A loop-mediated isothermal amplification test for yaws: a multi-country diagnostic accuracy evaluation.

BACKGROUND: To meet the WHO target of eradicating yaws by 2030, highly sensitive and specific diagnostic tools are needed. A multiplex Treponema pallidum-Haemophilus ducreyi loop-mediated isothermal amplification (TPHD-LAMP) test holds promise as a near-patient diagnostic tool for yaws and H ducreyi. We conducted a prospective evaluation in Cameroon, Côte d'Ivoire, Ghana, and the Republic of the Congo to determine the diagnostic accuracy of the TPHD-LAMP test, as well as to assess its acceptability, feasibility, and cost. METHODS: Active case searching within schools and communities was used to locate participants with clinically suspicious laws-like lesions. Individuals with serologically confirmed active yaws provided paired lesion swabs between March, 2021, and April, 2023. For each participant, one swab was tested with the TPHD-LAMP at a local district laboratory and the other with reference quantitative PCR (qPCR) tests conducted at national reference laboratories. The primary outcome was TPHD-LAMP test sensitivity and specificity compared with qPCR. Laboratory technicians were interviewed using a multiple-choice survey to gauge acceptability and feasibility of the TPHD-LAMP test. Costs of each test were calculated. FINDINGS: Of 3085 individuals with at least one suspected yaws lesion, 531 (17%) were serologically confirmed. We enrolled 493 participants with seropositive yaws and a further 32 with negative serology. The sensitivity of the TPHD-LAMP test for detecting T pallidum was 63% (95% CI 56-70) and the specificity was 66% (95% CI 61-71). Sensitivity and specificity for T pallidum improved to 73% (63-82; p=0·0065) and 75% (68-80; p=0·0003), respectively, in H ducreyi-negative samples. Interviews highlighted challenges in user-friendliness and practicality of the TPHD-LAMP test. The cost of the test per sample was one third of that of qPCR, although the TPHD-LAMP test entailed higher costs to establish the assay. INTERPRETATION: This was the first multi-country diagnostic evaluation of a molecular test for yaws. The TPHD-LAMP testing, in its current form, falls short of the WHO target product profile criteria for yaws diagnostics. These findings highlight the importance of assessing new diagnostics in real-world conditions to ensure their suitability for programmatic use. FUNDING: The EDCTP2 programme supported by the EU.

Sequence typing of Haemophilus ducreyi isolated from patients in the Namatanai region of Papua New Guinea: Infections by Class I and Class II strain types differ in ulcer duration and resurgence of infection after azithromycin treatment.

Haemophilus ducreyi (HD) is an important cause of cutaneous ulcers in several endemic regions, including the Western Pacific Region, especially among children. An HD sequence typing on swab samples taken from 1,081 ulcers in the Namatanai district of Papua New Guinea, during the pilot study for treatment of yaws, has been performed using the Grant typing system. Of the 363 samples that tested positive for the 16S rDNA of HD, the dsrA sequences of 270 samples were determined. Altogether they revealed 8 HD strain types circulating in Namatanai, including seven strain types of Class I (I.3, I.4, I.5, I.9, I.10, I.11, I.12) and one strain of Class II (II.3); four Class I types (I.9, I.10, I.11, I.12) were novel. The southern region of Namatanai (Matalai Rural) was identified as the region with the lowest genotype diversity and with most infections caused by HD Class II. The middle and northern subdistricts were affected mainly by HD Class I. Analysis of patient characteristics revealed that Class II HD infections were more often represented by longer-lasting ulcers than Class I HD infections. An increase in the prevalence of the I.10 strain was found after azithromycin administration compared to the untreated population at baseline likely reflecting higher infectivity of HD Class I, and more specifically strain type I.10.

Coinfection of a yaws patient with two closely related Treponema pallidum subsp. pertenue strains: A rare event with potential evolutionary implications.

The etiological agent of yaws is the spirochete Treponema pallidum (TP) subsp. pertenue (TPE) and infects the children of Papua New Guinea, causing ulcerative skin lesions that impairs normal growth and development. Closely related strains of Treponema pallidum subsp. pertenue, JE11, and TE13 were detected in an ulcer biospecimen derived from a 5-year-old yaws patient. Cloning experiments validated the presence of two distinct but similar genotypes, namely TE13 and JE11, co-occurring within a single host. While coinfection with highly related TPE strains has only limited epidemiological and clinical relevance, this is the first documented coinfection with genetically distinct TP strains in a single patient. Similar coinfections in the past were explained by the existence of over a dozen recombinant loci present in the TP genomes as a result of inter-strain or inter-subspecies recombination events following an anticipated scenario of TP coinfection, i.e., uptake of foreign DNA and DNA recombination.

