PLOS ONE
RESEARCH ARTICLE
Evaluation of the fluorescent-thin layer
chromatography (f-TLC) for the diagnosis of
Buruli ulcer disease in Ghana
Richard K. Amewu 1ID *, Gideon Atinga Akolgo 1ID , Millicent Esi Asare 1ID , Zigli Abdulai1,
Anthony S. Ablordey 2ID , Kingsley Asiedu
3
1 Department of Chemistry, University of Ghana, Accra, Ghana, 2 Department of Bacteriology, Noguchi
Memorial Institute for Medical Research, University of Ghana, Accra, Ghana, 3 Department of Control of
Neglected Tropical Diseases, World Health Organization, Geneva, Switzerland
a1111111111 * ramewu@ug.edu.gh
a1111111111
a1111111111
a1111111111
a1111111111 Abstract
Background
Buruli ulcer is a tissue necrosis infection caused by an environmental mycobacterium called
OPEN ACCESS
Mycobacterium ulcerans (MU). The disease is most prevalent in rural areas with the highest
Citation: Amewu RK, Akolgo GA, Asare ME, rates in West and Central African countries. The bacterium produces a toxin called mycolac-
Abdulai Z, Ablordey AS, Asiedu K (2022) Evaluation
tone which can lead to the destruction of the skin, resulting in incapacitating deformities with
of the fluorescent-thin layer chromatography (f-
TLC) for the diagnosis of Buruli ulcer disease in an enormous economic and social burden on patients and their caregivers. Even though
Ghana. PLoS ONE 17(8): e0270235. https://doi. there is an effective antibiotic treatment for BU, the control and management rely on early
org/10.1371/journal.pone.0270235 case detection and rapid diagnosis to avert morbidities. The diagnosis of Mycobacterium
Editor: Maria Stefania Latrofa, University of Bari, ulcerans relies on smear microscopy, culture histopathology, and PCR. Unfortunately, all
ITALY the current laboratory diagnostics have various limitations and are not available in endemic
Received: October 22, 2021 communities. Consequently, there is a need for a rapid diagnostic tool for use at the commu-
Accepted: June 6, 2022 nity health centre level to enable diagnosis and confirmation of suspected cases for early
treatment. The present study corroborated the diagnostic performance and utility of fluores-
Published: August 2, 2022
cent-thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer.
Copyright: © 2022 Amewu et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which Methodology/Principal findings
permits unrestricted use, distribution, and The f-TLC method was evaluated for the diagnosis of Buruli ulcer in larger clinical samples
reproduction in any medium, provided the original
than previously reported in an earlier preliminary study Wadagni et al. (2015). A total of 449
author and source are credited.
patients suspected of BU were included in the final data analysis out of which 122 (27.2%)
Data Availability Statement: All relevant data are
were positive by f-TLC and 128 (28.5%) by PCR. Using a composite reference method gen-
within the paper and its Supporting Information
files. erated from the two diagnostic methods, 85 (18.9%) patients were found to be truly infected
with M. ulcerans, 284 (63.3%) were uninfected, while 80 (17.8%) were misidentified as
Funding: 1. The author RKA received funding
(RE76) from the World Health Organization, https:// infected or noninfected by the two methods. The data obtained was used to determine the
www.who.int. The funder provided training and discriminatory accuracy of the f-TLC against the gold standard IS2404 PCR through the
initial resources for the study. 2. The author RKA
analysis of its sensitivity, specificity, positive (+LR), and negative (–LR) likelihood ratio. The
also received funding (RBF2) from Anesvad.
https://www.anesvad.org/en. The funder provided positive (PPV) and negative (NPV) predictive values, area under the receiver operating
equipment and supplies for the study. characteristic curve Azevedo et al. (2014), and diagnostic odds ratio were used to assess
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 1 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Competing interests: The authors have no the predictive accuracy of the f-TLC method. The sensitivity of f-TLC was 66.4% (85/128),
competing interest. specificity was 88.5% (284/321), while the diagnostic accuracy was 82.2% (369/449). The
AUC stood at 0.774 while the PPV, NPV, +LR, and–LR were 69.7% (85/122), 86.9% (284/
327), 5.76, and 0.38, respectively. The use of the rule-of-thumb interpretation of diagnostic
tests suggests that the method is good for use as a diagnostic tool.
Conclusions/Significance
Larger clinical samples than previously reported had been used to evaluate the f-TLC
method for the diagnosis of Buruli ulcer. A sensitivity of 66.4%, a specificity of 88.5%, and
diagnostic accuracy of 82.2% were obtained. The method is good for diagnosis and will help
in making early clinical decisions about the patients as well as patient management and
facilitating treatment decisions. However, it requires a slight modification to address the
challenge of background interference and lack of automatic readout to become an excellent
diagnostic tool.
Introduction
Buruli ulcer is a tissue necrosis infection caused by an environmental mycobacterium called
Mycobacterium ulcerans (MU). The evolution of MU includes the acquisition of the virulence
plasmid pMUM001 that encodes genes involved in the production of mycolactone, a macro-
lide cytotoxin with immunosuppressive properties responsible for BU pathology. M. ulcerans
causes destruction of the skin, often followed by debilitating deformities [1–4]. The global
health concern for Buruli ulcer as revealed by the WHO classification of Neglected tropical dis-
eases (NTDs) shows that BU is a top-ranking emerging NTD [5]. It is estimated that nearly
3,000 cases of BU are reported annually from 14 out of 33 countries; for instance, 2,260 new
cases were reported in the year 2019 [6]. The highest rate of disease is in West and Central
African countries such as Benin, Côte d’Ivoire, the Democratic Republic of Congo, and
Ghana. In Africa, the disease is most prevalent in rural areas [7] and more severe among
impoverished inhabitants [8]. It constitutes about 30% of ulcer cases seen at highly endemic
health facilities in Africa even though considerable under-reporting still exists [9]. Other ulcer-
ative lesions such as cutaneous tuberculosis and mycobacterium marinum, venous ulcers, cuta-
neous leishmaniasis, neurogenic ulcer, yaws, tropical ulcer, fungal lesions, and squamous cell
carcinoma can be mistaken for Buruli ulcer, particularly when they are around the ankles [10].
Children under the age of 15 years are commonly affected by BU which is often accompa-
nied by long-term disability and social stigma [11]. Buruli ulcer disease poses deleterious con-
sequences with enormous economic and social burdens on patients and their caregivers [12].
Even though mortality due to the disease is low, morbidity is high with significant complica-
tions, contractures, functional limitations, and social participation restrictions [13, 14], espe-
cially among the often-affected children [9].
Despite the discovery of Buruli ulcer more than a century ago with rapid progression since
the 1980s, the mode of transmission of the disease remains uncertain. However, with the intro-
duction of antibiotic treatment, BU control and management have relied heavily on early case
detection preferably in the WHO Category I and II stages, followed by rapid diagnosis and
prompt initiation of antibiotic treatment [15]. The mainstay of BU drug treatment consists of
a combination of antibiotics administered within eight weeks. The current World Health
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 2 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Organization (WHO)-recommended regimen is rifampicin (RIF) (10 mg/kg once daily, oral
tablet) combined with streptomycin (STR) (15 mg/kg once daily, intramuscular injection).
