Hindawi Publishing Corporation
Canadian Journal of Infectious Diseases and Medical Microbiology
Volume 2016, Article ID 5310718, 10 pages
http://dx.doi.org/10.1155/2016/5310718
Review Article
Clinical and Laboratory Diagnosis of Buruli Ulcer Disease:
A Systematic Review
Samuel A. Sakyi,1,2 Samuel Y. Aboagye,1
Isaac Darko Otchere,1,3 and Dorothy Yeboah-Manu1,3
1Department of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
2Department of Molecular Medicine, School of Medical Sciences, Kwame Nkrumah University of Science and Technology (KNUST),
Kumasi, Ghana
3Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Accra, Ghana
Correspondence should be addressed to Samuel A. Sakyi; samasamoahsakyi@yahoo.co.uk
Received 14 March 2016; Accepted 25 May 2016
Academic Editor: Aim Hoepelman
Copyright © 2016 Samuel A. Sakyi et al.This is an open access article distributed under theCreative CommonsAttribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Buruli ulcer (BU) is a necrotizing cutaneous infection caused byMycobacterium ulcerans. Early diagnosis is crucial to
prevent morbid effects and misuse of drugs. We review developments in laboratory diagnosis of BU, discuss limitations of available
diagnostic methods, and give a perspective on the potential of using aptamers as point-of-care. Methods. Information for this
review was searched through PubMed, web of knowledge, and identified data up to December 2015. References from relevant
articles and reports fromWHO Annual Meeting of the Global Buruli Ulcer initiative were also used. Finally, 59 articles were used.
Results. The main laboratory methods for BU diagnosis are microscopy, culture, PCR, and histopathology. Microscopy and PCR
are used routinely for diagnosis. PCR targeting IS2404 is the gold standard for laboratory confirmation. Culture remains the only
method that detects viable bacilli, used for diagnosing relapse and accrued isolates for epidemiological investigation as well as
monitoring drug resistance. Laboratory confirmation is done at centers distant from endemic communities reducing confirmation
to a quality assurance.Conclusions. Current efforts aimed at developing point-of-care diagnostics are saddledwithmajor drawbacks;
we, however, postulate that selection of aptamers against MU target can be used as point of care.
1. Introduction is low, morbidity and subsequent functional disability can
be severe [5–8]. The main virulence factor responsible for
Buruli ulcer disease (BUD) is a neglected tropical dis- the pathology of BUD is mycolactone. Mycolactone, an
ease caused by the environmental pathogen Mycobacterium immunosuppressive and cytotoxic macrocyclic polyketide, is
ulcerans (MU). The disease is characterized by necrotizing,
ulcerative lesions of subcutaneous fat and the overlying skin widely distributed within infected human lesions and has
and is prevalent in poor regions of Africa, the Americas, Asia, been postulated as a marker for diagnosis of BUD [9]. The
and the Western Pacific [1]. The exact mode of transmission social and economic burden of BUD can be high, particularly
ofMU remains unclear, but accruing data suggests that, prob- in impoverished rural regions. The disease affects both sexes
ably, different modes of transmission occur in different geo- equally and all age groups, but it is particularly common in
graphic areas and epidemiological settings [2]. BUD begins children under the age of 15 [10].
with a preulcerative stage characterized by a firm nontender Previously, BUD was treated by wide surgical excision
nodule, edema, or plaque with large areas of indurated skin, followed by skin grafting; however, a study initiated byWHO
which is then followed by ulceration due to extensive skin cell and conducted in Ghana indicated that BU lesions can be
destruction leading to the typically undermined edges [3, 4]. sterilized by treatment with streptomycin and rifampicin
If left untreated, self-healing may occur which often leads to [11]. Following that, the mainstay treatment protocol for
loss of vital organs and contractures. Even though mortality BU is daily oral rifampicin plus intramuscular injection of
2 Canadian Journal of Infectious Diseases and Medical Microbiology
streptomycin for 56 days, reducing surgery as an adjunct for 353 articles identified from various
correction of deformities [3, 12].With the introduction of this 234 duplicates and unrelatedarticles
antimycobacterial treatment, confirmation of clinically sus- databases 
pected cases is evenmore crucial for the clinical management
of BU to prevent misdiagnosis and hence administration 119 abstracts reviewed
of unnecessary antibiotics. Previous reports of individuals
treated for BUbutwere later found not to be BUby laboratory
confirmation are available in literature [13–15].
