Rapid diagnostic testing for onchocerciasis in Maridi (South Sudan) before and after improving elimination strategies: a repeated cross-sectional survey

Background Maridi County is an onchocerciasis-endemic area in South Sudan. Annual community-directed treatment with ivermectin (CDTi) was instituted in Maridi since the early 2000s, but with low coverage. In 2021, the CDTi programme was strengthened to a six-monthly programme. Additionally, the community-based vector control strategy “Slash and Clear” has been implemented since 2019 at the Maridi Dam, the only blackfly breeding site in the area. This study assessed the effect of these reinforced onchocerciasis elimination interventions on the Onchocerca volvulus seroprevalence among young children, an indicator of ongoing transmission. Methods Baseline and follow-up serosurveys were conducted in Maridi in 2019 (prior to strengthening onchocerciasis elimination efforts) and 2023, respectively. During both surveys, children aged three to nine years were recruited from five study sites situated at different distances from the Maridi Dam. Ov16 antibodies were detected via rapid diagnostic tests (RDTs) using whole blood obtained by finger-pricking the participants. Baseline and follow-up Ov16 prevalence rates were calculated and compared. Results In 2019, the Ov16 seroprevalence among children aged three to nine years was 24.5% compared to 30.6% in 2023 (p=0.22). Both surveys found a particularly high Ov16 seroprevalence in the study site closest to the Maridi Dam (35.0% in 2019 and 44.0% in 2023, p=0.52). The Ov16 seroprevalence had a non-significant decreasing trend in the three-year-old children, from 12.5% (3/24) in 2019 to 8.8% (3/34) in 2023 (p=0.65). Conclusion The persistent Ov16 RDT seropositivity among three-year-old children in 2023 indicates ongoing O. volvulus transmission. Therefore, further strengthening of the onchocerciasis elimination programme is required. The study highlights the utility of RDTs in monitoring onchocerciasis transmission in highly endemic settings.


Introduction
Onchocerciasis is a neglected tropical disease primarily found in Africa, caused by the filarial nematode Onchocerca volvulus and transmitted through bites of infectious blackflies (Simulium spp.) 1 .Onchocerciasis is known to cause dermatitis and blindness ("river blindness") and is associated with a clinical spectrum of seizures and disorders grouped under the termonchocerciasis-associated epilepsy (OAE) 2 .In areas where there is high ongoing O. volvulus transmission, children are at risk of developing OAE, with seizure onset typically occurring between the ages of three to 18 years 2 .Elimination of onchocerciasis depends mainly on community-directed treatment with ivermectin (CDTi) in at-risk communities, which can be supplemented with activities to eradicate the blackflies 3 .
Onchocerciasis was first reported in Sudan in 1947 by Kirk et al.Ivermectin distribution started in South Sudan in 1996 through the Southern Sudan Onchocerciasis Control Programme, but annual CDTi was only formally initiated in 2005 4,5 .Moreover, there have been several years of low coverage treatment due to insecurity, and community drug distributors' high attrition rate, which, in combination with high onchocerciasis transmission potential, has allowed the disease to remain endemic most of the country 4-6 .Although CDTi was interrupted in 2020 because of the COVID-19 pandemic, it has been re-introduced biannually (six-monthly) since 2021.
Currently, the World Health Organisation (WHO) recommends using serodiagnostic tools based on the detection of antibodies specific to a 16KDa species-specific antigen of O. volvulus (Ov16), utilising either a rapid diagnostic test or an Enzyme-Linked Immunosorbent Assay (ELISA) platform 7 .The antigen Ov16 serves as an early marker of onchocerciasis infection, so seroconversion to Ov16 can be used to detect pre-patent infection, which is not possible with skin snips 8 .Since its discovery in 1991, the use of Ov16 has emerged as the gold standard for onchocerciasis serology, with sensitivities in the 80-89% range and specificity values in the 97-99% range depending on the assay used and the sample collection assessed 9,10 .
In 2018, a house-to-house survey in Maridi County, an onchocerciasis-endemic area in Western Equatoria State, South Sudan, showed a very high epilepsy prevalence of 43.8 per 1000 individuals 11 .Notably, in Kazana 2, the site closest to the major blackfly breeding site in the area (Maridi Dam), an epilepsy prevalence of 119.0 per 1000 individuals was noted 11 .In December 2019, onchocerciasis transmission was assessed in Maridi in three sites at different distances from the Maridi Dam via an Ov16 serosurvey in children three to nine years of age.The overall seroprevalence of the children was 24.5% 12 .As these findings indicated high onchocerciasis transmission, a community-based vector control strategy, "Slash and Clear", has been implemented at least once a year at the Maridi Dam since December 2019 (Figure 1) 13 .This strategy consists of clearing the trailing vegetation to eliminate the blackfly breeding site and reduce biting density in the nearby communities.
In February 2023, a follow-up O. volvulus serosurvey was conducted among children aged three to nine years in Maridi to evaluate possible changes in transmission patterns.This paper presented the effect of strengthening the onchocerciasis elimination interventions as demonstrated by seroprevalence in children aged three to nine years using the Ov16 SD BIOLINE rapid diagnostic test (RDT).

Ethical standards
Ethical approval for this study was obtained from the University of Antwerp's ethics committee (Ref: BUN B300201940004) and from the ethics committee of the Ministry of Health of South Sudan (MOH/RERB 56/2022).All participants were

Amendments from Version 1
As a result of the comments of the reviewers, we have revised our manuscript.We have chosen for a more streamlined way to present numbers and percentages throughout the document.Resulting in a changed lay-out in Table 1 and Table 2. Additionally, we included specifics on the WHO recommendations regarding O. volvulus diagnosis using Ov16 based rapid diagnostic tests and Enzyme-linked immunosorbent Assay (ELISA).More information was added on the study site and Onchocerciasis control interventions performed in South Sudan.Including past studies on epilepsy and Onchocerciasis prevalence and the prevalence in relation to the Maridi Dam.Furthermore, we added background information on the occurrence of skin disease in the area.Finally, we refined our discussion by referring to a modelling study presenting an initial rise in prevalence after vector control.Providing an additional potential explanation on the increased seroprevalence in the three to nine years old age category in Maridi, South Sudan.
Any further responses from the reviewers can be found at the end of the article recruited only after informed parental consent was granted (and assent for children aged seven years and above).The research procedures were in compliance with the declaration Helsinki, and all personal data were treated confidentially.

Study setting
South Sudan is known to have multiple endemic hotspots of onchocerciasis, including Maridi County in the Western Equatoria State (Figure 2).Maridi County is home to over 115,000 individuals 14 and is crossed by the Maridi River, upon which a dam was built in the 1950s.The dam spillway was identified as the sole blackfly breeding site in Maridi 12 , driving onchocerciasis transmission in the neighbouring villages.This study was conducted between 2019 (baseline) and 2023 (follow-up) in five villages, of which Kazana 1, Kazana 2, and Hai-Matara are situated in the proximity of the dam (high-transmission zone; HTZ) and Hai-Tarawa and Hai-Gabat are located further away from the dam (low-transmission zone; LTZ) (Figure 3).

Study procedures
A cross-sectional study was conducted before and after strengthening the control interventions against onchocerciasis.The procedures of the baseline survey (2019) have been described in detail previously 12 .Several weeks before the follow-up survey in 2023, the research team contacted the community leaders to inform them about the study and obtain their consent and collaboration.Five local healthcare workers were trained to perform the Ov16 SD BIOLINE RDT and administer a questionnaire to the parents of the children.One day before participant recruitment, community mobilisers notified villagers about the study and of the site(s) chosen in each village (schools, churches, etc.) to test the eligible children (three to nine years old).On the day of the study in each study site, the mobilisers used megaphones, and healthcare workers went from house to house to gather more participants.All children for whom informed consent was provided by a parent/guardian were enrolled in the study (assent was obtained from children aged seven to nine years).
A short questionnaire was administered to the parent/guardian and the child to collect relevant information about the child's history (age, sex, village of residence, previous ivermectin intake, itching/skin lesions, epilepsy).Thereafter, all the participating children were finger-pricked with a single-use, retractable lancet of 2.00 millimetres (Ergo lance).Whole blood from each finger prick was used for Ov16 rapid diagnostic testing (RDT) (SD Bioline, Inc, Gyeonggi-do, South Korea).The Ov16 RDT test was read after 30 minutes and performed in the field following the manufacturers' instructions.

