Improving Buruli ulcer control Wadagni, Anita

University of Groningen

Improving Buruli ulcer control Wadagni, Anita

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10.33612/diss.171907958

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2021

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Wadagni, A. (2021). Improving Buruli ulcer control: steps towards decentralized care. University of Groningen. https://doi.org/10.33612/diss.171907958

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Chapter 1

Introduction and outline of the thesis

Buruli ulcer, a Neglected Tropical Disease, is a chronic infectious disease of the skin, subcutaneous tissues and bone; it is caused by Mycobacterium ulcerans [1]. It represents, after tuberculosis and leprosy, the third most common mycobacterial disease in

immunocompetent patients. Buruli ulcer manifests as non-ulcerative lesion (nodule, plaque or edema) that can progress, in the absence of treatment, to a large ulcer [1-3]. All of the pathology observed is the result of one single virulence factor: a secreted toxin, called mycolactone [3, 4]. Although generally not fatal, people with BU might be left with cosmetic or functional deformity that can last a lifetime if effective treatment is unavailable or

delayed; such deformities in turn may result in long-term functional disability [1-3, 5], stigma [6] and restriction in social participation [7].

First descriptions of Buruli ulcer

Chronic ulcers resulting from a new mycobacterial infection were first reported by

researchers from the university of Melbourne, Victoria, Australia, in 1948 [8]. Patients aged 2.5, 13, 16, 45 and 51 years had been identified with chronic ulcers in a hospital in

Bairnsdale, over a period of five years. Biopsies were sent to the pathologist in Melbourne, and abundant extracellular acid-fast bacilli were noted in the biopsy specimens. First attempts to culture the organism failed; rats were however successfully inoculated, and these rats showed signs of infection that could in turn be passed on to other rats. Specimens from the sixth patient were eventually successfully cultured in vitro, on egg-enriched

culture media, at around 30-33°C. This sixth patient, 26 years old, came from a rural area

200 miles away from Bairnsdale. Four of these patients had lesions on the lower leg, two

had lesions on their arm. In the lesions of these patients, many other organisms including

streptococci and staphylococci were occasionally found, but the presence of mycobacteria

was invariably present. Wide excision of ulcer margins and subsequent skin grafting

resulted in healing. Material from the wound on one of the patients was injected

subcutaneously into a rat that subsequently developed swollen, ulcerated limbs and a

sloughed-off tail, with numerous acid-fast bacilli in these lesions [8]. Subsequently, a whole

array of different experimental animals was inoculated, and their pathology resembled

closely the lesions observed in human patients. In the then-called Belgian Congo – now

called, DR Congo – cases of chronic skin ulceration caused by mycobacteria were described,

with the first case noted as early as 1942, from eastern parts of the country [9-12]. Janssens

who commented on the report by van Oye and Ballion [9], citing the report by MacCallum

[8], observed that antibiotics including streptomycin failed, and expressed his conviction

that resection of the ulcers with wide margins, followed by skin grafting, was the only way

to obtain cure [9]. Clancey et al. first described M. ulcerans infection from Uganda [13]. In

Australia, the lesion was called ‘Bairnsdale ulcer’ after the place where the first cases were

identified. Later, it was called ‘Buruli ulcer’, a name derived from the Buruli district near the

Nile river, where refugee camps hosted refugees that had fled the Hutu-Tutsi civil war from

the neighboring country Rwanda [14]. Ulcerative lesions with undermined edges, typical for Buruli ulcer, were earlier described from Uganda by Albert Cook, a missionary doctor

working in the Mengo Hospital near Kampala, in the end of the 19

century [15].

As explained above, M. ulcerans was first described as the cause of Buruli ulcer from Australia. Although the authors did not mention the name of the organism, they first discovered to be the cause of Buruli ulcer, they named the organism M. ulcerans later, in a personal communication with Fenner [16]. Buruli ulcer was diagnosed for the first time in Benin in 1977 [17]. In an early report from Benin, the need for surgical treatment was emphasized [18].

Epidemiology

As of today, Buruli ulcer has been reported in over 33 countries around the world. Most cases have been reported from West and Central Africa - especially from Benin, Côte d’Ivoire, Ghana, Cameroon, Democratic Republic of the Congo, and Nigeria [1-3]. In West Africa, many cases have been reported from Benin [17, 19-21]. The disease is endemic in the south of the country along the Ouémé and Couffo rivers [21, 22]. The National Program for the Control of Leprosy and Buruli Ulcer relies on four reference centers (CDTUB) for the care of patients. These centers are located in the various endemic departments (Allada, Lalo, Pobè and Zagnanado). The epidemiology of Buruli ulcer has been fluctuating; it has emerged in some areas and disappeared from areas once highly endemic [1, 23-27].

M. ulcerans reservoir and transmission

The mode, or modes, of transmission have not been convincingly identified to date [1]. It is widely believed and assumed that M. ulcerans infection has an as yet incompletely

understood environmental reservoir ^{[28, 29]} , and infection may result from direct

inoculation subcutaneously, either by trauma like thorn pricks [11], or insect bites [30-32].