Prevalence and risk factors associated with Haemophilus ducreyi cutaneous ulcers in Cameroon.

Epidemics of yaws-like cutaneous ulcers are regularly documented in children in the tropics. They occur mainly in poor and remote communities without access to health facilities. The integration of molecular tools into yaws control efforts has made it possible to describe Haemophilus ducreyi (HD) as a major cause of cutaneous ulcers. The objective of this study was to determine the prevalence of HD as cause of cutaneous ulcers, investigate its presence in asymptomatic individuals and identify associated risk factors. A cross-sectional study was conducted in yaws endemic districts of Cameroon. Participants included people presenting yaws-like ulcers and asymptomatic individuals. Swab samples were collected from each participant and tested for HD and Treponema pallidum (TP) using an established qPCR method. Additionally, demographic, habitat, proximity, and hygiene characteristics were collected using a structured questionnaire. A total of 443 individuals participated in the study, including 271 ulcer cases and 172 asymptomatic contacts. The prevalence of HD in ulcers was 30.3% (Confidence Interval (CI) 95% [24.8-35.7]) and the prevalence of asymptomatic HD carriage was 8.6% (CI95% [4.5-12.9]). TP was also detected in our sample among ulcer cases but in lower proportion (5.2% CI95% [2.5-7.8]) compared to HD. The adjusted logistic regression model showed that women were as much at risk of having HD cutaneous ulcer as men regardless of age. Physical proximity to a confirmed ulcer case was the major factor identified favouring HD transmission. HD ulcers were more likely to be present on Bantu individuals compared to Baka as well as HD colonization. These findings highlight HD as the most common cause of cutaneous ulcers in yaws-endemic communities in Cameroon. The exact implications of detecting HD on intact skin are not yet clear. Further studies are needed to understand the significance of this carriage in the spread dynamics of the disease.

Antimicrobial susceptibility of Treponema pallidum subspecies pallidum: an in-vitro study.

BACKGROUND: The increasing incidence of syphilis and the limitations of first-line treatment with penicillin, particularly in neurosyphilis, neonatal syphilis, and pregnancy, highlight the need to expand the therapeutic repertoire for effective management of this disease. We assessed the in-vitro efficacy of 18 antibiotics from several classes on Treponema pallidum subspecies pallidum (T pallidum), the syphilis bacteria. METHODS: Using the in-vitro culture system for T pallidum, we exposed the pathogen to a concentration range of each tested antibiotic. After a 7-day incubation, the treponemal burden was evaluated by quantitative PCR targeting the T pallidum tp0574 gene. The primary outcome was the minimum inhibitory concentration (MIC) at which the quantitative PCR values were not significantly higher than the inoculum wells. We also investigated the susceptibility of macrolide-resistant strains to high concentrations of azithromycin, and the possibility of developing resistance to linezolid, a proposed candidate for syphilis treatment. FINDINGS: Amoxicillin, ceftriaxone, several oral cephalosporins, tedizolid, and dalbavancin exhibited anti-treponemal activity at concentrations achievable in human plasma following regular dosing regimens. The experiments revealed a MIC for amoxicillin at 0·02 mg/L, ceftriaxone at 0·0025 mg/L, cephalexin at 0·25 mg/L, cefetamet and cefixime at 0·0313 mg/L, cefuroxime at 0·0156 mg/L, tedizolid at 0·0625 mg/L, spectinomycin at 0·1 mg/L, and dalbavancin at 0·125 mg/L. The MIC for zoliflodacin and balofloxacin was 2 mg/L. Ertapenem, isoniazid, pyrazinamide, and metronidazole had either a poor or no effect. Azithromycin concentrations up to 2 mg/L (64 times the MIC) were ineffective against strains carrying mutations associated to macrolide resistance. Exposure to subtherapeutic doses of linezolid for 10 weeks did not induce phenotypic or genotypic resistance. INTERPRETATION: Cephalosporins and oxazolidinones are potential candidates for expanding the current therapeutic repertoire for syphilis. Our findings warrant testing efficacy in animal models and, if successful, clinical assessment of efficacy. FUNDING: European Research Council.

Two Streptococcus pyogenes emm types and several anaerobic bacterial species are associated with idiopathic cutaneous ulcers in children after community-based mass treatment with azithromycin.

