Yaws recurrence in children at continued risk of infection.

BACKGROUND: In yaws-endemic areas, children with Treponema pallidum subsp. pertenue infection may suffer recurrent episodes due to either reinfection or relapse. However, the possibility of infection with other cutaneous ulcer causative agents and difficulties in interpreting standard laboratory results challenges the estimation of yaws recurrence rates.
METHODS: We estimated the rates of yaws recurrences in the Lihir Island (Papua New Guinea) using two approaches: passive surveillance based on a retrospective screening of electronic medical records of cutaneous ulcers diagnosed using serological testing between 2005 and 2016, and active surveillance conducted during a cross-sectional prevalence study which included PCR analyses of ulcers of all suspected cases of yaws. The risk of recurrent infection was assessed based on data from the passive surveillance analysis and using two Cox regression models (crude and multivariate), stratified by year of index episode. Data gathered from the active surveillance was used to characterize the recurrences and no hypothesis testing was performed.
RESULTS: The electronic medical records included 6,125 patients (7,889 ulcer episodes) with documented serological results of cutaneous ulcers of which1,486 were diagnosed with yaws. Overall, 1,246/6,125 patients (20.3%) presented more than once with a cutaneous ulcer, and 103/1,486 (6.7%) patients had multiple episodes of yaws. The risk of yaws recurrence significantly increased with age and was higher in patients with ≥3 recurrent episodes. In the active surveillance, we identified 50 individuals with recurrent cutaneous ulcer that had PCR results available for both the index and recurrent episode. Of 12 individuals with T. pallidum in the index ulcer, 8 (66%) had T. pallidum in subsequent assessments, relapse related to macrolide-resistance was identified in two of these cases.
CONCLUSIONS: Our results confirm the need for active follow-up of yaws patients after treatment, particularly children and individuals with a history of recurrence.

Introduction

Yaws, caused by Treponema pallidum subsp. pertenue, is an important public health problem in many countries worldwide. The disease predominantly affects the skin and bone, and most cases occur in children. Following treatment, skin lesions heal and titres of serological markers of infection, such as the Rapid Plasma Reagin (RPR), fall. However, in yaws-endemic areas, children present with recurrent cutaneous ulcers from either reinfection caused by ongoing contact with other infected individuals in their community or, more rarely, caused by relapse, often due to antibiotic-resistance [1]. Because yaws is commonly diagnosed using serology, reinfection is indistinguishable from relapse.

Epidemiological reports of yaws, in which cases of early active disease are rarely found in adults, suggest that human beings may develop some degree of immunity to reinfection [2]. Studies in experimental models have shown that animals experimentally infected with T. pallidum and subsequently treated can develop some degree of acquired immunity when re-challenged [3,4]. In this context, protection against reinfection is influenced by the duration of the original infection prior to treatment and seemed not to change after curative treatment with a lapse of time for re-exposure [5]. At the same time, within-host immune evasion by T. pallidum is well documented, and humans may experience multiple clinical episodes of syphilis and yaws, suggesting that immunity is non-sterilising and may be either transient or potentially strain-specific [6-8]. Better understanding the risk of recurrence of yaws may help shed light both on immunity to T.pallidum and inform programmatic surveillance efforts.

There is very little clinical data about whether natural reinfection with T. p. pertenue occurs after treatment. The clinical picture is complicated because of the occurrence of latent yaws infection, during which individuals have reactive serology but no clinical evidence of the disease. In yaws-endemic regions, children may present with skin ulcers caused by a range of etiological agents; therefore, serological tests cannot differentiate between active infectious ulcer caused by T. p. pertenue and a co-infection with latent yaws and a cutaneous ulcer caused by another agent. In particular, Haemophilus ducreyi, a gram-negative bacteria, has been found to be a major cause of skin ulcers in yaws endemic regions of the tropics [9] and is commonly found in individuals with reactive serology for yaws.

In this study, we used data from both passive and active surveillance to identify rates of recurrence, of cutaneous ulceration, either reinfection or relapse, due to yaws and other aetiologies in a highly endemic region of Papua New Guinea.

Methods

Ethics statement

The Medical Research Advisory Committee of the Papua New Guinea National Department of Health approved the study protocol (number 12.36). All participants (or their parent or guardian in case of children) provided oral informed consent. Additionally, written informed consent was obtained from individuals with suspected active yaws before the etiologic study. In case of children, the parent or guardian provided written consent and the children consented orally.