Recently, the injectable STR has been shown to be replaceable with oral clarithromycin for
smaller lesions for the last four weeks of treatment of the eight-week regimen [11, 16–18].
Currently, the diagnosis of BU relies on direct detection of acid-fast bacilli (AFB) by smear
microscopy, the culture of M. ulcerans, histopathology, and PCR targeting the multicopy inser-
tion sequence IS2404 [19–21]. However, all the current laboratory diagnostics have various
limitations. Nonetheless, the current “gold” standard diagnostic tool for BU is the highly spe-
cific and sensitive PCR method that targets the insertion sequence IS2404. This method has
major drawbacks such as the requirement of well-equipped reference laboratories, the require-
ment of sophisticated instrumentation and facilities, highly skilled and experienced personnel,
and strict quality control which strongly limit the routine implementation of the technique
[22]. There is also the challenge of delayed diagnostic results for Buruli ulcer patients which
contributes to morbidity and increased economic hardship [23]. However, since the best treat-
ment outcome is achieved when the disease is diagnosed early, the World Health Organization
(WHO) prioritizes a diagnostic test that is rapid, inexpensive, sensitive, specific, and suitable
for in-field use [6]. In addition, the diagnostic test should have the potential of being deployed
in BU endemic areas outside of a primary care facility, and be able to return a laboratory con-
firmation of at least 70% of the cases [24].
Fluorescent-thin layer chromatography (f-TLC) is a method developed by Kishi and co-
workers based on the detection of mycolactone [25]. The method relies on the chemical deriv-
atization of mycolactone by complexing the 1,3-diol units in the structure with 2-naphthyl-
boronic acid to form two cyclic boronates [25].
This study sought to corroborate the diagnostic performance and utility of the fluorescent-
thin layer chromatography (f-TLC) method for the diagnosis of BU disease. The method
focused on the detection of mycolactone as a biomarker in clinical samples of patients who
reported to health facilities with lesions suspected to be Buruli ulcer.
Materials and method
Ethical approval
Ethical approval for the study and the subsequent continuous enrolment of suspected cases
was obtained from the local ethical review board of the Noguchi Memorial Institute for Medi-
cal Research (NMIMR), University of Ghana (Legon). In addition, written consent was
obtained from all adult participants and legal guardians or parents of any child participant pro-
vided informed written consent on the child’s behalf.
Setting
The study was carried out in f-TLC and PCR references laboratories at the Department of
Chemistry, and NMIMR respectively both at the University of Ghana, Legon from November
2014 to December 2020.
Inclusion/exclusion criteria
All patients presenting at various health facilities from known endemic communities who had
a skin lesion suspected to be that of Buruli ulcer disease (defined as a non-ulcerative nodule, a
plaque, localized swelling, and/or an ulcerated lesion in an individual residing in or having
spent at least one night in a known M. ulcerans endemic area, without an obvious cause to the
lesion such as acute trauma) and who had received a clinical diagnosis of the disease were
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 3 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
eligible and thus, enrolled for the study. Clinical data were prospectively collected, before the
results of laboratory examination were available. Participation was voluntary and patients who
were excluded were those who were unwilling to participate, patients reporting for review, and
those with unrelated causes of ailment to Buruli ulcer.
Patients
Patients were recruited from Buruli ulcer treatment centres from November 2014 to
December 2020 across various health facilities in endemic communities if they had a skin
lesion suspected to be caused by M. ulcerans infection. Samples were collected by fine-needle
aspirates (FNA) or swabs according to whether the lesion was non-ulcerated or ulcerated,
respectively.
A total of 494 eligible patients with presumptive BU lesions were recruited for the study. Of
these, a total of 449 were included in the final data analysis because they had complete datasets
on both f-TLC and PCR. Of these, 222 (49.4%) were males and 227 (50.6%) were females with
a male to female ratio of 1:1.02 (Fig 1). The age range of the patients was 1–94 with a mean age
of 43 years. The general characteristics of the patients are shown in Table 1.
Fig 1. Flowchart of diagnostic tests performed on patients included in the final analysis.
https://doi.org/10.1371/journal.pone.0270235.g001
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 4 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Table 1. Characteristics of all suspected cases of Buruli ulcer cases submitted for diagnosis.
Characteristics Percentage
Included in final data analysis 449 100.0
Mean Age 43 –
Age, (range) 1–94 –
Sex N Percentage
Male 222 49.4
Female 227 50.6
Total 449 100.0
Type of lesion
Ulcer 425 94.7
Nodule 9 2.0
Oedema 10 2.2
Plaque 3 0.7
Osteomyelitis 2 0.4
Total 449 100.0
Specimen type
FNA 18 4.0
Swab 431 96.0
Total 449 100.0
Category
I 152 33.9
II 84 31.8
III 67 24.5
Not indicated 43 9.8
Total 449 100.0
https://doi.org/10.1371/journal.pone.0270235.t001
Standardized sample collection and transport
Samples were collected according to standardized procedures as previously described [26, 27].
In many cases, the diagnostic samples were collected during the patients’ initial presentation
to the health facility. Swab samples were taken by circling the entire undermined edges of
ulcerative lesions. Fine needle aspirates (FNA) were collected from the center of non-ulcerative
lesions or undermined edges of ulcerative lesions including necrotic tissue [28, 29]. To facili-
tate sampling, standardized specimen collection bags (double zip-lock plastic bags) including
swabs, syringes, and needles, aluminum foil, O-ring seal plastic vials with transport media (1
mL absolute ethanol for swab and FNA samples for f-TLC samples), and data entry forms
(BU01 laboratory data entry form [30]) were sent to every sample collection site. For the f-
TLC method, fine needle aspirate (FNA) or swab samples were collected in O-ring seal plastic
vials (Fisher Scientific) containing 1 mL absolute ethanol, wrapped in aluminum foil for
onward transport [31]. According to earlier reports, the mycolactone is stable in absolute etha-
nol [32] and sensitive to UV light [33] hence the samples were transported cold and in the
dark to the laboratory.
For PCR samples, fine needle aspirates were kept in 1 mL phosphate-buffered saline (PBS)
and swabs were stored dry in sterile tubes, and placed in specimen collection zip-lock plastic
bags [34]. All samples for both f-TLC and PCR tests were transported in an ice chest with ice
packs from the field to the Department of Chemistry, and Bacteriology Department of
NMIMR, University of Ghana, Legon for f-TLC and PCR analyses, respectively. Upon the
arrival of PCR samples at NMIMR these were stored at 4–8˚C until further processing where
definite BU diagnosis was obtained by insertion sequence IS2404 PCR.
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 5 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Laboratory diagnostics
DNA extraction from clinical specimens. PCR specimens were processed at NMIMR.
