Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan.

BACKGROUND: Amebiasis, caused by Entamoeba histolytica, is spreading in developing countries and in many developed countries as a sexually transmitted infection. Here, we evaluated the efficacy of serological screening to identify asymptomatic E. histolytica infection as a potential epidemiological control measure to limit its spread. METHODOLOGY/PRINCIPAL
FINDINGS: This cross-sectional study was carried out between January and March 2021 in an HIV-negative men who have sex with men (MSM) cohort at the National Center for Global Health and Medicine. Serological screening was performed using a commercially available ELISA kit. For seropositive individuals, we performed stool polymerase chain reaction (PCR) to determine current E. histolytica infection. We performed E. histolytica serological screening of 312 participants. None had a history of E. histolytica infection prior to the study. The overall E. histolytica seropositivity was 6.7% (21/312), which was similar to that found by the rapid plasma reagin test (17/312). We identified current infection in 8 of 20 seropositive participants (40.0%) by stool PCR.
CONCLUSIONS/SIGNIFICANCE: Our serological screening approach constitutes a potentially practical epidemiological strategy. Active epidemiological surveys, in combination with an effective screening strategy for asymptomatically infected individuals, should be applied to help reduce sexually transmitted E. histolytica infections.

Introduction

Amebiasis is an intestinal protozoa infection caused by Entamoeba histolytica, which is the second most common cause of parasite-related deaths worldwide and is particularly found in developing countries [1]. It is also a growing concern in some developed countries in East Asia and Europe, where E. histolytica infection is increasingly prevalent as a sexually transmitted infection [2-4]. In Japan, men who have sex with men (MSM) are reported to be at especially high risk for sexually transmitted E. histolytica infection [5,6]. Life-threatening cases of E. histolytica infection are accumulating in these countries [7-9]. Moreover, many of these cases were not diagnosed until autopsy [9]. This is likely because E. histolytica infection is a neglected disease; thus, it is rarely suspected in the clinical setting when a patient has acute abdominal symptoms. Hence, an effective epidemiological strategy to reduce E. histolytica infections is urgently needed. In developing countries, transmission typically occurs as a result of unsanitary conditions; however, transmission can also directly occur between people through sexual contact [10]. Furthermore, most cases of E. histolytica are asymptomatic [11]. Indeed, seroprevalence data has shown that asymptomatic infection occurs among sexually active individuals [12,13] who act as a reservoir for sexual transmission. Polymerase chain reaction (PCR) using stool samples is the best method for detecting E. histolytica infection [11]; however, it is expensive, time-consuming and requires complicated procedures and is thus not ideal as a screening method. Moreover, the handling of stool samples at voluntary counselling and testing centres in developed countries is inconvenient; most sexually transmitted infection (STI) screening tests at these centres are performed using blood samples. Although the screening utility of serology for asymptomatic E. histolytica infected carriers has not been assessed in previous studies, our recent data strongly suggest that serological testing is highly sensitive for detecting symptomatic infectious diseases and asymptomatic E. histolytica infection [14].

Here, we prospectively performed serological testing for HIV-negative men who have sex with men (MSM) and confirmed E. histolytica infection by PCR for those with positive serology. We also assessed the utility of serological screening for the identification of asymptomatic E. histolytica infection.

Methods

Ethics statement

This study was approved by the ethics committee of the Center (NCGM-G-002091-00), and all participants provided written informed consent in accordance with the Declaration of Helsinki. All participants gave written informed consent for the study.

Study population

This cross-sectional study was carried out between January and March 2021 in an HIV-negative MSM cohort at the Sexual Health Clinic of the National Center for Global Health and Medicine (NCGM) [15]. This cohort was a single-centre prospective study. It was established to perform HIV screening and serological testing for syphilis and rectal Chlamydia trachomatis and Neiserria gonorrhoeae every 3 months for HIV-negative MSM in 2017. Inclusion criteria of the HIV-negative cohort were MSM, aged ≥16 years old, those who have anal sexual intercourse. People diagnosed with HIV at enrolment were excluded from the cohort and were referred to an HIV-positive clinic, the AIDS Clinical Center at NCGM, or other medical institutions.

