LAMP4yaws: Treponema pallidum, Haemophilus ducreyi loop mediated isothermal amplification - protocol for a cross-sectional, observational, diagnostic accuracy study.

INTRODUCTION: Yaws, caused by the bacterium Treponema pallidum subsp. pertenue, is a neglected tropical disease targeted for eradication by 2030. Improved diagnostics will be essential to meet this goal. Diagnosis of yaws has relied heavily on clinical and serological tools. However, the presence of coendemic cutaneous skin ulcer diseases, such as lesions caused by Haemophilus ducreyi (HD), means these techniques do not provide a reliable diagnosis. Thus, new diagnostic tools are needed. Molecular tools such as PCR are ideal, but often expensive as they require trained technicians and laboratory facilities, which are often not available to national yaws programmes. METHODS AND ANALYSIS: The LAMP4yaws project is a cross-sectional, observational, diagnostic accuracy study of a combined Treponema pallidum (TP) and HD loop mediated isothermal amplification (TPHD-LAMP) test performed under real world conditions in three endemic countries in West Africa. Individuals with serologically confirmed yaws will be recruited in Cameroon, Côte d'Ivoire and Ghana. Each participant will provide paired swabs, one of which will be sent to the respective national reference laboratory for yaws quantitative PCR and the other will be tested for both TP and HD using the TPHD-LAMP test at local district laboratories. Sensitivity and specificity of the TPHD-LAMP test will be calculated against the reference standard qPCR. We will also assess the acceptability, feasibility and cost-effectiveness of the test. We anticipate that results from this study will support the adoption of the TPHD-LAMP test for use in global yaws eradication efforts. ETHICS AND DISSEMINATION: We have received ethical approval from all relevant institutional and national ethical committees. All participants, or their parents or guardians, must provide written informed consent prior to study enrolment. Study results will be published in an open access journal and disseminated with partners and the World Health Organization. TRIAL REGISTRATION NUMBER: NCT04753788. © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

A multicountry evaluation of the new Treponema pallidum and Haemophilus ducreyi loop mediated isothermal amplification (TPHD-LAMP) test performed in a large ‘real-world’ programmatic setting.

Capacity building and strengthening of in-country teams to aid with future yaws eradication efforts.

Extensive social science analysis will allow us to highlight barriers to achieving yaws eradication and enable us to determine if the new LAMP test is acceptable, feasible and costeffective for use in national yaws programmes.

The TPHD-LAMP is not a true point of care test and currently requires laboratory facilities to carry out the test.

The study is only being carried out in three of 15 yaws endemic countries. With a large burden of yaws in Asia and the Western Pacific, an evaluation in these populations may be required to ensure local context, including genetic variation, does not affect the performance of the TPHD-LAMP test.

Introduction

Yaws is a neglected tropical disease (NTD) caused by the spirochete bacterium Treponema pallidum subsp. pertenue (TPE), and is closely related to the causative agent of syphilis, Treponema pallidum subsp. pallidum.1 2 Yaws mainly affects children aged 5–15 years1 living in endemic areas. It is mostly found in rural, remote communities in the tropics with scarce access to health facilities and sanitation.3 The disease is spread through direct skin-to-skin contact.4 Yaws presents in stages: primary yaws appears as a single papilloma or ulcer, and is highly infective. After the initial active infection, a period of latency ensues where the patient appears clinically healed but is still seroreactive for the disease. If untreated, the patient may develop secondary yaws, affecting the lymph nodes and bones. This may be followed by further periods of latency and eventually if left untreated, up to 10% of cases may develop tertiary disease which can result in disfiguring and destructive lesions, which can lead to life-long deformity and disability.4

Under the new NTD roadmap 2021–2030, yaws is targeted for eradication by 2030.5 It is anticipated that this goal will be achieved through mass drug administration (MDA) with azithromycin, an antibiotic shown to be effective against yaws infection as a single oral dose treatment. Azithromycin is distributed to entire endemic communities, where there is at least one single case of serologically or molecularly confirmed yaws, known as Total Community Treatment (TCT). After an initial round of TCT, eradication efforts move to targeted treatment of remaining cases and their close contacts (Total Targeted Treatment). Clinical diagnosis of yaws can be challenging as other pathogens can cause skin ulcers with similar characteristics to yaws. The most common of these is Haemophilus ducreyi (HD), which can be responsible for over 50% of the ulcers clinically diagnosed as yaws.6 Currently, molecular confirmation through PCR is the gold-standard diagnosis for yaws but is often unavailable at the district level in yaws-endemic countries.