Each swab was transferred into a tube containing 2 mL Milli-Q purified water (Millipore Cor-
poration, Billerica, MA) and gently vortexed for 5 sec, and then removed. Sample suspension
portions of 250 μL were transferred to separate new sterile Eppendorf tubes containing 250 μL
of lysis buffer (1.6 M GuHCl, 60 mM Tris pH 7.4, 1% Triton X-100, 60 mM EDTA, Tween-20
10%), 50 μL proteinase-K and 250 μL glass beads. The mixtures were incubated horizontally in
a shaker (200 rpm) at 60˚C overnight. To capture the DNA, 40 μL of diatomaceous earth solu-
tion (10 g diatomaceous earth obtained from Sigma Aldrich Chemi GmbH in 50 ml of H2O
containing 500 μL of 37% (wt/vol) HCl) was added to the suspensions and incubated at 37˚C
with shaking (200 rpm) for 60 min. The mixtures were centrifuged at 14,000 rpm for 10 sec
and the resulting pellets were washed twice with 900 μL of 70% ethanol (2–8˚C) followed by
900 μL of acetone. The pellets were dried at 50˚C for 20 min and resuspended in 100 μL Milli-
Q purified water and centrifuged at 14,000 rpm for 10 sec. The purified DNA was used as tem-
plates for IS2404 PCR assays to detect M. ulcerans [34].
Polymerase Chain Reaction (PCR) for IS2404. Standard PCR targeting IS2404 was per-
formed according to the protocol described by Stinear et al. [35]. All PCR assays included neg-
ative extraction controls, positive, negative (no template), and inhibition controls.
Mycolactone extraction and fluorescent-thin layer chromatography (f-TLC) tech-
nique. Mycolactone detection by f-TLC analysis was done according to the protocol devel-
oped and published elsewhere with minor modifications (S1 File) [25, 32]. Ethanol containing
the dissolved sample was filtered through a cotton plug into a glass vial. The sample container
was further rinsed with 1 mL ethyl acetate, which was added to the glass vial through a cotton
plug, and the contents were evaporated to dryness under reduced pressure of about 10–15
mmHg using a rotary evaporator. To separate any contaminating solid from liquid, 100 μL
hexane/ether (1:1) solution was added to the glass vial, rinsed, and transferred by micro-
syringe into a clean glass vial which was air-dried. After evaporation, 50 μL hexane/ether (1:1)
was added to the dry sample and 10 μL of the resuspended sample was spotted onto a 3.3×6.6
cm fluorescent-dye free TLC plate (TLC Silica gel 60, EMD Millipore, Darmstadt, Germany;
Gibbstown, NJ, USA) alongside 50 ng synthetic mycolactone A/B standard in ethyl acetate and
a co-spot of 10 μL sample with 50 ng synthetic mycolactone A/B. The plate was developed in
chloroform: hexane: methanol in a ratio of 5:4:1 until the leading edge reached the top of the
plate, air-dried, and dipped in 0.1 M 2-naphthylboronic acid solution in acetone, then heated
for 60 seconds at 100˚C on a hot plate. The glass side of the plate was wiped with acetone on a
paper towel. The plate was placed on a UV lamp with a 365 nm filter. The fluorescent band at
retention factor 0.23 from the patient sample was compared to that of the standards to confirm
the presence of mycolactone. The f-TLC plates were independently read by two laboratory
analysts before reporting the results. The laboratory analysts who worked on both the f-TLC
and PCR specimens were blinded to certain relevant clinical information (except for age, gen-
der, and type and localization of the lesion) as well as diagnostic test results of each other. This
was to ensure that there was no diagnosis bias.
Data analysis
The data were recorded on a standardized BU report form, double-entered into a Microsoft
Excel (Microsoft Office Inc for Windows, USA) database (S2 File), and analyzed using Med-
Calc Version 19.1 (Vienna, Austria,) and Microsoft Excel (Microsoft Corporation) software
packages for Windows. Descriptive statistics were used to obtain general descriptive informa-
tion such as the mean and ranges from the data.
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 6 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Diagnostic test parameters including sensitivity, specificity, diagnostic odds ratio (DOR), posi-
tive predictive value (PPV), and negative predictive value (NPV) were used to determine the dis-
criminatory accuracy of the f-TLC method for the diagnosis of Buruli ulcer in comparison with
the “Gold” standard PCR method. The choice of PCR as the gold standard is because it is the best
available method in terms of sensitivity and specificity for diagnosing Buruli ulcer [36]. Also, the
PCR method has been used as the gold standard in similar previous studies [29, 31, 37].
Also, a composite reference standard (CRS) method was generated from the two methods
(f-TLC and PCR) and used to calculate the sensitivity, specificity, and predictive values of each
of the methods. The CRS was defined as a method that is positive or negative for BU by both
methods (f-TLC and PCR). This gives the method 100% hypothetical sensitivity, specificity,
accuracy, positive and negative predictive values [38]. The sensitivity, specificity, and predic-
tive values of each of the 3 methods were then calculated using the formulas.
TP
Sensitivity ¼ � 100 ð1Þ
ðTPþ FNÞ
TN
Specif icity ¼ � 100 ð2Þ
ðTNþ FPÞ
TP
PPV ¼ � 100 ð3Þ
ðTPþ FPÞ
TN
NPV ¼ � 100 ð4Þ
ðTNþ FNÞ
TPþ TN
Accuracy ¼ � 100 ð5Þ
ðTPþ TNþ FPþ FNÞ
where TP = true positive, FP = false positive, TN = true negative, and FN = false negative.
Sensitivity was defined as the probability that a truly infected individual will test positive
and specificity as the probability that a truly uninfected individual will test negative. Taking
PCR as the gold standard, the performance of the f-TLC diagnosis method was evaluated to
generate diagnostic accuracy summary statistics including receiver-operator characteristics
(ROC) test, and Youden J. statistics [39] using MedCalc Version 19.1.
Results
The diagnostic value of two methods (f-TLC and PCR) for the diagnosis of BU in Ghana were
compared. A third method called the CRS which was generated from the other two methods
(f-TLC and PCR) was also contrasted [38]. Of the total of 449 samples, 128 (28.5%) were posi-
tive for PCR whilst 321 (71.7%) were negative. Of the total, 122 (27.2%) were positive for f-
TLC, and 327 (72.8%) were negative for f-TLC. Using a composite reference (gold standard)
method generated from the two diagnostic methods, only 85 (18.9%) patients were found to
be truly infected with M. ulcerans, 284 (63.3%) were truly uninfected while 80 (17.8%) were
misidentified as infected or noninfected by the two methods (Table 2).
Next, the true positive (TP), false positive (FP), true negative (TN), and false negative (FN)
values of the f-TLC test in comparison with PCR as the “gold” standard were calculated. 85 out
of the 449 suspected cases were properly classified as true positives representing 18.9%. This
means that 18.9% of suspected BU patients that were diseased and confirmed by gold standard
PCR also tested positive for f-TLC. Also, the true negatives (TN) stood at 63.3% (284/449)
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 7 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Table 2. Diagnostic results with each method.