Laboratory diagnosis of BU is multifaceted and has 87 full text articles retrieved and 32 articles did not meet
evolved over the years.There are currently fourmainmethods reviewed inclusion criteria
that are being used for the laboratory confirmation of BUD
and includemicroscopy for detecting acid-fast bacilli, culture
to isolate viable organism, PCR for detecting pathogen
specific DNA which is usually IS2404, and histopathology. 59 articles with original laboratory diagnosis/
The WHO recommends two laboratory tests to confirm detection/confirmation of Buruli ulcer were included
BUD. However, in endemic settings, one may consider one in final review
positive test result from PCR or microscopy appropriate for
the confirmation of clinical diagnosis because of the high Figure 1: Schematic selection of review articles.
positive predictive values for PCR (100%) and microscopy
(97%) [3, 16]. In this review, we describe developments in
the field of laboratory diagnosis of BUD, discuss applications the following: (1) swabs should be collected by circling the
and limitations of currently available diagnostic methods, entire undermined edge of ulcerative lesions to maximize
and provide data on positivity and sensitivity ratios. This cell collection as MU is not uniformly distributed in the
review further gives a perspective on the potential of selecting ulcers [21]. A good sample collection can be achieved through
aptamers against MU targets for the development of a point- collection of at least two swabs per lesion; (2) FNA should be
of-care diagnostics for BUD. collected from the weakest part of the lesion to increase the
chance of collecting MU cells; and (3) tissue samples from
ulcerative lesions should be taken from the edge of the lesion,
2. Methods preferably below the end of the undermined edge, and should
2.1. Search Strategy and Selection Criteria. Searched infor- contain necrotic tissue. For nonulcerative lesions, tissue
mation for this review was done through PubMed, web samples should be collected from the center of the lesion.
of knowledge and Embase databases, and identified data Tissue samples must always contain subcutaneous adipose
up to December, 2015. References from relevant articles tissue. All the samples including FNA should be evaluated
together with other published data from the WHO website by microscopy, PCR, and cultivation [22, 23]. Laboratory
and unpublished data presented at annual WHO advisory confirmation of osteomyelitis cases requires whole bone
group meetings on Buruli ulcer were also used.The literature samples (e.g., from amputation specimens) or curetted bone
search was done using the following keywords: Mycobac- samples [17, 24, 25]. Table 1 summarizes the various types of
terium ulcerans, laboratory diagnosis and confirmation, and specimen and transport media used for diagnosing BUD.
methods for BU diagnosis and BU.
3.2. Microscopy. Microscopy is a quick, comparatively sim-
ple, and low-cost approach for the laboratory confirmation
2.2. Assessment and Data Extraction. Articles in the full-text of suspected BUD cases and can be done with FNA, tissue,
review were classified as containing original laboratory diag- or swabs specimen. Microscopic diagnoses by direct smear
nostic methods for Buruli ulcer including sample collection examination with Ziehl-Neelsen staining to detect the pres-
methods, microscopy, culture, molecular techniques (PCR ence of acid-fast bacilli are done using the quantification
and its offshoots), and histopathology. Figure 1 illustrates how of smears in accordance with the method locally used for
the review articles were searched and selected. the diagnosis of TB [25]. The technological simplicity and
requirement of low infrastructure allow microscopy to be
3. Results and Discussion conducted at all levels of health care delivery, even in less
resourced countries. However, recorded sensitivity in litera-
3.1. Samples for Laboratory Confirmation of Buruli Ulcer. ture is quite low and therefore undermines the overreliance
Samples for laboratory diagnosis of BUD include swabs and of microscopy for case confirmation. Studies in Ghana and
tissue specimens [17] from punch biopsies, surgical excision Benin which used microscopy as a first-line diagnosis of BU
[18], and fine needle aspirates (FNA) [19]. FNA and tissues reported positivity rates between 40% and 78% [24, 37, 40].