BACKGROUND: In yaws-endemic areas, two-thirds of exudative cutaneous ulcers (CU) are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD); one-third are classified as idiopathic ulcers (IU). A yaws eradication campaign on Lihir Island in Papua New Guinea utilizing mass drug administration (MDA) of azithromycin initially reduced but failed to eradicate yaws; IU rates remained constant throughout the study. Using 16S rRNA gene sequencing, we previously determined that Streptococcus pyogenes was associated with some cases of IU. Here, we applied shotgun metagenomics to the same samples we analyzed previously by 16S rRNA sequencing to verify this result, identify additional IU-associated microorganisms, and determine why S. pyogenes-associated IU might have persisted after MDA of azithromycin. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced DNA extracted from 244 CU specimens separated into four groups based upon microorganism-specific PCR results (HD+, TP+, TP+HD+, and TP-HD- or IU). S. pyogenes was enriched in IU (24.71% relative abundance [RA]) specimens compared to other ulcer sub-groups, confirming our prior results. We bioinformatically identified the emm (M protein gene) types found in the S. pyogenes IU specimens and found matches to emm156 and emm166. Only ~39% of IU specimens contained detectable S. pyogenes, suggesting that additional organisms could be associated with IU. In the sub-set of S. pyogenes-negative IU specimens, Criibacterium bergeronii, a member of the Peptostreptococcaceae, and Fusobacterium necrophorum (7.07% versus 0.00% RA and 2.18% versus 0.00% RA, respectively), were enriched compared to the S. pyogenes-positive sub-set. Although a broad range of viruses were detected in the CU specimens, none were specifically associated with IU. CONCLUSIONS/SIGNIFICANCE: Our observations confirm the association of S. pyogenes with IU in yaws-endemic areas, and suggest that additional anaerobic bacteria, but not other microorganisms, may be associated with this syndrome. Our results should aid in the design of diagnostic tests and selective therapies for CU.

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.

Ficus septica exudate, a traditional medicine used in Papua New Guinea for treating infected cutaneous ulcers: in vitro evaluation and clinical efficacy assessment by cluster randomised trial.

BACKGROUND AND OBJECTIVES: Infected cutaneous ulcers are major health problems for children living in rural areas of Papua New Guinea. The inaccessibility of affected populations and lack of access to basic healthcare, make a local plant-based therapy an attractive treatment option. We assessed Ficus septica exudate in biological assays relevant to wound healing. We then carried out a clinical trial to determine the exudate's efficacy in healing small cutaneous ulcers compared with Savlon antiseptic cream, and soap and water washing. METHODS: Pre-clinical in vitro assessment of the exudate was carried out using assays to monitor the pro-inflammatory responses of M1 macrophages and neutrophils, antibacterial assays using known ulcer pathogens, an Ames test for mutagenicity and LC-MS chemical analysis of the exudate. An open label cluster-randomised clinical trial was performed, enrolling participants from three different clusters with skin lesions less than 1 cm in diameter. Each cluster comprising 50 participants was randomly assigned to one of three treatment arms namely topical exudate, topical Savlon antiseptic cream, and standard care (soap and water treatment), all administered daily for 2 days. The primary outcome was clinical healing/improvement measured at days 7 and 14, assessed by three dermatologists using blinded photographs. The primary analysis was assessed as non-inferiority of F. septica treatment based on the risk difference for healing/improvement. RESULTS: In vitro, the exudate which is rich in the alkaloid ficuseptine, was found to be non-mutagenic whilst also inhibiting pro-inflammatory responses and exhibiting antibacterial activity. When administered to participants enrolled in the clinical trial, no significant differences were observed between the healing efficacy of F. septica exudate and the two comparator treatments (Savlon antiseptic cream and soap/water treatment). At day 14, but not at day 7, the efficacy of F. septica exudate for healing/improving the ulcers was non-inferior to Savlon antiseptic cream or water/soap treatment. CONCLUSIONS: F. septica exudate is non-mutagenic and has both bactericidal and anti-inflammatory properties. When applied topically to small cutaneous ulcers, the exudate has a healing effect that is non-inferior to Savlon antiseptic cream and standard treatment with soap and water at day 14. Our findings, which should be confirmed in larger clinical trials, have important public health implications.

Safety of mass drug coadministration with ivermectin, diethylcarbamazine, albendazole, and azithromycin for the integrated treatment of neglected tropical diseases: a cluster randomized community trial.