In the campaign for yaws eradication, it is vital that all endemic communities are identified. In post-MDA surveillance, it is key that no yaws re-emergence is missed or misdiagnosed. While PCR is a highly sensitive and specific tool for yaws diagnosis and, therefore, recommended by WHO to aid with eradication, it depends on trained laboratory staff and requires well-equipped laboratory facilities. This is often not available in remote yaws endemic settings with little infrastructure. A combined Treponema pallidum (TP) and HD loop mediated isothermal amplification test (TPHD-LAMP)7 8 has been developed for simultaneous detection of these two primary causes of yaws-like lesions. This test is user-friendly and does not require costly thermocyclers. Therefore, it is suitable for use in local hospital laboratories close to the patient, to test for yaws and HD. The TPHD-LAMP test has been previously evaluated in a laboratory setting8 using swab samples collected during yaws studies in Papua New Guinea and Ghana with promising results: sensitivities of 84.7% (95% CI 72.5% to 92.4%) for TP and 91.6% (95% CI 85.8% to 95.3%) for HD were reported. The respective specificities were 95.7% (95% CI 92.0% to 97.8%) and 84.7% (95% CI 77.4% to 90.1%). The sensitivities for both targets were reduced in mixed infection samples: 68.4% (95% CI 43.5% to 86.4%) for TP and 73.7% (95% CI 48.6% to 89.9%) for HD. However, the test is yet to be evaluated in a real-world setting. The TPHD-LAMP test can also potentially be adapted to include TP macrolide resistance detection, a feature which is hugely important in light of the recent detection of macrolide-resistant TPE strains, which emerged after MDA campaigns in Papua New Guinea.9 10

Methods

Study design

Study sites, population and participant eligibility

The study will take place in three countries in West Africa: Cameroon, Côte d’Ivoire and Ghana. These three countries have been selected as they contribute to the highest recorded prevalence of yaws in Africa.11 12 In each country, national disease reporting data have been used to select districts that are suspected or known to be highly endemic for yaws, facilitating rapid fulfilment of the required number of yaws cases for the study. The number of selected villages/communities will depend on the prevalence of the disease and the length of time needed to reach our target sample size. Recruitment will cease whenever the desired sample size is reached.

Aims

The primary aim of the project is to perform a cross-sectional, observational, diagnostic accuracy evaluation of the novel TPHD- LAMP test to assess its value as a diagnostic tool for use in yaws eradication and surveillance programmes. This will include testing the TPHD-LAMP test’s diagnostic accuracy against the reference standard real-time qPCR tests performed at national reference laboratories, and performing social science and health economic studies to assess its feasibility for deployment in yaws-endemic areas.

Objectives

Primary objective

(1) Assess the diagnostic accuracy (sensitivity and specificity) of the TPHD-LAMP test for TP and HD diagnosis under programmatic conditions in yaws-endemic areas in Ghana, Côte d’Ivoire and Cameroon in order to determine its suitability as a diagnostic tool in yaws eradication programmes.

Perform acceptability and feasibility and health economic studies to see if it is realistic to use the TPHD-LAMP test to aid with eradication efforts and/or surveillance.

Develop an external quality assurance (EQA) scheme for the molecular diagnosis of yaws and HD.

Data and sample collection for objective 1

Participant enrolment will take place between April 2021 and September 2022. Recruitment will cease prior to this if the desired sample size is met. Laboratory procedures will be conducted concurrently with the aim to complete all assays by November 2022. In each district (figure 1), we will use active case finding to identify eligible participants for the study. Primarily, we will include those seropositive for treponemal and non-treponemal antibodies as this is the best indicator of an active yaws infection. Recruitment will consist of rapidly screening participants living in communities with reported cases of suspected or confirmed yaws. Individuals will be invited to participate, based on the following inclusion and exclusion criteria.

Figure 1

Highlighted districts indicate where active case searching will take place in Cameroon (A), Côte d’Ivoire (B) and Ghana (C)

Able to give written informed consent if over the age of consent (≥18 years in Côte d’Ivoire and Ghana, ≥21 years in Cameroon), or parent/guardian provides written consent.

