Background: Buruli ulcer is a disease of the skin and soft tissue caused by the pathogen Mycobacterium ulcerans. Mycolactone, a lipid toxin has been identified as the main bacterial virulence factor for the disease. This toxin has been shown to be responsible for the immunosuppression and tissue necrosis which is characteristic of the disease. There is currently no vaccine for Buruli ulcer disease. In this study, the systemic immune responses of M. ulcerans infected patients to polyketide synthase domains were investigated. The effect of paradoxical reactions on the Th1 and Th2 responses of M. ulcerans infected patients were also evaluated.
Methods: This was a prospective observational study. All clinically suspected cases of Buruli ulcer were confirmed by standard PCR. Interferon gamma (interferon-γ) secretion following whole blood stimulation with polyketide synthase domains(PKS) using heparinised blood samples from patients with PCR confirmed Buruli ulcer, endemic controls and non-endemic controls were investigated using the ELISA. The effectiveness of 12 recombinant antigens and Ag85Aulc as potential vaccines were tested using the ELISA method. Also, concentrations of cytokines (interferon-γ, IL-5) in overnight supernates of whole blood assays in Buruli ulcer patients who developed paradoxical reaction, after stimulation with M. ulcerans sonicate antigens were measured using ELISA.
Results: The results show that responses to antigens were generally high (above 80% responders) with the exception of ACP3 where active Buruli ulcer cases had 47% and endemic controls 71% responders. The highest percentage responders in both participant groups were observed in reaction to Ksalt (100% responders) and ER (100% responders) antigens. A higher proportion of endemic controls responded with higher interferon-γ responses than the Buruli ulcer cases to all the PKS domain antigens and to DNA encoding mycolyltransferase Ag85A of M. ulcerans (Ag85Aulc). There was no response to any of the antigens in all but one of the control participants from non-endemic areas. There was a trend to increasing interferon-γ responses with treatment; this was not statistically significant except for ACP2 and ATac2. Patients with non-ulcerative lesions mounted generally higher interferon-γ responses than those with ulcerative disease. There was a positive but not statistically significant association between interferon-γ responses and time to healing of the patients to the PKS antigens.
Although patients who developed paradoxical reactions mounted a lower interferon-γ response [median (range) 754.32 (50.93-4190.4) pg/ml] at baseline compared to patients who had no paradoxical reactions [1246.93 (81.11-6969) pg/ml], there was no statistically significant difference between the two groups. By contrast, these two groups of patients elicited comparable median interleukin-5 (IL-5) response levels 35.61 (24.79-67.83) pg/ml vs 35.52 (11.82-993.90) pg/ml). Patients who developed paradoxical reaction mounted a consistently low Th1 and Th2 responses when compared with patients who did not develop paradoxical reactions. Th1 and Th2 responses of patients who developed paradoxical reaction improved with treatment.

Conclusion: These results suggests that the immune response of patients to PKS domains was lower compared to that of the contacts, which could be suggestive to the immunosuppressive effect of mycolactone on immune response. ER and Ksalt were the most immunogenic antigens. A vaccine made up of the most immunogenic plasmid DNA encoding mycolactone polyketide synthase domains and Ag85Aulc is an interesting possibility that require further study. We have confirmed that paradoxical reaction has an effect on the immune response of patients.

1.0 Background
Buruli ulcer is a skin disease which affects the soft tissues and is caused by infection with a slow growing pathogen, Mycobacterium ulcerans (Mu) (Demangel et al., 2009). Cases have been reported from 33 countries from tropical, subtropical and temperate climates in Africa, South America and the Western pacific regions respectively (WHO, 2014). Globally, over 5000 cases are consistently reported annually from 15 out of the 33 countries. Of the number, children below the age of 15 years are most infected with 48% from Africa, 10% from Australia, and 19% from Japan (WHO, 2014). Majority of the cases reported from sub-Saharan Africa are from poor rural communities (WHO, 2014). Even though, there is a strong association between incidence rate of the disease with stagnant or flowing water bodies, the specific mode of transmission remains elusive (Bratschi et al., 2014). The disease usually manifests itself as a painless nodule, a firm plaque, or oedematous lesion which soon ulcerates with characteristic undermined edges (Etuaful et al., 2005). However, late presentation of lesion results in scars with contractures and disabilities when found over joints and at times amputation (Huygen et al., 2009).

Reliable diagnosis of Mu infection is of significant importance to the success of clinical studies of Buruli ulcer. The gold standard confirmatory test for Mu is Polymerase chain reaction (PCR) for the IS2404 repeat sequence. This is undertaken in the Buruli ulcer laboratory at the Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) on routine bases. Studies have been published proving the high sensitivity of PCR on DNA extracted from punch biopsies and subsequently from fine needle aspirate samples (Phillips et al., 2005; Phillips et al., 2009; Eddyani et al., 2009).