Overview of study setting

The study was conducted in Lihir Island in the New Ireland Province of Papua New Guinea. According to 2016 census data, Lihir has an estimated population of 22,000 inhabitants. In Lihir, public healthcare is delivered through aid posts spread across the island attended by nurses and the Lihir Medical Centre (LMC), a modern private rural hospital run by physicians and nurses. Aid posts lack essential laboratory tests; therefore, diagnose skin diseases clinically only, whereas the LMC can perform tests, such as the RPR. Diagnoses and treatments of skin ulcers performed out of public health campaigns are afforded by patients. We previously estimated that the overall incidence of yaws in the population was 1% per year, with an incidence among children aged between 1–15 years of 7% [10]. The study objectives were to determine the overall incidence of recurrences and to characterize the aetiology patterns of recurrences. Therefore, we used two approaches for case detection: passive surveillance based on screening of hospital electronic records and active surveillance of skin ulcers during a public health intervention for yaws elimination that included molecular analyses of skin ulcers.

Passive surveillance

We retrospectively screened hospital electronic medical records from the LMC to identify cases of recurrent yaws. Although yaws can be treated clinically in other aid posts spread across the island, serological analyses can only be performed at LMC. All outpatients diagnosed with infectious cutaneous ulcers of any aetiology between Jan 1, 2005 and Jun 1, 2016 were screened.

A case of cutaneous ulcer was identified by the presence of the diagnostic code listed in either the first or second diagnostic position of an outpatient medical encounter (ICD-9 codes 707.1 and 707.9). We excluded cases of non-infectious ulcers, including diabetic, venous, and post-trauma ulcers. Serological testing is the routine diagnostic method to confirm yaws at LMC. In this centre, a presentation of cutaneous ulcer together witha positive Treponema pallidum haemagglutination assay (TPHA) and RPR ≥ 1:8 are considered diagnostic of active yaws. Cutaneous ulcers with recorded RPR test results < 1:8 are classified as non-yaws ulcers. Ulcer cure is not routinely confirmed by RPR test, and patients are not actively followed-up after treatment. Therefore, we considered recurrent yaws in all individuals who had a previous entry in the electronic records with serologicallyconfirmed yaws ulcer, achieved clinical remission after treatment, and experienced a recurrent episode of cutaneous ulcer that met one of the following criteria of RPR titre change compared with the index episode: (1) 4-fold or greater titre increase within the 12 months following treatment or (2) absence of 4-fold or greater decline between 12 and 96 months after treatment (Fig 1).

Fig 1

Definition of recurrence in the passive and active surveillance analysis.

We retrieved clinical information about every patient on a specifically designed database, including the date of visit, RPR titre, beginning of follow-up, months since previous ulcer episode, number and date of ulcer episodes, months since previous yaws episode, and number and date of yaws episodes. We also retrieved demographical information, including age, gender, the village of origin, and distance to the hospital. We assessed the accessibility to the hospital based on the distance between the patient’s place of residence and the hospital. Roads in Lihir are non-paved tracks, and the land is covered with woody vegetation. The vehicle travelling time from the remotest village is 1.5 hours with longer times during the rainy season. The distance (Km) between a given village and healthcare centre was grouped as follows: 0 to <5, 5 to <15, 15 to <25, and ≥25 Km.

Active surveillance

We used data collected as part of a prospective study of yaws elimination [1]. Briefly, the study consisted of repeated clinical surveys for active yaws, serologic surveys for latent yaws, and molecular analyses; surveys were conducted before and 6 and 12 months after mass drug administration (MDA) with azithromycin. During the initial MDA and at each subsequent survey, we swabbed the ulcers of all suspected cases of yaws for polymerase chain reaction (PCR) testing. Antimicrobial therapy with single dose azithromycin 30mg/Kg up to 2g was administered at the time of each visit. We defined molecularly confirmed recurrence when an individual had a new episode of PCR-confirmed yaws after clinical remission in the previous survey (Fig 1). The following possible scenarios of repeated episodes of cutaneous ulcers where also considered: T. pertenue DNA detected in the index episode but not in the second one, T. pertenue DNA detected in the second episode but not in the first one, and no T. pertenue DNA detected in either of the two episodes.