INTRODUCTION: Neglected tropical diseases control programmes run separately. For settings with more than one endemic disease, combined mass drug administration (MDA) has potential practical advantages compared with separate programmes but needs confirmation of safety. We assessed the safety of combined MDA for multiple neglected tropical diseases using ivermectin, diethylcarbamazine, albendazole (IDA) and azithromycin (AZI). METHODS: We conducted an open-label, cluster-randomized trial involving individuals living in 34 wards (smaller administrative division) in two study sites, Namatanai District and Lihir Island, Papua New Guinea. We randomly assigned wards to the combined treatment arm (which received a single dose of the triple combination IDA and a single dose of AZI at the same visit) or the control arm (which received IDA separately followed by AZI separately one week after). All participants underwent safety assessments one day after drug administration. Methodology for collecting the adverse events (AEs) was a general question (in Namatanai) and individual questions about specific AEs (in Lihir). The primary endpoint was the prevalence of AEs. Safety of combined treatment was taken to be non-inferior to that of IDA if the upper limit of the two-sided CI for the difference in rates was equal or lower than 5%. FINDINGS: The study enrolled 15,656 participants. Of those enrolled, 7,281 (46.3%) received the combined regimen and 8,375 (53.3%) received standard treatment with IDA for lymphatic filariasis between Nov 1, 2018, and Apr 15, 2019. Of the individuals in the control group, 4,228 (50.5%) attended a second visit one week apart to receive AZI for yaws. In Namatanai, the proportion of AEs was similar in the combined group (0.8%) compared to the IDA group (1.3%, difference 0.5% [95CI -2.5% to 1.4%]) or the AZI group (3.6%, d -2.8% [95CI -8.6% to 2.8%]). In Lihir, the proportion of AEs was higher in the combined group (23.0%) compared to the IDA group (12.2%, d 10.8% [95% CI 1.5% to 20.2%]) or the AZI group (11.1%, d 11.9% [95% CI 2.7% to 21.1%]).We observed 21 (0.3%) grade-2 AEs in the combined treatment group, 33 (0.4%) in the IDA separately group, and 18 (0.2%) in the AZI separately group. No participants required treatment for any AE. We observed no deaths, serious AEs, or AEs of special interest. INTERPRETATION: In the largest trial so far involving coadministration of regimens based on IDA and AZI, the combination was safe and feasible in a population of more than 15,000 people. Combined MDA based on these two regimens opens up new potential for the control of neglected tropical diseases in the Western Pacific region.

Streptococcus pyogenes Is Associated with Idiopathic Cutaneous Ulcers in Children on a Yaws-Endemic Island.

Exudative cutaneous ulcers (CU) in yaws-endemic areas are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD), but one-third of CU cases are idiopathic (IU). Using mass drug administration (MDA) of azithromycin, a yaws eradication campaign on Lihir Island in Papua New Guinea reduced but failed to eradicate yaws; IU rates remained constant throughout the campaign. To identify potential etiologies of IU, we obtained swabs of CU lesions (n = 279) and of the skin of asymptomatic controls (AC; n = 233) from the Lihir Island cohort and characterized their microbiomes using a metagenomics approach. CU bacterial communities were less diverse than those of the AC. Using real-time multiplex PCR with pathogen-specific primers, we separated CU specimens into HD-positive (HD+), TP+, HD+TP+, and IU groups. Each CU subgroup formed a distinct bacterial community, defined by the species detected and/or the relative abundances of species within each group. Streptococcus pyogenes was the most abundant organism in IU (22.65%) and was enriched in IU compared to other ulcer groups. Follow-up samples (n = 31) were obtained from nonhealed ulcers; the average relative abundance of S. pyogenes was 30.11% in not improved ulcers and 0.88% in improved ulcers, suggesting that S. pyogenes in the not improved ulcers may be azithromycin resistant. Catonella morbi was enriched in IU that lacked S. pyogenes As some S. pyogenes and TP strains are macrolide resistant, penicillin may be the drug of choice for CU azithromycin treatment failures. Our study will aid in the design of diagnostic tests and selective therapies for CU.IMPORTANCE Cutaneous ulcers (CU) affect approximately 100,000 children in the tropics each year. While two-thirds of CU are caused by Treponema pallidum subspecies pertenue and Haemophilus ducreyi, the cause(s) of the remaining one-third is unknown. Given the failure of mass drug administration of azithromycin to eradicate CU, the World Health Organization recently proposed an integrated disease management strategy to control CU. Success of this strategy requires determining the unknown cause(s) of CU. By using 16S rRNA gene sequencing of swabs obtained from CU and the skin of asymptomatic children, we identified another possible cause of skin ulcers, Streptococcus pyogenes Although S. pyogenes is known to cause impetigo and cellulitis, this is the first report implicating the organism as a causal agent of CU. Inclusion of S. pyogenes into the integrated disease management plan will improve diagnostic testing and treatment of this painful and debilitating disease of children and strengthen elimination efforts.

A Cluster-Randomized Trial of Hydroxychloroquine for Prevention of Covid-19.