Using Whole Genome Sequencing approaches of M. ulcerans, it has been suggested that humans might be involved in shedding M. ulcerans into the environment, resulting in new endemic foci of transmission [33, 34]. Direct transmission between humans has been considered exceedingly unusual [35, 36].

Host susceptibility, protection and prevention

Not all individuals, exposed to M. ulcerans, develop disease; many healthy people living in endemic areas show immune recognition of M. ulcerans without developing disease [37-39].

A genetic polymorphism detected in a specific gene (SLC11A1) has been shown to play a role in susceptibility to develop Buruli ulcer disease [40]. Subsequent studies addressing genetic susceptibility to develop Buruli ulcer have been reviewed [41].

Environmental exposure of skin to areas close to slow-flowing water bodies and marshes

has been identified as risk factor for Buruli ulcer [42], and wearing long pants provided

some protection [43]. Fetching water is a risk factor that could be prevented by installing water wells providing piped water; in Benin, the incidence of Buruli ulcer declined in villages where water well had been drilled [44].

A vaccine would be an alternative approach to prevent Buruli ulcer [45]. In animal models, BCG vaccination provides some protection against Buruli ulcer when challenged with experimental M. ulcerans infection [46, 47]. Earlier studies in humans suggested a protective effect as well [48, 49] but a more recent study did not confirm these findings [50].

Pathogenesis; the role of mycolactone

As mentioned above, mycolactone plays a dominant role in human disease following infection with M. ulcerans. The presence of a secreted toxin was suspected long ago [51].

When a culture filtrate of M. ulcerans is injected into the subcutis of a guinea pig, lesions developed that were similar to lesions observed following inoculation of M. ulcerans [52, 53]. In 1999, the chemical structure of mycolactone was first described [4]. In vivo, the mycolactone molecule is constructed by combining several building blocks; the core and side chain are synthesized by three polyketide synthase enzymes encoded by a large plasmid - pMUM001 [54, 55], while three additional cell wall-bound enzymes (MLSA1, MLSA2 and MLSB) are necessary to join the building blocks of the toxin [56]; these additional enzymes are produced by genes mup045, mup038 and mup053. The biological effects of mycolactone are three-fold. First, it causes necrosis and apoptosis [57]. The second effect is a down-regulation of the host immune defense - partly as a result of apoptosis of immune cells, by inhibiting Sec61, an important transporting complex for secretory proteins into the endoplasmatic reticulum [58-60]. The third effect is an effect on sensory nerves. Buruli ulcer starts as a painless lesion, and only when treatment results in a clean wound, pain returns. First it was believed that this lack of pain sensation was

exclusively caused by nerve damage due to destruction of Schwann cells [61-63], but pain sensation may also be reduced as a result of impaired nerve conduction in sensory nerves [64]. Interaction of mycolactone with the type 2 angiotensin 2 receptor pathway is involved in this impaired nerve conduction [65].

Mycolactone does not only act locally, but there is an important systemic effect as well [66- 68]. It takes several weeks after surgical excision or antimicrobial treatment before

mycolactone has disappeared, and for immune responses to return to normal [69-71].

Typically, the return of normal immune defense coincides with the end of eight weeks of antimicrobial treatment, around the time that clinically, Buruli ulcer lesions may (slightly) deteriorate, a phenomenon called paradoxical reaction [40, 72]. These paradoxical

reactions are characterized by intense inflammation in lesions and a worsening clinical

condition [73-75]. These reactions should not be mistaken as failure to respond to

treatment.

Diagnosis

The clinical diagnosis is fairly reliable if made by experienced clinicians in endemic regions [76, 77]. The first laboratory test to confirm the diagnosis of Buruli ulcer was

histopathologic evidence of the presence of acid-fast bacilli in subcutaneous tissues, or coalescent necrosis of subcutaneous fat tissue. The first attempts of cultivation were

unsuccessful, but finally, culture succeeded at a temperature around 33°C [8]. In DR Congo, M. ulcerans was also successfully cultured on a Löwenstein slope at a temperature around 30° - 32°C [12]. Many years later, in Australia, a PCR assay was developed based on a species-specific 1,109-bp DNA non-coding fragment (Insertion Segment 2404, or IS2404);

at least 50 copies of this insertion fragment are present in each M. ulcerans genome [78]. M.

ulcerans DNA detection has been improved and IS2404 appeared to be specific for M.

ulcerans [79, 80]. PCR appears to be the most sensitive diagnostic test for Buruli ulcer, among the four standard laboratory methods - IS2404 PCR, direct microscopy,

histopathology and culture [81-83].

