Yaws elimination in Ecuador: Findings of a serological survey of children in Esmeraldas province to evaluate interruption of transmission.

BACKGROUND: The WHO roadmap for neglected tropical diseases includes yaws eradication requiring certification of elimination of transmission in all endemic and formerly endemic countries worldwide. A community-based programme for yaws control was considered to have achieved elimination of the infection in the endemic focus in Ecuador after 1993. We did a serosurvey of children in this focus to provide evidence for interruption of transmission.
METHODS: Survey of serum samples collected from children aged 2 to 15 years living in the formerly endemic and in geographically contiguous areas. A convenience sample of sera collected between 2005 were 2017 from non-yaws studies, were analyzed using immunochromatic rapid tests to screen (OnSite Syphilis Ab Combo Rapid Test) for Treponema pallidum-specific antibodies and confirm (DPP Syphilis Screen and Confirm) seroreactivity based on the presence antibodies to treponemal and non-treponemal antigens.
RESULTS: Seroreactivity was confirmed in 6 (0.14%, 95% CI 0.06-0.30) of 4,432 sera analyzed and was similar in formerly endemic (0.11%, (95% CI 0.01-0.75) and non-endemic (0.14%, 95% CI 0.06-0.34) communities. All seroreactors were of Afro-Ecuadorian ethnicity and most were male (4/6) and aged 10 or more years (5/6), the latter possibly indicating venereal syphilis. Only 1 seroreactor lived in a community in the Rio Santiago, that was formerly hyperendemic for yaws.
CONCLUSION: We observed very low levels of treponemal transmission in both formerly endemic and non-endemic communities which might be indicative of congenital or venereal syphilis and, if yaws, would likely be insufficient to maintain transmission of this endemic childhood infection. Additional surveys of children aged 1 to 5 years are planned in Rio Santiago communities to exclude yaws transmission.

Introduction

Yaws, caused by the spirochete Treponema pallidum subspecies pertenue, is a chronic debilitating disease of skin, cartilage, and bone, transmitted during childhood through skin-to-skin contact. Yaws was targeted for eradication in Latin America and other world regions in the 1950s but remained endemic in a geographically isolated region of Esmeraldas Province in North-Western coastal Ecuador [1]. During a yaws survey of this region in 1988, active cases were identified in a geographically restricted area in a single District [2]. Mass treatment was given with benzathine penicillin according to WHO recommendations [3]. This was accompanied by a control programme of clinical and serological resurveys at 5-year intervals with mass treatments for communities with active cases. Surveillance was done by community health workers during inter-survey periods with treatment of cases and contacts [4]. No active cases were detected after the 1993 survey and active surveillance was maintained to 1998 when a repeat survey showed no active cases and negligible low-titer seropositivity, indicating likely interruption of yaws transmission [5].

There has been renewed interest in yaws eradication since 2012 when WHO launched a roadmap for the control of neglected tropical diseases [6]. A clinical trial showing high effectiveness of oral azithromycin in treating clinical yaws [7] led to the Morges strategy for the eradication of yaws. This strategy was based on mass treatments with azithromycin of communities with active cases (or total community treatment) followed by resurveys with treatment of cases and contacts [8].

Worldwide eradication will require certification of elimination of transmission in all currently endemic and formerly endemic countries including Ecuador. A meeting of experts (representing universities, a non-governmental organization [NGO], the Ministry of Public Health [MSP] and the Pan American Health Organization) was held in Quito, Ecuador, on 30 May 2018, to review gaps in information to support the interruption of yaws transmission. At the meeting it was decided to use validated rapid diagnostic tests for T. pallidum following WHO guidelines [9] to screen stored sera to provide evidence for interruption of transmission. Sera collected after 1998 from children living in the formerly endemic yaws focus, and in geographically close areas in three Districts in Esmeraldas province, were therefore screened.

Methods

Ethics statement

The protocols of each of the three surveys used for the present analysis were approved by local ethics committees in Ecuador as described [10,12,13]. The protocol for this retrospective analysis of stored samples was approved by the Ethics Committee of the Universidad Central del Ecuador (Reference 001-SEISH-UCE-20). Informed written consent was obtained from the child’s parent or guardian for participation in each of the three studies. Informed consent in all three studies included storage and analysis of blood samples for future studies.