BACKGROUND: Current strategies for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are limited to nonpharmacologic interventions. Hydroxychloroquine has been proposed as a postexposure therapy to prevent coronavirus disease 2019 (Covid-19), but definitive evidence is lacking. METHODS: We conducted an open-label, cluster-randomized trial involving asymptomatic contacts of patients with polymerase-chain-reaction (PCR)-confirmed Covid-19 in Catalonia, Spain. We randomly assigned clusters of contacts to the hydroxychloroquine group (which received the drug at a dose of 800 mg once, followed by 400 mg daily for 6 days) or to the usual-care group (which received no specific therapy). The primary outcome was PCR-confirmed, symptomatic Covid-19 within 14 days. The secondary outcome was SARS-CoV-2 infection, defined by symptoms compatible with Covid-19 or a positive PCR test regardless of symptoms. Adverse events were assessed for up to 28 days. RESULTS: The analysis included 2314 healthy contacts of 672 index case patients with Covid-19 who were identified between March 17 and April 28, 2020. A total of 1116 contacts were randomly assigned to receive hydroxychloroquine and 1198 to receive usual care. Results were similar in the hydroxychloroquine and usual-care groups with respect to the incidence of PCR-confirmed, symptomatic Covid-19 (5.7% and 6.2%, respectively; risk ratio, 0.86 [95% confidence interval, 0.52 to 1.42]). In addition, hydroxychloroquine was not associated with a lower incidence of SARS-CoV-2 transmission than usual care (18.7% and 17.8%, respectively). The incidence of adverse events was higher in the hydroxychloroquine group than in the usual-care group (56.1% vs. 5.9%), but no treatment-related serious adverse events were reported. CONCLUSIONS: Postexposure therapy with hydroxychloroquine did not prevent SARS-CoV-2 infection or symptomatic Covid-19 in healthy persons exposed to a PCR-positive case patient. (Funded by the crowdfunding campaign YoMeCorono and others; BCN-PEP-CoV2 ClinicalTrials.gov number, NCT04304053.).

Yaws re-emergence and bacterial drug resistance selection after mass administration of azithromycin: a genomic epidemiology investigation.

BACKGROUND: In a longitudinal study assessing the WHO strategy for yaws eradication using mass azithromycin treatment, we observed resurgence of yaws cases with dominance of a single JG8 sequence type and emergence of azithromycin-resistant Treponema pallidum subspecies pertenue (T p pertenue). Here, we analyse genomic changes in the bacterial population using samples collected during the study. METHODS: We did whole bacterial genome sequencing directly on DNA extracted from 37 skin lesion swabs collected from patients on Lihir Island, Papua New Guinea, between April 1, 2013, and Nov 1, 2016. We produced phylogenies and correlated these with spatiotemporal information to investigate the source of new cases and the emergence of five macrolide-resistant cases. We used deep amplicon sequencing of surveillance samples to assess the presence of minority macrolide-resistant populations. FINDINGS: We recovered 20 whole T p pertenue genomes, and phylogenetic analysis showed that the re-emerging JG8 sequence type was composed of three bacterial sublineages characterised by distinct spatiotemporal patterns. Of five patients with resistant T p pertenue, all epidemiologically linked, we recovered genomes from three and found no variants. Deep sequencing showed that before treatment, the index patient had fixed macrolide-sensitive T p pertenue, whereas the post-treatment sample had a fixed resistant genotype, as did three of four contact cases. INTERPRETATION: In this study, re-emergence of yaws cases was polyphyletic, indicating multiple epidemiological sources. However, given the genomic and epidemiological linkage of resistant cases and the rarity of resistance alleles in the general population, azithromycin resistance is likely to have evolved only once in this study, followed by onward dissemination. FUNDING: Wellcome and Provincial Deputation of Barcelona.

Etiological Characterization of the Cutaneous Ulcer Syndrome in Papua New Guinea Using Shotgun Metagenomics.