are used for analysis of nonulcerative lesions, whilst all Tissue smears prepared from ground samples can also
other specimen types can be collected from ulcerative tissues be used for microscopy as well as from material hitherto
[20]. However, with the advent of chemotherapy, FNA and subjected to decontamination procedures for culture. Never-
swabs are becoming the preferred sample for laboratory con- theless, according to a recent study in Benin, grounding of tis-
firmation. Recommendations for sample collection include sue does not increase the sensitivity of tissue smears (56.7%)
Canadian Journal of Infectious Diseases and Medical Microbiology 3
Table 1: Summary of types of specimen and transport media for BU diagnosis.
Materials for
diagnosis Types Country of origin Reference
Swabs Ghana Yeboah-Manu et al. [26]; de Souza et al. [27]
Togo Bretzel et al. [28]
Ghana de Souza et al. [27]; Phillips et al. [29]
Punch biopsy Australia O’Brien et al. [30]
Togo Bretzel et al. [28]
Specimen Benin Ruf et al. [31]
Biopsy Ghana Stienstra et al. [32]
Ghana Ablordey et al. [33]; Yeboah-Manu et al. [26]
Fine needle aspirate Togo Bretzel et al. [28]
Benin Eddyani et al. [19]
Whole bone or curetted bone samples Ghana Herbinger et al. [17]; Bretzel et al. [24]
Modified Dubos medium (P5 medium) Ghana Stienstra et al. [32]; Yeboah-Manu et al., [34]
Liquid Middlebrook 7H9 broth Benin Eddyani et al. [19]; Dobos et al. [35];
Transport media 10% OADC augmented with PANTA Ghana Wansbrough-Jones and Phillips [9]
Solid transport media (STM) Benin Eddyani et al. [19]
Liquid Nitrogen Ghana Rondini et al. [21]; Beissner et al. [36]
Oxalic acid Ghana Mensah-Quainoo et al. [37]; Yeboah-Manu et al. [34]
Decontamination N-Acetyl-cysteine-NaOH technique Ghana Schunk et al. [8]
methods
Reversed Petroff technique Ghana O’Brien et al. [30]
Benin Eddyani et al. [19]
Commercial Ghana de Souza et al. [27]
In-house Ghana Ablordey et al. [33]
DNA extraction Modified Boom DNA extraction procedure Ghana Durnez et al. [38]; Affolabi et al. [39]
method Commercial Maxwell 16 DNA extraction Ghana Affolabi et al. [39]
One tube cell lysis (OT) Ghana Durnez et al. [38]
FastPrep procedure Ghana Durnez et al. [38]
compared with direct smears prepared from unground tissue been observed [8]. Culturing MU from clinical samples is
(sensitivity, 59.4%) [40].Whilst ZN staining is used inmost of difficult and has a low sensitivity of about 35–60% [45]. The
the studies, some other studies have suggested that Kinyoun bacteria are extremely slow growing (6–8 weeks) and culture
and auramine-rhodamine staining techniques can also be media are repeatedly contaminated with other faster growing
applied to MU [8, 40]. species [7, 12, 26, 46]. This makes cultures unsuitable for
quick laboratory confirmation and is limited to laboratory
3.3. Cultivation of Mycobacterium ulcerans from Clinical facilities with class II safety cabinets. The contamination
Specimen. Isolation of viable MU by culture is the final effect of fast growing species are, however, counteracted by
proof method among the diagnostics; however, due to the decontaminating the sample with either an acid and or a
technological and infrastructure demand such as biosafety base to remove the unwanted fast growers using protocols
cabinets, cultures are done mainly at research centers of such as the modified Petroff method (sodium hydroxide) [8],
endemic and northern countries. Cultivation of MU from and the reversed Petroff technique (“Fortep” technique) [44].