Verbal assent if under the age of consent.

Presenting with a yaws-like lesion.

Resident in yaws-endemic district.

Participants aged 6 months and older.

Does not consent/unwilling to participate.

Refusal of village chief (for village inclusion), or refusal of individual and/or guardian (for individual inclusion).

Permanent disability that prevents or impedes study participation and/or comprehension.

Participants under 6 months of age.

Exclusion criteria

Screening for the primary objective will consist of a visual examination of a participant’s arms, legs, head and torso to look for the presence of lesions consistent with yaws, such as papilloma which tend to have a yellow crust, or yaws ulcers which are typically round, have raised edges and are not painful. All screening will be performed by healthcare workers trained to recognise yaws and perform serological tests. Following written informed consent, participants presenting with yaws-like lesions will undergo an SD Bioline (Abbott, USA) test which detects long-lived anti-treponemal antibodies; these markers generally remain positive for life after exposure to a treponemal infection. If reactive, a Chembio Dual Path Platform test (Chembio Diagnostics, New York, USA) will be performed which will allow for simultaneous detection of both treponemal and non-treponemal antibodies the latter of which are a more reliable indicator of current infection. Results of all serological tests will be recorded in Open Data Kit (ODK) data collection forms. Participants reactive for both treponemal and non-treponemal antibodies will be invited to enrol in the study (figure 2). In addition, up to 10% of participants with negative serology for either test will be randomly selected using the Open Data Kit (ODK) data collection forms and invited to enrol, allowing us to sample from children with negative serology that may not have seroconverted yet, for example, if they are early in an infection (in an early infection, there may be detectable bacteria using molecular diagnostic tests but individuals may not have yet mounted a serological response).

Figure 2

Schematic of the LAMP4yaws recruitment strategy. To find 6000 suspected of ways, we expect to need to screen around 60 000 people. Of these, we expect one-third to be positive for treponemal antibodies and around 10% of individuals to be positive for both treponemal and non-treponemal antibodies. These participants will be invited to enrol in the study.

For each participant, we will collect two swabs from their largest yaws-like lesion. If presenting with a yaws papilloma, a curette will be used to lift the scab first. If needed, saline solution will be used to moisten the wound. The two swabs will be simultaneously rolled over a 1 cm2 area of the lesion for five seconds. Both swabs will then be stored separately in 500 µL of lysis buffer (10 mM Tris-HCL, pH 8.0; 100 mM EDTA, pH 8.0; 0.5% SDS)13 within cool boxes, until transport to a refrigerator at the local district laboratory, within 48 hours of collection. A lesion assessment will be carried out, including recording the size, shape and other physical features of the lesion. We will ask if the participant has suffered with these yaws-like lesions before and if they have received treatment for the current presentation.

All participants with yaws-like lesions will be offered treatment with 30 mg of azithromycin/kg (max 2 g),14 regardless of whether they choose to enrol in the study or their serology results. As per WHO guidelines,14 after 2–4 weeks, all participants with serologically-confirmed yaws will be followed up to ensure clinical resolution. Any participants with suspected treatment failure (those presenting with lesions that have not healed, or are worse), will be treated with injectable benzathine penicillin as per WHO guidelines14 and two additional swabs will be collected to test for mutations conferring macrolide resistance in TPE.

Sample size considerations

To measure a 95% sensitivity of the TPHD-LAMP test (±3%), a total of 210 qPCR positive TP cases are needed.15 A similar number of cases are required to assess sensitivity and specificity for HD. We calculated the number of participants we would need to screen and enrol to achieve our target of 210q PCR-positive cases based on previously available data and assumptions: (1) 10% of suspected cases have positive rapid-diagnostic-test (RDT) serology results16 and (2) approximately 35% of RDT-positive cases have qPCR-detectable TP,17 with a similar number positive for HD.18 19 Even in endemic districts of West Africa, previous case searches report suspected childhood yaws prevalence of ≤10%. Our active case searches, therefore, need to screen approximately 60 000 individuals to identify an average 600 serology-positive and 210 qPCR-positive cases for primary analysis (figure 2).