Statistical analysis

Variables collected from outpatient records were described as medians, interquartile ranges, and percentages. Patients were considered to be at risk for recurrence from the day of an index episode until the end of the study period (01 Jun 2016). Serologically-confirmed recurrent episodes were identified by passive case detection, assuming no migrations or deaths during the follow-up period. Crude and multivariable Cox conditional regression models applying the Prentice, Williams, and Peterson approach (PWP) were estimated to assess the risk for recurrent infections per subject over time [11]. The models, built using a robust variance estimate, were stratified by the year of index episode, allowing the baseline hazard function to differ throughout the entire follow-up period, which is appropriate for long periods that may be affected by environmental, political, economic changes as well as by the health-seeking behaviour of the population. Although the models estimated with the PWP method are recommended to stratify according to the number of previous episodes, we used this information as a covariate in the model to account for change in the hazard for each additional episode. Since the number of previous episodes (ulcers or yaws) and the time between episodes changed over time during the follow-up period, we tested for time-varying coefficients of those covariates in the adjusted model. Interactions between the number of episodes and elapsed time between episodes were also considered. Analyses regarding the findings of active surveillance were descriptive and no hypothesis testing was performed. We used the same recurrence criteria as for the case definition in passive surveillance analysis (i.e., 4-fold or greater RPR titre increase). All analyses, data manipulation, and implementations were done in Stata ver16 [12].

Results

Passive case detection

Overall, 8,598 diagnoses of cutaneous ulcers were reported in the electronic records from LMC during the study period (Fig 2A). We excluded 388 episodes that lacked documented serological results, 139 patients with non-infectious ulcers, and 182 that lacked a documented date of birth. The resulting study dataset consisted of 7,889 episodes among 6,125 patients. Of them, 1,486 patients were classified as having had a serologically-confirmed yaws ulcer. Overall, 1,246/6,125 (20.3%) individuals presented more than once to LMC with a cutaneous ulcer, and 103/1,486 (6.9%) presented more than once with serologically-confirmed yaws. The demographic and clinical characteristics of patients at diagnosis of the primary and recurrent yaws episodes were similar (Table 1). Patients had a median age of 9.7 years, 57% were male, and more than half had an RPR titre between 1:32–1:64.

Fig 2

Study profile A: Passive case detection through hospital records review; cases were confirmed by a serological test.

B: Active case detection in mass drug administration setting; cases were confirmed by PCR.

Table 1

Characteristics of patients included in the passive case detection analysis (i.e., retrospective screening of electronic health records).

		Number of Yaws-episode
		Primary	First recurrence	Second recurrence	Third recurrence
		(N = 1486)	(N = 103)	(N = 7)	(N = 2)
Male a		850 (57%)	56 (54%)	6 (86%)	1 (50%)
Age (years) b		9.7 (6.1–14.0)	10.2 (7.8–11.9)	9.9 (9.2–12.8)	11.4 (10.1–12.6)
Age (years) a	0-<5	248 (17%)	9 (9%)	0 (0%)	0 (0%)
	5-<15	918 (62%)	85 (83%)	7 (100%)	2 (100%)
	15-<25	167 (11%)	6 (6%)	0 (0%)	0 (0%)
	≥25	153 (10%)	3 (3%)	0 (0%)	0 (0%)
Distance to hospital a	<5Km	588 (40%)	50 (49%)	5 (71%)	2 (100%)
	5-10Km	299 (20%)	26 (25%)	1 (14%)	0 (0%)
	>10Km	283 (19%)	16 (16%)	1 (14%)	0 (0%)
	Unknown	316 (21%)	11 (11%)	0 (0%)	0 (0%)
RPR titre at primary episode a	1:8 to 1:16	558 (38%)	22 (21%)	0 (0%)	0 (0%)
	1:32 to 1:64	771 (52%)	66 (64%)	6 (86%)	2 (100%)
	1:128 or higher	157 (11%)	15 (15%)	1 (14%)	0 (0%)
Number of previous ulcer episodes a	0	1348 (91%)	0 (0%)	0 (0%)	0 (0%)
	1	114 (8%)	76 (74%)	0 (0%)	0 (0%)
	2	18 (1%)	22 (21%)	2 (29%)	0 (0%)
	≥3	6 (0%)	5 (5%)	5 (71%)	2 (100%)
Months since index yaws episode b			25.3 (18.9–41.0)	50.0 (37.6–52.5)	61.5 (49.2–73.7)
Months since previous ulcer episode c		18.2 (6.6–37.2) [138]	23.7 (13.9–33.8) [103]	24.8 (15.3–27.5) [7]	16.4 (11.6–21.2) [2]
Months since previous yaws episode b			25.3 (18.9–41.0)	26.6 (15.3–31.6)	16.4 (11.6–21.2)

a n (Column percentage) of the various categories of variables distribution

b Median (IQR)

c Median (IQR) [n]