swabs and punch biopsies is normally transported inMiddle- In a decontamination protocol study conducted in Ghana,
brook 7H9 broth supplemented with polymyxin B, azlocillin, three different decontamination procedures were evaluated
amphotericin B, nalidixic acid, and trimethoprim (PANTA, and concluded that a simple oxalic acid decontamination
Becton Dickinson Biosciences, NJ, USA). Additional sup- method produces high recovery rates [26, 34].
plementation with 0.5% agar yields a semisolid transport Notwithstanding these drawbacks, cultures are consid-
medium (STM) and preserves positive samples for up to ered the only currently available valid confirmatory test for
21 days [25, 41]. Although a number of culture media have detection of viable bacilli in clinically suspected relapses
been evaluated [34, 42, 43], Lowenstein-Jensen is considered and patients with nonhealing lesions after antimycobac-
the most appropriate medium for MU [42, 44]. Cultures are terial treatment [24]. Furthermore, cultures are required
typically positive within 9–12 weeks of incubation at 29–33∘C. for speciation, susceptibility testing, and other downstream
Yet still, longer incubation times of up to 9 months have applications [41]. Culture positivity ratios of 3–80% and
4 Canadian Journal of Infectious Diseases and Medical Microbiology
sensitivities of 45–70% have been reported [6, 37, 46, 47].The PCR and real-time PCR targeting the insertion element
isolation of acid-fast bacilli fromBUDpatients alone does not IS2404.The insertion sequence IS2404 is present in high copy
offer adequate proof of the presence of MU. A cohort study numbers in the MU genome and it is considered as the gold
in Ghana, indicated that a number of patients harbor other standard because it has the highest sensitivity [56] and results
nontuberculous mycobacteria [37]. It is thus imperative that are accessible within a short time. A positive PCR result is
a confirmation of cultured isolates should be done. The main considered sufficient evidence to commence antimycobacte-
methods that have been used for isolate confirmation include rial treatment; moreover, real-time PCR is being considered
sequence analysis and/or PCR detection of the insertion for monitoring antimycobacterial treatment. However, the
sequences IS2404, IS2606, ketoreductase gene of the giant technique is expensive, requires sophisticated laboratory, and
plasmid, rpoB gene, the 16S–23S ribosomal RNA (rRNA) expertise, a strict quality control, and does not distinguish
internal transcribed spacer gene, the 16S rRNA gene, VNTR, between viable and nonviable organism [3, 57]. A WHO
and the 65-kDa hsp gene, [17, 48–51]. report further encourages endemic countries to confirm at
least 50% of all cases of PCR, either locally or with an external
3.4. Histopathology. Histopathology as a diagnostic method PCR reference laboratory [16].
for BUD provides a fairly rapid result with a very high DNA extraction is a crucial step in PCR processes and
sensitivity (about 90%) [25]. It is also useful in establishing different methods involving in-house as well as commercial
differential diagnosis and monitoring response to treatment. kits are being used. Methods involving mechanical homog-
Histopathological analysis is carried out on tissue specimens enization in a digestion buffer followed by proteinase K
in 10% neutral or buffered (pH 7.4) formalin stained with digestion and purification by the guanidinium thiocyanate-
hematoxylin and eosin, Ziehl-Neelsen, or Kinyoun, and diatoms methods have been applied successfully [29, 58].
auramine-rhodamine. Distinctive histopathological features Durnez et al. compared two adapted extraction methods,
of BUD comprise the presence of acid-fast bacilli, (AFB) the modified Boom (MB) DNA extraction procedure with
hyperplasia of the epidermis, elastolysis, inflammation, vas- a commercial Maxwell 16 DNA extraction procedure (M16,
cular variations of the dermis, and fat necrosis of the subcutis Promega, WI, USA), based on enzymatic lysis and paramag-
[25, 44]. In nonulcerated lesions, the epidermis is unbroken netic separation, and demonstrated the superiority of theMB
but hyperplastic.The upper dermis is intact but shows several in terms of IS2404 PCR sensitivity with clinical samples [38].