Study populations

Samples were collected between 2005 and 2017 as part of two observational studies of allergy in children in the Districts of Eloy Alfaro, San Lorenzo, and Quininde in Esmeraldas Province: the first study was a cross-sectional survey of school age children aged 6 to 15 years (predominantly of Afro-Ecuadorian ethnicity in the Districts of Eloy Alfaro and San Lorenzo including formerly endemic and non-endemic communities) using updated censuses of each community with blood sampling between 2005 and 2009 [10] while the second was a population-based birth cohort (predominantly of mestizo ethnicity in the District of Quininde in geographically close but non-endemic communities) in which serum samples collected between 2014 and 2017 were analysed at 8 to 9 years [11,12]. Sera from a survey of indigenous Awa children aged 2 to 15 years in the District of San Lorenzo (of unknown endemicity for yaws) collected in 2014 and 2015 were analyzed also [13]. Frozen sera from these studies were selected based on living in a previously endemic community for yaws [2], location of yaws cases from historical records of the Ecuadorian Ministry of Public Health, and geographical proximity to these locations.

Serological analysis

Stored sera were tested for the presence of antibodies to treponemal and non-treponemal antigens using two rapid immunochromatic lateral flow diagnostic tests as recommended by WHO [9]. Screening of all samples was done using OnSite Syphilis Ab Combo Rapid Test (CTK Biotech, San Diego, CA), that detects T. pallidum-specific antibodies, and confirmation of positives was done using DPP Syphilis Screen and Confirm (ChemBio Diagnostics, Hauppauge, NY) that detects antibodies to T. pallidum and non-treponemal antigens, following the manufacturers’ instructions. A positive confirmatory test showed a positive test for specific antibodies to both T. pallidum and non-treponemal antigens (referred to here as seroreactors or active infections). Individuals whose sera had T. pallidum-specific antibodies only were interpreted as having past or treated infections. Rapid tests were read by eye in a well-lit area with natural light. All positive or invalid screening and confirmatory tests were repeated twice to confirm results. The rapid tests were purchased by the Pan American Health Organization (PAHO) and supplied through the Ministry of Public Health and a training course was provided on the use of the rapid tests through PAHO.

Statistical analysis

This study used a convenience sample based on availability of archived samples collected from previous non-yaws surveys done in the region and 4,500 rapid screening tests were provided through the Ecuadorian MPH. Lists of available sera samples from children aged 2 to 15 years living in communities in the formerly endemic or contiguous areas was drawn up, anonymized, and random samples were selected using Stata (version 11). Seroreactive individuals were defined using the results of the confirmatory test as having specific antibodies to both T. pallidum and non-treponemal antigens. All other individuals were defined as seronegative. Seroreactivity in endemic and non-endemic samples was compared with a population estimate of 0.37% [14] using the binomial probability test with a two-tailed P value. A separate analysis looked at individuals with either evidence of active (i.e seroreactors) or past/treated infections (i.e presence of antibodies only to T. pallidum antigens).

Results

A total of 4,432 serum samples were screened using the ‘OnSite Syphilis Ab Combo Rapid Test’: 53 samples (1.2%) gave a positive result (S1 Table). All were positive on repeat testing. Confirmation of positivity on these samples was done using ‘DPP Syphilis Screen and Confirm assay’ giving the following results: 33 negatives; 2 false positives (positive only for antibodies to non-trepenomal antigens); 12 positives just for antibodies to T. pallidum antigen (representing previously treated or cured T. pallidum infections); and 6 were seroreactors and positive for antibodies to T. pallidum and non-treponemal antigens (representing active T. pallidum infections). Findings of seroreactivity in communities from formerly endemic and non-endemic communities in Esmeraldas Province with age and sex distributions are shown in Table 1, and the locations of these communities are shown in Fig 1. Sera samples from only 3 of the formerly endemic communities were not available for this analysis (Fig 1).

Table 1

Seroreactivity to T. pallidum using the confirmatory rapid test (DPP Syphilis Screen and Confirm) in 4,432 schoolchildren aged 2 to 15 years living in formerly endemic* and non-endemic communities for yaws.