Training

Prior to participant enrolment, standard operating procedures (SOPs) for all aspects of the study will be developed. All healthcare workers and laboratory staff will receive study-specific training according to the relevant SOPs. This will include an introduction to the protocol and study rationale as well as Good Clinical Research Practice and Good Clinical Laboratory Practice sessions. Trainees will be introduced to the study documentation and data entry forms and will be shown how to correctly perform the informed consent procedure. Healthcare workers will be trained in conducting serological tests and interpreting the results as well as lesion swab collection and sample storage. Laboratory technicians in the district laboratories will be shown how to store and process samples, including DNA extraction and the TPHD-LAMP test. We will also explain the importance of maintaining a clean environment within which to work, as well as teaching about important sources of contamination, how to avoid and detect this (including the use of no template controls and negative extraction controls) and how to respond if any issues with the laboratory techniques arise. In each national reference laboratory, the qPCR tests will be validated prior to use. Training will be provided in-person by the international study coordinator (BLH), the field coordinator (CG-B), work package lead on capacity building (ST) and the study coordinators in each country (LAB, KAH and ST).

Laboratory analysis

TPHD-LAMP conducted at district laboratories

The TPHD-LAMP test will be conducted at selected local district hospitals or health centres (table 1). We will provide the local teams with all the necessary equipment, reagents and consumables required to perform the test.

Table 1

District and reference laboratories involved in sample processing

Country	Cameroon	Côte d’Ivoire	Ghana
Reference laboratories	Centre Pasteur du Cameroun	Institut Pasteur de Côte d’Ivoire	Noguchi Memorial Institute for Medical Research
District laboratories	To be confirmed*	Divo District laboratoryTabo District laboratoryYamoussoukro District Laboratory	Tetteh Quarshie Memorial HospitalAsuboi Health CentreAdeiso Health Centre

*District laboratories in Cameroon will be selected as the study proceeds, dependant on successful case finding.

When samples reach the district laboratory, DNA will be extracted from a 200 µL sample of swab lysis buffer using the MAST ISOPlex DNA/RNA extraction kit and eluted into 80 µL RNAse free water. This kit has been chosen because it is a magnetic bead-based extraction that forgoes the need for fast centrifugation. After extraction, the DNA eluate is stored at 2°C–8°C until the TPHD-LAMP test is performed, within 1 week.

To perform the TPHD-LAMP test,8 5 µL of DNA is mixed with reaction buffer, Bst Polymerase and a dye mix. The test is then incubated at 64°C in a MAST Tubescanner for 60 min. To ensure the test is performing correctly, that samples have been successfully extracted and that contamination has been avoided, a negative extraction control (extracted lysis buffer), a positive control (a plasmid containing both pathogens’ target gene sequence) and a no template control (NTC) of double-distilled water (ddH20) will be included in each run. The TubeScanner machine controls the temperature and reads fluorescent signals of a multiplex mediator probe. The result output for each target is positive or negative and is generated within 1 hour. Tests which fail quality control (one or more of the controls showing discordant results) will be repeated and troubleshooting will occur if the problems persist. Results will be immediately entered into an ODK electronic data entry form. Results will not be relayed to participants as the test has not received regulatory approval and is therefore for research purposes only.

Reference standard qPCRs conducted at national reference laboratories

One of each lesion swab sample will be transported to the national reference laboratory in each country (table 1) and stored at 2°C–8°C until processing. Here, DNA will be extracted using the Qiagen DNA mini Qiamp kit (Qiagen, Germany). Again, the sample will consist of 200 µL of lysis buffer and DNA will be eluted into 100 µL of RNAse free water.

First, 5 µL of DNA will be tested using a qPCR targeting the human endogenous gene RNAse P.20 21 This will allow for confirmation that the sample contains human tissue and has been extracted successfully. Samples positive for the RNAse P gene will then be tested for TP and HD using two separate PCR reactions, again 5 µL of DNA will be used in each reaction. The polA gene and the V8 region of the 16S ribosomal RNA gene will be targeted for TP and HD, respectively.22 These targets have been selected as they have been widely used for the detection of TPE and HD.22 All samples will be tested twice within one assay and if results for any sample are discordant, the qPCR will be repeated for that specimen. Sample results will be considered positive or negative for each target and these results will be recorded on an ODK form for real-time analysis. Plasmids containing both gene targets, a negative extraction control (extracted lysis buffer) and an NTC (ddH20) will be included in each qPCR run and if any discordant results of controls are recorded the assay will be repeated.