Data analysis
Study data was checked daily and uploaded to the REDCap secure online platform.The final datasets were exported from REDCap, cleaned in Excel spreadsheets, then transferred to the R software version 4.2.2 for analysis.Continuous variables were summarised as median with interquartile range (IQR), while categorical variables were expressed as percentages.The findings of the baseline ( 2019) 12 and follow-up (2023) surveys were compared using the Chi-squared test (or Fisher's exact test when appropriate), and 95% confidence intervals (CIs) were produced using the Wilson score with continuity correction.

Ov16 seroprevalence in Maridi 2023
During the 2023 O. volvulus seroprevalence study in Maridi, 248 children aged three to nine years were recruited.The median age was six years (IQR: 4-8), and 114 were males (46.0%).Ninety-three of the participating children (37.5%) had some form of dermatitis, as evidenced by itching and/or visible skin lesions.Furthermore, four children (1.6%) were identified as having epilepsy.

Ov16 seroprevalence in Maridi over time
Comparing baseline and follow-up Ov16 RDT results, the overall Ov16 seroprevalence increased non-significantly from 24.3% in 2019 to 30.6% in 2023 (p=0.22)(Figure 5).The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, was measured at 8% in 2023 and was thus lower than the level of 12.5% measured among three-year-olds in 2019.However, the difference was not significant (p=0.68).Conversely, the Ov16 seropositivity rose non-significantly from 26.7% in 2019 to 34.1% in 2023 among children aged four years and above (p=0.16).Transmission zone-specific Ov16 seroprevalence did not significantly change across surveys (HTZ: p= 0.70; LTZ: p= 0.18; Table 2).

Discussion
In 2023, an Ov16 seroprevalence of 30.7% was found among children aged three to nine years in Maridi, representing a non-significant increase from the 24.3% seroprevalence documented in 2019 12 (p=0.98).As the elimination measures were only introduced three years ago, children between the ages of four and nine years in the 2023 cohort had already been highly exposed to onchocerciasis before strengthening the control interventions.An increase in the seroprevalence in the age group of three to nine years old children was to be expected, particularly because of the very high pre-intervention endemicity 15 .Indeed, some of the children might harbour prepatent infection before the start of the intervention, which may break out in the course of the intervention.A force-of-infection model for onchocerciasis has shown that upon interrupting O. volvulus transmission by vector control, the microfilarial prevalence of the zero to four and five to nine year old age groups continues to rise during the first five years, after which it starts falling 16 .The fact that the Ov16 seroprevalence among the three-year-olds did not decrease significantly may also be related to the small sample size, short follow-up duration since the strengthening of the control interventions and the lack of CDTi in 2021.Indeed, mathematical modelling has suggested that a one-year CDTi interruption may significantly impact onchocerciasis elimination prospects in settings such as Maridi, with short CDTi histories and high onchocerciasis endemicity 17,18 .The one-year CDTi interruption in 2020 and the consistently low CDTi coverage rates achieved in Maridi (40.8% in 2017 11 and 56.6% in 2021 19 ) explain why some three-year-old children became infected with O. volvulus.Achieving a CDTi coverage of at least 80% of the total population is strongly recommended for the successful elimination of onchocerciasis transmission, particularly in areas with a high prevalence of the disease 20 .
The high Ov16 seroprevalence among young children in Maridi reveals that the onchocerciasis elimination measures must be further strengthened to protect children from developing onchocerciasis-associated morbidities.Over one-third (37.5%) of the children had some form of dermatitis, as evidenced by itching and/or visible skin lesions.A significantly higher proportion of children with dermatitis tested Ov16 RDT positive (43.5%) compared to their peers without dermatitis (22.6%, p<0.001), suggesting the dermatitis was likely caused by an active O. volvulus infection in a large proportion of children.In resource-constrained settings such as many parts of sub-Saharan Africa, when children present with dermatitis and itching, scabies should be considered as a possible diagnosis.However, the absence of characteristic scabies lesions was noted in the children included in the study.Additionally, since scabies is treatable with ivermectin, its

Yankum Dadzie
Emory University, Atlanta, Georgia, USA This is an interesting paper reporting on the effect of using a new onchocerciasis diagnostic tool to follow up an onchocerciasis intervention in an uncontrolled area, comparable to an onchocerciasis naïve area with a high endemicity.Every observation is thus intriguing whilst the interpretation of what is being observed needs to be done cautiously, particularly as regards the application of the new diagnostic tool.
This study is well set out and appears well conducted.However, the paper must address two areas/points which constitute a setback for the paper's strength.The first point is the use of some words or phrases in the paper which are not scientifically appropriate and give the wrong information on the situation in question.The second is about some points in the discussion of the results which portray an opinion which do not seem to be backed by evidence.
The following concern comments on the first point with the use of inappropriate words or phrases: Introduction: Line 7-10, replace 'Prevention' with 'Elimination' and continue with the sentence and in the 10 th line substitute 'control' for 'eradicate'.The last sentence of the introduction is confusing as what it states is not was what was done.I suggest the following formulation: "This paper presented the effect of strengthening the onchocerciasis elimination interventions as demonstrated by seroprevalence in children aged 3-9 years using the Ov16 SD BIOLINE rapid diagnostic test (RDT)".

Results:
Under the subtitle 'Ov16 seroprevalence in Maridi over time', the following sentence in lines 3-5, 'The seroprevalence among the three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, non-significantly decreased from 12.5% in 2019 to 8.8% in 2023 (p=0.68)',needs to be reformulated.The three-yearolds born after implementation of more robust onchocerciasis elimination measures could be found only in 2023 and could therefore not have contributed to the RDT seroprevalence in 2019 to enable showing a reduced level in 2023.As the measurements are cross-sectional values the sentence should read correctly thus: The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and biannual CDTi measured in 2023 8% and was thus lower than the level of 12.5% measured among three-year-olds in 2019 but the difference was not significant, (p=0.68).
My main concern is with the "discussion" and it is specifically about the interpretation of the results from the study.The first paragraph of the discussion points out the increase, albeit insignificant, of the seroprevalence in the 4-9-year-olds following enhanced onchocerciasis measures and interprets the 'increased seroprevalence as 'an indication of intensification of transmission'.Such an interpretation, "increase in transmission" cannot be correct as the enhanced intervention measures clearly must have had increased effect entomologically as well as epidemiologically, through twice a year CDTi even if the coverage was not optimal yet, on the transmission.The entomological impact of "slash and clear" has been reported in the ref.no 5.The publication, "A force-of-infection model for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area by Remme, J et al. in the Bulletin of the World Health Organization, 64: 667-681 (1986) on which the onchocerciasis mathematical model is based, shows that at the onset of intervention of vector control, which interrupts transmission almost immediately, the mf prevalence of age groups in years 0-4, 5-9, continues to rise during the first five years after which it starts falling.It is only in the adults that there is a fall of mf prevalence which is seen in the first three years.A similar effect is found with CDTi where it is even more accentuated the higher the pre-intervention endemicity level of the community.It may be recalled that impact assessment of CDTi in APOC countries did not occur until after five years of CDTI when impact would be expected.The notion was based on this observation.Recently, Lont et al. have shown that there are basic similarities in the effect of application of mf prevalence and seroprevalence in their publication (Lont et al., 2017 1 ).The increase in the seroprevalence recorded in the age group of 3-9 years old children is normal and it is to be expected in that age group particularly when dealing with communities with very high pre-intervention endemicity.It may be noted that some of the young might harbour prepatent infection before the start of the intervention and which may break out in the course of the intervention as Lont et al. discuss in their paper.
As regards to the above considerations, certain statements in the discussion will have to be reviewed and I would like to point them out: 1 st paragraph, line 3-5 "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".The sentence may be deleted as what has been found is to be expected and importantly does not suggest an absence of impact.The continuation is correct as it takes a few years before interruption of transmission can occur showing also the absence of new infections or new seroconversions as applicable to CDTi.
Line 10, "may indicate an intensification of O. volvulus transmission" may be deleted for the same reason as above.
Paragraph 2 is well discussed.However, I would like to point out a striking mitigating factor which has either been downplayed or overseen.It is true that no CDTi was carried out in 2020 because of Covid-19 with the negative implications for which Hamley's good work has been quoted to highlight the effect on this study.However, a good look at the first-year results following the first slash and clear activity as read from the paper by Raimon et al. suggests that Maridi focus possibly did not do as badly as it has been described in this paper.A proper analysis of the events could very well indicate that there could have been more reduction in transmission in the year 2020 effected by the slash and clear than what could have been achieved by annual CDTI alone before the slash and clear was introduced.
In the penultimate line of the paragraph, kindly substitute 'eliminate blackflies' for 'eradicate blackflies'.
Paragraph 5: I wonder whether after the foregoing discussions it is still worth retaining this statement, particularly the second half of the statement."A sufficiently large sample size of 3-year-old children needs to be Ov16 RDT tested before and after interventions to evaluate the short-term effect of strengthening an onchocerciasis elimination programme".