According to the multivariate analysis, the occurrence of three or more previous ulcer episodes was significantly associated with the risk of recurrence (Table 2). The hazard ratio (HR) of yaws recurrence decreased progressively with age, being children in the 0 to <5 years group associated with the highest risk of recurrence. The time-varying coefficient analysis showed that the effect of the number of previous episodes and time since the last episode did not change over time (HR 1.00, p = 0.81) for all the covariates. Likewise, we did not find meaningful interactions between time from the last episode and the number of previous episodes, neither between the time since the last yaws episode, and time from the last ulcer. Distance to the hospital and the RPR titre at primary episode did not show a significant contribution to the multivariate model.

Table 2

Multivariate model for predicting recurrence among patients included in the passive case detection analysis of individuals from electronic health records.

Covariate		Crude Model a			Adjusted Model b
		Hazard Ratio	p-value	Overall	Hazard Ratio	p-value	Overall
		(95%CI)		p-value	(95%CI)		p-value
Male		0.95 (0.64–1.41)	0.7987		0.87 (0.60–1.25)	0.4363
Age (years)	0-<5	1		0.0011	1		0.0009
	5-<15	0.69 (0.45–1.06)	0.0928		0.65 (0.44–0.97)	0.0338
	15-<25	0.26 (0.10–0.66)	0.0045		0.26 (0.10–0.65)	0.0043
	≥25	0.11 (0.03–0.45)	0.002		0.11 (0.03–0.45)	0.0023
Distance hospital	<5Km	1		0.0273	1		0.1439
	5-10Km	0.82 (0.52–1.30)	0.3955		0.85 (0.55–1.31)	0.4589
	>10Km	0.53 (0.30–0.94)	0.0292		0.62 (0.37–1.05)	0.0783
	Unknown	0.43 (0.22–0.83)	0.0118		0.55 (0.29–1.04)	0.0676
RPR titre at primary episode	1:08 to 1:16	1		0.2977	1		0.1038
	1:32 to 1:64	0.74 (0.47–1.16)	0.1867		0.67 (0.43–1.02)	0.0644
	1:128 to 1:512	0.57 (0.23–1.42)	0.2259		0.50 (0.21–1.17)	0.1085
Number of previous ulcer episodes	0	1		<0.0001	1		0.0208
	1	1.91 (1.20–3.04)	0.0065		1.46 (0.54–3.92)	0.4524
	2	2.56 (1.10–5.94)	0.0289		1.93 (0.66–5.64)	0.2286
	≥3	6.03 (2.78–13.09)			4.13 (1.64–10.43)	0.0026
Number of previous yaws episodes	0	1		0.0019	1		0.714
	1	2.13 (1.29–3.51)	0.003		0.75 (0.23–2.48)	0.6345
	≥2	5.89 (1.88–18.46)	0.0023		1.14 (0.29–4.44)	0.847
Months since previous ulcer episode c		1.02 (1.01–1.03)	0.0001		1.01 (0.97–1.04)	0.7109
Months since previous yaws episode c		1.03 (1.02–1.04)			1.01 (0.97–1.06)	0.5047

RPR: Rapid Plasma Reagin

a Cox regression stratified by year of primary episode (one model per covariate)

b Cox regression stratified by year of primary episode and forcing all covariates into the model

c Hazard Ratio per unit increase

Active case detection

During nine biannual community surveys, we diagnosed 1,701 individuals with 1,807 cutaneous ulcers; lesion samples from 844 (46.7%) cutaneous ulcers were tested by PCR. Ninety-nine (5.8%) individuals had a total of 111 recurrent episodes: 90 individuals with one recurrent ulcer, six individuals with two recurrences, and three individuals with three recurrences. (Fig 2B) PCR results of lesion swabs for both the index and recurrent episodes were available for 50/99 individuals, who accounted for 61/111 recurrent ulcers. Table 3 shows the aetiology of the index and recurrent episodes of cutaneous ulcer. Of 12 individuals with T. pallidum in the index ulcer (alone or dual infection), 8 (66.6%) had T. pallidum in subsequent assessments. Correspondingly, of 33 people with H. ducreyi in the index ulcer (alone or dual infection), 20 (60.6%) had H. ducreyi in subsequent assessment.

Table 3

Etiology of recurrent cutaneous ulcers according to the type of the index episode in patients identified during a public health intervention for yaws eradication (active case detection).

Data correspond to 61 ulcers detected among 50 individuals with PCR available.