Communities screened	Sample	Sex(Male/female)%	Median age (range) yrs.	Seroreactivity (%)
				N	% [95%CI]
All areas	4,432	51/49	10 (2–15)	6	0.14 [0.06–0.30]
Formerly endemic region    All communities    Rio Santiago        Playa de Oro        Angostura        Playa Tigre/Playa Nueva/Zapote         Palma Real/Guayabal        Chanuzal/Pailon/Picadero        Selva Alegre        Timbire/El Porvenir        Las Antonias        Rocafuerte    Rio Cayapas        San Miguel        Mafua        Zapallo Grande        Telembi    Rio Zapallito        Boca de Zapallito    Rio Onzole        Colon        Santo Domingo	947401445373112212127313315787445131103	54/4658/4264/3651/4965/3545/5551/4952/4856/4442/5849/5153/4755/4557/4360/4052/4861/39	11 (5–15)10 (7–15)10 (5–14)9 (6–15)11 (7–15)11 (7–15)11 (7–15)10 (7–15)9 (7–15)10 (6–13)11 (7–15)9 (7–15)12 (7–15)12 (7–15)10 (7–15)11 (7–15)11 (7–15)	10000010000000000	0.11 [0.01–0.75]000000.82 [0.11–5.59]0000000000
Contiguous regions    All communities    District of Eloy Alfaro        Rio Cayapas            8 communities        Rio Santiago            3 communities        Rio Onzole            3 communities        Others            19 communities    District of San Lorenzo        16 communities    District of Quininde        10 communities	3,4853043401551,0591,154473	50/5054/4653/4748/5251/4947/5349/51	10 (2–15)10 (6–15)11 (6–15)10 (6–15)11 (6–15)10 (2–15)8 (8–13)	5010400	0.14 [0.06–0.34]00.29 [0.04–2.06]00.38 [0.14–1.00]00

*Endemicity defined by presence of active yaws lesions in 1988 survey [2].

Fig 1

Map of formerly endemic and non-endemic sample communities in Esmeraldas Province.

Shown are locations of formerly endemic (red circles) and non-endemic (yellow circles) communities, and formerly endemic communities not sampled (red stars). The map was built using ArcGIS version 10.2.2 (ESRI, California, USA).

Seroreactivity was observed in 0.14% (95% CI 0.06–0.30) of all children tested: only 1 (0.11%, 95% CI 0.01–0.75) seroreactive individual (detected in 2005 in Selva Alegre, Rio Santiago) was detected in formerly endemic compared to 5 (0.14%, 95% CI 0.06–0.34) in non-endemic communities (Table 1). Compared to a population estimate of T. pallidum seropositivity of 0.37% derived from women of reproductive age from coastal Ecuador [14], seroreactivity was significantly less in non-endemic (P = 0.024) but not endemic (P = 0.278) communities. All sero-reactors were of Afro-Ecuadorian ethnicity and most were male (4/6) and aged 10 or more years (5/6) (Table 2). Most sera were collected between 2005 and 2009 (83.1% of samples): sampling in formerly endemic areas was done between 2005 and 2009 while sampling in non-endemic areas was done between 2005 and 2017 (S2 Table). The last detected seroreactive was from a sample collected in 2007.

Table 2

Characteristics of seroreactive individuals using the confirmatory rapid test (DPP Syphilis Screen and Confirm).

Age (years)	Ethnicity	Community	Region	Sex	Sample year	Formerly endemic
10	Afro-Ecuadorian	Canchimalero	Eloy Alfaro	Male	2007	No
8	Afro-Ecuadorian	Cuerval	Eloy Alfaro	Male	2006	No
12	Afro-Ecuadorian	Maldonado	Rio Santiago (Eloy Alfaro)	Female	2005	No
15	Afro-Ecuadorian	Rompido	Eloy Alfaro	Male	2006	No
12	Afro-Ecuadorian	Santa Rosa	Eloy Alfaro	Male	2006	No
14	Afro-Ecuadorian	Selva Alegre	Rio Santiago (Eloy Alfaro)	Female	2005	Yes