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Onchocerciasis epidemiology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 19 Feb 2024

Amber Hadermann
Response to the reviewer: Rapid diagnostic testing for onchocerciasis in Maridi (South Sudan) before and after improving elimination strategies: a repeated cross-sectional survey Reviewer The reported data indicate that despite the annual treatment with ivermectin (CDTI) and the vector control strategy "Slash and Clear" the Ov16 response did not decrease but showed insignificant increase in 4-9-year-old children (about 27% to 34%) but an insignificant decrease in 3-year-old children (about 12% to 9%).The cross-sectional survey indicates the ongoing Onchocerca transmission demanding strengthening of the onchocerciasis elimination programme.The work is clearly and accurately presented.The authors cited current references except the indications noted below.The study design is appropriate, the authors exhibit sufficient details of the analysis -except the constraint below -including the statistics and applied methods which in part should be better introduced.The article merits indexing for scientists involved in onchocerciasis control.The source data are available.The conclusions drawn adequately are supported by the results.However, the title appears not to represent the documented data as their conclusion and should be improved.Thus, not the applied diagnostic test pertains the core statement of the article, furthermore the notification "improving elimination strategies" rather indicates a success in onchocerciasis control.The title more preferably should read: "Continuance of onchocerciasis transmission to children in a highly endemic area in South Sudan after ivermectin treatment and vector control" or "Continuance of onchocerciasis transmission to children in the highly endemic Maridi county (South Sudan) after ivermectin treatment and vector control strategy requiring strengthening of onchocerciasis elimination programmes"

Objections:
The authors should not state "Maridi Dam, the only blackfly breeding site in the area" since this statement cannot be accurate, there surely are other breeding sites. 1.

Response: The rivers in the Maridi central area were extensively investigated for the presence of breeding sites by Lakwo et al. (2020), and the Maridi dam was found to be the single black fly breeding site in the area. The reference of the paper by Lakwo et al. is included in the reference list. We agree this is a unique situation for an onchocerciasis endemic area. Our population-based studies showed that the Maridi Dam explains the reason why an "epilepsy epidemic" started in Maridi. Nevertheless, we now state "Maridi Dam, the major blackfly breeding site in the area".
The authors stated that the "annual CDTi was introduced in South Sudan in the early 2000s", however, they in addition should refer to the detection and occurrence of onchocerciasis in Sudan.Thus, onchocerciasis had been reported in 1947 by Kirk, in 1985 by Williams et al..

Identification of an Onchocerca volvulus cDNA encoding a low-molecular-weight antigen uniquely recognized by onchocerciasis patient sera. 1990 Feb;39(1):135-45. Cama VA, McDonald C, Arcury-Quandt A, Eberhard M, Jenks MH, Smith J, et al. Evaluation of an OV-16 IgG4 Enzyme-Linked Immunosorbent Assay in Humans and Its Application to Determine the Dynamics of Antibody Responses in a Non-Human Primate Model of Onchocerca volvulus Infection. Am J Trop Med Hyg. 2018;99(4):1041-8. Golden A, Steel C, Yokobe L, Jackson E, Barney R, Kubofcik J, et al. Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools. PLoS One. 2013;8(7):e69231.
The note of the recommendation of the Ov16 diagnostic test by the WHO in the text should include the respective reference (WHO, 2016 1 ).
The author correctly accentuated the limitation of the presented study by (i) the small sample size (n=248), (ii) the short follow-up duration, and (iii) the impact of the interrupted ivermectin treatment by Covid-19 pandemic.Thus, this study needs follow-up survey. 1.

Response: Yes, we are planning a follow-up survey.
Reviewer 2: Kiswendsida Thierry Guiguemde The authors worked on the importance of using rapid tests for the monitoring and control of onchocerciasis.Which is very interesting.However, a few comments could help improve the manuscript.

Methods: Study procedures:
1.There were community mobilizers to spread the information.It would be good to specify who injected the children for the test and whether they were trained beforehand.

Response: We have adapted the text to fit the suggested standards.
Discussion: 1.A significantly higher proportion of children with dermatitis tested Ov16 RDT positive (43.5%) compared to their peers without dermatitis (22.6%, p<0.001), suggesting the dermatitis was likely caused by an active O. volvulus infection in a large proportion of children.
Other causes of dermatitis in children in the study area must be clarified and discussed.

Response: We now include in the discussion: "In resource-constrained settings, when African children present with dermatitis and itching, scabies should be considered as a possible diagnosis. However, the absence of characteristic scabies lesions was noted in the children included in the study. Additionally, since scabies is treatable with ivermectin, its prevalence typically decreases in regions where mass ivermectin distribution occurs."
2. More elements of discussion should be provided.Studies have shown limitations in the detection of antibodies against the OV-16 antigen.This is after discrepancies in the results with skin snip method according to WHO standards for the detection of microfilariae in the blood.It is important to specify this and give possible reasons.Carrying out the skin snip method in children over 5 with dermatitis could provide more precision on the cause of dermatitis.

Response: Indeed, the addition of skin snips would have added more information on active disease in the children. However, skin snip testing is increasingly not very well accepted by local populations because it is an invasive procedure. The use of rapid diagnostic tests is not to diagnose an O. volvulus infection on an individual level but to identify whether transmission is ongoing or not. Furthermore, in the age of ivermectin, the use of skin snip microscopy is becoming redundant. The number of microfilariae in the skin after ivermectin use is low and often below the detection limit. Finally, the fact that Ov16 antibodies can be detected even before the production of microfilariae make it a much better proxy for recent onchocerciasis transmission as it can identify pre-patent infections in recently infected children.
Reviewer 3 : Yankum Dadzie This is an interesting paper reporting on the effect of using a new onchocerciasis diagnostic tool to follow up an onchocerciasis intervention in an uncontrolled area, comparable to an onchocerciasis naïve area with a high endemicity.
Every observation is thus intriguing whilst the interpretation of what is being observed needs to be done cautiously, particularly as regards the application of the new diagnostic tool.
This study is well set out and appears well conducted.However, the paper must address two areas/points which constitute a setback for the paper's strength.The first point is the use of some words or phrases in the paper which are not scientifically appropriate and give the wrong information on the situation in question.The second is about some points in the discussion of the results which portray an opinion which do not seem to be backed by evidence.
The following concern comments on the first point with the use of inappropriate words or phrases: Introduction: Line 7-10, replace 'Prevention' with 'Elimination' and continue with the sentence and in the 10 th line substitute 'control' for 'eradicate'.
The last sentence of the introduction is confusing as what it states is not was what was done.I suggest the following formulation: "This paper presented the effect of strengthening the onchocerciasis elimination interventions as demonstrated by seroprevalence in children aged 3-9 years using the Ov16 SD BIOLINE rapid diagnostic test (RDT)". 1.