Background: Treponema pallidum subsp pertenue and Haemophilus ducreyi are causative agents of cutaneous ulcer (CU) in yaws-endemic regions in the tropics. However, a significant proportion of CU patients remain polymerase chain reaction (PCR) negative for both bacterial agents. We aimed to identify potential additional etiological agents of CU in a yaws-endemic region. Methods: This population-based cohort study included children in Lihir Island (Papua New Guinea) examined during a yaws eradication campaign in October 2013-October 2014. All consenting patients with atraumatic exudative ulcers of >1 cm diameter were enrolled. Lesional swabs were collected for real-time PCR testing for T. pallidum subsp pertenue and H. ducreyi. We then performed shotgun whole DNA metagenomics sequencing on extracted DNA and taxonomically assigned shotgun sequences using a human microbiome reference. Results: Sequence data were available for 122 samples. Shotgun sequencing showed high classification agreement relative to PCR testing (area under the curve for T. pallidum/H. ducreyi was 0.92/0.85, respectively). Clustering analysis of shotgun data revealed compositional clusters where the dominant species (median relative abundance ranged from 32% to 66%) was H. ducreyi (23% of specimens), T. pallidum subsp pertenue (16%), Streptococcus dysgalactiae (12%), Arcanobacterium haemolyticum (8%), and Corynebacterium diphtheriae (8%). Sample clustering derived from ulcer microbial composition did not show geographical patterns. Conclusions: These data suggest a diverse etiology of skin ulcers in yaws-endemic areas, which may help design more accurate diagnostic tools and more effective antimicrobial treatment approaches to the cutaneous ulcer syndrome.

Multiple Class I and Class II Haemophilus ducreyi Strains Cause Cutaneous Ulcers in Children on an Endemic Island.

Background: Together with Treponema pallidum subspecies pertenue, Haemophilus ducreyi is a major cause of exudative cutaneous ulcers (CUs) in children. For H. ducreyi, both class I and class II strains, asymptomatic colonization, and environmental reservoirs have been found in endemic regions, but the epidemiology of this infection is unknown. Methods: Based on published whole-genome sequences of H. ducreyi CU strains, a single-locus typing system was developed and applied to H. ducreyi-positive CU samples obtained prior to, 1 year after, and 2 years after the initiation of a mass drug administration campaign to eradicate CU on Lihir Island in Papua New Guinea. DNA from the CU samples was amplified with class I and class II dsrA-specific primers and sequenced; the samples were classified into dsrA types, which were geospatially mapped. Selection pressure analysis was performed on the dsrA sequences. Results: Thirty-seven samples contained class I sequences, 27 contained class II sequences, and 13 contained both. There were 5 class I and 4 class II types circulating on the island; 3 types accounted for approximately 87% of the strains. The composition and geospatial distribution of the types varied little over time and there was no evidence of selection pressure. Conclusions: Multiple strains of H. ducreyi cause CU on an endemic island and coinfections are common. In contrast to recent findings with T. pallidum pertenue, strain composition is not affected by antibiotic pressure, consistent with environmental reservoirs of H. ducreyi. Such reservoirs must be addressed to achieve eradication of H. ducreyi.

Re-emergence of yaws after single mass azithromycin treatment followed by targeted treatment: a longitudinal study.

BACKGROUND: Yaws is a substantial cause of chronic disfiguring ulcers in children in at least 14 countries in the tropics. WHO's newly adopted strategy for yaws eradication uses a single round of mass azithromycin treatment followed by targeted treatment programmes, and data from pilot studies have shown a short-term significant reduction of yaws. We assessed the long-term efficacy of the WHO strategy for yaws eradication. METHODS: Between April 15, 2013, and Oct 24, 2016, we did a longitudinal study on a Papua New Guinea island (Lihir; 16 092 population) in which yaws was endemic. In the initial study, the participants were followed for 12 months; in this extended follow-up study, clinical, serological, and PCR surveys were continued every 6 months for 42 months. We used genotyping and travel history to identify importation events. Active yaws confirmed by PCR specific for Treponema pallidum was the primary outcome indicator. The study is registered with ClinicalTrials.gov, number NCT01955252. FINDINGS: Mass azithromycin treatment (coverage rate of 84%) followed by targeted treatment programmes reduced the prevalence of active yaws from 1·8% to a minimum of 0·1% at 18 months (difference from baseline -1·7%, 95% CI, -1·9 to -1·4; p<0·0001), but the infection began to re-emerge after 24 months with a significant increase to 0·4% at 42 months (difference from 18 months 0·3%, 95% CI 0·1 to 0·4; p<0·0001). At each timepoint after baseline, more than 70% of the total community burden of yaws was found in individuals who had not had the mass treatment or as new infections in non-travelling residents. At months 36 and 42, five cases of active yaws, all from the same village, showed clinical failure following azithromycin treatment, with PCR-detected mutations in the 23S ribosomal RNA genes conferring resistance to azithromycin. A sustained decrease in the prevalence of high-titre latent yaws from 13·7% to <1·5% in asymptomatic children aged 1-5 years old and of genetic diversity of yaws strains from 0·139 to less than 0·046 between months 24 and 42 indicated a reduction in transmission of infection. INTERPRETATION: The implementation of the WHO strategy did not, in the long-term, achieve elimination in a high-endemic community mainly due to the individuals who were absent at the time of mass treatment in whom yaws reactivated; repeated mass treatment might be necessary to eliminate yaws. To our knowledge, this is the first report of the emergence of azithromycin-resistant T p pertenue and spread within one village. Communities' surveillance should be strengthened to detect any possible treatment failure and biological markers of resistance. FUNDING: ISDIN laboratories, Newcrest Mining Limited, and US Public Health Service National Institutes of Health.