Sample size estimation

Sample size estimation to assess the seropositivity of E. histolytica among a sexually active MSM population in the study site was performed using Power Analysis & Sample Size 2021 (NCSS Statistical Software, LLC, Utah, USA). The minimum number of necessary samples was estimated as 255 participants. The following numbers were used for the calculation: confidence level 95%, precision, half width 5%, population proportion 21.3%, and population size 24,452 people. The population proportion was estimated using the previously reported seropositivity of E. histolytica among HIV-positive MSM [12]. The population size (MSM at study location) was calculated based on the data of 1.2% of Japanese males having sex with men during their life span [16], and that of 2,037,693 males between 21 and 50 years old living in the metropolitan Tokyo area (https://www.toukei.metro.tokyo.lg.jp/juukiy/2021/jy21q10601.htm#kubu).

Serum anti-E. histolytica testing

The presence of anti-E. histolytica antibody was detected using a commercially available ELISA kit (Entamoeba histolytica IgG-ELISA; GenWay Biotech, Inc., San Diego, CA. USA). All procedures were performed according to the manufacturer’s instructions. In brief, diluted serum samples (100X dilution in IgG sample diluent) as well as 5 control samples, consisting of 1 substrate blank, 1 negative control, 2 cut-off controls, and 1 positive control, were applied to 96-well plates pre-treated with E. histolytica antigen and incubated at 37°C for 1 hour. After washing the plates using washing solution, 100 μL of E. histolytica Protein A conjugate was added to all wells except the substrate blank and incubated for 30 minutes in the dark. After a second wash, TMB (3,3’,5,5’-Tetramethylbenzidine) substrate solution was added to all wells. After a 15-minute incubation, 100 μL of stop solution was applied to the plates, and absorbance of the specimen was then read at 450/620 nm using a spectrometer. The ELISA titer was calculated by employing correction to obtain the cut-off value [formula used for the correction: units = (sample absorbance value × 10) / (cut-off absorbance value)]. Positive results were interpreted as 11 units or higher.

Identification of Entamoeba from stool samples

For seropositive participants, stool samples were obtained and examined by stool ova and parasite examination (O&P), which consisted of direct microscopic examination for trophozoites and formalin-ether sedimentation for cyst forms stained with iodine. A single-round conventional PCR (cPCR) assay for the detection of three Entamoeba species (E. histolytica, E. dispar, and E. moshkovskii) was carried out. Stool specimens (approximately 0.2 g) were weighed and subjected to DNA extraction using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). DNA extraction was performed according to the manufacturer’s instructions. The DNA was eluted in 100 μL of elution buffer (Qiagen) and stored at −80°C until further analysis. The primer set was designed based on signature sequences in the small-subunit rRNA of each species, of which the utility was confirmed in a previous study [17]. The primer set consisted of the same forward primer (EntaF, 5′-ATGCACGAGAGCGAAAGCAT-3′) in combination with three reverse primers, one for each of the three species (EhR, 5′-GATCTAGAAACAATGCTTCTCT-3′; EdR, 5′-CACCACTTACTACC-3′; EmR, 5′-CACCACCACTTACTATCCCTACC-3′). Entamoeba species were differentiated based on the sizes of the PCR products (a 166-bp PCR product for E. histolytica, a 752-bp PCR product for E. dispar, and a 580-bp PCR product for E. moshkovskii). Finally, the results were confirmed by DNA sequencing. Sanger sequencing was performed with a BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific Inc., Tokyo, Japan), and then analysed on an ABI 3730xl DNA Analyzer (Thermo Fisher Scientific Inc., Tokyo, Japan).

Measurements of other STI testing

Hepatitis B surface antigen, core antibody, and hepatitis C antibody were tested by a chemiluminescent enzyme immunoassay (CLEIA)-based HISCL analyser with HISCL kits (Sysmex Corp. Japan). Serum rapid plasma reagin test (RPR) [“Sankoh” (EIDIA Co, Tokyo)] and Treponema pallidum latex hemagglutination assay (TPHA) were performed. The diagnosis of syphilis was based on serum RPR ≥8 and positive TPHA results. A nucleic acid amplification test (Bio Medical Laboratories, Inc., Tokyo, Japan) was used to detect Chlamydia trachomatis and Neiserria gonorrhoeae.