Analysis of any positivity to T. pallidum antigens including treated/cured infections could provide useful information about the history of T. pallidum infection in a population, given that these antibodies can persist for many years. The positivity rate in the whole sample was 0.41% (95% CI 0.26–0.64): 0.53% (95% CI 0.22–1.26) in formerly endemic and 0.37% (95% CI 0.22–0.64) in non-endemic communities (S3 Table). There was some evidence of an increase in antibody positivity to T. pallidum antigens by age (S1A Fig) that was clearer in non-endemic (S1C Fig) than endemic communities (S1B Fig). No positives were detected among the youngest 132 children aged 2 to 6 years. Although most communities in the formerly endemic area had no evidence of positivity, 4 did with positivity rates ranging 0.76% to 2.70%. The upper level of the 95% confidence intervals indicates positivity of up to 16.85%. Among non-endemic communities geographically contiguous with formerly endemic communities in Rio Santiago and Rio Onzole, positivity varied 0.17% to 1.47%. There were no positives among a sample of Awa Amerindians in the District of San Lorenzo. Characteristics of all individuals with positive tests for antibodies to T. pallidum antigens are shown in S4 Table. All positives were of Afro-Ecuadorian ethnicity, 12/18 were aged greater than 10 years, 13/18 were boys, 5/18 were from formerly endemic communities, and 16/18 were from communities in the District of Eloy Alfaro. Five communities on the Rio Santiago accounted for half the positives.

Discussion

Yaws is a chronic debilitating neglected tropical disease that has been targeted for worldwide eradication by 2030 [15]. Yaws was formerly endemic in a small geographically restricted and isolated focus in Esmeraldas Province in north-western coastal Ecuador [2]. A control programme implemented from 1988 was assumed to have eliminated the infection [5] although elimination has not yet been formally certified. We present here data from a serosurvey of a sample of 4,432 schoolchildren aged 2 to 15 years to detect possible evidence of yaws transmission in formerly endemic and non-endemic communities in this region of Esmeraldas Province. The survey, done using two rapid diagnostics tests recommended by WHO for the surveillance of yaws, showed low rates of seroreactivity that were similar in formerly endemic (0.11%) and non-endemic (0.14%) communities. All seroreactives were of Afro-Ecuadorian ethnicity and most were boys aged 10 or more years.

The control programme for yaws in Esmeraldas Province was implemented in the late 1980s. A survey of villages with sporadic official reports of yaws cases in 1988 identified a prevalence of 16.5% with active yaws lesions and seropositivity of 96.3% [2]. The survey was accompanied by mass treatment with intramuscular benzathine penicillin according to level of endemicity following WHO recommendations [3]. Yaws was subsequently incorporated into the surveillance activities of a community health programme centred around a network of community health workers (CHWs) or ‘health promoters’. Surveillance was based on continuous active case finding by CHWs in which confirmed cases and close contacts were treated with intramuscular benzathine penicillin. Clinical and serological surveys for yaws were repeated at 5-year intervals (in 1993 and 1998) by health teams (from Vicariato Apostolico de Esmeraldas or CECOMET in coordination with MSP) in previously affected communities [5]. Mass treatments were given to those communities still with active cases. The second survey in 1993 identified 16 active cases in only 5 communities representing 1.4% of those examined and seropositivity of 4.7% [5]. No active cases were detected after 1993 and a repeat survey in 1998 showed a seroprevalence of 3.5%: those with positive serology at this time were presumed to have had previously treated syphilis or yaws with low-level antibody titers of less than 1:8 [5]. Active surveillance for yaws after 1998 was continued through CHWs but no new active cases were confirmed, indicating presumed interruption of yaws transmission [5]. CHWs were key to the implementation and execution of this control programme. CHW training was based on essential techniques and instruments of community epidemiology [16]. Yaws control activities were integrated within other activities that included surveillance, management, and control of malaria, tuberculosis, onchocerciasis, chronic non-communicable diseases, and maternal and child health.