PCR f-TLC CRS
N (%) N (%) N (%)
Positive samples 128 (28.5) 122 (27.2) 85 (18.9)
Negative samples 321 (71.7) 327 (72.8) 284 (63.3)
Misidentified as infected or non-infected – – 80 (17.8)
Total 449 (100.0) 449 (100.0) 449 (100.0)
PCR: polymerase chain reaction; f-TLC: fluorescent-thin layer chromatography; CRS: composite reference standard;
N: number; %: percent
https://doi.org/10.1371/journal.pone.0270235.t002
indicating that the suspected cases who were non-diseased, tested negative by both f-TLC and
gold stand PCR methods. The false positive (FP) and false negative (FN) values were 37 (8.2%)
and 43 (9.6%) respectively.
Furthermore, the sensitivity, specificity, positive predictive value (PPV), negative predictive
value (NPV), accuracy, and likelihood ratio with 95% confidence intervals (CIs) of the f-TLC
method were calculated. The f-TLC method for the detection of mycolactone reported an
overall sensitivity of 66.4% (95% CI: 57.9–74.0) and a specificity of 88.5% (95% CI: 84.5–91.5).
The overall positive and negative predictive values were 69.7% (62.4–76.1) and 86.9% (83.8–
89.4) respectively. The accuracy of the f-TLC method compared to the PCR was 82.2%. When
the analysis was performed according to the type of sample, the sensitivity of the f-TLC for
FNA in comparison to PCR was 87.5% while that of swabs was 65.0% at their respective 95%
confidence intervals (CIs) (Table 3). In addition, when the results were analysed according to
the type of lesion, a specificity of 73% was obtained for ulcers while oedema, nodules, plaque,
and osteomyelitis gave 100%. On the other hand, sensitivities of 65.3%, 100%, 67% were
obtained for ulcers, oedema, and nodules respectively.
Evaluating the two diagnostic methods (f-TLC and PCR) over the composite reference stan-
dard (CRS) as the new gold standard, which was generated from the two diagnostic methods,
85 patients were found to be truly positive for Buruli ulcer and 284 people were found to be
truly negative giving a hypothetical 100% sensitivity, specificity PPV, NPV, and accuracy. Both
methods (f-TLC and PCR) reported a sensitivity of 66.4%, specificity of 88.5%, PPV of 69.7%,
NPV of 86.9%, and diagnostic accuracy of the f-TLC method for the detection of mycolactone
in the diagnosis of BU stood at 82.2% (Table 4).
Assessing the effectiveness of the f-TLC method for the diagnosis of Buruli ulcer, the
receiver operating characteristic (ROC) analysis was used to estimate the area under the diag-
nostic curve [40], ranging from 0.5, a worthless test, to 1, a perfect test Fig 2. The AUC of the
ROC is a credible metric for evaluating the effectiveness of the f-TLC diagnostic test to distin-
guish between those with BU disease and those without it. The ROC curve provides a graphical
representation of the overall performance of the f-TLC to discriminate between those with dis-
ease and those without the disease.
The f-TLC was shown to have an AUC = 0.774 ± 0.023 (95% CI: 73.3–81.2) and the significance
level is p< 0.0001 with a Youden’s index value of 0.549. The positive likelihood ratio was 5.76
(95% CI: 4.2–8.0) while the negative likelihood ratio was 0.38 (95% CI: 0.3–0.5) and the diagnostic
odds ratio (DOR) value is 15.17. Youden’s index for f-TLC diagnostic test was 0.549 (Table 5).
Discussion
The need to obtain an early, rapid, and sensitive diagnosis is significantly increasing hence we
considered a diagnostic method known as the fluorescent-thin layer chromatography (f-TLC)
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 8 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Table 3. Sensitivity, specificity, PPV, NPV, and accuracy of f-TLC compared to PCR according to sample type.
Analysis according to the method of sample collection
FNA
PCR + PCR – Total Sensitivity [95% CI] Specificity [95% CI] PPV [95% CI] NPV [95% CI] Accuracy [95% CI]
f-TLC + 7 1 8 87.5 [47.4–99.7] 90.0 [55.5–99.8] 87.5 [51.7–97.9] 90.0 [58.7–98.3] 88.9 [65.3–98.6]
f-TLC – 1 9 10
Total 8 10 18
Swab
f-TLC + 78 36 114 65.0 [55.8–73.5] 88.4 [84.3–91.8] 68.4 [60.8–75.2] 86.8 [83.6–89.3] 81.9 [77.9–85.4]
f-TLC – 42 275 317
Total 120 311 431
Overall
f-TLC + 85 37 122 66.4 [57.5–74.5] 88.5 [84.5–91.8] 69.7 [62.4–76.1] 86.9 [83.8–89.4] 82.2 [78.3–85.6]
f-TLC – 43 284 327
Total 128 321 449
Analysis according to the type of lesion
Ulcer
f-TLC + 79 37 116 65.3 [56.1–73.7] 72.7 [68.2–76.9] 40.5 [35.7–45.5] 88.0 [85.1–90.4] 71.1 [67.1–74.8]
f-TLC – 42 267 309
Total 121 304 425
Oedema
f-TLC + 4 0 4 100.00 [39.8–100.0] 100.00 [54.1–100.0] 100.0 100.0 100.0 [69.2–100.0]
f-TLC – 0 6 6
Total 4 6 10
Nodule
f-TLC + 2 0 2 66.7 [9.4–99.2] 100.00 [54.1–100.0] 100.0 85.7 [54.8–96.8] 88.9 [51.8–99.7]
f-TLC – 1 6 7
Total 3 6 9
Plague
f-TLC + 0 0 0 100.0 [29.2–100.0] 100.0
f-TLC – 0 3 3
Total 0 3 3
Osteomyelitis
f-TLC + 0 0 0 100.0 [15.8–100.0] 100.0
f-TLC – 0 2 2
Total 0 2 2
f-TLC: fluorescent-thin layer chromatography; PCR: polymerase chain reaction; PPV: positive predictive value; NPV: negative predictive value
https://doi.org/10.1371/journal.pone.0270235.t003
Table 4. Sensitivity, specificity, PPV, NPV, accuracy, and likelihood ratio of f-TLC compared to PCR.
Diagnostic TP FP TN FN Sensitivity Specificity PPV NPV Accuracy
PCR 85 37 284 43 66.4% 88.5% 69.7% 86.9% 82.2%
f-TLC 85 37 284 43 66.4% 8.5% 69.7% 86.9% 82.2%
CRS 85 0 284 0 100.0% 100.0% 100.0% 100.0% 100.0%
TP: true positive; FP: false positive; TN: true negative; FN: false negative; f-TLC: fluorescent-thin layer chromatography; PCR: polymerase chain reaction; CRS:
composite reference standard; PPV: positive predictive value; NPV: negative predictive value
https://doi.org/10.1371/journal.pone.0270235.t004
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 9 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Fig 2. Receiver operating characteristics (ROC) curve of f-TLC showing the relationship between sensitivity (true positive) and 1 –specificity (true
negative).
https://doi.org/10.1371/journal.pone.0270235.g002
Table 5. Diagnostic performance criteria for f-TLC diagnosis of Buruli ulcer using PCR as a gold standard.