stages of degeneration with infiltration of inflammatory Another study compared semiautomated DNA extraction
cells. There is also clotting necrosis of the lower dermis, method using Maxwell kit with a modified Boom method
subcutaneous tissue, and underlying fascia with oedema. and observed thatMaxwell extractionmethod, performed on
Vasculitis is common in the subcutaneous tissue. The ZN nondecontaminated suspensions, is the best for themolecular
stain reveals large numbers of extracellular AFB in clusters, diagnosis ofMU [39]. Other promisingmethods include heat
confined to the necrotic areas. In ulcerative lesions, ulcers and alkaline lysis by NaOH and sodium dodecyl sulphate fol-
are undermined with reepithelialization of the edges of the lowed by phenol-chloroform purification [26, 34, 59]. Many
lesion and undersurface of the superimposing flap of the commercially available kits particularly Gentra systems and
dermis. Neighboring epidermis is usually hyperplastic with Puregene Genomic DNA purification kits have successfully
AFB located at the base of the central slough and necrotic been used with proteinase k to extract DNA from swabs,
subcutaneous tissue [25]. Many studies have suggested that FNA, and tissue samples [17, 24, 44, 47]. It is recommended
histopathology can identify about 30% additional cases than that DNA extraction is performed in a separate area using
other confirmatory tests combined, mainly from paucibacil- dedicated reagents and equipment to reduce the possibility
lary late or healing stages of the disease [20, 24, 47, 52]. of contamination.
However, histopathological features cannot always provide Samples for PCR can be processed within hours to a
clear-cut identification, as granulomas diffuse mixed cellular day without prior storage in transport media [8] or stored
∘
infiltrates and dense lymphocyte aggregates in the locality at −20 C until processing or stored in transport buffers
of vessels during antibiotic treatment [53]. Moreover, the which is compatible with the extraction method [29, 58].
method is expensive to perform and requires a sophisti- Many studies used transport media enriched with OADC,
cated laboratory and highly trained personnel. Furthermore, supplemented with PANTA and 0.5% agar [22, 42, 48, 57,
the technique is invasive as it requires 3mm to 4mm in 60, 61]. Transport of samples in liquid nitrogen has also
diameter punch biopsies. Figure 2(a)(A and B) indicates been reported [21], dried swabs are also being used for
epidermal hyperplasia and necrotic subcutis with fat cell DNA extraction, and positive PCR has been achieved after
ghost, respectively, whilst Figure 2(b) indicates acid-fast stain two weeks. PCR can also be done on paraffin-embedded
of lesion specimen showing characteristic clusters of AFB in tissue specimens using xylene-based deparaffinization for 10
the preulcerative stage. minutes at room temperature [16, 54].The initial primer design used for detectingMU insertion
sequence IS2404 was MU1 and MU2 for amplification of a
3.5. Polymerase Chain Reaction (PCR). Polymerase chain 569 bp fragment.These primers were burdenedwith spurious
reaction (PCR) methods have been developed for BU diag- banding and were improved with MU5 and MU6 primers
nosis based on the insertion sequence IS2404 [54], 16S rRNA which amplify the 492 bp fragment [54]. This was tested
gene [45], and the hsp-65 gene [55]. The most routinely with a panel of 45 mycobacteria and other organisms and
used PCR methods are conventional single-step gel-based obtained 100% specificity and detection sensitivity of at least
Canadian Journal of Infectious Diseases and Medical Microbiology 5
(A)
(B)
(a) (A) Epidermal hyperplasia. (B): Necrotic subcutis with fat cell ghosts
(b) Acid-fast stain of lesion specimen showing definitive clusters of AFBs inside the necrotic subcutis in the
preulcerative stage
Figure 2: Histopathological images of Buruli ulcer disease.
