Introduction

Skin conditions affect almost 900 million people each year worldwide and are the third most common reason of medical consultation [1–3]. Among them, yaws is a skin neglected tropical disease (NTD) for which 50 million cases were reported in 1952. Following integration into public health programs, the disease burden was drastically reduced and re-emerged towards the end of the 1970s [4,5]. Among the 16 countries currently endemic for yaws, the 2024 data show that 152,164 suspected yaws cases were reported from 10 countries, with 996 confirmed cases in seven countries. Cameroon is among the nine Africans countries endemic for yaws. In 2024, 1620 suspected cases were reported in Cameroon, but none were confirmed. In contrast, over the three previous years (2021–2023), a cumulative total of 123 confirmed cases were reported [6]. Ghana and Papua New Guinea alone accounted for more than 80% of the reported cases in previous reports [6]. The discrepancy between number of suspected and confirmed yaws cases highlights the gap in the diagnosis. This is problematic because most yaws cases reported to the World health Organization (WHO) are based on the clinical diagnosis. Yaws is difficult to diagnose based on clinical presentation alone, necessitating serological and molecular tools for confirmation. A substantial gap in diagnostics persists because few of these endemic countries have a diagnostic platform and most data are still clinically based [7]. Furthermore, existing laboratory data (e.g., molecular diagnostics) at the country level come from research projects, with samples often analysed outside the endemic countries. This could explain therapeutic failures that stem from challenge of distinguishing between yaws and clinically similar skin ulcers. Treatments targeted specifically for yaws may be ineffective against other similar skin ulcers [8]. As for Africa, Cameroon, Côte d’Ivoire, Congo and Ghana are endemic countries for yaws and have been part of a project to evaluate a Point-Of-Care molecular test [9,10]. This research, which is more conclusive and more decisive, shows that reported cases of yaws are not always caused by Treponema pallidum subspecies pertenue. In some cases, it is another aetiology such as Haemophilus ducreyi or a bacterial co-infection with Treponema pallidum subspecies pertenue in low proportion in many published studies [8,11–13].

Five years into the 2030 NTD roadmap, it is necessary to investigate areas where yaws notifications have been reported. This allows to achieve the objectives set by laboratory confirmation of cases and appropriate treatment. Previous research has identified about 15 bacterial species in the microbiome of skin ulcers similar to yaws, in which some return at varying carriage rates. In 2019 in Papua New Guinea, 15 bacteria were recorded, five of which were strongly involved in the appearance of yaws-like conditions. The micro-organisms included Haemophilus ducreyi with a predominance of 23%, Treponema pallidum subspecies pertenue for a rate of 16%, Streptococcus dysgalactiae (12%), Arcanobacterium haemolyticum (8%) and Corynebacterium diphteriae (8%). Theshotgun metagenomics were used and these results suggest a varied aetiology for skin ulcers in yaws endemic areas [14]. In 2024, in Ghana and the Solomon Islands Staphylococcus spp. and Streptococcus spp. were detected in skin conditions that could be confused with yaws or Haemophilus ducreyi ulcers. These data could not be interpreted as a definitive causation. The study show that 15 bacteria are also recorded in the microbiome of these ulcers. They include Campylobacter ureolyticus with 42% detection, Streptococcus pyogenes at 36%, Arcanobacterium haemolyticum at 34%, and Corynebacterium diphtheriae present at 21% [15]. In 2021 and 2023 in Cameroon, two studies highlighted the predominance of Haemophilus ducreyi in the appearance of skin lesions similar to yaws. Haemophilus ducreyi was identified at 49.6% compared to 23.4% for Treponema pallidum subspecies pertenue in the first study [11]. In 2023 in 14 districts of Cameroon, 30.3% of Haemophilus ducreyi and 5.2% of Treponema pallidum subspecies pertenue [12].

In addition, environmental factors such as water, insects, and fomites have been identified as reservoirs and matrices of the bacteria responsible for yaws-like skin ulcers. In 2017 in Papua New Guinea, 90% of flies were carriers of Haemophilus ducreyi and half of these same flies carried Treponema pallidum subspecies pertenue. Similarly, out of six bed sheets were collected by swabbing 10x10 cm across five separate areas during this research in Papua New Guinea. Two sheets were found to carry Haemophilus ducreyi against a bed sheet containing Treponema pallidum subspecies pertenue [16]. In Cameroon, 8.6% of asymptomatic participants carrying the Haemophilus ducreyi were recorded in 2023 (10). Haemophilus ducreyi was detected on both clothing (13.3%) and in flies (27%) while Treponema pallidum (detection at species level) was tested positive in flies (2.6%) [17]. Fly-to-human mechanical transmission of these bacteria has not been definitively proven. While the bacteria have been detected in flies, the precise epidemiological importance of flies or fomites in transmission remains unknown.