Index episode		Recurrent episode				Total
		T.p.pertenue only detected	H.ducreyi only detected	Dual infection detected	Negative in all tests
	T.p.pertenue only detected	4	0	0	0	4
	H.ducreyi only detected	2	14	3	6	25
	Dual infection detected	3	2	1	2	8
	Negative in all tests	1	9	3	11	24
Total		10	25	7	19	61

Of eight patients with PCR-confirmed recurrent yaws, 3 (37.5%) had evidence of serological failure (i.e., ≥4-fold increase in RPR titre), 4 (50.0%) increased RPR titre, albeit not reaching the recurrence criteria, and 1 (12.5%) had a 4-fold reduction in RPR titre consistent with serological cure (Table 4). All eight patients with recurrent yaws had wild type 23S rRNA sequences in the index ulcer. According to the PCR analysis of recurrent ulcers, two cases had A2059G mutations associated with resistance to macrolide, and five were wild-type strains; the 23s rRNA gene could not be amplified in one case.

Table 4

Clinical and laboratory findings of 8 cases of PCR-confirmed yaws reinfection among individuals identified during a public health intervention for yaws eradication (active case detection analysis).

	Baseline episode of yaws							RPR nadir	Repeated episode of yaws
Study no	Age (years)	Gender	Type of lesion (size)	Duration of current episode in weeks	RPR titre	Strain genotype	Macrolide resistance		Months between baseline and repeat yaws	Type of lesion (size)	Duration of current episode in weeks	RPR repeat	Strain genotype	Macrolide resistance
1	9	F	ulcer (2.5 cm)	3	1:08	JG8	WT	NR	6	papilloma (1 cm)	12	1:16	JG8	WT
4	2	M	ulcer (3 cm)	3	1:08	JG8	WT	No data	6	ulcer (5 cm)	2	1:128	JG8	not amplified
5	10	F	ulcer (2 cm)	3	1:64	Not amplified	WT	No data	6	ulcer (2 cm)	3	1:04	JG8	WT
6	10	M	ulcer (3 cm)	3	Negative	JG8	WT	No data	6	ulcer (1 cm)	1	1:32	JG8	WT
7	10	F	ulcer (1 cm)	1	1:16	JG8	WT	No data	6	ulcer (2 cm)	1	1:32	JG8	WT
8	10	M	ulcer (4 cm)		1:02	JG8	WT	NR	12	papilloma (1cm)	6	1:64	JG8	WT
2	10	M	ulcer (2 cm)	2	1:16	JG8	WT	No data	6	papilloma (3 cm)	32	1:16	JG8	A2059G
3	12	M	ulcer (1 cm)	2	1:08	Not amplified	WT	NR	30	ulcer (3 cm)	15	1:16	JG8	A2059G

M: male. F: female. NR: not reported. RPR: Rapid Plasma Reagin. WT: wild-type

Discussion

In this study, we found that up to 20% of patients who presented to a health care facility in the Lihir island of Papua New Guinea with a cutaneous ulcer experienced recurrent episode within the 6–36 months following treatment. A low–albeit substantial (7%)–proportion of patients with yaws had serologically-confirmed recurrent yaws. Nearly all individuals with recurrent yaws were children aged 15 years or younger. To date, the epidemiology of ulcer recurrence has been barely investigated in yaws; however, our findings are in line with those reported for other treponemal infections such as syphilis (recurrence range from 4%- 9%) [13-16]. Our active search study, in which aetiology could be confirmed by PCR, revealed that among cases with T. pallidum at baseline that had a recurrence, this was often related to reinfection with the same microorganism.

Several observations in our study argue against the development of fully protective immunity in yaws. First, patients with a higher cumulative number of treated serologically-confirmed yaws ulcers had an increased hazard of subsequent serologically-confirmed yaws ulcers. This effect may be favoured by behavioural determinants, such as treated patients returning to environments with a high force of reinfection–therefore, increased likelihood of reinfection–, or individual-level risk factors (e.g., genetics) that predispose them to yaws disease. Second, we provide evidence that children are re-infected with the same strain of T.p. pertenue (e.g., JG8), arguing against strain-specific immunity. Of note, we have previously used whole-genome sequencing to demonstrate that strains based on multi-locus sequence typing (MLST) may be further sub-divided [17]. Therefore, we cannot exclude the possibility of sub-lineage-specific immunity. Lower rates of yaws in adults might be influenced by innate differences manifested with age, age-related behavioural exposure, and age-dependent immunological effects, which we could not detect in this study. Further data, such as establishing whether re-infection occurs with strains which are identical by whole genome sequencing, may be needed to assess whether acquired resistance to reinfection with yaws can develop independently of age-related behavioural factors. Our data also demonstrated repeated infection with H. ducreyi in consecutive episodes and suggests that patients do not develop immunity to H. ducreyi.