Parameter Accuracy +LR [95% CI] -LR [95% CI] AUC (ROC) [95% CI] SE p value DOR Youden index
f-TLC 82.2 5.76 [4.2 - 8.0] 0.38 [0.3 - 0.5] 0.774 [73.3–81.2] 0.023 < 0.001 15.17 0.549
AUC: area under curve and ROC: receiver operative characteristic. p is significant at 0.05. +LR: Positive likelihood ratio. -LR: Negative likelihood ratio. SE: Standard
error. DOR: Diagnostic odds ratio
https://doi.org/10.1371/journal.pone.0270235.t005
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 10 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
which depends on the interaction between mycolactone and a boronic acid to give off fluores-
cence that can be detected on a TLC plate.
It has been demonstrated to be a technique capable of detecting mycolactone in M. ulcerans
both in a mouse footpad model [32] and infected human skin samples [31]. This study was
designed to evaluate the diagnostic accuracy of the f-TLC for the detection of mycolactone in
clinical samples against PCR as the “gold” standard in larger field studies following a successful
preliminary study of the technique by Wadagni et al. [31]. In the present study, PCR was used
as the “gold” standard diagnostic method to compare the results obtained by f-TLC. A false
negative rate of 9.6% (43/449) was found for the f-TLC method while a false positive value was
8.2% (37/449). False negative results were suspected to be partly because of low concentration
of mycolactone in lesion specimens, poor extraction, and decongestion efficiency of mycolac-
tone from clinical samples, and/or the high background interference due to co-extracted
human lipids [41]. A few of the patients who were recruited for the study had previously visited
some traditional healers who had given them some topical mixtures to apply. Organic com-
pounds extracted from the mixtures into the lesions sometimes co-elute with the mycolactone
on the TLC which gave fluorescent spots similar to the mycolactone [42]. Consequently, false
negative patients do not get the indicated anti-mycobacterial treatment. This is because there
is a part of the population that returns home without a correct diagnosis and treatment with
the confirmed presence of the bacteria. As a result, this could have important implications for
public health, and possibly mortality [43, 44]. Also, 284 out of 449 suspected BU cases which
represented 63.3% returned true negative values. This is a true reflection of the fact that lesions
wrongly clinically diagnosed and hence suspected to be BU will give negative results in the
actual laboratory confirmation test [43].
The sensitivity of f-TLC against gold standard PCR was observed at 66.4%, the specificity
was calculated to be 88.5% and the PPV and NPV were calculated to be 69.7% and 86.9%
respectively. Generally, the f-TLC was found to be more specific (88.5%) than sensitive
(66.4%). The specificity of 88.5% obtained in this current evaluation study is comparable to the
specificity of 85.7%, however, the sensitivity (66.4%) was lower than the sensitivity of 73.2%
reported in an earlier preliminary study be it with a limited number of suspected cases [31].
When the analysis of the results was performed on swab and FNA samples from ulcerative and
pre-ulcerative lesions respectively, we observed sensitivity differences. The sensitivity of the f-
TLC for FNA in comparison to PCR was 87.5% while that of swabs was 65.0%. Generally, the
FNA samples were cleaner because they have less co-extracted human tissues hence lower
background overlap.
The sensitivity of the f-TLC method in the present study was greater than those reported by
microscopy (30–60%) or culture (35–60%). Another method reported by Sakyi and coworkers
for the detection of mycolactone involved the use of aptamers. In their study, they reported
specificity of 100% and sensitivity of 50% [45]. Thus, the f-TLC is comparable to microscopy,
culture, and aptamer binding ELONA test but less than histology (82%), and PCR (92–98%)
[36, 45, 46].
However, our results fall short of the WHO threshold for a diagnostic tool of laboratory
confirmation of at least 70% of the cases [24]. Using a composite reference method generated
from the two diagnostic methods, 85 (18.9%) patients were found to be truly infected and 284
(63.3%) truly uninfected, while 80 (17.8%) were misidentified as infected or noninfected. The
composite reference gold standard method had a hypothetical 100% sensitivity, specificity,
PPV, NPV, and accuracy compared to a sensitivity of 66.4%, specificity of 88.5%, PPV of
69.7%, NPV of 86.9%, and a diagnostic accuracy of 82.2% reported for both methods (f-TLC
and PCR). It is indicated that a positive likelihood ratio (PLR) of more than 10 (+LRs >10)
and a negative likelihood ratio (NLR) less than 0.1 (-LRs <0) are considered to provide
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 11 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
convincing diagnostic evidence. Additionally, +LRs>5 provide strong diagnostic evidence to
rule in diagnoses; whereas -LRs <0.2 provide strong diagnostic evidence to rule out diagnoses,
in the majority of cases [47]. In the present study, the +LR was 5.76 which determined Buruli
ulcer suspected patients had a nearly 6-fold higher chance of a positive test compared to the
healthy ones. However, the -LR was 0.38 for mycolactone detection using f-TLC. This sug-
gested that if the outcome of the f-TLC method for the diagnosis of Buruli ulcer was negative,
38% of the patients may still be positive for Buruli ulcer, and 38% of patients may be
misdiagnosed.
On the predictive accuracy of the f-TLC used in Buruli ulcer diagnosis in the current study,
it is worth noting that PPVs are functions of disease prevalence [48]. The PPV of 69.7% was
lower compared to the NPV which was relatively higher at 86.9%. This observation suggests
that the likelihood of having Buruli when the f-TLC method is positive is only about 7 in every
10 suspected cases while the likelihood of being non-BU when the f-TLC test is negative is very
high (almost 9 in every 10 suspected BU cases).
As shown in Fig 2, the AUC of the current diagnosis by the f-TLC in comparison with the
PCR test was 77.4% which suggests that almost 8 out of every 10 suspected cases of BU were
correctly classified by the f-TLC technique. This means that the f-TLC method is an effective
tool to discriminate between those with and without Buruli ulcer. Youden index (J) was carried
out to measure the medical usefulness and precision of a diagnostic test for Buruli ulcer detec-
tion. From this measurement, f-TLC is a useful diagnostic test with a Youden’s index of 0.549.
This index is a function of sensitivity and specificity (J = Specificity + Sensitivity– 1), ranging
between 0 and 1. The index has a value of zero if the test reports the same proportion of posi-
tive tests for both the control group and the disease group. It has value unity when neither false
positives nor false negatives emerge from the test [39, 49, 50].
Ray et. al. proposed a rule-of-thumb interpretation of a diagnostic test whereby an excellent
diagnostic test should possess an AUC and accuracy greater than 0.90, +LR >10, and -LR<
0.1, respectively. On the other hand, a diagnostic test has good discriminative properties when
AUC and accuracy are higher than 0.75 with a +LR value between 5 and 10, and a -LR between
0.1–0.2. Whereas a poor diagnostic value had AUC (0.50–0.75), +LR (1–5), -LR (0.20–1), and
accuracy (0.50–0.75) respectively while a test was considered to have no diagnostic value when
AUC is 0.50 and below with both +LR and -LR being 1 respectively [51]. The results of the cur-
rent evaluation gave diagnostic accuracy such as sensitivity (66.4%), specificity (88.5%), AUC
(0.774), +LR (5.76), -LR (0.38) and accuracy (82.2%) respectively. Based on these results, the f-
TLC method for mycolactone detection can be classified as a good diagnostic.