Maternal Smoking: A Life Course Blood Pressure Determinant?

Introduction: Exposure to maternal smoking early in life may affect blood pressure (BP) control mechanisms. We examined the association between maternal smoking (before conception, during pregnancy, and 4 years after delivery) and BP in preschool children. Methods: We evaluated 4295 of Generation XXI children, recruited at birth in 2005-2006 and reevaluated at the age of 4. At birth, information was collected by face-to-face interview and additionally abstracted from clinical records. At 4-year follow-up, interviews were performed and children's BP measured. Linear regression models were fitted to estimate the association between maternal smoking and children's BP. Results: Children of smoking mothers presented significantly higher BP levels. After adjustment for maternal education, gestational hypertensive disorders, and child's body mass index, children exposed during pregnancy to maternal smoking presented a higher systolic BP (SBP) z-score (ß = 0.08, 95% confidence interval [CI] 0.04 to 0.14). In crude models, maternal smoking was associated with higher SBP z-score at every assessed period. However, after adjustment, an attenuation of the association estimates occurred (ß = 0.08, 95% CI 0.03 to 0.13 before conception; ß = 0.07, 95%CI 0.02 to 0.12; ß = 0.04, 95%CI -0.02 to 0.10; and ß = 0.06, 95%CI 0.00 to 0.13 for the first, second, and third pregnancy trimesters, respectively; and ß = 0.07, 95%CI 0.02 to 0.12 for current maternal smoking). No significant association was observed for diastolic BP z-score levels. Conclusion: Maternal smoking before, during, and after pregnancy was independently associated with systolic BP z-score in preschool children. This study provides additional evidence to the public health relevance of maternal smoking cessation programs if early cardiovascular health of children is envisaged. Implications: Using observational longitudinal data from the birth cohort Generation XXI, this study showed that exposure to maternal smoking-before pregnancy, during pregnancy, and 4 years after delivery-was associated with a systolic BP-raising effect in children at the age of 4. The findings of this study add an important insight into the need to support maternal smoke-free environments in order to provide long-term cardiovascular benefit, starting as early as possible in life.

Effectiveness of single-dose azithromycin to treat latent yaws: a longitudinal comparative cohort study.

BACKGROUND: Treatment of latent yaws is a crucial component of the WHO yaws eradication strategy to prevent relapse and the resulting transmission to uninfected children. We assessed the effectiveness of single-dose azithromycin to treat patients with latent yaws. METHODS: This population-based cohort study included children (age <20 years) living on Lihir Island, Papua New Guinea, with high-titre (rapid plasma reagin titre ≥1:8) latent or active yaws, between April, 2013, and May, 2015. Latent yaws was defined as lack of suspicious skin lesions or presence of ulcers negative for Treponema pallidum subsp pertenue on PCR, and active yaws was defined as ulcers positive for T pertenue on PCR. All children received one oral dose of 30 mg/kg azithromycin. The primary endpoint was serological cure, defined as a two-dilution decrease in rapid plasma reagin titre by 24 months after treatment. Treatment of latent yaws was taken to be non-inferior to that of active yaws if the lower limit of the two-sided 95% CI for the difference in rates was higher than or equal to -10%. This study is registered with ClinicalTrials.gov, number NCT01955252. FINDINGS: Of 311 participants enrolled, 273 (88%; 165 with latent yaws and 108 with active yaws) completed follow-up. The primary endpoint was achieved in 151 (92%) participants with latent yaws and 101 (94%) with active yaws (risk difference -2·0%, 95% CI -8·3 to 4·3), meeting the prespecified criteria for non-inferiority. INTERPRETATION: On the basis of decline in serological titre, oral single-dose azithromycin was effective in participants with latent yaws. This finding supports the WHO strategy for the eradication of yaws based on mass administration of the entire endemic community irrespective of clinical status. FUNDING: Newcrest Mining Limited and ISDIN laboratories.

Single-Dose Azithromycin for the Treatment of Haemophilus ducreyi Skin Ulcers in Papua New Guinea.