Data analysis plan for the primary outcome

The national reference laboratory qPCR results will be considered the reference standard result. We will calculate the sensitivity and specificity of the new test as well as the positive and negative predictive values. These will be calculated separately for both pathogens. A secondary analysis on mixed infection samples will also be conducted. Data analysis will be performed using R V.4.0.223 or above.

Social science and health economic methods

We will investigate how cost-effective the TPHD-LAMP test is, as well as the feasibility and ease with which it can be conducted at local district laboratories. The health economic analysis will involve costing all aspects of both diagnostic tests, from sample collection to results generation, including shipping and transport costs, and staff time and salaries. We will perform a knowledge attitudes and practice questionnaire with community members, healthcare workers, traditional healers and other key stakeholders. This will allow us to highlight potential barriers to yaws eradication as well as local beliefs regarding yaws diagnosis and treatment and perceptions of the disease. Separate questionnaires have been designed for healthcare workers, district laboratory technicians and reference laboratory staff to determine how feasible and/or acceptable the TPHD-LAMP test is in its current form. All questionnaires will be completed in ODK. Data will be analysed using a mixture of quantitative and qualitative measures, which will be covered elsewhere.

External quality assurance

A second objective of the project is to develop and use an EQA scheme for TPE and HD diagnosis. The EQA scheme in the LAMP4Yaws project comprises three components: in-country laboratory monitoring, re-testing of a subset of samples at partner institutions, and proficiency testing (PT). For in-country laboratory monitoring, the Study Coordinator (BLH) and capacity building lead (ST) will be involved in the initial laboratory skills training and assay validation at both the reference and district laboratories and will also participate in monitoring visits to all laboratories involved in the project. For the second component, DNA extracts of a subset of 20% of samples collected during the study will be retested with both the TPHD-LAMP and qPCR tests at a yaws-specialised laboratory at the Friedrich-Loeffler-Institut. In case of substantial variances, an on-site evaluation will be performed.

For the final EQA component we will develop PT panels for the reference laboratories performing the qPCRs and for the district laboratories performing the TPHD-LAMP tests. For this we will create two plasmids (one with the TP polA gene target and one with the HD 16SrRNA gene target). Following amplification, plasmids will be quantified using digital PCR, tested for long-term stability and the limits of detection on both the TPHD-LAMP and qPCR platforms. Cotton swabs will be spiked with HEK293 cells to simulate the human cell background of a clinical sample. Subsequently, a panel of seven swabs will be prepared, with swabs containing either one, both or none of the plasmids in different concentrations and combinations. The participating laboratories will receive a blinded PT panel transported at ambient temperature. Each laboratory will be bi-annually re-evaluated using a new set of blinded samples. The basic criteria for successful participation will be the correct identification of the NTC and the high positive samples in all triplicates, as well as an appropriate SD within the triplicates. We will implement a 3-grade evaluation system, where the participants can obtain perfect results, fulfil the minimum requirements, or fail. Only laboratories which have fulfilled the minimum requirements will be able to start or proceed with the testing of clinical samples. If a laboratory fails the PT, it will undergo a detailed quality assessment to identify critical points to take appropriate corrective measures before repeating the PT.

Ethics

We have ethical approval from the London School of Hygiene & Tropical Medicine (LSHTM) ethics committee (Reference: 21633, 19 August 2021) as well as local and national committees in each country. We were granted approval to conduct the study from Cameroon’s National Ethics committee for Human Research (22 December 2020), the National Research Ethics Committee in Côte d’Ivoire (16 September 2020) and in Ghana by the Noguchi Memorial Institute for Medical Research NMIMR institutional review board (6 November 2020) and Ghana health service National Research Ethics Committee (29 April 2021). Prior to any study procedures, written informed consent will be obtained from participants or from parents or guardians if the participant is under the age of consent. Following national recommendations, informed consent will be conducted in English or French, with the assistance of a community representative translating into the local language, if necessary. If a person is illiterate, the study information and consent procedure will be clearly explained to them by a member of the study team or a local translator. Children over ten years old will also be required to give verbal assent for enrolment. If any participants wish to withdraw from the study at any point post-enrolment, that participant’s information will be completely eliminated. Consent forms, approved by all relevant ethical review boards, will be available in English and French, as appropriate (see online supplemental material 1).

