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Yaws, a neglected tropical disease caused by the bacterium Treponema pallidum subspecies pertenue, manifests as ulcerative skin lesions. Nucleic acid amplification tests, like loop-mediated isothermal amplification (LAMP), are versatile tools to distinguish yaws from infections that cause similar skin lesions, primarily Haemophilus ducreyi. We developed a novel molecular test to simultaneously detect T. pallidum and H. ducreyi based on mediator displacement LAMP. We validated the T. pallidum and H. ducreyi LAMP (TPHD-LAMP) by testing 293 clinical samples from patients with yaws-like lesions. Compared with quantitative PCR, the TPHD-LAMP demonstrated high sensitivity and specificity for T. pallidum (84.7% sensitivity, 95.7% specificity) and H. ducreyi (91.6% sensitivity, 84.8% specificity). This novel assay provided rapid molecular confirmation of T. pallidum and H. ducreyi DNA and might be suitable for use at the point of care. TPHD-LAMP could support yaws eradication by improving access to molecular diagnostic tests at the district hospital level.

Children in parts of Africa, the South Pacific, and Southeast Asia frequently develop cutaneous ulcers caused by two bacteria: Haemophilus ducreyi (HD) and Treponema pallidum subspecies pertenue (causative agent of yaws). The World Health Organization (WHO) aims to eradicate yaws using mass administration of azithromycin. This also leads to a temporary decrease in ulcers caused by HD followed by a rebound suggesting an ongoing reservoir of infection. The aim of this study was to investigate whether HD could spread through the environment or animals. Alongside detection of human cases of cutaneous ulcers from villages in Cameroon, we additionally collected samples from animals (dogs, cats, flies), fomites (bedsheets, clothing, benches, doors), and water sources (marigots and lakes). DNA was extracted and tested for HD and T. pallidum using two specific qPCR assays. HD was not detected in any of the environmental samples but it was on both clothing (13.3%) and in flies (27%). Flies also tested positive for T. pallidum, but at a lower rate (2.6%). These results suggest that flies and some fomites may contribute to the transmission of HD. Future research should focus on determining whether either of these are capable of carrying live bacteria that can cause onward transmission.

A high-resolution view of the immune infiltrate due to infection with an extracellular bacterial pathogen in human skin has not yet been defined. Here, we used the human skin pathogen Haemophilus ducreyi in a human challenge model to identify on a single cell level the types of cells that are present in volunteers who fail to spontaneously clear infection and form pustules. We identified 13 major cell types. Immune cells and immune-activated stromal cells were enriched in pustules compared to wounded (mock infected) sites. Pustules formed despite the expression of multiple pro-inflammatory cytokines, such as IL-1β and type I interferon. Interferon stimulation was most evident in macrophages, which were proximal to the abscess. The pro-inflammatory response within the pustule may be tempered by regulatory T cells and cells that express indoleamine 2,3-dioxygenase, leading to failure of the immune system to clear H. ducreyi .

Haemophilus ducreyi was historically known as the causative agent of chancroid, a sexually-transmitted disease causing painful genital ulcers endemic in many low/middle-income nations. In recent years the species has been implicated as the causative agent of nongenital cutaneous ulcers affecting children of the South Pacific Islands and West African countries. Much is still unknown about the mechanism of H . ducreyi transmission in these areas, and recent studies have identified local insect species, namely flies, as potential transmission vectors. H . ducreyi DNA has been detected on the surface and in homogenates of fly species sampled from Lihir Island, Papua New Guinea. The current study develops a model system using Musca domestica , the common house fly, as a model organism to demonstrate proof of concept that flies are a potential vector for the transmission of viable H . ducreyi . Utilizing a green fluorescent protein (GFP)-tagged strain of H . ducreyi and three separate exposure methods, we detected the transmission of viable H . ducreyi by 86.11% ± 22.53% of flies sampled. Additionally, the duration of H . ducreyi viability was found to be directly related to the bacterial concentration, and transmission of H . ducreyi was largely undetectable within one hour of initial exposure. Push testing, Gram staining, and PCR were used to confirm the identity and presence of GFP colonies as H . ducreyi . This study confirms that flies are capable of mechanically transmitting viable H . ducreyi , illuminating the importance of investigating insects as vectors of cutaneous ulcerative diseases.