Response: Suggested alterations done.
Results: Under the subtitle 'Ov16 seroprevalence in Maridi over time', the following sentence in lines 3-5, 'The seroprevalence among the three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, non-significantly decreased from 12.5% in 2019 to 8.8% in 2023 (p=0.68)',needs to be reformulated.The three-year-olds born after implementation of more robust onchocerciasis elimination measures could be found only in 2023 and could therefore not have contributed to the RDT seroprevalence in 2019 to enable showing a reduced level in 2023.As the measurements are crosssectional values the sentence should read correctly thus: The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi measured in 2023 8% and was thus lower than the level of 12.5% measured among three-year-olds in 2019 but the difference was not significant, (p=0.68). 1.

Response: Text altered. We now state" The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, was measured at 8% in 2023 and was thus lower than the level of 12.5% measured among three-year-olds in 2019. However, the difference was not significant (p=0.68)."
My main concern is with the "discussion" and it is specifically about the interpretation 1.
of the results from the study.The first paragraph of the discussion points out the increase, albeit insignificant, of the seroprevalence in the 4-9-year-olds following enhanced onchocerciasis measures and interprets the 'increased seroprevalence as 'an indication of intensification of transmission'.Such an interpretation, "increase in transmission" cannot be correct as the enhanced intervention measures clearly must have had increased effect entomologically as well as epidemiologically, through twice a year CDTi even if the coverage was not optimal yet, on the transmission.The entomological impact of "slash and clear" has been reported in the ref.no 5.The publication, "A force-of-infection model for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area by Remme, J et al. in the Bulletin of the World Health Organization, 64: 667-681 (1986) on which the onchocerciasis mathematical model is based, shows that at the onset of intervention of vector control, which interrupts transmission almost immediately, the mf prevalence of age groups in years 0-4, 5-9, continues to rise during the first five years after which it starts falling.It is only in the adults that there is a fall of mf prevalence which is seen in the first three years.A similar effect is found with CDTi where it is even more accentuated the higher the pre-intervention endemicity level of the community.It may be recalled that impact assessment of CDTi in APOC countries did not occur until after five years of CDTI when impact would be expected.The notion was based on this observation.Recently, Lont et al. have shown that there are basic similarities in the effect of application of mf prevalence and seroprevalence in their publication (Lont et al., 2017 1 ).The increase in the seroprevalence recorded in the age group of 3-9 years old children is normal and it is to be expected in that age group particularly when dealing with communities with very high pre-intervention endemicity.It may be noted that some of the young might harbour prepatent infection before the start of the intervention and which may break out in the course of the intervention as Lont et al. discuss in their paper.As regards to the above considerations, certain statements in the discussion will have to be reviewed and I would like to point them out: 1 st paragraph, line 3-5 "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".The sentence may be deleted as what has been found is to be expected and importantly does not suggest an absence of impact.The continuation is correct as it takes a few years before interruption of transmission can occur showing also the absence of new infections or new seroconversions as applicable to CDTi.Response: We thank you for these important comments.We eliminated the sentence "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".We now included in the discussion "An increase in the seroprevalence in the age group of 3-9 years old children was to be expected, particularly because of the very high pre-intervention endemicity.Indeed some of the children might harbour prepatent infection before the start of the intervention which may break out in the course of the intervention.A force-of-infection model for onchocerciasis has shown that even during interruption of O. volvulus transmission by vector control, the microfilarial prevalence of the 0-4 and 5-9 year old age groups continues to rise during the first five years after which it starts falling."And we added the following two references Lont YL, Coffeng LE, de  Line 10, "may indicate an intensification of O. volvulus transmission" may be deleted for the same reason as above. 1.

Response: We deleted the the text mentioning the ' intensification of O. volvulus transmission"
Paragraph 2 is well discussed.However, I would like to point out a striking mitigating factor which has either been downplayed or overseen.It is true that no CDTi was carried out in 2020 because of Covid-19 with the negative implications for which Hamley's good work has been quoted to highlight the effect on this study.However, a good look at the first-year results following the first slash and clear activity as read from the paper by Raimon et al. suggests that Maridi focus possibly did not do as badly as it has been described in this paper.A proper analysis of the events could very well indicate that there could have been more reduction in transmission in the year 2020 effected by the slash and clear than what could have been achieved by annual CDTI alone before the slash and clear was introduced. 1.

Response: We agree that the strengthening of the onchocerciasis elimination program decreased the O. volvulus transmission in the area. This was strongly suggested by the results of a longitudinal population-based study that showed that the incidence of onchocerciasis-associated epilepsy, including nodding syndrome, decreased in Maridi. This paper has now been published and is included among the references. Jada SR, Amaral LJ, Lakwo T, Carter JY, Rovarini J, Bol YY, Logora MY, Hadermann A, Hopkins A, Fodjo JNS, Colebunders R. Effect of onchocerciasis elimination measures on the incidence of epilepsy in Maridi, South Sudan: a 3-year longitudinal, prospective, population-based study. Lancet Glob Health. 2023 Aug;11(8):e1260-e1268. doi: 10.1016/S2214-109X(23)00248-6.
In the penultimate line of the paragraph, kindly substitute 'eliminate blackflies' for 'eradicate blackflies'.
Paragraph 5: I wonder whether after the foregoing discussions it is still worth retaining this statement, particularly the second half of the statement."A sufficiently large sample size of 3-year-old children needs to be Ov16 RDT tested before and after interventions to evaluate the short-term effect of strengthening an onchocerciasis elimination programme".

Results:
Ov16 seroprevalence in Maridi 2023 1. "During the 2023 O. volvulus seroprevalence study in Maridi, 248 children aged three to nine years were recruited.The median age was six years (IQR: 4-8), and 114 (46.0%) were males.Ninetythree (37.5%) of the participating children had some form of dermatitis, as evidenced by itching and/or visible skin lesions.Furthermore, four (1.6%) children were identified as having epilepsy2." To make reading easier, it is good to harmonize the way you write the numbers.Either write in letters or write in numbers as for the 248 children.It must be applied throughout the entire manuscript.

○
Put the percentage after the number and the specimen.Example: Ninety-three of the participating children (37.5%) instead of "Ninety-three (37.5%) of the participating children".four children (1.6%) were identified instead of four (1.6%) children were identified.

Discussion:
1.A significantly higher proportion of children with dermatitis tested Ov16 RDT positive (43.5%) compared to their peers without dermatitis (22.6%, p<0.001), suggesting the dermatitis was likely caused by an active O. volvulus infection in a large proportion of children.
Other causes of dermatitis in children in the study area must be clarified and discussed.
2. More elements of discussion should be provided.Studies have shown limitations in the detection of antibodies against the OV-16 antigen.This is after discrepancies in the results with skin snip method according to WHO standards for the detection of microfilariae in the blood.It is important to specify this and give possible reasons.Carrying out the skin snip method in children over 5 with dermatitis could provide more precision on the cause of dermatitis.