Data storage and security

All participants recruited will be randomly assigned a study identification number and only coded data will be analysed. Data on the: (1) number of screened individuals, (2) number of treponemal and non-treponemal antibody rapid tests conducted and (3) number of serologically confirmed cases, will be shared with national programme managers at regular intervals throughout the study. Data collected through ODK will be stored on the LSHTM ODK Central Server. Access to this will be limited to the principal investigator (MM), the international study coordinator (BLH) and the field coordinator (CG-B). Data will be made available to study personnel on reasonable request. Any data downloaded from ODK central will be stored on institutional network drives with firewalls and security measures in place. Hard copies of signed consent forms will be stored in secured offices at the reference laboratory in each country.

Dissemination

The findings of this study will be reported back to all the relevant project stakeholders. The results of the study will be published in an Open Access journal. If this study’s findings indicate that the new TPHD-LAMP test could be deployed successfully to aid with yaws eradication, we will engage with WHO and national and international networks to ensure relevant policy makers, public health advisors and researchers are aware of the study and its findings.

Patient and public involvement

There was no patient and public involvement in the design of this study protocol.

Discussion

Under the new NTD road map 2021–2030, yaws and dracunculiasis are the only two diseases targeted for eradication. Currently, PCR is the most sensitive and specific tool for yaws diagnosis. However, it is not always available in endemic countries, especially in the most remote and rural areas where the disease is more prevalent. To achieve eradication, sensitive diagnostic tools that can detect a singular case of yaws are needed. This will be important both for deciding whether to implement MDA and for surveillance post-MDA. These diagnostic methods will also need to be highly specific so as to not mis-diagnose yaws resulting in unnecessary interventions, and potentially leading to the development of antimicrobial resistance.

The LAMP4yaws study will assess the accuracy of the TPHD-LAMP by conducting an evaluation of the test performed at local rural laboratories compared with the reference standard qPCR conducted at national reference laboratories. The TPHD-LAMP test has shown promising results during a laboratory evaluation but it is also important to assess its performance under programmatic conditions. Isothermal tests, such as LAMP, offer a solution to the problems faced by PCR, as they are cheap, easy to perform and can be operated close to the patient. Having a test that can be conducted locally, allows for prompt detection of yaws cases, which will be particularly important post-MDA to prevent onward transmission of remaining cases. The establishment of the EQA scheme and strengthening of in-country yaws diagnostic capacity will be an invaluable aid for future eradications efforts in our three study countries in West Africa, as well as other yaws-endemic countries globally.

There are some limitations to this study. First, the LAMP4yaws test is not a true point of care test as it must be performed within a basic laboratory facility. In its current form it also requires a cold chain. Power shortages in the district laboratories may not be uncommon, thus conducting the TPHD-LAMP assay could be hindered in these conditions. The TPHD-LAMP test also relies on reagents produced by a single company based in Europe. If the TPHD-LAMP is to be adopted by national programmes, the infrastructure to access these products easily at a low cost will be essential. The acceptability and feasibility and cost-benefit analysis will allow us to highlight which challenges require overcoming before the test can be successfully deployed.

If the TPHD-LAMP is shown to be useful, further work would include evaluating the test in other yaws endemic countries, such as Papua New Guinea and Solomon Islands, where the highest burden of yaws is found. This will allow us to ensure difference in local context and genetic variation of TPE will not affect the performance of the TPHD-LAMP. A recombinase polymerase amplification test for TP and HD has also recently been developed.24 Similarly, this test relies on isothermal amplification, which can be conducted close to the patient and has performed well in a laboratory evaluation. Pursuing a clinical evaluation in a programmatic setting will also be useful.

Finally, the detection of azithromycin resistant TPE strains in a post-MDA setting in Papua New Guinea9 10 has the potential to derail eradication efforts, which are primarily based on mass treatment with this antibiotic. The LAMP test is currently under development to include multiplex detection of TP and the point mutations that confer resistance. If resistance emerges, its prompt detection is vital to prevent transmission of these strains. A successful evaluation of this test when available could be key to allowing prompt detection of these cases.

The need for new point of care diagnostics for yaws has been on the public health agenda for years. However, this need has become more urgent since the documented emergence of azithromycin resistance. Successful evaluation of the TPHD-LAMP test, demonstrating high sensitivity and specificity, could provide an invaluable tool for the future of yaws eradication.