Background Treponema pallidum subspecies pertenue and Haemophilus ducreyi cause skin ulcers in impoverished communities. Historical serologic records from Ghana focus on T. pallidum , omitting potential H. ducreyi cases. The objective of this study was to investigate, for the first time, the presence and distribution of T. p .subsp. pertenue and/or H. ducreyi in skin lesions in two previously unstudied areas in Western North Region of Ghana. Methods We conducted observational cross-sectional surveys and screened skin lesions in the Aowin municipality and Suaman district in Western North Region, Ghana. Suspected yaws cases were tested for 1) treponemal and non-treponemal antibodies using a finger-prick blood test and 2) Treponema pallidum and H. ducreyi DNA in lesion swabs using polymerase chain reaction (PCR). GPS coordinates were recorded for all tests administered near patients’ schools or residences. Results Of the 11,917 individuals examined, 1,699 patients with suspected yaws consented to testing. In the Aowin municipality, the seroprevalence of current treponemal infections was 10.8%, while the prevalence of yaws and H. ducreyi , confirmed by PCR, was 2.3% and 4.3%, respectively. High RPR titers correlated with active yaws infection. However, T. pallidum DNA was confirmed in lesions with low RPR titers (≤1:4). Communities with high seropositivity rates were more likely to have PCR-confirmed yaws cases. No evidence of resistance to azithromycin was observed in T. p. subsp. pertenue isolates. Dual infections with H. ducreyi were uncommon. No treponemal-positive cases were identified in the Suaman district. Conclusions Serological surveillance without molecular testing may undermine efforts to interrupt yaws transmission. More comprehensive community surveillance detecting yaws and H. ducreyi infections is critical in developing strategies to achieve the WHOs yaws eradication goal.

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. 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. 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. 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 causes chancroid, which facilitates transmission of human immunodeficiency virus type 1. To better understand the biology of H. ducreyi we developed a human inoculation model. In the present article, we describe clinical outcomes for 267 volunteers who were infected with H. ducreyi. There was a relationship between papule formation and estimated delivered dose. The outcome (either pustule formation or resolution) of infected sites for a given subject was not independent; the most important determinants of pustule formation were sex and host effects. When 41 subjects were infected a second time, their outcomes segregated toward their initial outcome, confirming the host effect. Subjects with pustules developed local symptoms that required withdrawal from the study after a mean of 8.6 days. There were 191 volunteers who had tissue biopsy performed, 173 of whom were available for follow-up analysis; 28 (16.2%) of these developed hypertrophic scars, but the model was otherwise safe. Mutant-parent trials confirmed key features in H. ducreyi pathogenesis, and the model has provided an opportunity to study differential human susceptibility to a bacterial infection

Haemophilus ducreyi is a major cause of skin ulcers in the tropics. On an endemic island, multiple strains of H. ducreyi cause infection, coinfections are common, and mass treatment with azithromycin did not exert selection pressure on the organism. Abstract 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.

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

A randomized, double-blind, placebo-controlled clinical trial was conducted in Nairobi, Kenya, to compare single-dose ciprofloxacin with a 7-day course of erythromycin for the treatment of chancroid. In all, 208 men and 37 women presenting with genital ulcers clinically compatible with chancroid were enrolled. Ulcer etiology was determined using culture techniques for chancroid, serology for syphilis, and a multiplex polymerase chain reaction for chancroid, syphilis, and herpes simplex virus (HSV). Ulcer etiology was 31% unmixed chancroid, 23% unmixed syphilis, 16% unmixed HSV, 15% mixed etiology, and 15% unknown. for 111 participants with chancroid, cure rates were 92% with ciprofloxacin and 91% with erythromycin. For all study participants, the treatment failure rate was 15%, mostly related to ulcer etiologies of HSV infection or syphilis, and treatment failure was 3 times more frequent in human immunodeficiency virus—infected subjects than in others, mostly owing to HSV infection. Ciprofloxacin is an effective single-dose treatment for chancroid, but current recommendations for empiric therapy of genital ulcers may result in high treatment failure due to HSV infection.