The Akonolinga health district in Cameroon is known for its endemicity to Buruli ulcer [18,19]. Notification data shows 21 cases of yaws recorded in March 2023 (with a total of 69 suspected cases during the year 2023) based on clinical diagnosis (data from the Akonolinga health district). Exploring yaws-like idiopathic ulcers is crucial. This research aimed to strengthen the hypothesis of a bacterial co-detection or even a bacterial involvement other than Treponema pallidum subspecies pertenue in the appearance of yaws-like ulcers in the Akonolinga health district, as well as the potential sources of bacterial spread through animals and environment.

Methods

Nucleic acid amplification

Sample processing.

All samples were removed from the freezer and allowed to thaw at room temperature. For the swabs collected, each 1.5 mL microtube was vortexed, and 200 µL of suspension was taken for DNA extraction. The water samples were centrifuged at 4000 rpm for 10 minutes. The supernatant was then decanted, and the resulting pellet was resuspended in

500 µL of lysis buffer (10mM Tris pH 8.0, 0.1M EDTA pH 8.0, 0.5% SDS) prior to DNA extraction. The pooled flies were ground using a mortar and a pestle, after which 200 µL of the vortexed homogenate was used for DNA extraction.

DNA extraction.

DNA was extracted using the Qiagen QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Briefly, 200 μL of lysed sample after vortexing were processed by adding 20 μL of proteinase K and incubating the mixture at 56°C for 1 hour. Following incubation, 200 μL of AL buffer were added and another incubation at 70°C for 10 minutes was done. A 200 μL of absolute ethanol was added and a centrifugation followed. The mixture was then transferred to spin columns and washed sequentially with 500 μL of AW1 and 500 μL of AW2. DNA was eluted by adding 100 μL of molecular-grade water to the spin column, and the resulting DNA was stored at -20°C. The procedure was the same for all collected samples.

Polymerase chain reactions.

For Treponema pallidum subspecies pertenue, Staphylococcus aureus and Arcanobacterium haemolyticum, DNA amplification was performed by end-point PCR targeting the Tp0548, nuc and aln genes, respectively (see Table 1 for more details). Then, the amplified products were detected on agarose gel electrophoresis. When Tp0548 gene was detected, a second PCR for subspecies pertenue was required by targeting TprL gene. Prior to this step, sample quality, presence of host DNA, and absence of amplification inhibitors in the extracted DNA were assessed by amplifying a 268 bp fragment of the ß2-microglobulin (beta-2-M) gene, a housekeeping gene using the primers beta-2-M_F (5’-CAACTTCATCCACGTTCACC-3’) and

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Table 1. Polymerase chain reactions conditions.

https://doi.org/10.1371/journal.pntd.0014579.t001

beta-2-M_R (5’-GAAGAGCCAAGGACAGGTA-3’) [20]. Haemophilus ducreyi was detected by real-time PCR by amplifying the hhda gene, which codes for haemolysin.

For each target, in-house positive controls consisting of known DNA extracts were used. The negative controls consisted of negative DNA extraction control (molecular grade water processed alongside the samples to monitor the extraction process) and a no-template control (molecular grade water).

The different amplification programs for each target are recorded in the Table 1:

Data analysis.

All data were entered into Excel, and simple descriptive statistics were used to summarize sample and patient characteristics, as well as analytical results by amplification. The Statistical Package for Social Sciences software (version 22.0, SPSS Inc., Chicago, IL, USA) was used for the data analysis. All categorical variables were compared using the Fisher’s exact test. A p-value ˂ 0.05 was considered significant.

Results

Study population, yaws-like ulcer prevalence and detection of bacteria in specimens

We screened 5,931 children, including 3,215 boys (54.21%) and 2,716 girls (45.79%) from 98 villages and 21 schools. From them, we obtained 28 participants with ulcers similar to yaws, with a median age of 9 years, and 28 contacts aged between 5 and 15 years (Table 2). We collected potential transmission agents including 6 water samples, 5 dog samples and 4 pool of flies (more details in the S1 Table). The prevalence of skin ulcers clinically similar to yaws was 0.5%. During active case searching, eight suspected cases of Buruli ulcers were identified and were reported to the health district for the management. Additional information on reported Buruli ulcers is available in the S2 Table.

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Table 2. Characteristics of population screened and bacteria detected in yaws-like ulcers in Akonolinga health district in Cameroon.

https://doi.org/10.1371/journal.pntd.0014579.t002

Haemophilus ducreyi, Arcanobacterium haemolyticum and Treponema pallidum subspecies pertenue were not detected in any human samples (symptomatic or asymptomatic), nor were they found in environmental matrices, flies, or domestic animals.