Is the work clearly and accurately presented and does it cite the current literature?
Yes

Is the study design appropriate and does the work have academic merit? Yes
Are sufficient details of methods and analysis provided to allow replication by others?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Parasitology
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Response: We have now added more information about the Ov16 test and included in the introduction: "Currently, the World Health Organisation (WHO) recommends using serodiagnostic tools based on the detection of antibodies specific to a 16KDa speciesspecific antigen of O. volvulus (Ov16), utilising either a rapid diagnostic test or an Enzyme-Linked Immunosorbent Assay (ELISA) platform. The antigen Ov16 serves as an early marker of onchocerciasis infection, so seroconversion to Ov16 can be used to detect pre-patent infection, which is not possible with skin snips. Since its discovery in 1991, the use of Ov16 has emerged as the gold standard for onchocerciasis serology, with sensitivities in the 80-89% range and specificity values in the 97-99% range depending on the assay used and the sample collection assessed. We also added the following references: World Health Organisation. Onchocerciasis: diagnostic target product profile to support preventive chemotherapy, 2021 https://www.who.int/publications/i/item/9789240024496 Lobos E, Altmann M, Mengod G, Weiss N, Rudin W, Karam M. Mol Biochem Parasitol. Identification of an Onchocerca volvulus cDNA encoding a low-molecular-weight antigen uniquely recognized by onchocerciasis patient sera. 1990 Feb;39(1):135-45. Cama VA, McDonald C, Arcury-Quandt A, Eberhard M, Jenks MH, Smith J, et al. Evaluation of an OV-16 IgG4 Enzyme-Linked Immunosorbent Assay in Humans and Its Application to Determine the Dynamics of Antibody Responses in a Non-Human Primate Model of Onchocerca volvulus Infection. Am J Trop Med Hyg. 2018;99(4):1041-8. Golden A, Steel C, Yokobe L, Jackson E, Barney R, Kubofcik J, et al. Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools. PLoS One. 2013;8(7):e69231.
The note of the recommendation of the Ov16 diagnostic test by the WHO in the text should include the respective reference (WHO, 2016 1 ).
The author correctly accentuated the limitation of the presented study by (i) the small sample size (n=248), (ii) the short follow-up duration, and (iii) the impact of the interrupted ivermectin treatment by Covid-19 pandemic.Thus, this study needs follow-up survey. 1.

Response: Yes, we are planning a follow-up survey.
Reviewer 2: Kiswendsida Thierry Guiguemde The authors worked on the importance of using rapid tests for the monitoring and control of onchocerciasis.Which is very interesting.However, a few comments could help improve the manuscript.

Methods: Study procedures:
1.There were community mobilizers to spread the information.It would be good to specify who injected the children for the test and whether they were trained beforehand.

Results:
Ov16 seroprevalence in Maridi 2023 1. "During the 2023 O. volvulus seroprevalence study in Maridi, 248 children aged three to nine years were recruited.The median age was six years (IQR: 4-8), and 114 (46.0%) were males.Ninety-three (37.5%) of the participating children had so To make reading easier, it is good to harmonize the way you write the numbers.Either write in letters or write in numbers as for the 248 children.It must be me form of dermatitis, as evidenced by itching and/or visible skin lesions.Furthermore, four (1.6%) children were identified as having epilepsy2."applied throughout the entire manuscript.Put the percentage after the number and the specimen.Example: Ninety-three of the participating children (37.5%) instead of "Ninety-three (37.5%) of the participating children".four children (1.6%) were identified instead of four (1.6%) children were identified.

Response: We have written the text to fit the following standards: "Spell out numbers one through nine, except in the case of units of measure or time. For these, and for values of 10 and higher, use Arabic numerals. Always spell out numbers at the beginning of a sentence if the sentence cannot be rearranged to avoid starting with a number." -Springer. Therefore, we have chosen to keep our way of writing numbers the same throughout this document. However, we have adapted the placement of percentages as suggested.
2. The overall Ov16 RDT seroprevalence was 76/248 (30.7%).The overall Ov16 RDT seroprevalence was 76/248 (30.7%).
The overall Ov16 RDT seroprevalence is in percentage, putting 30.7% (76/248).Correct everywhere in the text the way of writing.

Response: We have adapted the text to fit the suggested standards.
Discussion: 1.A significantly higher proportion of children with dermatitis tested Ov16 RDT positive (43.5%) compared to their peers without dermatitis (22.6%, p<0.001), suggesting the dermatitis was likely caused by an active O. volvulus infection in a large proportion of children.
Other causes of dermatitis in children in the study area must be clarified and discussed.

Response: We now include in the discussion: "In resource-constrained settings, when African children present with dermatitis and itching, scabies should be considered as a possible diagnosis. However, the absence of characteristic scabies lesions was noted in the children included in the study. Additionally, since scabies is treatable with ivermectin, its prevalence typically decreases in regions where mass ivermectin distribution occurs."
2. More elements of discussion should be provided.Studies have shown limitations in the detection of antibodies against the OV-16 antigen.This is after discrepancies in the results with skin snip method according to WHO standards for the detection of microfilariae in the blood.It is important to specify this and give possible reasons.Carrying out the skin snip method in children over 5 with dermatitis could provide more precision on the cause of dermatitis.

Response: Indeed, the addition of skin snips would have added more information on active disease in the children. However, skin snip testing is increasingly not very well accepted by local populations because it is an invasive procedure. The use of rapid diagnostic tests is not to diagnose an O. volvulus infection on an individual level but to identify whether transmission is ongoing or not. Furthermore, in the age of ivermectin, the use of skin snip microscopy is becoming redundant. The number of microfilariae in the skin after ivermectin use is low and often below the detection limit. Finally, the fact that Ov16 antibodies can be detected even before the production of microfilariae make it a much better proxy for recent onchocerciasis transmission as it can identify pre-patent infections in recently infected children.
Reviewer 3 : Yankum Dadzie This is an interesting paper reporting on the effect of using a new onchocerciasis diagnostic tool to follow up an onchocerciasis intervention in an uncontrolled area, comparable to an onchocerciasis naïve area with a high endemicity.
Every observation is thus intriguing whilst the interpretation of what is being observed needs to be done cautiously, particularly as regards the application of the new diagnostic tool.
This study is well set out and appears well conducted.However, the paper must address two areas/points which constitute a setback for the paper's strength.The first point is the use of some words or phrases in the paper which are not scientifically appropriate and give the wrong information on the situation in question.The second is about some points in the discussion of the results which portray an opinion which do not seem to be backed by evidence.
The following concern comments on the first point with the use of inappropriate words or phrases: Introduction: Line 7-10, replace 'Prevention' with 'Elimination' and continue with the sentence and in the 10 th line substitute 'control' for 'eradicate'.The last sentence of the introduction is confusing as what it states is not was what was done.I suggest the following formulation: "This paper presented the effect of strengthening the onchocerciasis elimination interventions as demonstrated by seroprevalence in children aged 3-9 years using the Ov16 SD BIOLINE rapid diagnostic test (RDT)".

Response: Suggested alterations done.
Results: Under the subtitle 'Ov16 seroprevalence in Maridi over time', the following sentence in lines 3-5, 'The seroprevalence among the three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, non-significantly decreased from 12.5% in 2019 to 8.8% in 2023 (p=0.68)',needs to be reformulated.The three-year-olds born after implementation of more robust onchocerciasis elimination measures could be found only in 2023 and could therefore not have contributed to the RDT seroprevalence in 2019 to enable showing a reduced level in 2023.As the measurements are crosssectional values the sentence should read correctly thus: The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi measured in 2023 8% and was thus 1.
lower than the level of 12.5% measured among three-year-olds in 2019 but the difference was not significant, (p=0.68).

Response: Text altered. We now state" The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, was measured at 8% in 2023 and was thus lower than the level of 12.5% measured among three-year-olds in 2019. However, the difference was not significant (p=0.68)."
My main concern is with the "discussion" and it is specifically about the interpretation of the results from the study.The first paragraph of the discussion points out the increase, albeit insignificant, of the seroprevalence in the 4-9-year-olds following enhanced onchocerciasis measures and interprets the 'increased seroprevalence as 'an indication of intensification of transmission'.Such an interpretation, "increase in transmission" cannot be correct as the enhanced intervention measures clearly must have had increased effect entomologically as well as epidemiologically, through twice a year CDTi even if the coverage was not optimal yet, on the transmission.The entomological impact of "slash and clear" has been reported in the ref.no 5.The publication, "A force-of-infection model for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area by Remme, J et al. in the Bulletin of the World Health Organization, 64: 667-681 (1986) on which the onchocerciasis mathematical model is based, shows that at the onset of intervention of vector control, which interrupts transmission almost immediately, the mf prevalence of age groups in years 0-4, 5-9, continues to rise during the first five years after which it starts falling.It is only in the adults that there is a fall of mf prevalence which is seen in the first three years.A similar effect is found with CDTi where it is even more accentuated the higher the pre-intervention endemicity level of the community.It may be recalled that impact assessment of CDTi in APOC countries did not occur until after five years of CDTI when impact would be expected.The notion was based on this observation.Recently, Lont et al. have shown that there are basic similarities in the effect of application of mf prevalence and seroprevalence in their publication (Lont et al., 2017 1 ).The increase in the seroprevalence recorded in the age group of 3-9 years old children is normal and it is to be expected in that age group particularly when dealing with communities with very high pre-intervention endemicity.It may be noted that some of the young might harbour prepatent infection before the start of the intervention and which may break out in the course of the intervention as Lont et al. discuss in their paper.