Serological tests cannot distinguish between syphilis and yaws. The confirmatory test has been shown to be highly specific with high positive and negative predictive values [17]. Clinically active and latent (indicated here by serological evidence of active infection) yaws represent the reservoir of infection required to sustain transmission. No clinical data were available to determine presence of possible yaws lesions. Serologic evidence of active infections (i.e. seroreactors) was present in only 6 of all 4,432 (0.14%) children tested and in only 1 of 947 (0.11%) children from the formerly endemic communities. All these samples were collected before 2008. An explanation for a positive confirmatory test could be explained by congenital or sexually transmitted syphilis, the latter in minors if sexually abused. Rates of active infections (based on presence of antibodies to both T.pallidum and non-treponemal antigens) with syphilis in women of reproductive age have been estimated at 0.37% in women from coastal Ecuador [14]. If no treatment is given during pregnancy, as might be expected in most of the survey communities, transmission rates to offspring can reach 100% [18]. There are no reliable epidemiological data on congenital syphilis for the study area. The observation that the majority of seropositives occurred among those aged greater than 10 years could favour venereal transmission of syphilis as the primary cause of positive tests. We examined rates of positivity for antibodies to T. pallidum antigens, that persist for many years even after cure, as an indicator of the history of treponemal infections in these communities. T. pallidum pertenue is highly sensitive to several antibiotics [19], the wide use of which for other indications, could have modified the seroepidemiology of the infection in this setting. A gradual increase in positivity was observed with greater age in non-endemic but not formerly endemic communities–such a pattern could indicate either low-level transmission of yaws or sporadic cases of venereal syphilis with risk increasing with age. However, the fact that no active clinical cases of yaws have been reported since 1993 despite continued surveillance argues strongly against active transmission of yaws, although more attenuated forms of disease [20] might be overlooked.

It is unclear how large an infectious reservoir in humans is required to maintain transmission of yaws. Yaws transmission likely requires a sufficiently large reservoir of individuals with latent yaws (with repeated infectious relapses) living in sufficiently close contact with susceptible individuals in suitable social and environmental conditions. Such conditions would be present in populations living in severe poverty with close physical contact and poor hygiene in humid tropical climates. Previous studies have suggested that a ratio of 6 latent to 1 active cases may be typical for endemic transmission [21]. However, our data indicate that no such reservoir presently exists and social and economic conditions have altered dramatically over the past 20 years in the formerly endemic area—including widespread access to clean water, sanitation, housing, education, and basic health services including antibiotics–to such a degree that transmission even if initiated would be unlikely to be sustained. It seems improbable that such small numbers of seroreactors with active infections (i.e. 6/4,432) would be sufficient to maintain active transmission. Sporadic cases could conceivably result from contact with non-human primates, that have been shown to harbour T. pallidum pertenue-like strains of spirochetes [22]. Such contacts are known to be very infrequent in this region.

The Ecuador control and elimination strategy was similar to that proposed subsequently by WHO in 2012 as the Morges strategy [8]. WHO criteria for certification of yaws elimination include absence of: 1) confirmed cases detected for 3 consecutive years through high coverage with active surveillance; and 2) transmission as measured by serosurveys with evidence of continuous negative serological tests for at least 3 consecutive years in samples of asymptomatic children aged 1 to 5 years [9,14] (i.e. those born since the implementation of elimination strategies). In this study, we evaluated seroreactivity in children aged 2 to 15 years from formerly endemic and geographically close communities–all seroreactive children had been born after the last reported case in the community where they lived [5]. Current WHO guidelines for certification of elimination provide no recommendations for how to deal with low rates of seroreactivity in settings where yaws transmission may have been eliminated but where congenital syphilis is present. This is complicated by the fact that populations that have suffered endemic yaws tend to be extremely poor, live in geographically isolated settings, and have limited access to health care. Data on rates of congenital and venereal syphilis are likely to be inadequate in most settings but screening for syphilis during pregnancy is widely implemented. We recommend that in the absence of local epidemiological data on rates of syphilis in pregnant women in such populations, that the latest national data is used to estimate background rates of congenital syphilis (or venereal syphilis if children up to 15 years are included in such surveys) to define the upper limits of serological thresholds of concern. Seroreactivity ‘mapping’ as done here could then be used to identify communities where rates are higher than might be expected and which might require additional epidemiological studies.