Statistical analyses

Comparisons of the qualitative data were carried out with the Chi-square test, and analysis of variance (ANOVA) was used for comparisons of quantitative data. Statistical significance was defined as a two-sided P value < 0.05. All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA, USA).

Results

In total, serological testing for E. histolytica was performed for 312 asymptomatic HIV-negative MSM (Fig 1). Of these, 91.3% had only male-to-male sexual contact, while the other 8.7% had bisexual contact (Table 1 and S1 Data). More than half of the participants (158/312) had experienced STIs prior to the present study, although none had a history of treatment for E. histolytica infection based on a medical self-declaration form. The overall seropositivity of E. histolytica was 6.7% (21/312) (Fig 2A and S2 Data). This was the same positivity as found by RPR testing 5.4% (17/312), in which only four people showed high RPR titres (R.U. > 16.0). Additionally, E. histolytica seropositivity was significantly higher than that of hepatitis B surface antigen and hepatitis C virus antibody. The E. histolytica seropositivity was positively correlated with age (Fig 2B). There was no significant correlation between the E. histolytica seropositivity and sexual preferences of participants (S2 Data). As expected, E. histolytica seropositivity was relatively high among participants with positive serology for Treponema pallidum hemagglutination or hepatitis B core antibody and among those with Chlamydia trachomatis and/or Neisseria gonorrhoeae infection (Fig 2C).

Fig 1

Study workflow.

Table 1

Characteristics of study participants undergoing a screening test for anti-E. histolytica antibody.

Median [IQR] or % (N)	All (N = 312)
Age	34 [28–41]
Sexual partners
Male only	91.3% (282/309)
Male and female	8.7% (27/309)
Insertive/receptive
Insertive only	19.5% (60/308)
Receptive only	26.9% (83/308)
Both	51.0% (157/308)
No insertive sex	2.3% (8/308)
Number of sexual partners within 6 months	5 [3–10]
Condom use (%)	60 [20–90]
Past treatment of any STIs	50.6% (158/312) *
Past treatment of amebiasis	0%

Abbreviations: IQR, inter quartile range; N, number; STIs sexually transmitted infections; AmebaAb, anti-Entamoeba histolytica antibody.

*List of past STIs consisted of syphilis (n = 51 cases), Chlamydia trachomatis infection (n = 40 cases), condyloma acuminata (n = 33 cases), Neiserria gonorrhoeae (n = 26 cases), hepatitis B virus infection (n = 22 cases), pubic lice (n = 22 cases), genital herpes infection (n = 11 cases), hepatitis A virus infection (n = 4 cases), Mycoplasma genitalium infection (n = 2 cases), and giardiasis (n = 2 cases) (S3 Data).

Fig 2

Entamoeba histolytica seropositivity and screening results of other sexually transmitted infections.

(A) Seropositivity of sexually transmitted infections (solid bars) compared with that of Entamoeba histolytica (clear bar) by Fisher’s exact test. (B) E. histolytica seropositivity by age group. The ratio of PCR-positive cases to seropositive cases is indicated by the solid bar. (C) E. histolytica seropositivity in those with and without other sexually transmitted infections. Error bars indicate 95% confidence intervals. Abbreviations: AmebaAb, anti-Entamoeba histolytica antibody; RPR, rapid plasma regain; TPHA, Treponema pallidum hemagglutination; HBsAg, hepatitis B surface antigen; HBcAb, hepatitis B core antibody; HCVAb, hepatitis C virus antibody; PCR, polymerase chain reaction; Ct, Chlamydia trachomatis; Ng, Neisseria gonorrhoeae; REF, reference data; NS, not significant.

Next, to assess current asymptomatic infections among individuals who were seropositive for E. histolytica, we performed O&P and PCR of the stool samples to identify the pathogen. One of the 21 seropositive individuals refused stool examination; therefore, we examined a total of 20 stool samples. None of these seropositive individuals had abdominal symptoms at the time of stool sampling. O&P identified cysts in 20% (4/20) of the seropositive participants (cysts in three cases and cysts and trophozoites in one case). PCR identified E. histolytica DNA in 40.0% (8/20) of the seropositive participants. Interestingly, one person with cysts had a negative PCR result; this was concluded to be a false-positive by O&P. Thus, we finally identified 8 cases of asymptomatic E. histolytica infection in 20 seropositive participants of the 312 HIV-negative MSM cohort.