1.
As regards to the above considerations, certain statements in the discussion will have to be reviewed and I would like to point them out: 1 st paragraph, line 3-5 "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".The sentence may be deleted as what has been found is to be expected and importantly does not suggest an absence of impact.The continuation is correct as it takes a few years before interruption of transmission can occur showing also the absence of new infections or new seroconversions as applicable to CDTi.Response: We thank you for these important comments.We eliminated the sentence "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".We now included in the discussion "An increase in the seroprevalence in the age group of 3-9 years old children was to be expected, particularly because of the very high pre-intervention endemicity.Indeed some of the children might harbour 1.

Response: We deleted the the text mentioning the ' intensification of O. volvulus transmission"
Paragraph 2 is well discussed.However, I would like to point out a striking mitigating factor which has either been downplayed or overseen.It is true that no CDTi was carried out in 2020 because of Covid-19 with the negative implications for which Hamley's good work has been quoted to highlight the effect on this study.However, a good look at the first-year results following the first slash and clear activity as read from the paper by Raimon et al. suggests that Maridi focus possibly did not do as badly as it has been described in this paper.A proper analysis of the events could very well indicate that there could have been more reduction in transmission in the year 2020 effected by the slash and clear than what could have been achieved by annual CDTI alone before the slash and clear was introduced. 1.

Response: We agree that the strengthening of the onchocerciasis elimination program decreased the O. volvulus transmission in the area. This was strongly suggested by the results of a longitudinal population-based study that showed that the incidence of onchocerciasis-associated epilepsy, including nodding syndrome, decreased in Maridi. This paper has now been published and is included among the references. Jada SR, Amaral LJ, Lakwo T, Carter JY, Rovarini J, Bol YY, Logora MY, Hadermann A, Hopkins A, Fodjo JNS, Colebunders R. Effect of onchocerciasis elimination measures on the incidence of epilepsy in Maridi, South Sudan: a 3-year longitudinal, prospective, population-based study. Lancet Glob Health. 2023 Aug;11(8):e1260-e1268. doi: 10.1016/S2214-109X(23)00248-6.
In the penultimate line of the paragraph, kindly substitute 'eliminate blackflies' for 'eradicate blackflies'.

1.
Response: Text altered.Paragraph 5: I wonder whether after the foregoing discussions it is still worth retaining this statement, particularly the second half of the statement."A sufficiently large sample size of 3-year-old children needs to be Ov16 RDT tested before and after interventions to evaluate the short-term effect of strengthening an onchocerciasis elimination programme". 1.

Response: We now state "A sufficiently large sample size of 3-year-old children needs to be Ov16
RDT tested and a longer follow-up is needed to investigate the effect of strengthening an onchocerciasis elimination programme."

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Parasitology, microbiology, immunology, diagnostics, pathogen-hostinteraction I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Trop Med Hyg. 2018;99(4):1041-8. Golden A, Steel C, Yokobe L, Jackson E, Barney R, Kubofcik J, et al. Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools. PLoS
One. 2013;8(7):e69231.The note of the recommendation of the Ov16 diagnostic test by the WHO in the text should include the respective reference (WHO, 2016 1 ).
The author correctly accentuated the limitation of the presented study by (i) the small sample size (n=248), (ii) the short follow-up duration, and (iii) the impact of the interrupted ivermectin treatment by Covid-19 pandemic.Thus, this study needs follow-up survey. 1.

Response: Yes, we are planning a follow-up survey.
Reviewer 2: Kiswendsida Thierry Guiguemde The authors worked on the importance of using rapid tests for the monitoring and control of onchocerciasis.Which is very interesting.However, a few comments could help improve the manuscript.

Methods: Study procedures:
1.There were community mobilizers to spread the information.It would be good to specify who injected the children for the test and whether they were trained beforehand.2.More elements of discussion should be provided.Studies have shown limitations in the detection of antibodies against the OV-16 antigen.This is after discrepancies in the results with skin snip method according to WHO standards for the detection of microfilariae in the blood.It is important to specify this and give possible reasons.Carrying out the skin snip method in children over 5 with dermatitis could provide more precision on the cause of dermatitis.

Response: Indeed, the addition of skin snips would have added more information on active disease in the children. However, skin snip testing is increasingly not very well accepted by local populations because it is an invasive procedure. The use of rapid diagnostic tests is not to diagnose an O. volvulus infection on an individual level but to identify whether transmission is ongoing or not. Furthermore, in the age of ivermectin, the use of skin snip microscopy is becoming redundant. The number of microfilariae in the skin after ivermectin use is low and often below the detection limit. Finally, the fact that Ov16 antibodies can be detected even before the production of microfilariae make it a much better proxy for recent onchocerciasis transmission as it can identify pre-patent infections in recently infected children.
Reviewer 3 : Yankum Dadzie This is an interesting paper reporting on the effect of using a new onchocerciasis diagnostic tool to follow up an onchocerciasis intervention in an uncontrolled area, comparable to an onchocerciasis naïve area with a high endemicity.
Every observation is thus intriguing whilst the interpretation of what is being observed needs to be done cautiously, particularly as regards the application of the new diagnostic tool.
This study is well set out and appears well conducted.However, the paper must address two areas/points which constitute a setback for the paper's strength.The first point is the use of some words or phrases in the paper which are not scientifically appropriate and give the wrong information on the situation in question.The second is about some points in the discussion of the results which portray an opinion which do not seem to be backed by evidence.
The following concern comments on the first point with the use of inappropriate words or phrases: Introduction: Line 7-10, replace 'Prevention' with 'Elimination' and continue with the sentence and in the 10 th line substitute 'control' for 'eradicate'.
The last sentence of the introduction is confusing as what it states is not was what was done.I suggest the following formulation: "This paper presented the effect of strengthening the onchocerciasis elimination interventions as demonstrated by seroprevalence in children aged 3-9 years using the Ov16 SD BIOLINE rapid diagnostic test (RDT)". 1.

Response: Suggested alterations done.
Results: Under the subtitle 'Ov16 seroprevalence in Maridi over time', the following sentence in lines 3-5, 'The seroprevalence among the three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, non-significantly decreased from 12.5% in 2019 to 8.8% in 2023 (p=0.68)',needs to be reformulated.The three-year-olds born after implementation of more robust onchocerciasis elimination measures could be found only in 2023 and could therefore not have contributed to the RDT seroprevalence in 2019 to enable showing a reduced level in 2023.As the measurements are crosssectional values the sentence should read correctly thus: The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi measured in 2023 8% and was thus lower than the level of 12.5% measured among three-year-olds in 2019 but the difference was not significant, (p=0.68).