0.1 genome equivalents [59, 62, 63]. Primers used in nested Fyfe et al. developed two TaqMan Multiplex real-time PCR
IS2404-based PCR include MU1 and MU2 for amplification assays targeting three independent repeated sequences in
of a 569 bp fragment of IS2404 and PGP3 and PGP4 for the M. ulcerans genome, two multicopy insertion sequences
amplification of a 217 bp product [32, 57]. Primers PU4F and (IS2404, IS2606), and a multicopy sequence encoding the
PU7Rbio with a modified PCR protocol for amplification of a ketoreductase B domain (KR-B) [22]. Affolabi et al. compared
154 bp product of IS2404 have also been described [29, 58]. a single-step PCR, a nested PCR, and a real-time quantitative
For real-time PCR, TaqMan primer sequences are mostly PCR on 74 surgical specimens from patients with clinically
used. suspected Buruli ulcer and observed that real-time PCR
Most endemic countries are tropical and hence the devel- after the modified Boom extraction method and a single-run
opment of a dry reagent based PCR (DRB-PCR) which uses PCR assay after the Maxwell extraction method, performed
lyophilized reagents (PuReTaq Ready-To-Go-Beads, Amer- on nondecontaminated suspensions, are the best for the
sham, UK) and primers have been employed to simplify the molecular diagnosis of BUD [39]. Guimaraes-Peres et al.
process and reduce incidence of false positives [47, 56] and assessed two nested PCRs, the nested IS2404-based PCR and
requirement for elaborate infrastructure for PCR. Specific the nested 16S rRNA gene-based PCR, and observed that
real-time PCR assay allows quantitative valuation and distri- the 16S rRNA gene-based PCR was positive for both MU
bution of MU in BUD lesions and has exhibited much higher and M. marinum; they suggested that the use of IS2404-
sensitivity than the conventional single-run gel-based IS2404 based PCR showed better specificity, required less time, and
PCR. Moreover, the enhanced TaqMan real-time PCR assay was less costly than the 16S rRNA gene-based PCR [57].
shows 12.5% higher diagnostic sensitivity compared with cul- Stienstra et al. also evaluated the IS2404-based nested PCR
tures; the assay reduces contamination and turnaround times to detect MU from 143 BUD patients in Ghana. They further
for diagnosis andhas beenused routinely inAustralia [61, 64]. compared it with culture and histopathology results and
6 Canadian Journal of Infectious Diseases and Medical Microbiology
recommended that small tissue samples might be sufficient of DRB-PCR or cPCR and cLAMP (62.64% and 52.86%)
for case confirmation in future studies [32]. Phillips et al. also were comparable and there was no significant difference
used IS2404 PCR with punch biopsy specimen and obtained between the sensitivity of the assays (DRB-PCR and cPCR,
a positivity ratio of 98% from 70 clinically diagnosed BUD 86.76%; cLAMP, 83.82%).Moreover, the sensitivity of cLAMP
patients [29]. Among 162 clinically diagnosed BUD patients (95.83%) and the sensitivity of DRB-LAMP (91.67%) were
with ulcerative lesions from Cameroon, 83% were confirmed comparable. However, all the reported studies used sophis-
by IS2404 PCR [32]. In another study inDemocratic Republic ticated equipment which cannot be employed in the field and
of Congo, IS2404 PCR was used to diagnose 51 BUD patients there is the need for further work to use simpler equipment
with positivity ratio of 75% [6]. In a similar study in Ghana, in low-resourced laboratory settings; moreover, obtaining
DRB-PCR was used to clinically confirm 67% out of a purified DNA, as well as generating isothermal conditions,
cohort of 161 BUD patients. In this study, the positivity remains a major challenge for the use of the LAMP method
ratio for swab samples was 66%; analysis of tissue samples under field conditions [33].
produced 57% positive results for ulcerative and 63% for Another approach has been serological assays; however,
nonulcerative lesions [24]. In another cohort study of 230 currently available identified MU specific antigens such as
clinically diagnosed BUD patients from Ghana, DRB-PCR the one detecting 85kda protein cannot differentiate between
positivity ratios of 61% were determined for both swab and BU patients and exposed control individuals [69–71]. MUL-
tissue samples [47]. 3720 protein has been identified as a promising target for
In a related study in Togo, out of 202 suspected BUD antigen capture-based detection assays. It is highly expressed
cases, 109 BUD patients (54%) were PCR confirmed over a by MU and has no orthologs in other pathogenic mycobac-
period of three years [28]. These findings indicate that PCR teria. However, quest to use anti-MUL 3720 antibodies in
is considered the most sensitive method for the laboratory a sandwich-ELISA format was found to be of insufficient
confirmation of BUD; however, protracted persistence of sensitivity to make it suitable for the development of antigen
mycobacterial DNA in patients on antimycobacterial treat- capture-based diagnostic tests [72]. Thin layer chromatog-
ment makes PCR not applicable for monitoring of treatment raphy for detecting mycolactone in clinical specimen has
success [17]. also been employed. TLC is comparatively simple but can be
In an attempt to overcome the drawback of PCR, Beiss- complicated by the presence of other lipids in the specimen.