Fluorescent-thin layer chromatography (f-TLC) is cheap, yet the technique is robust, and
requires simple instrumentation, while still giving results in a short time for the diagnosis of
BU [25, 32]. For instance, the total cost is US$15,000.00 comprising equipment and laboratory
supplies. During the lifespan set up, only supplies will be required while the equipment can be
used for several years. In a typical setting where more than 500 samples are run in a year, the
average cost per sample is estimated to be less than a dollar compared to the conventional PCR
method of approximately US$ 6.0 per sample [52]. Besides, the cost of reagents required to run
the equipment is significantly lower compared to PCR. In addition, the turnaround time for
the results using the f-TLC method is considerably lower (approximately 1 hour) as compared
to about 6 hours for the conventional PCR [31, 52].
The f-TLC method has the potential of augmenting existing methods, particularly in
endemic communities, and improving upon the timeliness of BU diagnosis. Most importantly,
its potential deployment in rural settings including district hospitals will significantly impact
the overall well-being of the BU patients. In addition, by adopting the f-TLC method, the num-
ber of patients to be diagnosed shall increase resulting in a positive impact on the health of the
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 12 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
patients. The method will help in making early clinical decisions as well as patient manage-
ment and facilitate treatment decisions. Furthermore, the f-TLC method can be used to moni-
tor the course of the BU disease or treatment response, detect disease recurrence, and
determine the necessity of readiness for discharge.
The study limitations however are that the f-TLC does not have any potential presence of
laboratory contaminations however, it has been established that this could occur in PCR test-
ings. Thus, basing the sensitivity of PCR at 100% could affect the actual performance of the f-
TLC method. Secondly, the interpretation of the f-TLC analysis is either “a YES or a NO” with
no graduation in between. This suggests that some decisions on the analysis results could be
wrong if the protocol is not strictly adhered to. Also, once a wrong interpretation has been
made, subsequent decisions including treatment will be wrongly applied.
Conclusion
Larger clinical samples than previously reported had been used to evaluate the f-TLC method
for the diagnosis of Buruli ulcer. A sensitivity of 66.4%, a specificity of 88.5%, and diagnostic
accuracy of 82.2% were obtained. The use of the rule-of-thumb interpretation of diagnostic
tests suggests that the method is ideal for the diagnosis of Buruli ulcer. The f-TLC method has
a short turnaround time, is cost-effective, and is easy to use. The method will help in making
early clinical decisions about the patients as well as patient management and facilitate treat-
ment decisions. It however requires a slight modification by addressing the challenge of back-
ground interference and lack of automatic readout to become an excellent diagnostic tool. The
resultant method can easily be deployed in rural settings where the disease is predominantly
endemic as a potential point-of-care tool.
Supporting information
S1 File. Schematic diagram of F-TLC analysis of swab or FNA samples.
(PDF)
S2 File. Raw data of participants included in the study.
(XLSX)
S3 File. Flow diagram of participants included in the validation of the index test (f-TLC).
(PDF)
S4 File. STARD (Standards for the reporting of diagnostic accuracy studies) checklist.
(DOCX)
Acknowledgments
The authors would like to thank the World Health Organization and ANESVAD for generous
funding of this work. The authors are also thankful to Professor Kishi Yoshito of Harvard Uni-
versity for the donation of the synthetic mycolactone for the study as well as the training of the
research team. The authors are also grateful to all the patients and workers at the various health
facilities where the samples came from.
Author Contributions
Conceptualization: Richard K. Amewu, Anthony S. Ablordey.
Formal analysis: Richard K. Amewu, Gideon Atinga Akolgo, Zigli Abdulai.
Funding acquisition: Richard K. Amewu, Kingsley Asiedu.
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 13 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
Methodology: Richard K. Amewu, Gideon Atinga Akolgo, Millicent Esi Asare, Zigli Abdulai,
Anthony S. Ablordey, Kingsley Asiedu.
Project administration: Richard K. Amewu.
Resources: Richard K. Amewu, Anthony S. Ablordey, Kingsley Asiedu.
Supervision: Richard K. Amewu.
Writing – original draft: Richard K. Amewu, Gideon Atinga Akolgo, Millicent Esi Asare,
Zigli Abdulai, Anthony S. Ablordey, Kingsley Asiedu.
Writing – review & editing: Richard K. Amewu, Gideon Atinga Akolgo, Millicent Esi Asare,
Zigli Abdulai, Anthony S. Ablordey, Kingsley Asiedu.
References
1. Bayley AC. Buruli ulcer in Ghana. Br Med J. 1971; 2(5758):401–2. https://doi.org/10.1136/bmj.2.5758.
401-c PMID: 5575986
2. Connor DH, Lunn HF. Mycobacterium ulcerans infection (with comments on pathogenesis). Int J Lepr.
1965; 33(3):Suppl:698–709.
3. Hayman J. Mycobacterium ulcerans infection. Lancet. 1991; 337(8733):124.
4. Demangel C, Stinear TP, Cole ST. Buruli ulcer: reductive evolution enhances pathogenicity of Myco-
bacterium ulcerans. Nature Reviews Microbiology. 2009; 7(1):50–60. https://doi.org/10.1038/
nrmicro2077 PMID: 19079352
5. WHO. WHO | Buruli ulcer. WHO. 2019.
6. WHO. Ending the neglect to attain the sustainable development goals: a road map for neglected tropical
diseases 2021–2030. World Health Organization; 2020.
7. WHO. Distribution of Buruli ulcer, worldwide, 2015. 2015.
8. Portaels F, Johnson P, Meyers WM, Organization WH, Initiative GBU. Buruli ulcer: diagnosis of Myco-
bacterium ulcerans disease: a manual for health care providers. World Health Organization; 2001.
9. Yotsu RR, Suzuki K, Simmonds RE, Bedimo R, Ablordey A, Yeboah-Manu D, et al. Buruli Ulcer: a
Review of the Current Knowledge. Curr Trop Med Rep. 2018; 5(4):247–56. https://doi.org/10.1007/
s40475-018-0166-2 PMID: 30460172
10. Guarner J, Bartlett J, Whitney EA, Raghunathan PL, Stienstra Y, Asamoa K, et al. Histopathologic fea-
tures of Mycobacterium ulcerans infection. Emerg Infect Dis. 2003; 9(6):651–6. https://doi.org/10.3201/
eid0906.020485 PMID: 12780997
11. Omansen TF, Almeida D, Converse PJ, Li SY, Lee J, Stienstra Y, et al. High-Dose Rifamycins Enable
Shorter Oral Treatment in a Murine Model of Mycobacterium ulcerans Disease. Antimicrob Agents Che-
mother. 2019; 63(2).
12. Mulder AA, Boerma RP, Barogui Y, Zinsou C, Johnson RC, Gbovi J, et al. Healthcare seeking behav-
iour for Buruli ulcer in Benin: a model to capture therapy choice of patients and healthy community mem-
bers. Trans R Soc Trop Med Hyg. 2008; 102(9):912–20. https://doi.org/10.1016/j.trstmh.2008.05.026
PMID: 18617207
13. Barogui Y, Johnson RC, van der Werf TS, Sopoh G, Dossou A, Dijkstra PU, et al. Functional limitations
after surgical or antibiotic treatment for Buruli ulcer in Benin. Am J Trop Med Hyg. 2009; 81(1):82–7.