Our study has several limitations. Our results might underestimate the actual burden of recurrence because we could not ensure completeness of follow-up, and some subjects may have migrated, been attended in one of the peripheral aid-posts in the Island after the index episode or be reluctant (or have difficulties) to visit LMC. Second, a false-positive RPR test can occur and may have biased the estimate of recurrent yaws ulcers. We used a treponemal test together with the RPR test with a high titre threshold (>1:8) to define yaws, which is unlikely to be related to common causes of false-positive results [18]. Also, we cannot completely rule out treatment failures, which can occur because of multiple factors; however, considering the efficacy of azithromycin [19] and that therapy was administered under supervision, we did not expect treatment failures to account for a substantial contribution to new ulcers. Our passive case detection analysis is also based only on serologically-confirmed yaws which is not completely reliable. However, our findings are supported by our active case finding exercise where molecular confirmation was used.

Our results confirm the need for active follow-up of young children diagnosed with cutaneous ulcers, with particular attention to those with younger age and previous history of recurrences. This follow-up, currently conducted to monitor azithromycin resistances, should also identify recurrences, since it would provide an opportunity to identify contacts of yaws patients and plan interventions beyond the case. As a significant burden of yaws transmission may not all occur within the household [20,21], there is a need to develop effective strategies to ensure all household and community contacts are identified and treated to reduce the risk of reinfection and break chains of transmission within communities.

Dataset of active ulcer cases included in the analysis.

(ODS)

Click here for additional data file.

20 Oct 2021

Dear Dr. González-Beiras,

Thank you very much for submitting your manuscript "Yaws recurrence in children at continued risk of infection" for consideration at PLOS Neglected Tropical Diseases and thank you for your patience during the longer than usual period for peer review. 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), all reviewers found merit in your manuscript, and thus we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. Particularly I recommend you pay close attention to the clarifications around ethics highlighted by Reviewer 3.

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Associate Editor

PLOS Neglected Tropical Diseases

Ana LTO Nascimento

Deputy Editor

PLOS Neglected Tropical Diseases

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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: The objectives of the study were clearly tested. Hypothesis testing was not indicative for this study.

The study design was appropriate with good description of the study participants (some clarifications have been asked; included in the attachment)

Sample size was adequate. Few suggestions for statistical analyses have been included (see attachment)

No major ethical issues. The authors should however provide a statement on ethical considerations for the study

Minor Revisions required

Reviewer #2: (No Response)

Reviewer #3: - The hypothesis is not clear stated in the background. The background misses stating the link about the knowledge gap on immunity to re-infection and how this study (estimating recurrence with these particularly methods) can contribute to it.

Explaining in the background, that these first estimations of recurrence (as a first step) can start pointing towards the type of immunity (saying that you will use of molecular methods), while giving clear recommendations on the type of surveillance and follow up needed (in the discussion-recommendations), may clarify the justification of conducting this study and its added value.

- The appropriateness of the study design could be better clarified, by explaining why these two methods are chosen (i.e. in the absence of a formal surveillance system), and how they correlate to each other (in time, in target study population), and the added value of putting both approaches together in a study.

- The study population is not clearly mentioned (and should be specified for the two surveillance approaches included). The study setting mentions Lihir island, but in the Passive surveillance description, it is only mentioned the Lihir Medical Centre (LMC) consulting patients, so it should be described which is the target population of LMC in comparison with Lihir population (i.e. also if there are any other health posts-centers in the island which were treating ulcers during the study period? and if different characteristics in access), if the treatment was for free for everybody (or certain age groups), etc. All this orients towards accessibility, and potentially completeness-representativeness of the passive surveillance data and its interpretations.

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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: Analysis plan and results clearly presented.

Quality of the figure (flow diagram) provided should be improved

Minor revisions requested (See detailed comments in attachment)

Reviewer #2: (No Response)

Reviewer #3: - Clarity of the results:

In the passive surveillance, are there any differences among patients with positive and negative serology (in terms of demography, number of ulcers, distance to centre)?