Staphylococcus aureus was detected in 9 participants with yaws-like ulcers out of the 28 sampled (32.14%) and in 6 asymptomatic individuals (21.43%) with p = 0.547. Detection of Staphylococcus aureus was significantly higher in males (42.85%) compared to females (9.52%), p = 0.031. At least one case of skin ulcer associated with Staphylococcus aureus was recorded in 4 of the 13 health areas screened with varying proportions: Mengang, Akonolinga urbain, Emvane-sso and Yeme-Yeme (see Table 2). Colonization of Staphylococcus aureus in asymptomatic people has been identified in three health areas: Mengang, Djoudjoua and Yeme-Yeme.

Discussion

In the 2030 WHO roadmap for yaws eradication, a crucial aspect is the laboratory confirmation of suspected cases [25,26]. This investigation was carried out in the Akonolinga health district, known for its endemicity to Buruli ulcer, in order to determine whether yaws is co-endemic, and alternatively to look for other pathogens as aetiological agents of skin ulcers clinically similar to yaws.

We found a 0.5% prevalence of skin ulcers similar to yaws in the health district which is low compared to other yaws endemic health districts in Cameroon such as Djoum, Mbang, Abong-Mbang, Sangmelima, and Yokadouma with prevalence over 1% [10]. None of the 28 suspected cases of yaws were PCR-confirmed. This finding aligns with previous studies conducted across nine health districts out of 12 where yaws has been reported endemic but tested negative [10].

With the exception of Staphylococcus aureus, with similar detection rate in symptomatic participants compared to asymptomatic individuals, the other bacteria were not detected in the samples collected. These results are similar to the findings in 2024 and 2019 in yaws-endemic countries [14,15], where approximately 26% and 81% of samples were tested positive to Staphylococcus aureus in Papua New Guinea, Ghana and the Solomon Islands respectively. Those baseline studies performed broad molecular sequencing, whereas the current study utilized targeted PCR. Staphylococcus aureus is a common pathogen in chronic wounds, with various detection rates across diverse geographical regions; ranging from 30% to 93% [27–29]. The statistics show that Staphylococcus aureus was more detected in boys than girls, suggesting that boys have higher colonisation rates than females. There was no significant difference between symptomatic and asymptomatic participants. In agreement with the aforementioned research, and given the absence of a significant difference between symptomatic and asymptomatic participants, presence of Staphylococcus aureus warrants in-depth investigation into its pathogenic role, carriage and prevalence in yaws-like lesions in Cameroon.

In addition, some bacteria detected in these tropical lesions such as Arcanobacterium haemolyticum, Haemophilus ducreyi and Treponema pallidum subspecies pertenue [9,14,15] have not been identified in this part of central Cameroon. None of the targeted bacteria were detected in the matrices studied (flies, waters, dogs). Given the exceptionally small animal and environmental sample sizes, it remains a challenge to determine whether these matrices serve as constant reservoirs of the agents responsible for tropical skin ulcers. However, the studies conducted on the Lihir Islands detected Haemophilus ducreyi and Treponema pallidum subspecies pertenue in human and environmental samples [16]. In Cameroon, DNA of these bacteria have been evidenced in flies and fomites [17].

The main limitation of this work could be the small volume of water analysed for environmental pathogen detection compared to similar studies that used larger sample volumes [30,31]. The relatively small size of the clinical lesions within the screened population may be another limitation that affects statistical analyses. However, the findings can be also interpreted as impact of previous public health efforts to fight against diseases in the health district.

Our results demonstrates that Treponema pallidum subspecies pertenue and Haemophilus ducreyi are not the sole aetiological agents of skin lesions clinically similar to yaws. These findings highlight the need to to a re-evaluate suspected cases and surveillance of similar emerging tropical ulcers. Additionally, the integrated approach facilitated the detection and management of Buruli ulcer cases in the health district, thereby strengthening the NTD surveillance system.

Conclusion

This work shows that only Staphylococcus aureus was detected in similar proportions among both symptomatic participants and healthy controls in the Akonolinga health district. It actually suggests that no definitive microbial causal agent has been identified. Skin ulcers clinically resembling yaws in this health district are driven by aetiologies other than Treponema pallidum subspecies pertenue, highlighting the urgent need for wider diagnostic tools to prevent the misallocation of national NTD resources.

Supporting information

Acknowledgments

We thank the EDCTP (European and Developing Countries Clinical Trials Partnership)/CANTAM (Central Africa Network for Tuberculosis, AIDS/HIV and Malaria) for the material support. The authors acknowledge the health workers of the Akonolinga health district for their contribution, school officials and communities for their willingness to participate in this study.