ner et al. developed a MU specific RNA-based viability This step was informed by a study that demonstrated the
assay combining a 16S rRNA reverse transcriptase real-time presence of intact mycolactone in punch biopsies before
PCR (RT-qPCR) to determine bacterial viability with an and during antibiotic therapy using thin layer chromatog-
IS2404 quantitative real-time PCR (qPCR) for increased raphy and mass spectrophotometry [73]. The group further
specificity and concurrent quantification of bacilli [36]. This provided proof of concept that indicated assays based on
technique has previously been applied for the detection of mycolactone detection in serum and ulcer exudates can
viable mycobacteria in patients with tuberculosis and leprosy form the basis of BU diagnostic tests. Fluorescent TLC had
[65, 66]. Conversely, the current test format requires well sensitivity of 73.2% and specificity of 85.7% when compared
equipped laboratory with real-time PCR facilities and the with PCR [68, 74]. A method using a boronate-assisted
costs per test limit its applicability. The reliance on PCR for fluorogenic chemosensor in TLC was employed by Converse
diagnostic and research purposes in the field of BU requires et al., to selectively detectmycolactonewhen visualized under
the continued demonstration of its accuracy, reliability, and UV light. They concluded that F-TLC may offer a new tool
reproducibility. To this effect, Eddyani et al. established a for confirmation of suspected clinical lesions and may be
multicenter external quality assessment program for PCR more specific than smearmicroscopy, faster than culture, and
detection of BUD in clinical and environmental samples simpler than PCR [75]. Recently, Wadagni and colleagues
and reported an improved performance among participating evaluated fluorescent thin layer chromatography (fTLC) for
laboratories [67]. detection of mycolactone in skin samples from patients with
Buruli ulcer and compared them with samples from non-
3.6. Diagnostic Methods in Development. There is the need Buruli ulcer lesions that gave a negative result in the standard
for simpler diagnostic that is both sensitive and specific PCR test for MU [76]. However, further studies are needed
and can be used at the point of care. The loop mediated to determine the feasibility of detecting mycolactone from
isothermal amplification (LAMP) technique has previously samples obtained routinely. Table 2 summarizes the various
been evaluated in many diseases, including malaria, and diagnostic techniques and their positivity ratios.
has been employed. The reported protocol employs four
sets of primers, targeting sequences of the mycolactone
encoding plasmid [27]. To overcome the requirement of cold- 4. Conclusion and Future Perspective
chains for transport and storage of reagents, Beissner et al.
[68] recently establish a dry-reagent-based LAMP (DRB- Molecular techniques for the diagnosis of BUD have proven
LAMP) assay employing lyophilized reagents and clinically to be effective. Notably, real-time PCR offers a consistent
validated 140 clinical samples from 91 suspected BUD cases quantitative and rapid tool for diagnosis and can be used
by routine assays, that is, IS2404 dry-reagent-based (DRB) for monitoring of treatment response of BUD. The devel-
PCR, conventional IS2404 PCR (cPCR), and IS2404 qPCR, opment and application of reverse transcriptase PCR assays
compared to cLAMP. Case confirmation and positivity rates for the detection of viable MU would provide a valuable
Canadian Journal of Infectious Diseases and Medical Microbiology 7
Table 2: Summary of various diagnostic techniques for BU.