Discussion

In the present study, serological testing to identify E. histolytica infection was performed for HIV-negative MSM individuals. The overall seropositivity (6.7%) was between that found in HIV-positive individuals (21.3%) [12] and that at a voluntary counselling and testing centre in Tokyo (2.6%) [13], even though no participants had a previous treatment history of E. histolytica infection at inclusion. This indicates that E. histolytica infection is a common STI among HIV-negative MSM individuals. We also identified current E. histolytica infection in 40% of seropositive individuals. This finding is consistent with a previous study that found a 38.9% (7/18) serological testing specificity against colonoscopically identified asymptomatic amoebic colitis [17]. On the basis of the results calculated by dividing 1 by the positive ratio of each test, 37.1 serologic tests followed by 2.5 stool PCR tests were required for the identification of one case of asymptomatic infection in this study population. This is the first study showing that mass-screening by serology can identify new cases of asymptomatic E. histolytica infection in a high-risk population.

There are some limitations to this study. First, we performed stool PCR testing only for seropositive participants because of limited funding. We were unable to assess serology false-negatives; however, our previous study using colonoscopy found that the false-negative rate is low (1.9%, 1/53) [18]. The sensitivity and specificity of serological testing to identify asymptomatic infection (effectiveness of the serological screening strategy for asymptomatic E. histolytica infection) should be confirmed by a future prospective analysis study, which performs stool PCR and serology for all participants. Second, owing to the small sample size, the epidemiological impact of the applied screening strategy for identifying asymptomatically infected individuals could not be assessed. Our serological screening approach provides a potential strategy for the epidemiological control of re-emerging sexually transmitted E. histolytica infection. However, active epidemiological surveys to identify high-risk populations are also essential for the future epidemiological control of sexually transmitted E. histolytica infection.

In conclusion, we identified eight patients with E. histolytica infection from 312 asymptomatic HIV-negative MSM individuals by serological screening. Active epidemiological surveys, in combination with an effective screening strategy to identify asymptomatically infected individuals, should be considered for the future control of this re-emerging communicable disease.

Comparison of characteristics between antibody positive and negative participants.

(DOCX)

Click here for additional data file.

E. histolytica seropositivity and sexual preferences of participants.

There were no significant correlations between the seropositivity and sexual preferences by Fisher’s exaxt test or ANOVA test. Error bars indicate 95% confidence intervals. Abbreviations: E. histolytica, Entamoeba histolytica; STI, sexually transmitted infection; Tx, treatment history; NS, not significant.

(TIF)

Click here for additional data file.

Data set of characteristics and test results of study participants.

(XLSX)

Click here for additional data file.

8 Dec 2021

Dear Dr. Watanabe,

Thank you very much for submitting your manuscript "Effectiveness of serological testing to detect asymptomatic Entamoeba histolytica infection: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Aysegul Taylan Ozkan, Ph.D., M.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Aysegul Taylan Ozkan

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: This is an descriptive study for MSM people where the authours have found a seropositive for E. histolytica . 40% of the seropositive patients also positive for E. histolytica in their stool samples

L-117,118 “In addition to stool ova and parasite examination (O&P)”- This line the authours has to clarify

L-123 “eluted in 100 mL of elution buffer”- pleasae acheck this line

L-131,132 “Finally, the results were confirmed by DNA sequencing”- should include briefly the procedures or a reference of the method..

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: L-143 “none had a history of E. histolytica infection”- How did the author confirm this? There is no precise statement.

L-147 “hepatitis B surface antigen and hepatitis C virus antibody”- Which methods were utilized to find those? Should include in methods section.

L-175,176 “One of the 21 seropositive individuals refused stool examination”- therefore, author examined a total of 20 stool samples. In this case, PCR detected 40% E. histolytica, yet the author computed all 21 samples in table 1 –should it be revised

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The manuscript does not provide any justification how E. histolytica infections correlate with STI’s.

