and commercial) instead of an ELISA technique for screening and a more sensitive and specific Western blot technique for confirmation?

Authors: In the laboratory routine, the macro-ELISA method is used for the diagnosis of different helminthiasis, allowing to perform distinct tests simultaneously according to the clinician’s suspicion. Toxocara kit is used only for confirmation in those cases with inconclusive results as it is considered to have a higher sensitivity and specificity. The authors agree with the Reviewer concerning the WB method which are currently in optimization to be included in the routine serodiagnosis of Toxocariasis.    

3.4 How are these two ELISA tests complementary?

Authors: Considering the higher sensitivity (>95%) of the commercial kit, it is an advantageous for a more solid result as compared with the macro-ELISA/TES antigen prepared in house which sensitivity is 85-90%.

3.5 Which Toxocara standardized antigens are used in the commercial kit?

Authors: According to the manufacturer, it is a synthetic antigen produced from T. canis

3.6 What is the sensitivity and specificity of each kit?

Authors: The authors thank the reviewer. The macro-ELISA (TES in house antigen) has sensitivity (85-90%) and specificity (93.3%), while the commercial kit (Bioactiva Diagnostica) reports sensitivity and specificity above 95%.

3.7 Results section: Do you know the number of patients treated? In whom toxocariasis was considered by clinicians.

Authors: The authors have no information regarding the patients that were treated. In fact, despite the request included in the patient chart and the laboratory result, the authors rarely have feedback from clinicians. It is important to consider, that based on the clinical suspicion, other complementary diagnostic procedures (echography, TAC, other imaging) are also performed to achieve a definitive diagnosis. 

3.6 Discussion: It would be interesting to specify that a positive serology does not mark an infection but a potential serological scar. There is no tool currently available to distinguish between the two.

Authors: The authors thank the reviewer for this comment. This particularity was already mentioned in the limitations of the study (“Although ELISA is widely used, is not the gold standard test. ELISA could generate false positive results due to cross-reactivity with other helminths (especially A. lumbricoides) and it has lack of ability to differentiate between active and past infections (line 348).” Nevertheless, another sentence was added to the text: “Therefore, a positive serology does not mark an infection but a potential serological scar, and no tool is currently available to distinguish between the two.”

Reviewer 4 Report

After reading the last version of manuscript “Human toxocariasis in Portugal – an overview of a neglected 2 zoonosis over the last decade (2010-2020)”. I have found that the authors made the suitable changes, so the article has improved substantially.

However, I have a comment

- In line 109, please check  “followed by incubation of 2 ml of sera diluted at 1:500 (IgG)”.  Is it probably 100 or 200 µl instead of 2 ml?

Author Response

Author's Reply to the Review Report (Reviewer 4)

Comments and Suggestions for Authors: After reading the last version of manuscript “Human toxocariasis in Portugal – an overview of a neglected 2 zoonosis over the last decade (2010-2020)”. I have found that the authors made the suitable changes, so the article has improved substantially. However, I have a comment: In line 109, please check “followed by incubation of 2 ml of sera diluted at 1:500 (IgG)”.  Is it probably 100 or 200 µl instead of 2 ml?

Authors: The authors thank the reviewer for this comment. As it is a macro ELISA, we used 2 ml of a dilution of 1:500 (4 µl of sera in 1996 µl of PBST). Nevertheless, the sentence was changed accordingly.

Round 2

Reviewer 2 Report

Thank you for your responses. I accept the paper in present form.

Author Response

Comments and Suggestions for Authors: Thank you for your responses. I accept the paper in present form.

Authors: The authors thank the reviewer for his/her feedback.

Reviewer 3 Report

I have no more comment.