Techniques Number +ve −ve Positivity ratio (%) Geographic origin Reference
39 23 16 58.9% Australia O’Brien et al. [30]
31 7 24 22.5% Benin
202 43 159 21.3% Togo Bretzel et al. [28]
24 11 13 45.8% Ghana Beissner et al. [36]
99 78 21 78.8% Ghana Mensah-Quainoo et al. [37]
Microscopy 41 32 9 78.0% Ghana Yeboah-Manu et al. [34]
44 15 29 34.1% Ghana Rondini et al. [21]
65 19 46 29.2% Benin/Ghana Guimaraes-Peres et al. [57]
164 38 126 23.2% Cameroon Noeske et al. [60]
36 22 14 61.1% DRC Phanzu et al. [6]
94 28 66 29.8 Ghana Bretzel et al. [28]
33 — 33 — Australia O’Brien et al. [30]
143 56 87 39.2% Ghana Stienstra et al. [32]
Culture 41 32 9 78.0% Ghana Yeboah-Manu et al. [34]
97 77 20 79.4% Ghana Mensah-Quainoo et al. [37]
65 22 43 33.8% Benin/Ghana Guimaraes-Peres et al. [57]
12 12 — 100.0% Benin Ruf et al. [31]
Histopathology 143 78 65 54.5% Ghana Stienstra et al. [32]
36 27 9 75.0% DRC Phanzu et al. [6]
30 21 9 70.0% Ghana Ablordey et al. [33]
26 23 3 88.5% Australia O’Brien et al. [30]
143 107 36 74.8% Ghana Stienstra et al. [32]
202 109 93 54.0% Togo Bretzel et al. [28]
IS2404 PCR 24 18 6 75.0% Ghana Beissner et al. [36]
65 55 10 84.6% Benin/Ghana Guimaraes-Peres et al. [57]
162 135 27 83.3% Cameroon Noeske et al. [60]
36 27 9 75.0% DRC Phanzu et al. [6]
94 62 32 66.0% Ghana Bretzel et al. [28]
DRB-PCR 230 139 91 60.6% Ghana Siegmund et al. [47]
18 15 3 83.3% Ghana Beissner et al. [36]
Real-time qPCR 44 29 15 65.9% Ghana Rondini et al. [21]
74 44 30 59.5% Benin Affolabi et al. [39]
21 21 0 100.0% Ghana Stienstra et al. [32]
Nested PCR 65 52 13 80.0% Benin/Ghana Guimaraes-Peres et al. [57]
74 33 41 44.6% Benin Affolabi et al. [39]
Others
20 6 14 30.0% Ghana de Souza et al. [27]
LAMP assay 30 9 21 30.0% Ghana Ablordey et al. [33]
20 13 7 65% Ghana de Souza et al. [27]
TLC 10 5 5 50.0% Ghana Sarfo et al. [73]
Serology 61 43 18 70.5% Ghana Dobos et al. [35]
Faecal 67 0 67 0.0% Ghana Sarfo et al. [74]
alternative for conventional mycobacterial cultures and thus Furthermore, the application of molecular species identi-
considerably improve the clinical management of BUD. fication assays, such as internal transcribed spacer length
Culture remains the only method that detects viable bacilli. polymorphism or PCR restriction analysis of partial rpoB or
However, low sensitivity, long generation time and failure to hsp-65 genes [45, 55, 56, 63], would allow the distinction of
distinguish between MU and other mycobacterial infections MU from other nontuberculous mycobacteria. Most of these
without extra confirmatory diagnostic tools, makes cultures DNA-based techniques are present only in referenced and
unsuitable to support clinical management decisions timely. specialized centers. Conscious efforts should be channeled
8 Canadian Journal of Infectious Diseases and Medical Microbiology
towards the formation of multicenter collaborative research antimycobacterial treatment in Ghana,” The American Journal
programs. This will ensure reliability and reproducibility of of Tropical Medicine and Hygiene, vol. 81, no. 1, pp. 75–81, 2009.
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feasible since the positivity and sensitivity ratios are influ- land, 2000.
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Competing Interests Society of Tropical Medicine and Hygiene, vol. 97, no. 2, pp. 159–160, 2003.
Authors declare that there are no competing interests. [14] J. Novales-Santa Coloma, G. Navarrete-Franco, P. Iribe, and L.
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