In conclusion, our data indicate evidence for the presence of very low levels of active treponemal infections, based on treponemal serology, among schoolchildren in a region of Ecuador where yaws was formerly endemic and where a successful control programme eliminated clinical yaws. Higher rates of active infections were observed in some communities in or contiguous with the formerly endemic area, but serological tests are unable to distinguish between yaws and venereal or congenital syphilis. It appears unlikely that such low rates of treponematosis, if truly T. pallidum pertenue, would be sufficient to sustain yaws transmission, thus indicating no evidence for active transmission of yaws in the communities where this study was done. Serosurveys of young children aged 1 to 5 years accompanied by detailed clinical and laboratory evaluation of seroreactive children, are now planned in communities in the Rio Santiago, to exclude the persistence of yaws transmission.

Results of screening tests for antibodies to T. pallidum antigens in 4,432 schoolchildren aged 2 to 15 years living in formerly endemic and non-endemic communities for yaws using OnSite Syphilis Ab Combo.

(DOCX)

Click here for additional data file.

Timing of sera collection by calendar year and seropositivity stratified by endemicity for yaws.

(DOCX)

Click here for additional data file.

Serological results using the confirmatory rapid test (DPP Syphilis Screen and Confirm) in 4,432 schoolchildren aged 2 to 15 years living in formerly endemic* and non-endemic communities for yaws.

Results show serological reactivity to T. pallidum antigens (any positive) further stratified into presence (active infections) or absence (past infections) of non-treponemal antibodies.

(DOCX)

Click here for additional data file.

Characteristics of all individuals with positivity for antibodies to T. pallidum antigens with and without antibodies to non-treponemal antigens using the confirmatory rapid test (DPP Syphilis Screen and Confirm).

(DOCX)

Click here for additional data file.

Bar charts showing changes in any antibody positivity to T. pallidum antigens by age in 4,432 schoolchildren aged 2 to 15 years.

A–age-positivity in all children and stratified into children living in formerly endemic (B) and non-endemic (C) communities. Numbers in brackets represent sample numbers for each age.

(TIF)

Click here for additional data file.

Raw data used for analyses.

(XLSX)

Click here for additional data file.

15 Mar 2022

Dear Dr. Cooper,

Thank you very much for submitting your manuscript "Yaws elimination in Ecuador: findings of a serological survey of children in Esmeraldas Province to evaluate interruption of transmission" 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. However, the reviewers raise a few concerns, including the lack of statistical analysis and the inconsistent use of definitions such as seropositivity. The reviewers also suggest that the manuscript be proofread more carefully. Overall, the reviews are generally positive and we encourage you to submit a modified 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,

Brian Stevenson, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Melissa Caimano, Ph.D.

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: The methods do not note plasma samples collected, but plasma samples are specifically mentioned in the introduction and results. Please clarify (either add plasma to methods or delete plasma elsewhere).

Reviewer #3: The study objectives are clear with rationale explained. The study populations are described, but the studies where samples were collected should be cited. The study design is appropriate but no sample size calculation has been included to demonstrate how the sample size was determined. This needs to be included to enable readers to see if there is adequate power behind the study. No statistical analysis plans have been presented. Only one ethics approval reference has been included despite saying these samples come from three distinct studies. All previous studies should have there ethics approval reference included.

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

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: My primary question relates to the broad time frame that the tested samples derived from. Since the positive samples were from before 2010, does that add confidence to your estimates of no/little yaws? Perhaps showing a distribution of the samples collected by year would be helpful. For example, are there no positive samples since 2010 because there are almost no samples from that time frame? Knowing that information will help us understand what the year of collection is really telling us about current yaws transmission.

Reviewer #3: • The table 1’s categories are unclear

• You said that there were two false positive results, I would disagree these are false positive as they were presumably positive during repeat testing using the OnSite Syphilis Ab Combo Rapid Test. Why do you believe the DPP test to be more accurate?

• The lateral flow tests are looking for antibodies but you mention results being positive for antigens.

• “Seropositivity was observed in 0.4% of all children tested (active 0.1% and past infections 0.3%)” this is confusing, these percentage should not be added.

• Statistics needed, you say “Rates of seropositivity using the confirmatory assay did not differ between formerly endemic and non-endemic samples of schoolchildren (0.4% - 0.1% active infections and 0.3% past infections)” but have not presented any statistics, it is also not clear what the seropositivity rate is in each community.

