Epidemiology of Haemophilus ducreyi Infections

The global epidemiology of Haemophilus ducreyi infections is poorly documented because of difficulties in confirming microbiological diagnoses. We evaluated published data on the proportion of genital and nongenital skin ulcers caused by H. ducreyi before and after introduction of syndromic management for genital ulcer disease (GUD). Before 2000, the proportion of GUD caused by H. ducreyi ranged from 0.0% to 69.0% (35 studies in 25 countries). After 2000, the proportion ranged from 0.0% to 15.0% (14 studies in 13 countries). In contrast, H. ducreyi has been recently identified as a causative agent of skin ulcers in children in the tropical regions; proportions ranged from 9.0% to 60.0% (6 studies in 4 countries). We conclude that, although here has been a sustained reduction in the proportion of GUD caused by H. ducreyi, this bacterium is increasingly recognized as a major cause of nongenital cutaneous ulcer

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

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

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

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

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

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

Prospective individual patient data meta-analysis of two randomized trials on convalescent plasma for COVID-19 outpatients

Data on convalescent plasma (CP) treatment in COVID-19 outpatients are scarce. We aimed to assess whether CP administered during the first week of symptoms reduced the disease progression or risk of hospitalization of outpatients. Two multicenter, double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when = 50 years and symptomatic for <= 7days were included. The intervention consisted of 200-300mL of CP with a predefined minimum level of antibodies. Primary endpoints were a 5-point disease severity scale and a composite of hospitalization or death by 28 days. Amongst the 797 patients included, 390 received CP and 392 placebo; they had a median age of 58 years, 1 comorbidity, 5 days symptoms and 93% had negative IgG antibody-test. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The odds ratio (OR) of CP for improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311); OR for hospitalization or death was 0.919 (CI 0.592-1.416). CP effect on hospital admission or death was largest in patients with <= 5 days of symptoms (OR 0.658, 95%CI 0.394-1.085). CP did not decrease the time to full symptom resolution

Refining the guidelines for the treatment and management of Cutaneous Ulcers through research in endemic population Papua New Guinea