Treatment has shifted from surgery to antibiotic therapy with the combination of rifampicin and streptomycin for 56 days which is more efficacious in healing all lesions caused by Mu disease. This therapy has been reported to reduce the recurrence rate from 6-47% after surgery to 0-2% after antibiotic treatment (Chauty et al., 2007; Sarfo et al., 2010). Although antibiotic therapy was shown to be effective in treatment of Mu infection (Gordon et al., 2010), a phenomenon called paradoxical reaction, has been reported in some of the patients. In this situation, patients develop clinical deterioration of lesion(s) following initiation of antibiotic therapy. It is characterized by the rapid worsening of ulcers and progression of non-ulcerated lesion forms into ulcers. This can also be reported when there is a formation of a new lesion. Paradoxical reaction occur during antibiotic treatment (Beissner et al., 2010). This reaction have now been proved to complicate up to 20% of patients on antibiotic therapy for Mu infection and can cause significant secondary tissue necrosis (Wanda et al., 2014). Exposure to mycobacterial antigens, a reduction in suppressor mechanisms, or improved host cell–mediated immunity have been implicated as responsible for this reaction (Nienhuis et al., 2012).

Research has shown that Human Immunodeficiency virus (HIV)-positive individuals on highly active antiretroviral therapy experience the worst episode of paradoxical reactions (Beissner et al., 2010). Buruli ulcer-HIV co-infection is an emerging management challenge for Buruli ulcer disease (O’Brien et al., 2014a). There is an increased incidence of several forms of Buruli ulcer disease in HIV infected individuals (Wanda et al., 2014). Co-infection with HIV is thought to result in more severe disease and slower healing times following treatment (O’Brien et al., 2014a). Nevertheless, the World Health Organisation (WHO) recommends that all co-infected patients be actively screened for tuberculosis before commencing Buruli ulcer disease treatment and before starting antiretroviral therapy (ART).

Additionally,cotrimoxazole preventive therapy followed by the combination antibiotic therapy for 8 weeks and ART should be administered to HIV co-infected patients living in an area with established prevalence of malaria and or bacterial infections (O’Brien et al., 2014a).

Through research there have been some advancements over the years to better understand Mu infection with respect to immunology. Previous studies have used whole bacteria or burulin, which is a crude, heat-killed bacterial sonicate as well as culture filtrate proteins to elucidate the cellular immune response against Mu (Huygen et al., 2009).

Patients with early Buruli ulcer disease elicited a delayed hypersensitivity response following intradermal injection of a crude preparation of burulin resulting in no immune response. However, 45 (76%) patients with healed lesions who were initially non-reactive, mounted favourable responses which is an indication of T-cell responsiveness (Dobos et al., 2000).

A Study was conducted on ten individuals with a history of previous Buruli ulcer disease and four patients with active disease in Australia. Peripheral blood mononuclear cells (PBMC`s) of test subjects was stimulated for 6 days with live M. ulcerans or live Mycobacterium bovis. Low levels of interferon gamma was produced compared to PBMCs from healthy tuberculin-positive study participants indicating that there was T-cell immune unresponsiveness to mycobacterial antigens (Gooding et al., 2001). In a similar study in French Guyana, PBMCs from patients were stimulated with whole killed M. ulcerans or M. bovis. Five patients presenting with early nodules showed more profound Th1 cytokine profile while those with ulcers had a Th2 cytokine profile (Bourreau et al., 2004).

In Ghana, a larger study was carried out using an overnight stimulation of whole blood with M ulcerans sonicate among BU patients and the pattern of interferon gamma (interferon-γ) production was consistent with the hypothesis that the development of a Th-1 response is a slowly developing process (Phillips et al., 2006). Using similar methods, it was shown in a subsequent study that a gradual but significant recovery of the interferon-γ response emerged with antibiotic treatment at week 4 and week 8 compared with baseline (Sarfo et al., 2009).

Culture filtrate antigens 423 and 425 induced a similar pattern but lower interferon-γ response than those against Mu sonicate (Phillips et al., 2006). An essential fraction of the secreted proteins in mycobacterial culture filtrates is the mycolyl transferase antigen 85 (Ag85). Antigen 85 is a 30-32 kDa family of three proteins (Ag85A, Ag85B and Ag85C) (Fakult and Sch, 2009) , which all have a mycolyl transferase enzymatic activity needed for the integrity of the cell wall. This cross-reactive antigen has been researched in detail (Tanghe et al., 2008). Most of the healthy subjects infected with M. tuberculosis or M. leprae and in BCG vaccinated mice after stimulation with purified antigen-85 from BCG elicited a profound T-cell proliferation and interferon-γ secretion (Wiker and Harboe, 1992). Also, in patients with Buruli ulcer, their PBMCs produced lower interferon-γ responses against Ag85 purified from BCG compared to that from healthy BCG vaccinated participants (Gooding et al., 2001).

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