• I think you need to be clear throughout the paper these are not active infections now, but at the time of the sero-survey.

• When you say “Most communities in the formerly endemic area had no evidence of T. pallidum seropositivity but 3 communities had evidence of past infections (seropositivity ranging 0.8-4.2%) of which 1 had evidence of an active infection” what do you mean by T.pallidum seropositivity, are you distinguishing this to a past infection? I think you need to be clearer in defining terms you go on to use throughout the paper.

• Table 2 doesn’t add much that hasn’t already been described in the text

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

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: Same as results - having a better understanding of the distribution of samples over the noted time frame will help us better understand the data in Table 2 - are no positive specimens since 2010 indicative of no transmission or were there not enough samples representing the time frame 2010-2017 to make any inference on recent yaws transmission.

Reviewer #3: You have concluded that the positive DPP tests found could be attributable to syphilis infection and that along with little evidence of clinical infections despite ongoing survelliance means yaws is likely to no longer be endemic. You have also mentioned how these data are important for declaring interrupted transmission and the future work that is needed to do this. Again the discussion needs proofing.

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

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 #2: (No Response)

Reviewer #3: This paper should be thoroughly proof read prior to re-submission as there are many grammatical errors and lots of very long sentences. It would benefit from the use of more scientific language in parts. Make sure you correctly use and are consistent with the terms: interruption of transmission, elimination and eradication. Include missing ethics references. Make sure terms are well defined.

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

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: Major comments:

Methods

- Were the DPP tests read by eye or by the electronic reader?

- A map would be extremely helpful to provide a clearer understandign of the overlap between the districts and communities from which samples are available in the current study and the presumed formerly endemic districts in esmereldas. THe map provided I think (although not 100% clear from the figure legend) shows where the samples came from and if they were endemic or not endemic previously but doesnt (as far as I can tell) give a sense of what proportion of formerly endemic communities are represented in the current study.

Results

Do I read the results correctly that of 53 tests positive on the initial treponemal anibody test (and which were positive on repeat testing) 33 were negative for the treponemal component of the DPP? This is quite unusual.

Please provide at least some demographics - age, gender etc.

Did the authors consider an age seroprevalence curve? It might be a useful supplementary figure. If there was active transmission of yaws one would anticipate a slow but consistent increase in treponemal positivity rates with age (as the test stays poisitve for life) whereas the pattern would look different in the context of venereal syphilis transmission.

There should be confidence intervals on the proportions presented throughout the manuscript.

Minor Comments:

Some typos - for example in the introduction "Frozen sera from these studies were selected based on living a previously endemic community for yaws" is missing a word.

Im not quite sure what is meant by the sentence:

". Only latent yaws with serological evidence of active infection can sustain transmission" do you mean treponemal antibody positivity alone doesnt find potentially infectious individuals?

In the sentence "that have been shown to harbour T. pallidum pertenue-like spirochetes " Im not sure it is correct to use the word like. Phylogenetically the organism in NHPs IS t.p.pertenue.

Reviewer #2: (No Response)

Reviewer #3: This study is important for declaring elimination in Ecuador. However, there are current limitations including the lack of statistical analysis and the presentation of results as well as clear and consistent use of definitions such as seropositivity. There needs to be inclusion of a sample size calc, statistical analysis plan and presentation of these results.

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

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: No

Reviewer #3: Yes: Becca L. Handley

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.

11 Apr 2022

Submitted filename: PNTD-D-22-00085_R_1_responses.docx

Click here for additional data file.

19 Apr 2022

Dear Dr. Cooper,

We are pleased to inform you that your manuscript 'Yaws elimination in Ecuador: findings of a serological survey of children in Esmeraldas Province to evaluate interruption of transmission' 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,

Brian Stevenson, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Melissa Caimano

Deputy Editor

PLOS Neglected Tropical Diseases

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

7 May 2022

Dear Dr. Cooper,

We are delighted to inform you that your manuscript, "Yaws elimination in Ecuador: findings of a serological survey of children in Esmeraldas Province to evaluate interruption of transmission," 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