PMID: 19556571
14. de Zeeuw J, Omansen TF, Douwstra M, Barogui YT, Agossadou C, Sopoh GE, et al. Persisting social
participation restrictions among former Buruli ulcer patients in Ghana and Benin. PLoS Negl Trop Dis.
2014; 8(11):e3303. https://doi.org/10.1371/journal.pntd.0003303 PMID: 25392915
15. Junghanss T, Johnson RC, Pluschke G. Mycobacterium ulcerans disease. 2014.
16. Nienhuis WA, Stienstra Y, Thompson WA, Awuah PC, Abass KM, Tuah W, et al. Antimicrobial treat-
ment for early, limited Mycobacterium ulcerans infection: a randomised controlled trial. Lancet. 2010;
375(9715):664–72. https://doi.org/10.1016/S0140-6736(09)61962-0 PMID: 20137805
17. Iddrisah FN, Yeboah-Manu D, Nortey PA, Nyarko KM, Anim J, Antara SN, et al. Outcome of Streptomy-
cin-Rifampicin treatment of Buruli Ulcer in two Ghanaian districts. Pan Afr Med J. 2016; 25(Suppl 1):13.
https://doi.org/10.11604/pamj.supp.2016.25.1.6203 PMID: 28149438
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 14 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
18. Phillips RO, Sarfo FS, Abass MK, Abotsi J, Wilson T, Forson M, et al. Clinical and bacteriological effi-
cacy of rifampin-streptomycin combination for two weeks followed by rifampin and clarithromycin for six
weeks for treatment of Mycobacterium ulcerans disease. Antimicrob Agents Chemother. 2014; 58
(2):1161–6. https://doi.org/10.1128/AAC.02165-13 PMID: 24323473
19. Yeboah-Manu D, Bodmer T, Mensah-Quainoo E, Owusu S, Ofori-Adjei D, Pluschke G. Evaluation of
decontamination methods and growth media for primary isolation of Mycobacterium ulcerans from sur-
gical specimens. J Clin Microbiol. 2004; 42(12):5875–6. https://doi.org/10.1128/JCM.42.12.5875-5876.
2004 PMID: 15583329
20. Portaels F, Agular J, Fissette K, Fonteyne P, De Beenhouwer H, De Rijk P, et al. Direct detection and
identification of Mycobacterium ulcerans in clinical specimens by PCR and oligonucleotide-specific cap-
ture plate hybridization. Journal of clinical microbiology. 1997; 35(5):1097–100. https://doi.org/10.1128/
jcm.35.5.1097-1100.1997 PMID: 9114387
21. Ablordey A, Swings J, Hubans C, Chemlal K, Locht C, Portaels F, et al. Multilocus variable-number tan-
dem repeat typing of Mycobacterium ulcerans. J Clin Microbiol. 2005; 43(4):1546–51. https://doi.org/10.
1128/JCM.43.4.1546-1551.2005 PMID: 15814964
22. Tabah EN, Johnson CR, Degnonvi H, Pluschke G, Röltgen K. Buruli Ulcer in Africa. In: Pluschke G,
Röltgen K, editors. Buruli Ulcer: Mycobacterium Ulcerans Disease. Cham: Springer International Pub-
lishing; 2019. p. 43–60.
23. Grietens KP, Boock AU, Peeters H, Hausmann-Muela S, Toomer E, Ribera JM. "It is me who endures
but my family that suffers": social isolation as a consequence of the household cost burden of Buruli
ulcer free of charge hospital treatment. PLoS Negl Trop Dis. 2008; 2(10):e321. https://doi.org/10.1371/
journal.pntd.0000321 PMID: 18923711
24. Beissner M, Phillips RO, Battke F, Bauer M, Badziklou K, Sarfo FS, et al. Loop-Mediated Isothermal
Amplification for Laboratory Confirmation of Buruli Ulcer Disease-Towards a Point-of-Care Test. PLoS
Negl Trop Dis. 2015; 9(11):e0004219. https://doi.org/10.1371/journal.pntd.0004219 PMID: 26566026
25. Spangenberg T, Kishi Y. Highly sensitive, operationally simple, cost/time effective detection of the
mycolactones from the human pathogen Mycobacterium ulcerans. Chem Commun (Camb). 2010; 46
(9):1410–2. https://doi.org/10.1039/b924896j PMID: 20162131
26. Beissner M, Huber KL, Badziklou K, Halatoko WA, Maman I, Vogel F, et al. Implementation of a national
reference laboratory for Buruli ulcer disease in Togo. PLoS Negl Trop Dis. 2013; 7(1):e2011. https://doi.
org/10.1371/journal.pntd.0002011 PMID: 23359828
27. Bretzel G, Huber KL, Kobara B, Beissner M, Piten E, Herbinger KH, et al. Laboratory confirmation of
Buruli ulcer disease in Togo, 2007–2010. PLoS Negl Trop Dis. 2011; 5(7):e1228. https://doi.org/10.
1371/journal.pntd.0001228 PMID: 21811641
28. Bretzel G, Siegmund V, Nitschke J, Herbinger KH, Thompson R, Fleischmann E, et al. External quality
assurance for the laboratory diagnosis of Buruli ulcer disease in Ghana. Trop Med Int Health. 2006; 11
(11):1688–93.
29. Bretzel G, Siegmund V, Nitschke J, Herbinger KH, Thompson W, Klutse E, et al. A stepwise approach
to the laboratory diagnosis of Buruli ulcer disease. Trop Med Int Health. 2007; 12(1):89–96. https://doi.
org/10.1111/j.1365-3156.2006.01761.x PMID: 17207152
30. WHO. Buruli ulcer: progress report, 2004–2008. Wkly Epidemiol Rec. 2008; 83(17):145–54. PMID:
18437758
31. Wadagni A, Frimpong M, Phanzu DM, Ablordey A, Kacou E, Gbedevi M, et al. Simple, rapid Mycobacte-
rium ulcerans disease diagnosis from clinical samples by fluorescence of mycolactone on thin layer
chromatography. PLoS Neglected Trop Dis. 2015; 9(11):e0004247/1. https://doi.org/10.1371/journal.
pntd.0004247 PMID: 26583925
32. Converse PJ, Xing Y, Kim KH, Tyagi S, Li SY, Almeida DV, et al. Accelerated detection of mycolactone
production and response to antibiotic treatment in a mouse model of Mycobacterium ulcerans disease.