As úlceras cutâneas (UC), lesões cutâneas com perda das camadas superficiais da pele, são comumente encontradas em países de baixo rendimento da África e do Pacífico Sul. Em certas áreas remotas e rurais, como as ilhas da Papua Nova Guiné, a prevalência e a morbidade de UC são muito altas, representando, portanto, uma importante causa de sofrimento e estigmatização em crianças. Certas UC, se não forem tratadas, podem resultar em deformidades permanentes que afetam a vida das pessoas e que estão associadas a pobreza. Embora vários agentes patogénicos possam causar UC, dois deles representam um grande problema de saúde pública nas ilhas do Pacífico Sul - bouba (Treponema pallidum pertenue) e Haemophilus ducreyi. Estes dois agentes patogénicos são a base desta tese e foram selecionados por variadas razões: em primeiro lugar, são as duas causas mais comuns de UC na região tropical do Pacífico Sul, onde os estudos para esta tese foram realizados. Em segundo lugar, estes são co endémicos, mas a sua interação como doenças infeciosas não foi estudada. Em terceiro lugar, por motivos epidemiológicos, clínicos e terapêuticos, as duas doenças são potencialmente tratáveis de forma integrada por meio do tratamento total da comunidade (TTC) com administração em massa de antibióticos (AMM) . A bouba é classificada pela Organização Mundial da Saúde (OMS) como uma doença tropical negligenciada (DTN) que afeta principalmente crianças de 6 a 15 anos de idade em comunidades com falta de água e sem saneamento, levando à desfiguração e incapacidade crónica. No passado, a bouba foi sempre considerada a causa mais comum de UC, mas estudos de coorte recentes conduzidos na Papua Nova Guiné (PNG), Ilhas Salomão e Vanuatu identificaram o H. ducreyi como o agente etiológico que causa mais de metade de todas as UCs nos trópicos. Em cenários endémicos de bouba rural, onde há ausência de diagnósticos moleculares, as UCs são classificadas com base nos resultados dos testes sorológicos da sífilis. Por exemplo, uma UC com um teste serológico positivo é classificada como bouba e tratada com azitromicina oral de dose única, enquanto uma UC com um resultado serológico negativo é classificada como úlceras não-bouba (por exemplo, H. ducreyi ou outros agentes patogénicos) e tratada com curso de 5-7 dias de diferentes antibióticos de amplo espectro. Esquemas de dosagem de múltiplos dias de tratamento e várias vezes por dia comprometem a adesão e satisfação do paciente.Recentemente, um alarmante ressurgimento de bouba foi observado em vários países nos trópicos. O maior número de casos é relatado na Papua Nova Guiné, Ilhas Salomão e Gana. A OMS reagiu ao ressurgimento de bouba conduzindo campanhas de eliminação em áreas altamente endémicas usando tratamento empírico de UC com azitromicina oral de dose única. No entanto, se somente a bouba for tratada durante as campanhas, as comunidades podem ter a perceção de que a persistência de outros tipos de UC estará relacionada com um desfecho negativo da estratégia. A recente descoberta de que H. ducreyi é um dos principais agentes causadores de UC reforça a necessidade de maior investigação sobre o assunto. É preciso explorar novas estratégias para controlar a UC por H. ducreyi juntamente com a bouba. Em 2012, um ensaio clínico realizado na Papua Nova Guiné mostrou que a azitromicina era eficaz para tratar a bouba ativa. No entanto, para nosso conhecimento, há dados limitados sobre a eficácia do AMM com azitromicina para curar a infeção latente da bouba ou para eliminar a UC H. ducreyi como um efeito secundário benéfico. A azitromicina tem a vantagem de ser administrada em dose oral única e a um preço muito acessível em comparação com outros regimes de antibióticos. Se comprovadamente eficaz para o tratamento de UC por H. ducreyi e para a bouba latente, teria implicações para o tratamento de pacientes individuais e para o uso de antibióticos em estratégias de saúde pública para controlar a UC nos trópicos. Além disso, compreender a transmissão da UC de H. ducreyi permitirá definir novas estratégias para controlar e deter a propagação da doença. Os artigos originais desta tese pretendem fornecer uma atualização sobre a epidemiologia mundial da UC e bouba de H. ducreyi, investigar a eficácia da azitromicina de dose única no tratamento de pacientes com UC por H. ducreyi e bouba latente, explorar a transmissão e reservatórios ambientais de H. ducreyi e apresentar dados de eficácia a longo prazo da estratégia de AMM para eliminar a bouba e reduzir a prevalência de H. ducreyi a nível comunitário.Cutaneous Ulcers (CU) presenting as skin lesions with loss of the superficial skin layers are commonly found in low-resource countries of Africa and the South Pacific. In certain remote and rural areas, such as the islands of Papua New Guinea, CU prevalence and morbidity is very high, hence representing an important cause of suffering and stigmatization in children. Certain CU conditions, if untreated, can result into permanent impairing deformities that will impact the person’s life and anchor that person into poverty. Although several pathogens may cause CU, two of them pose a major public health problem in the South Pacific islands, namely yaws (Treponema pallidum pertenue) and Haemophilus ducreyi and are the basis of this thesis. These two pathogens were selected for a number of reasons: first they are the two most common causes of CU in the tropical South Pacific region where the studies for this thesis were conducted. Second, these are co-endemic but its interaction as infectious diseases has not been studied. And third, because of epidemiological, clinical and therapeutic reasons the two diseases are potentially treatable in an integrated manner through total community treatment (TCT) with mass drug administration (MDA) of antibiotics. Yaws is classified by the World Health Organization (WHO) as a neglected tropical disease (NTD) that primarily affects children 6-15 years old in communities with poor water supplies and no sanitation, and leads to chronic disfigurement and disability. In the past yaws was always considered the most common cause of CU, yet recent cohort studies conducted in Papua New Guinea (PNG), Solomon Islands and Vanuatu have identified Haemophilus ducreyi as an aetiologic agent causing more than half of all CU in the tropics. In rural yaws endemic settings where there is absence of molecular diagnostics, CUs are classified on the basis of syphilis serological test results. For example, a CU with a positive serological test is classified as yaws and treated with single dose oral azithromycin, while a CU with a negative serological result is classified as non-yaws ulcers (e.g. H. ducreyi or other pathogens) and treated with a 5-7 day course of different wide-spectrum antibiotics.Multiple days courses of treatment and multiple times per day dosing schedules compromise patient compliance and satisfaction. Recently, an alarming resurgence of yaws has been observed in several countries in the tropics. The largest number of cases are reported in Papua New Guinea, Solomon Islands, and Ghana. WHO has responded to yaws resurgence by conducting Elimination Campaigns in highly endemic areas using empirical treatment of CU with single-dose oral azithromycin. If only yaws is treated during campaigns, communities might have the perception that persistence of other types of CU is related to a poor outcome of the strategy. The recent discovery that H. ducreyi was as a major causative agent of CU calls for further research on this matter. New strategies to control H. ducreyi CU together with yaws need to be explored. In 2012, a clinical trial conducted in Papua New Guinea showed that azithromycin was efficacious to treat active yaws. However, to our knowledge there is limited data on the efficacy of azithromycin MDA to cure latent yaws infection, or to eliminate H. ducreyi CU as a beneficial secondary effect. Azithromycin offers the advantage of single oral dose at a very cheap price as compared to other antibiotic regimens; if proven efficacious for H. ducreyi CU and latent yaws, this would have implications for the treatment of individual patients and for the use of antibiotics in public health strategies to control CU in the tropics. Furthermore, understanding the transmission of H. ducreyi CU will helps us define new strategies to control and stop the spread of the disease. This thesis original research articles aim to provide an update on the worldwide epidemiology of H. ducreyi CU and yaws, to investigate the efficacy of single-dose azithromycin to treat individual patients with H. ducreyi CU and latent yaws, to explore the transmission and environmental reservoirs of H. ducreyi, and to present long-term efficacy data of the MDA strategy to eliminate yaws and reduce the prevalence of H. ducreyi at a community level