- Clarity of figures and tables:

Title of figures and table should be self-explanatory (at this point they miss to inform about which how data was collected (if from passive-active or both), from where, when, etc)

To specify in the title of Figure 1 that relates to both active and passive surveillance (to clarify since it is inserted in the Passive surveillance section)

In Figure 1, the A and B in the graph (A is active surveillance) does not correspond with the legend (A is passive)

In Figure 1, in the Diagnoses of cutaneous ulcers from medical records, there is a group of patients who have PCR (not clear why), since in the section of the Methods only serological methods are defined in the Passive surveillance.

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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: The study has significant public health indications and offers good evidence on the need for follow-up of yaws cases. The conclusions are valid.

Some suggestions have been made with respect to study limitations (see attachment)

Minor revisions required

Reviewer #2: (No Response)

Reviewer #3: - The main conclusion stated is supported by the findings from both surveillance systems.

- Further details on the limitations of using such medical records as surveillance system should be mentioned (i.e. only completeness already mentioned, not any other of the surveillance attributes which may affect the number of recurrent consultations).

- The authors suggest absence of full protective immunity, but they could explain which methodologies are recommended to properly confirm this argument.

- The public health relevance of the recommendation (conducting active follow up) is there, but it could be better integrated in the public health context (not as an isolated recommendation). For instance, it could be mentioned under which strategy (in the context of eradication plan) is recommended the active follow up for this/similar settings (i.e. active follow up embedded on a Lihi passive surveillance with ulcers as weekly notifiable disease? Active follow up under weekly or monthly active surveillance? Active follow up after regular MDAs?).

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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: Minor revision required (see reviewer's attachment)

Reviewer #2: (No Response)

Reviewer #3: Minor modifications:

- To clarify which diagnostic codes were included under the passive surveillance – so it is possible to ensure the quality of the case definition used for the objective of the surveillance

- Accessibility is reflected in the analysis as Km to the health center in the passive surveillance analysis. But there are other aspects related to accessibility which are not considered. For instance, it would be useful to know whether treatment was offered for free, and any information on the type of care provided (24 hours service, etc), acceptability by population of the healthcare provided.

- For the active surveillance data– It would be useful to describe (or refer to in the text – if published elsewhere) how the active finding was done (so reader can understand how systematic was the population targeting and the coverage)

- It is not clear how a new episode of PCR-confirmed was defined. Was clinical remission after treatment followed, or any other time variables?

- The use of same recurrence criteria for the case definition in the active and passive surveillances should be highlighted in the methods (and not only at the results section, at the end of the active surveillance system). For clarity, it could be considered to have a definitions sub-section in the methods section.

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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: Overall, well written paper which adds significant evidence to the body of knowledge in the epidemiology of yaws and useful insights into recurrence of yaws.

Reviewer #2: Comments:

1. On the end of page 1/beginning of page w, the sentence beginning “studies in experimental models” is a bit confusing; can you clarify what “acquired protection to T. pallidum can develop in untreated animals and persist after treatment” means? What is meant by “acquired protection” – were they protected after a challenge? I think the next sentence explains it but it may be more clear just to drop that first sentence and expand on the theme of the paragraph – is it that there is a conflict between experimental models and humans in the generation of protective immunity?

2. At the end of that same paragraph the sentence “humans may experience multiple episodes of syphilis and yaws, suggesting that immunity is either transient or potentially strain-specific” seems fairly limited in the interpretation of multiple infections. Even long-lived immune responses many not offer sterilizing immunity (see recent serological data on trachoma where kids get multiply infected yet have good antibody responses).

3. Methods: Is it standard to have a definition of RPR titer >=1:8 as serological confirmation of yaws? Marks et al Lancet Global Health PMCID: PMC7116878 used an RPR titer of 1:2 as evidence of seroconversion, so it would be useful to give some justification for this decision. The discussion briefly calls this a high-titer cutoff (although the Marks et al paper seems to use 1:16 as a high titer cutoff – it would be helpful to have consensus on what RPR titer constitutes high titer).

4. Results, paragraph 1: 103/1486 does not = 60.7%. Assuming there is a typo somewhere here?

5. I am confused by the flow of Figure 1 – there seems to be an increasing number of people in the flow. IFor example, why is the flow going from a smaller number of patient with PCR available (50) to a larger number of patients with recurrent episodes (1246) with >4000 people excluded?