PLoS Negl Trop Dis. 2014; 8(1):e2618. https://doi.org/10.1371/journal.pntd.0002618 PMID: 24392174
33. Marion E, Prado S, Cano C, Babonneau J, Ghamrawi S, Marsollier L. Photodegradation of the Myco-
bacterium ulcerans toxin, mycolactones: considerations for handling and storage. PLoS One. 2012; 7
(4):e33600. https://doi.org/10.1371/journal.pone.0033600 PMID: 22514607
34. Ablordey A, Amissah DA, Aboagye IF, Hatano B, Yamazaki T, Sata T, et al. Detection of Mycobacte-
rium ulcerans by the loop mediated isothermal amplification method. PLoS Negl Trop Dis. 2012; 6(4):
e1590. https://doi.org/10.1371/journal.pntd.0001590 PMID: 22509415
35. Stinear T, Ross BC, Davies JK, Marino L, Robins-Browne RM, Oppedisano F, et al. Identification and
characterization of IS2404 and IS2606: two distinct repeated sequences for detection of Mycobacterium
ulcerans by PCR. J Clin Microbiol. 1999; 37(4):1018–23. https://doi.org/10.1128/JCM.37.4.1018-1023.
1999 PMID: 10074520
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 15 / 16
PLOS ONE Evaluation of f-TLC method for the diagnosis of Buruli Ulcer
36. Phillips R, Horsfield C, Kuijper S, Lartey A, Tetteh I, Etuaful S, et al. Sensitivity of PCR targeting the
IS2404 insertion sequence of Mycobacterium ulcerans in an Assay using punch biopsy specimens for
diagnosis of Buruli ulcer. J Clin Microbiol. 2005; 43(8):3650–6. https://doi.org/10.1128/JCM.43.8.3650-
3656.2005 PMID: 16081892
37. Yeboah-Manu D, Asante-Poku A, Asan-Ampah K, Ampadu ED, Pluschke G. Combining PCR with
microscopy to reduce costs of laboratory diagnosis of Buruli ulcer. Am J Trop Med Hyg. 2011; 85
(5):900–4. https://doi.org/10.4269/ajtmh.2011.11-0362 PMID: 22049046
38. Ojurongbe O, Adegbosin OO, Taiwo SS, Alli OAT, Olowe OA, Ojurongbe TA, et al. Assessment of clini-
cal diagnosis, microscopy, rapid diagnostic tests, and polymerase chain reaction in the diagnosis of
Plasmodium falciparum in Nigeria. Malaria research and treatment. 2013;2013.
39. Youden WJ. Index for rating diagnostic tests. Cancer. 1950; 3(1):32–5. https://doi.org/10.1002/1097-
0142(1950)3:1<32::aid-cncr2820030106>3.0.co;2-3 PMID: 15405679
40. Azevedo MF, Faucz FR, Bimpaki E, Horvath A, Levy I, de Alexandre RB, et al. Clinical and molecular
genetics of the phosphodiesterases (PDEs). Endocr Rev. 2014; 35(2):195–233. https://doi.org/10.
1210/er.2013-1053 PMID: 24311737
41. Sarfo FS, Le Chevalier F, Aka N, Phillips RO, Amoako Y, Boneca IG, et al. Mycolactone diffuses into
the peripheral blood of Buruli ulcer patients—implications for diagnosis and disease monitoring. PLoS
Negl Trop Dis. 2011; 5(7):e1237. https://doi.org/10.1371/journal.pntd.0001237 PMID: 21811642
42. Toutous Trellu L, Nkemenang P, Comte E, Ehounou G, Atangana P, Mboua DJ, et al. Differential Diag-
nosis of Skin Ulcers in a Mycobacterium ulcerans Endemic Area: Data from a Prospective Study in
Cameroon. PLoS Negl Trop Dis. 2016; 10(4):e0004385. https://doi.org/10.1371/journal.pntd.0004385
PMID: 27074157
43. Addison NO, Pfau S, Koka E, Aboagye SY, Kpeli G, Pluschke G, et al. Assessing and managing
wounds of Buruli ulcer patients at the primary and secondary health care levels in Ghana. PLoS Negl
Trop Dis. 2017; 11(2):e0005331. https://doi.org/10.1371/journal.pntd.0005331 PMID: 28245242
44. Berzosa P, de Lucio A, Romay-Barja M, Herrador Z, González V, Garcı́a L, et al. Comparison of three
diagnostic methods (microscopy, RDT, and PCR) for the detection of malaria parasites in representa-
tive samples from Equatorial Guinea. Malaria journal. 2018; 17(1):333. https://doi.org/10.1186/s12936-
018-2481-4 PMID: 30223852
45. Sakyi SA, Aboagye SY, Otchere ID, Liao AM, Caltagirone TG, Yeboah-Manu D. RNA aptamer that spe-
cifically binds to mycolactone and serves as a diagnostic tool for diagnosis of Buruli ulcer. PLoS
neglected tropical diseases. 2016; 10(10):e0004950. https://doi.org/10.1371/journal.pntd.0004950
PMID: 27776120
46. Siegmund V, Adjei O, Nitschke J, Thompson W, Klutse E, Herbinger KH, et al. Dry reagent-based poly-
merase chain reaction compared with other laboratory methods available for the diagnosis of Buruli
ulcer disease. Clin Infect Dis. 2007; 45(1):68–75. https://doi.org/10.1086/518604 PMID: 17554703
47. Pormohammad A, Riahi S-M, Nasiri MJ, Fallah F, Aghazadeh M, Doustdar F, et al. Diagnostic test
accuracy of adenosine deaminase for tuberculous meningitis: A systematic review and meta-analysis.
Journal of Infection. 2017; 74(6):545–54. https://doi.org/10.1016/j.jinf.2017.02.012 PMID: 28366687
48. Fagbamigbe AF. On the discriminatory and predictive accuracy of the RDT against the microscopy in
the diagnosis of malaria among under-five children in Nigeria. Malaria journal. 2019; 18(1):46. https://
doi.org/10.1186/s12936-019-2678-1 PMID: 30791912
49. Mazzu-Nascimento T, Morbioli GG, Milan LA, Donofrio FC, Mestriner CA, Carrilho E. Development and
statistical assessment of a paper-based immunoassay for detection of tumor markers. Analytica Chi-
mica Acta. 2017; 950:156–61. https://doi.org/10.1016/j.aca.2016.11.011 PMID: 27916120
50. Chavarrı́a-Bolaños D, Rodrı́guez-Wong L, Noguera-González D, Esparza-Villalpando V, Montero-Agui-
lar M, Pozos-Guillén A. Sensitivity, specificity, predictive values, and accuracy of three diagnostic tests
to predict inferior alveolar nerve blockade failure in symptomatic irreversible pulpitis. Pain Research and
Management. 2017;2017.
51. Ray P, Manach Yannick L, Riou B, Houle Tim T, Warner David S. Statistical Evaluation of a Biomarker.
Anesthesiology. 2010; 112(4):1023–40. https://doi.org/10.1097/ALN.0b013e3181d47604 PMID:
20234303
52. de Souza DK, Quaye C, Mosi L, Addo P, Boakye DA. A quick and cost effective method for the diagno-
sis of Mycobacterium ulcerans infection. BMC Infect Dis. 2012; 12:8. https://doi.org/10.1186/1471-
2334-12-8 PMID: 22257432
PLOS ONE | https://doi.org/10.1371/journal.pone.0270235 August 2, 2022 16 / 16