BACKGROUND: Haemophilus ducreyi (HD) and Treponema pallidum subspecies pertenue (TP) are major causative agents of cutaneous ulcer (CU) in the tropics. Azithromycin is recommended to treat sexually transmitted HD infections and has good in vitro activity against HD strains from both genital and skin ulcers. We investigated the efficacy of oral single-dose azithromycin on HD-CU. METHODS: We conducted a community-based cohort study in Lihir Island, Papua New Guinea, from October 2014 through May 2016. Consenting patients with skin ulcers >1 cm in diameter were eligible for this study and had collected a lesional swab for polymerase chain reaction (PCR). All participants were treated with single-dose azithromycin (30 mg/kg) and were followed up for assessment of clinical resolution. We retrospectively classified patients according to PCR results into HD, TP, and PCR-negative groups. The primary endpoint was healing rates of HD-CU at 14 days after treatment. RESULTS: We obtained full outcome data from 246 patients; 131 (53.3%) were HD PCR positive, 37 (15.0%) were TP positive, and 78 (31.7%) were negative for all tests. Healing rates were 88.5% (95% confidence interval [CI], .82-.93) in the HD group, 78.4% [95% CI, .63-.89] in the TP group, and 74.4% (95% CI, .64-.83) in the PCR-negative group. If we included the participants with improved ulcers, the healing rates increased to 94.7%, 97.3%, and 89.7% respectively. HD cases classified as not healed all converted to HD-negative PCR. CONCLUSIONS: Based upon clinical resolution and PCR conversion to HD negative, a single oral dose of azithromycin is efficacious for the treatment of HD-CU. These results have implications for the treatment of individual patients and for the use of antibiotics in public health strategies to control CU in the tropics.

Haemophilus ducreyi DNA is detectable on the skin of asymptomatic children, flies and fomites in villages of Papua New Guinea.

BACKGROUND: Haemophilus ducreyi and Treponema pallidum subsp. pertenue are major causes of leg ulcers in children in Africa and the Pacific Region. We investigated the presence of DNA (PCR positivity) from these bacteria on asymptomatic people, flies, and household linens in an endemic setting. METHODOLOGY/PRINCIPAL FINDINGS: We performed a cross-sectional study in rural villages of Lihir Island, Papua New Guinea during a yaws elimination campaign. Participants were asymptomatic subjects recruited from households with cases of leg ulcers, and from households without cases of leg ulcers. We rubbed swabs on the intact skin of the leg of asymptomatic individuals, and collected flies and swabs of environmental surfaces. All specimens were tested by PCR for H. ducreyi and T. p. pertenue DNA. Of 78 asymptomatic participants that had an adequate specimen for DNA detection, H. ducreyi-PCR positivity was identified in 16 (21%) and T. p. pertenue-PCR positivity in 1 (1%). In subgroup analyses, H. ducreyi-PCR positivity did not differ in participants exposed or not exposed to a case of H. ducreyi ulcer in the household (24% vs 18%; p = 0.76). Of 17 cultures obtained from asymptomatic participants, 2 (12%) yielded a definitive diagnosis of H. ducreyi, proving skin colonization. Of 10 flies tested, 9 (90%) had H. ducreyi DNA and 5 (50%) had T. p. pertenue DNA. Of 6 bed sheets sampled, 2 (33%) had H. ducreyi DNA and 1 (17%) had T. p. pertenue DNA. CONCLUSIONS/SIGNIFICANCE: This is the first time that H. ducreyi DNA and colonization has been demonstrated on the skin of asymptomatic children and that H. ducreyi DNA and T. p. pertenue DNA has been identified in flies and on fomites. The ubiquity of H. ducreyi in the environment is a contributing factor to the spread of the organism.

Yaws Osteoperiostitis Treated with Single-Dose Azithromycin.

AbstractThe etiologic agent of yaws, Treponema pallidum subsp. pertenue, causes a multistage infection transmitted by nonsexual contact with the exudates from active lesions. Bone lesions in the form of osteoperiostitis are common and occur in numerous bones simultaneously in early stages. Although a multinational eradication campaign with mass administration of intramuscular benzathine benzylpenicillin in the 1950s greatly reduced its global incidence, a resurgence of yaws has occurred since around 2000 in western and central Africa and the Pacific Islands. The finding that a single oral dose of azithromycin (30 mg/kg) was as effective as benzathine benzylpenicillin prompted renewed interest by World Health Organization in 2012 toward eradication of this infection by 2020. We previously reported the excellent response to benzathine benzylpenicillin therapy for yaws osteoperiostitis. Herein, we document a confirmed case of yaws with osteoperiostitis successfully treated with single-dose azithromycin and discuss the pathology of yaws periostitis and comment on the implications of this in light of the new campaign toward yaws eradication.