1.
Response: Text altered.We now state" The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, was measured at 8% in 2023 and was thus lower than the level of 12.5% measured among three-year-olds in 2019.However, the difference was not significant (p=0.68)." My main concern is with the "discussion" and it is specifically about the interpretation of the results from the study.The first paragraph of the discussion points out the increase, albeit insignificant, of the seroprevalence in the 4-9-year-olds following enhanced onchocerciasis measures and interprets the 'increased seroprevalence as 'an indication of intensification of transmission'.Such an interpretation, "increase in transmission" cannot be correct as the enhanced intervention measures clearly must have had increased effect entomologically as well as epidemiologically, through twice a year CDTi even if the coverage was not optimal yet, on the transmission.The entomological impact of "slash and clear" has been reported in the ref.almost immediately, the mf prevalence of age groups in years 0-4, 5-9, continues to rise during the first five years after which it starts falling.It is only in the adults that there is a fall of mf prevalence which is seen in the first three years.A similar effect is found with CDTi where it is even more accentuated the higher the pre-intervention endemicity level of the community.It may be recalled that impact assessment of CDTi in APOC countries did not occur until after five years of CDTI when impact would be expected.The notion was based on this observation.Recently, Lont et al. have shown that there are basic similarities in the effect of application of mf prevalence and seroprevalence in their publication (Lont et al., 2017 1 ).The increase in the seroprevalence recorded in the age group of 3-9 years old children is normal and it is to be expected in that age group particularly when dealing with communities with very high pre-intervention endemicity.It may be noted that some of the young might harbour prepatent infection before the start of the intervention and which may break out in the course of the intervention as Lont et al. discuss in their paper.As regards to the above considerations, certain statements in the discussion will have to be reviewed and I would like to point them out: 1 st paragraph, line 3-5 "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".The sentence may be deleted as what has been found is to be expected and importantly does not suggest an absence of impact.The continuation is correct as it takes a few years before interruption of transmission can occur showing also the absence of new infections or new seroconversions as applicable to CDTi.Response: We thank you for these important comments.We eliminated the sentence "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".We now included in the discussion "An increase in the seroprevalence in the age group of 3-9 years old children was to be expected, particularly because of the very high pre-intervention endemicity.Indeed some of the children might harbour prepatent infection before the start of the intervention which may break out in the course of the intervention.A force-of-infection model for onchocerciasis has shown that even during interruption of O. volvulus transmission by vector control, the microfilarial prevalence of the 0-4 and 5-9 year old age groups continues to rise during the first five years after which it starts falling."And we added the following two references Lont YL, Coffeng LE, de

Response: We deleted the the text mentioning the ' intensification of O. volvulus transmission"
Paragraph 2 is well discussed.However, I would like to point out a striking mitigating factor which has either been downplayed or overseen.It is true that no CDTi was carried out in 2020 because of Covid-19 with the negative implications for which Hamley's good work has been quoted to highlight the effect on this study.However, a good look at the first-year results following the first slash and clear activity as read from the paper by Raimon et al. suggests that Maridi focus possibly did not do as badly as it has been described in this paper.A proper analysis of the events could In the penultimate line of the paragraph, kindly substitute 'eliminate blackflies' for 'eradicate blackflies'.

1.
Response: Text altered.Paragraph 5: I wonder whether after the foregoing discussions it is still worth retaining this statement, particularly the second half of the statement."A sufficiently large sample size of 3-year-old children needs to be Ov16 RDT tested before and after interventions to evaluate the short-term effect of strengthening an onchocerciasis elimination programme".
regions where mass ivermectin distribution occurs." 2. More elements of discussion should be provided.Studies have shown limitations in the detection of antibodies against the OV-16 antigen.This is after discrepancies in the results with skin snip method according to WHO standards for the detection of microfilariae in the blood.It is important to specify this and give possible reasons.Carrying out the skin snip method in children over 5 with dermatitis could provide more precision on the cause of dermatitis.

Response: Indeed, the addition of skin snips would have added more information on active disease in the children. However, skin snip testing is increasingly not very well accepted by local populations because it is an invasive procedure. The use of rapid diagnostic tests is not to diagnose an O. volvulus infection on an individual level but to identify whether transmission is ongoing or not. Furthermore, in the age of ivermectin, the use of skin snip microscopy is becoming redundant. The number of microfilariae in the skin after ivermectin use is low and often below the detection limit. Finally, the fact that Ov16 antibodies can be detected even before the production of microfilariae make it a much better proxy for recent onchocerciasis transmission as it can identify pre-patent infections in recently infected children.
Reviewer 3 : Yankum Dadzie This is an interesting paper reporting on the effect of using a new onchocerciasis diagnostic tool to follow up an onchocerciasis intervention in an uncontrolled area, comparable to an onchocerciasis naïve area with a high endemicity.Every observation is thus intriguing whilst the interpretation of what is being observed needs to be done cautiously, particularly as regards the application of the new diagnostic tool.
This study is well set out and appears well conducted.However, the paper must address two areas/points which constitute a setback for the paper's strength.The first point is the use of some words or phrases in the paper which are not scientifically appropriate and give the wrong information on the situation in question.The second is about some points in the discussion of the results which portray an opinion which do not seem to be backed by evidence.
The following concern comments on the first point with the use of inappropriate words or phrases: Introduction: Line 7-10, replace 'Prevention' with 'Elimination' and continue with the sentence and in the 10 th line substitute 'control' for 'eradicate'.The last sentence of the introduction is confusing as what it states is not was what was done.I suggest the following formulation: "This paper presented the effect of strengthening the onchocerciasis elimination interventions as demonstrated by seroprevalence in children aged 3-9 years using the Ov16 SD BIOLINE rapid diagnostic test (RDT)".

Results:
Under the subtitle 'Ov16 seroprevalence in Maridi over time', the following sentence in lines 3-5, 'The seroprevalence among the three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi, non-1.
significantly decreased from 12.5% in 2019 to 8.8% in 2023 (p=0.68)',needs to be reformulated.The three-year-olds born after implementation of more robust onchocerciasis elimination measures could be found only in 2023 and could therefore not have contributed to the RDT seroprevalence in 2019 to enable showing a reduced level in 2023.As the measurements are cross-sectional values the sentence should read correctly thus: The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and bi-annual CDTi measured in 2023 8% and was thus lower than the level of 12.5% measured among three-year-olds in 2019 but the difference was not significant, (p=0.68).
Response: Text altered.We now state" The seroprevalence among three-year-olds, born after implementation of more robust onchocerciasis elimination measures like vector control and biannual CDTi, was measured at 8% in 2023 and was thus lower than the level of 12.5% measured among three-year-olds in 2019.However, the difference was not significant (p=0.68)." My main concern is with the "discussion" and it is specifically about the interpretation of the results from the study.The first paragraph of the discussion points out the increase, albeit insignificant, of the seroprevalence in the 4-9-year-olds following enhanced onchocerciasis measures and interprets the 'increased seroprevalence as 'an indication of intensification of transmission'.Such an interpretation, "increase in transmission" cannot be correct as the enhanced intervention measures clearly must have had increased effect entomologically as well as epidemiologically, through twice a year CDTi even if the coverage was not optimal yet, on the transmission.The entomological impact of "slash and clear" has been reported in the ref.no 5.The publication, "A force-of-infection model for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area by Remme, J et al. in the Bulletin of the World Health Organization, 64: 667-681 (1986) on which the onchocerciasis mathematical model is based, shows that at the onset of intervention of vector control, which interrupts transmission almost immediately, the mf prevalence of age groups in years 0-4, 5-9, continues to rise during the first five years after which it starts falling.It is only in the adults that there is a fall of mf prevalence which is seen in the first three years.A similar effect is found with CDTi where it is even more accentuated the higher the pre-intervention endemicity level of the community.It may be recalled that impact assessment of CDTi in APOC countries did not occur until after five years of CDTI when impact would be expected.The notion was based on this observation.Recently, Lont et al. have shown that there are basic similarities in the effect of application of mf prevalence and seroprevalence in their publication (Lont et al., 2017 1 ).The increase in the seroprevalence recorded in the age group of 3-9 years old children is normal and it is to be expected in that age group particularly when dealing with communities with very high pre-intervention endemicity.It may be noted that some of the young might harbour prepatent infection before the start of the intervention and which may break out in the course of the intervention as Lont et al. discuss in their paper.

1.
As regards to the above considerations, certain statements in the discussion will have to be reviewed and I would like to point them out: 1 st paragraph, line 3-5 "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".The sentence may be deleted as what has been found is to be expected and importantly does not suggest an absence of impact.The continuation is correct as it takes a few years before interruption of transmission can occur showing also the absence of new infections or new seroconversions as applicable to CDTi.
Response: We thank you for these important comments.We eliminated the sentence "These results suggest that the elimination measures did not significantly impact the seroprevalence of the study population".We now included in the discussion "An increase in the seroprevalence in the age group of 3-9 years old children was to be expected, particularly because of the very high pre-intervention endemicity.Indeed some of the children might harbour prepatent infection before the start of the intervention which may break out in the course of the intervention Line 10, "may indicate an intensification of O. volvulus transmission" may be deleted for the same reason as above. 1.