6. Discussion. I am a little confused by the actual conclusions about the recurrence of yaws. Due to the error I noted above it is not clear what % of cases seen were recurrent yaws in the passive case detection section. In the active case finding section, 8/12 PCR-confirmed in the index ulcer were PCR_confirmed as having recurrent ulcers. But the discussion says “A low ― albeit substantial (7%) ― proportion of patients with yaws had serologically confirmed recurrent yaws.” Does that mean the original numbers – 103/1486 – were correct and the 8/12 in active case finding was an anomaly? I am not seeing that you commented on this in the discussion. Is there an advantage to either approach?

7. Discussion: you say that treatment failure is highly unlikely, but could you support that with a reference or data from one of the studies that looked at that? I know that I could look it up but your argument will be stronger with just a sentence with data to support it.

8. Are you surprised at the finding: ‘Nearly half of individuals with recurrent yaws (43%) were children aged 15 years or younger.”? I admit that I am having trouble deriving this based on the Tables, which suggest the majoring of recurrent infections were in <15 year olds.

Reviewer #3: The study presents data on recurrence yaws in a well-followed population, through the use of two distintictive data collection approaches. It is useful data to share with public health institutions working with yaws in PNG and other setttings.

The study is well written and the efforts to bring further understanding on the epidemiology of yaws in Lihir island with existing data are meritable. However, it is no very clear whether looking at retrospective medical registries is the most appropriate method to properly assess recurrence. It is my understanding that the registry was not set up with the objective to monitor cases or recurrence, so the registry should be further explained (i.e. for example in terms of attributes of the surveillance system - see CDC, ECDC surveillance system evaluations). The choice of these two methods, how they complement each other, and their weaknesses for this objective (and the point discussed on fully protective immunity) can be further explained. Thus, it can be better assessed about the methodological implications of measuring recurrence through it.

In terms of recommendations, further reflection on which kind of routine surveillance system would be needed for yaws eradication in the country and in the island would be welcome. Considering the previous work on yaws in the island, authors could propose and advocate on how active follow up could be integrated in this setting

Reviewer #3:

Details on ethical procedures are missing, mainly related to the consent process. The information provided in the study says that “all participants provided informed consent” but it should specify :

- The different consent process for the outpatient clinic participants and for the active surveillance participants. Was informed consent avaliable for conducting a research from the patients included in the passive surveillance data? In case not, it should be specified and whether this has been specifically approved by the local ethical review or exempted from full review.

- To specify how the consent to conduct research was processed for participants below 18 (in both surveillances) – was the caregiver providing with such a consent? Or was the legal responsible of the children. All this should be justified according to PNG law

- Whether consent was oral or written, and in which language was provided.

- Was assent obtained from children to participate (if so, from each age - according to PNG law)?

If those details are included in other publications from this data, please refer to them

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Reviewer #1: Yes: Rafiq N. A. Okine

Reviewer #2: No

Reviewer #3: No

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Submitted filename: Yaws Paper Review .docx

Click here for additional data file.

18 Dec 2021

Submitted filename: YawsRecurrence_re-submission letter and Responses.docx

Click here for additional data file.

24 Jan 2022

Dear Dr. Gonzalez-Beiras,

We are pleased to inform you that your manuscript 'Yaws recurrence in children at continued risk of infection' 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,

Stuart Robert Ainsworth

Associate Editor

PLOS Neglected Tropical Diseases

Ana LTO Nascimento

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: Subject selection and classification: The authors have provided sufficient information with respect to the queries raised on subject classification particularly in relation to the definitions for recurrence.

Statistical analyses: Good justification was provided for the choice of PWP instead of frailty/random effects model and how within-subject heterogeneity was accounted for.

Ethical considerations: the authors have included some statements on ethical considerations. However with respect to the active surveillance, the statement on all children providing oral consent should be re-considered since ALL children may not have been able to provide oral consent depending on their age. If oral consent was done for specific age group for the active surveillance, this must be stated. Additionally, where assent was provided, this should be clearly stated.

Reviewer #3: (No Response)

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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: The corrections to the flow chart have been effected and the annotations properly aligned.

The suggestions with respect to the thresholds for the RPR titre levels have been effected 1.8 instead of 1.08.

ALL other corrections effected

Reviewer #3: (No Response)

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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 major revisions to discussions, conclusions or recommendations. Authors have adequately addressed initial review comments.

Reviewer #3: (No Response)

**********

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: N/A

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: N/A

Comments provided in initial review

Reviewer #3: (No Response)

**********

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: Yes: Rafiq N.A Okine

Reviewer #3: No

16 Mar 2022

Dear Dr González-Beiras,

We are delighted to inform you that your manuscript, "Yaws recurrence in children at continued risk of infection," 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.

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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