Since this study focused on active epidemiological surveys, the author should specify the number of people that tested positive for rapid plasma reagin (RPR) out of the 21 Eh seropositive samples.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: In Table 1, the proportions of individuals with or without antibody should be shown in each category. For example, in male only individuals as sexual partner, the proportion of antibody (+) should be calculated as 20/282 (7.1%) and that of antibody (-) should be 262/282 (92.9%). In individuals with male and female as sexual partner, he proportion of antibody (+) should be calculated as 1/27 (3.7%) and that of antibody (-) should be 26/27 (96.3%). As such, I request to recalculate the proportion of antibody (+) and (-) with making the total number of each category as a denominator.

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This study showed the effectiveness of serological test in screening asymptomatically infected individuals with Entamoeba histolytica in HIV-negative men who have sex with men in Japan as a cross-sectional study. The study is meaningful, but there are several points to be clarified or improved.

Major points

It is recommended to clearly describe the background.

1) Would you describe why HIV-negative men who have sex with men (MEM) can be a target for the screening of asymptomatic Entamoeba histolytica infection, in addition to the reason that “sexually active individuals”?

2) How was the cohort of HIV-negative MEM set? Can you show the outline of this cohort?

3) Do 312 participants represent all of HIV-negative MEM from the cohort? If 312 participants are a part of the cohort, how did the authors select 312 HIV-negative MEM from the cohort?

4) In Table 1, the proportions of individuals with or without antibody should be shown in each category. For example, in male only individuals as sexual partner, the proportion of antibody (+) should be calculated as 20/282 (7.1%) and that of antibody (-) should be 262/282 (92.9%). In individuals with male and female as sexual partner, he proportion of antibody (+) should be calculated as 1/27 (3.7%) and that of antibody (-) should be 26/27 (96.3%). As such, I request to recalculate the proportion of antibody (+) and (-) with making the total number of each category as a denominator.

Minor points

1) Line 102: Would you describe the name of E. histolytica antigen(s) and the concentration of the pre-coated antigen(s)?

2) Line 117: Would you describe “stool ova and parasite examination” more accurately or scientifically in detail?

3) Line 132: Would you describe the methods and results of the DNA sequencing, if the PCR products were confirmed by sequencing?

4) Line 143: Would you describe what kinds of STIs the 159 participants experienced?

5) Line 150-: Would you describe the abbreviations you described in the Fig 2B, like Treponema pallidum hemagglutination (TPHA) etc? The name of species should write down in Italic.

6) Line 152: Whould you clarify how the participants were “affected”?

7) Line 181-184: It is not easy to understand the meaning of the sentence.

Would you edit “to identify asymptomatic E. histolytica infection” --> “to identify at least an asymptomatic E. histolytica infection”

Reviewer #2: Overall this is a nice story on asymptomatic E. histolytica infection in mem sex with men.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Rashidul Haque

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

7 Jan 2022

Submitted filename: PNTD-D-21-01330_Point by point_Yanagawa.docx

Click here for additional data file.

15 Feb 2022

Dear Dr. Watanabe,

Thank you very much for submitting your manuscript "Effectiveness of serological testing to detect asymptomatic Entamoeba histolytica infection: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Aysegul Taylan Ozkan, Ph.D., M.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Aysegul Taylan Ozkan

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #3: 1. While the data may be interesting from the perspectives of E. histolytica infection remaining prevalent among MSM in developed countries, particularly in East Asia, there are major concerns regarding the study design.

2. The major weakness of this study was that only the participants who were seropositive for E. histolytica underwent PCR assay to identify intestinal infection with E. histolytica. Given the high sensitivity and specificity of PCR assay for detection of E. histolytica, PCR assay should be considered as gold standard for the diagnosis of intestinal infection with E. histolytica. All recruited subjects should undergo PCR assay of stool samples, followed by serologic assay to better understand the performance of serologic assay used in this study. With the understanding of the performance of serologic assay could the authors be able to examine the “effectiveness” of serologic assay in identifying high-risk individuals with E. histolytica infection. It is understandable that serologic screening would be cheaper and more simple and convenient to perform than PCR assay of stool samples from clinicians’ standpoint; however, to better examine the “effectiveness” or “cost-effectiveness” of serologic testing that is to be widely used for screening in the clinical settings, both PCR assay of stool samples and serologic testing of all recruited participants, not selected participants, should be performed side by side. While the authors discussed it as a limitation, the last several sentences (Lines 218-221) in Results and Conclusions could be incorrect without testing all participants with the use of PCR assay.