Response: We deleted the the text mentioning the ' intensification of O. volvulus transmission"
Paragraph 2 is well discussed.However, I would like to point out a striking mitigating factor which has either been downplayed or overseen.It is true that no CDTi was carried out in 2020 because of Covid-19 with the negative implications for which Hamley's good work has been quoted to highlight the effect on this study.However, a good look at the first-year results following the first slash and clear activity as read from the paper by Raimon et al.
suggests that Maridi focus possibly did not do as badly as it has been described in this paper.A proper analysis of the events could very well indicate that there could have been more reduction in transmission in the year 2020 effected by the slash and clear than what could have been achieved by annual CDTI alone before the slash and clear was introduced.

Figure 2 .
Figure 2. Location of Maridi County in Western-Equatorian State, South Sudan (produced in Scribble Maps).

Figure 3 .
Figure 3.The Maridi central area with the location of high-(Kazana 1 and 2 and Hai-Matara sites) and low-onchocerciasis transmission zones (Hai-Gabat and Hai-Tarawa sites) and the Maridi Dam (adapted from Colebunders et al. 11 ).

Figure 4 .
Figure 4. Ov16 rapid diagnostic test seroprevalence in 2023 per study site and age (years old).

1 :
Norbert BrattigThe study represents a local investigation on the success of onchocerciasis control endeavour in South Sudan.The authors analysed the antibody response in children to the diagnostic Onchocerca protein Ov16 in 2023 compared to 2019.
the 80-89% range and specificity values in the 97-99% range depending on the assay used and the sample collection assessed.We also added the following references: World Health Organisation.Onchocerciasis: diagnostic target product profile to support preventive chemotherapy, 2021 https://www.who.int/publications/i/item/9789240024496Lobos E, Altmann M, Mengod G, Weiss N, Rudin W, Karam M. Mol Biochem Parasitol.
no 5.The publication, "A force-of-infection model for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area by Remme, J et al. in the Bulletin of the World Health Organization, 64: 667-681 (1986) on which the onchocerciasis mathematical model is based, shows that at the onset of intervention of vector control, which interrupts transmission 1.

1 .
very well indicate that there could have been more reduction in transmission in the year 2020 effected by the slash and clear than what could have been achieved by annual CDTI alone before the slash and clear was introduced.Response: We agree that the strengthening of the onchocerciasis elimination program decreased the O. volvulus transmission in the area.This was strongly suggested by the results of a longitudinal population-based study that showed that the incidence of onchocerciasis-associated epilepsy, including nodding syndrome, decreased in Maridi.This paper has now been published and is included among the references.Jada SR, Amaral LJ, Lakwo T, Carter JY, Rovarini J, Bol YY, Logora MY, Hadermann A, Hopkins A, Fodjo JNS, Colebunders R. Effect of onchocerciasis elimination measures on the incidence of epilepsy in Maridi, South Sudan: a 3-year longitudinal, prospective, population-based study.Lancet Glob Health.2023 Aug;11(8):e1260-e1268.doi: 10.1016/S2214-109X(23)00248-6.

Table 2 . Ov16 rapid diagnostic test seroprevalence among three-to nine-year-olds per survey (2019/2023) and village.
* All p-values were obtained with a Chi-square test/Fisher's exact test.N.A.: not applicable due to a too-rare event; CI -confidence interval.prevalencetypicallydecreases in regions where mass ivermectin distribution is implemented 21 .This study demonstrates the feasibility of using rapid diagnostic tests for evaluating onchocerciasis transmission in highly endemic settings.Until recently, field Ov16 RDTs were overlooked in monitoring onchocerciasis transmission in endemic foci since the gold standard technique recommended by the World Health Organization (WHO) was Ov16 enzyme-linked immunosorbent assay (ELISA)22.However, in practice, there are logistical challenges to performing Ov16 ELISA (need for a cold chain, specialised equipment, and capacity), particularly in remote sites like Maridi.Meanwhile, studies in Cameroon 23 and the Democratic Republic of Congo 24 have found that Ov16 RDT done in the field was helpful in investigating onchocerciasis transmission patterns.Therefore, it is worth considering the widespread use of RDTs as a point-of-care approach for monitoring onchocerciasis in resource-limited sites.

Republic. Ann Trop Med Parasitol. 1959;53(1):97-102.
The authors stated the application of Ov16 rapid diagnostic tests by citing an earlier study of their research group, however, they should explain to the readers the significance and specificity of this O. volvulus antigen.Thus, O. volvulus-specific antibodies demonstrated in 1987 by Sisley et al., Ov16 antibodies by Higazi et al. in 2013 and 2016; Lobos et al. documented first the high specificity of Ov16 in 1991, later Richards et al. documented 2018 the specificity of the operational performance of O. volvulus Ov16 ELISA serological assay, followed by Weil et al. in 2000.

We have written the text to fit the following standards: "Spell out numbers one through nine, except in the case of units of measure or time. For these, and for values of 10 and higher, use Arabic numerals. Always spell out numbers at the beginning of a sentence if the sentence cannot be rearranged to avoid starting with a number." -Springer. Therefore, we have chosen to keep our way of writing numbers the same throughout this document. However, we have adapted the placement of percentages as suggested.
throughout the entire manuscript.Put the percentage after the number and the specimen.Example: Ninety-three of the participating children (37.5%) instead of "Ninety-three (37.5%) of the participating children".four children (1.6%) were identified instead of four (1.6%) children were identified.
good to harmonize the way you write the numbers.Either write in letters or write in numbers as for the 248 children.It must be me form of dermatitis, as evidenced by itching and/or visible skin lesions.Furthermore, four (1.6%) children were identified as having epilepsy2."applied Vlas SJ, Golden A, de Los Santos T, Domingo GJ, Stolk WA.PLoS Negl Trop Dis.2017 Jan 23;11(1):e0005314.doi: 10.1371/journal.pntd.0005314.eCollection 2017 Remme J, Ba O, Dadzie KY, Karam M. A force-ofinfectionmodel for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area.Bull World Health Organ.1986;64(5):667-81 Lobos et al. documented first the high specificity of Ov16 in 1991, later Richards et al. documented 2018 the specificity of the operational performance of O. volvulus Ov16 ELISA serological assay, followed by Weil et al. in 2000.
methods which in part should be better introduced.The article merits indexing for scientists involved in onchocerciasis control.The source data are available.The conclusions drawn 2013 and 2016;

procedure "Five local health care workers were trained to perform the Ov16 SD BIOLINE RDT and administer a questionnaire to the parents of the children."
Response:

We have written the text to fit the following standards: "Spell out numbers one through nine, except in the case of units of measure or time. For these, and for values of 10 and higher, use Arabic numerals. Always spell out numbers at the beginning of a sentence if the sentence cannot be rearranged to avoid starting with a number." -Springer. Therefore, we have chosen to keep our way of writing numbers the same throughout this document. However, we have adapted the placement of percentages as suggested.
Response:

We have adapted the text to fit the suggested standards.
Response:

We now include in the discussion: "In resource-constrained settings, when African children present with dermatitis and itching, scabies should be considered as a possible diagnosis. However, the absence of characteristic scabies lesions was noted in the children included in the study. Additionally, since scabies is treatable with ivermectin, its prevalence typically decreases in regions where mass ivermectin distribution occurs."
. A force-of-infection model for onchocerciasis has shown that even during interruption of O. volvulus transmission by vector control, the microfilarial prevalence of the 0-4 and 5-9 year old age groups continues to rise during the first five years after which it starts falling."And we added the following two references Lont YL, Coffeng LE, de Vlas SJ, Golden A, de Los Santos T, Domingo GJ, Stolk WA.PLoS Negl Trop Dis.2017 Jan 23;11(1):e0005314.doi: 10.1371/journal.pntd.0005314.eCollection 2017 Remme J, Ba O, Dadzie KY, Karam M. A force-ofinfectionmodel for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area.Bull World Health Organ.1986;64(5):667-81