WHO recommends microbiological confirmation of at least 70% of patients suspected to have Buruli ulcer. The implementation of this WHO recommendation requires the existence of simple, rapid and reliable laboratory tests. As mentioned above, PCR for IS2404, which has sensitivity of 92–95% has been established as the gold standard for Buruli ulcer case confirmation [79, 81, 84]. PCR requires training and laboratory skills, and it is expensive, and only available in specialized, centralized laboratories away from rural remote areas where Buruli ulcer is endemic [84]. Establishing and maintaining the required quality of PCR is challenging, with a variable level of performance of the laboratories currently

providing diagnostic services [85]. New diagnostic tools for Buruli ulcer that can be used in peripheral health facilities are in dire need, and numerous studies have been carried out to develop a simple diagnostic test. A DNA amplification method called Loop Mediated

Isothermal Amplification (also known as LAMP); and mycolactone detection by the fluorescent Thin-Layer Chromatography (fTLC); f-TLC may offer a new tool for confirmation of suspected Buruli ulcer cases [86-88].

Surgery versus drug treatment

As explained above, wide surgical excision of wound margins, followed by skin grafting has been the standard practice for Buruli ulcer since long [8, 9, 13, 18]. The concept that

surgery even with resection of a wide margin would be able to cure M. ulcerans infection was challenged by researchers who were able to show that in the edge of resected tissue, still viable M. ulcerans organisms or at least, PCR signals for IS204 were present [89, 90].

Although many patients could be cured with surgery alone, the recurrence rate was

variable, with some centers reporting acceptably low rates – 6% [91]; but other authors

reporting 17-18% [92, 93], 26% [94] and even up to 47% [95].

In vitro studies had consistently shown a convincing bacteriostatic effect for clarithromycin, and a bactericidal effect of rifampicin and streptomycin on M. ulcerans [96]. Early studies evaluating the response to antimicrobial treatment failed to show an appreciable effect of clofazimine [97] or a combination of dapsone and rifampicin [98]. In retrospect, the design of these studies did not incorporate our current knowledge of the profound

immunosuppressive effects of mycolactone, and its suppressive effect on tissue repair;

therefore, the follow-up might have been too short to detect a beneficial effect. A study evaluating the bacteriological response of a combination of streptomycin and rifampicin in patients with pre-ulcerative Buruli ulcer lesions – predominantly, nodules – showed that indeed, antimicrobial treatment alone was able to kill M. ulcerans in the nodules that were resected following at least 4 weeks of treatment [99]. In 2004, WHO therefore changed the provisional guidelines for the treatment of Buruli ulcer; the use of antimicrobial treatment – initially, a combination of 8 weeks of oral rifampicin (10 mg/kg) and streptomycin

intramuscularly (15 mg/kg) was now recommended. [1, 100]. Meanwhile, and especially for larger lesions, the role of surgery – if any - was left to the discretion of the attending physician.

Outline of the Thesis

What is the current epidemiology and disease severity in Benin?

A recent study on the evolution of epidemiological and clinical characteristics of BU over time and space, carried out in Lalo (one of the most endemic districts of Benin) from 2006 to 2017 revealed a decrease in rates prevalence of Buruli ulcer in this region with less severe cases diagnosed [101]. In Chapter 2, we report on the study of the epidemiology of Buruli ulcer at a national level in order to understand the current distribution and burden of the disease in Benin.

What is the effect of the implementation of a decentralized community-based treatment program on the management of Buruli ulcer?

For many years, the National Control Program for Buruli ulcer in Benin has implemented a vertical approach to Buruli ulcer control that relied on surgery and hospitalization. From 2004 onward, Buruli ulcer patients care began being integrated into the government health system [102]. Decentralized management of patients with Buruli ulcer has been available in three out of four endemic departments in the country, and that has resulted in improved early detection of Buruli ulcer. In the Zou department, an area with a high prevalence of severe cases of Buruli ulcer, the strategy of decentralization has not yet been implemented.

In Chapter 3, we describe the implementation of the decentralization program which is

based on an innovative approach, and we hypothesized that the decentralization of care

improves indicators of the detection and management of Buruli ulcer in the department

Zou.

Can the fluorescent Thin Layer Chromatography (f-TLC) to detect mycolactone be used for diagnostic confirmation of Buruli ulcer in endemic areas in Africa?

The secretion of mycolactone responsible for necrosis observed in Buruli ulcer and found in skin biopsies of mice and humans infected with Mycobacterium ulcerans [67, 88, 103].

Mycolactone has been detected in infected tissue by fluorescent thin layer chromatography and might therefore be used for laboratory confirmation [88]. In Chapter 4, we address the hypothesis that the fluorescent thin layer chromatography technic is effective in terms of sensitivity and specificity compared to PCR for the biological diagnosis of Buruli ulcer.

What is the impact of delaying the decision of surgery in patients with Buruli ulcer?

In Chapter 5, we report a randomized clinical trial to address the hypothesis that delaying the decision about surgery to 14 weeks after start of treatment – compared to usual care whereby a decision about surgery was taken at around week 8, the end of antimicrobial treatment, would result in a reduction in the need for resection surgery; we explored several different secondary endpoints including the time to healing; residual functional limitations; and overall duration of hospitalization.

In Chapter 6, we report a survey of surgical practice in different hospitals providing care for patients with Buruli ulcer in Benin and Ghana.

Chapter 7 provides a Discussion with Future Perspectives, and Chapter 8 summarizes the findings of the studies included in this Thesis.

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