3. The authors are encouraged to provide the sample size estimation. It is not clear that how many MSM had been recruited for STI studies at the voluntary counseling and testing site and how many agreed to participate in serologic and PCR testing in this cross-sectional survey.

4. While testing for gonorrhea, syphilis and chlamydia was performed every 3 months, it sounds that serologic testing for E. histolytica was only performed at baseline. Did the authors follow the participants using the same serologic assay to estimate the seroconversion rate?

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #3: 1. In Table 1, 3 out of 311 participants had had treatment of amebiasis. In the footnote, 4 individuals had had amebiasis as past sexually transmitted infections (STIs). The authors are encouraged to provide more information on these participants who either had had treatment or amebiasis as STIs because none were reported to have had previous E. histolytica infection in Results section. Moreover, the statement (Lines 227-228) in the first paragraph of Discussion is contradictory to the data presented in Table 1.

2. Were the ELISA units of the participants testing positive for E. histolytica by PCR assay higher than those of participants testing seropositive but negative by PCR assay?

3. Table 1 could be improved by providing data of “all participants”, “E. histolytica-seropositive participants”, and “E. histolytica-seronegative participants”, with p-values for the comparisons between the latter two groups.

4. In Table 1, should “number of sex within 6 months” be “number of sexual partners within 6 months”?

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #3: While the authors discussed it as a limitation, the last several sentences (Lines 218-221) in Results and Conclusions could be incorrect without testing all participants with the use of PCR assay.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #3: The authors performed a cross-sectional serologic survey of E. histolytica infection among 312 HIV-negative men who have sex with men (MSM) and had had no known previous history of E. histolytica infection in Tokyo. Polymerase-chain-reaction assay was performed only in those 20 individuals who tested seropositive for E. histolytica, in which 8 (40%) tested positive for E. histolytica, suggesting current infection. While the data may be interesting from the perspectives of E. histolytica infection remaining prevalent among MSM in developed countries, particularly in East Asia, there are major concerns regarding the study design.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

9 Mar 2022

Submitted filename: R2_PNTD-D-21-01330_4_Point by point responses.docx

Click here for additional data file.

27 Mar 2022

Dear Dr. Watanabe,

Thank you very much for submitting your manuscript "Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Aysegul Taylan Ozkan, Ph.D., M.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Aysegul Taylan Ozkan

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Fine

Reviewer #2: OK

Reviewer #3: The revision made in response to previous comments and queries is acceptable.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Fine

Reviewer #2: OK, Authors can take out the last sentence of the Results section and use it in the discussion section

Reviewer #3: The revision made in response to previous comments and queries is acceptable.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Fine

Reviewer #2: OK

Reviewer #3: The revision made in response to previous comments and queries is acceptable.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: No editorial suggestion

Reviewer #2: OK

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: No additional comment

Reviewer #2: The last sentence of the abstract section is not required authors may take it out. This sentence is also included in the result section but, authors can take it to the Discussion section

Reviewer #3: The authors have responded to the queries and comments raised in the second round of review. While there are weaknesses and limitations of the study design, the authors have revised the manuscript as much as they can in providing the data on the seroprevalence of E. histolytica infection among HIV-negative men who have sex with men. The results of the study have important clinical and public health implications when it comes to prevent transmission of E. histolytica infection among the at-risk population in a developed conutry.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Rashidul Haque, icddr,b, Dhaka, Bangladesh

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

30 Mar 2022

Submitted filename: R3_PNTD-D-21-01330_4_Point by point responses.docx

Click here for additional data file.

3 Apr 2022

Dear Dr. Watanabe,

We are pleased to inform you that your manuscript 'Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Aysegul Taylan Ozkan, Ph.D., M.D.

Deputy Editor

PLOS Neglected Tropical Diseases

Aysegul Taylan Ozkan

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

21 Apr 2022

Dear Dr. Watanabe,

We are delighted to inform you that your manuscript, "Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases