Emerging Infectious Diseases
[Volume 6 No.2 / March - April 2000]

Research

Serologic Response to Culture Filtrate Antigens of Mycobacterium
ulcerans during Buruli Ulcer Disease

Karen M. Dobos,*† Ellen A. Spotts,*† Barbara J. Marston,* C.
Robert Horsburgh Jr.,* and C. Harold King*
*Emory University School of Medicine, Atlanta, Georgia, USA;
and †Centers for Disease Control and Prevention, Atlanta, Georgia,
USA

Buruli ulcer (BU) is an emerging necrotic skin disease caused by
Mycobacterium ulcerans. To assess the potential for a
serodiagnostic test, we measured the humoral immune response of
BU patients to M. ulcerans antigens and compared this response
with delayed-type hypersensitivity responses to both Burulin and
PPD. The delayed-type hypersensitivity response generally
supported the diagnosis of BU, with overall reactivity to Burulin in
28 (71.8%) of 39 patients tested, compared with 3 (14%) of 21
healthy controls. However, this positive skin test response was
observed primarily in patients with healed or active disease, and
rarely in patients with early disease (p=0.009). When tested for a
serologic response to M. ulcerans culture filtrate, 43 (70.5%) of 61
BU patients had antibodies to these antigens, compared with 10
(37.0%) of 27 controls and 4 (30.8%) of 13 tuberculosis patients.
There was no correlation between disease stage and the onset of
this serum antibody response. Our findings suggest that serologic
testing may be useful in the diagnosis and surveillance of BU.

Buruli ulcer (BU) disease, caused by Mycobacterium ulcerans, is
characterized by severe necrotizing ulcers. The disease, which is
found worldwide but primarily in tropical climates (1,2), occurs
predominantly in children ages 5 to 14 (3) and is associated with
severe illness and permanent disabilities in >26% of patients (1,3).
Over the past decade, the incidence of BU disease has dramatically
increased in several West African countries, notably Ghana, Côte
d'Ivoire, Benin, and Togo (1,2). In some West African
communities, BU has replaced tuberculosis (TB) and leprosy as the
most prevalent mycobacterial disease, affecting up to 22% of the
population (4). However, the true global impact of the disease is
unknown because reliable tools for its rapid diagnosis and
surveillance are lacking. In addition, neither the mode of
transmission nor the environmental reservoir for M. ulcerans is
known, although observational studies suggest that M. ulcerans
infection is transmitted through the skin after contact with
contaminated water, vegetation (5), or insects (6).

Current treatment for cutaneous disease is primarily surgical.
Ulcerative lesions require wide debridement and skin grafting in
specialized health-care facilities, long hospital stays (7), and
increased cost to the health-care system and the community (7).
Antibiotics are ineffective once ulcers are present, possibly because
M. ulcerans is absent from lesions or subcutaneous tissues have
already been destroyed, preventing penetration of
antimycobacterial drugs (3). Antimicrobial therapy has not been
studied in preulcerative lesions because apparently patients rarely
visit primary health-care providers with painless nodular lesions
and diagnostic tests are not available to clearly identify M. ulcerans
infection before it progresses to clinical disease. Early
identification and surgical excision of these preulcerative nodular
lesions can be curative and does not require inpatient care (8). We
investigated the serologic response to the M. ulcerans culture
filtrate (MUCF) of BU patients at different stages of disease to
determine whether serodiagnosis is feasible as a tool for early
diagnosis and intervention of BU disease.

Table 1. Burulin skin test responses of Buruli ulcer patients and
healthy controls from the same villages


Persons were scored Burulin-only skin-test positive if the diameter
of induration at the site of Burulin injection was 10 mm at 48 or 72
hours and the diameter of induration at the site of PPD injection
was at least 3 mm smaller than in the Burulin response. Persons
were scored as only PPD positive if the diameter of induration at
the site of PPD injection was 10 mm at 48 or 72 hours and the
diameter of induration at the site of Burulin injection was at least 3
mm smaller than that in the PPD response. Persons were scored as
both Burulin and PPD skin-test positive if the diameters of
induration of both the Burulin and PPD injection sites were 10 mm
at 48 or 72 hours and the differences in the diameters of the zone of
induration between the two was <3 mm. Persons were scored as
skin test negative for both if the induration at both injection sites
was <10 mm at 48 or 72 hours.

Table 2. Serologic and skin test responses of Buruli ulcer patients
and healthy controls from the same villages

The Study

BU patients were identified through a case-control study conducted
in 1991 in the Daloa region of Côte d'Ivoire (3). Patients with early
ulcerative BU were identified by small, painless, or minimally
painful nodules or edema, or ulcerative lesions for <2 weeks.
Patients with ulcerative BU were identified by lesions that were
chronic (>2 weeks duration), were painless or minimally painful,
or had undermined edges. Patients with healed BU were identified
by stellate scars with retraction in areas of prior ulceration.
Descriptive and clinical data were obtained from all BU patients
and controls (3). Blood specimens were collected from 82 BU
patients and 164 healthy controls in the Daloa region.

Because TB is endemic in many of the areas where BU occurs, 13
TB patients from the United States were used as controls for cross-
reactivity to MUCF. Use of sera from these patients ensured the
lowest probability for exposure to M. ulcerans, so that only cross-
reactivity to antigens common to both M. tuberculosis and M.
ulcerans would be measured. TB patients were identified through
an ongoing study in Atlanta by clinical presentation, a positive
PPD response, and isolation of M. tuberculosis from sputum. All
specimens tested negative for HIV and were strongly reactive to
many of the culture filtrate antigens from M. tuberculosis (data not
shown). Informed consent was obtained for all participants, and
protocols were approved by the Institutional Review Boards of the
Centers for Disease Control and Prevention and the Ministry of
Health of Côte d'Ivoire for the BU study and the Institutional
Review Board at Emory University for the TB study.

Skin Testing

During sera collection, Burulin (9) and PPD (purified protein
derivative of M. tuberculosis, Connaught Laboratories, U.K.) were
administered intradermally into the flexor surface of the forearms
of 39 BU patients and 21 controls, and the diameter of induration
was recorded at 48 and 72 hours. The criterion for using Burulin
and PPD dual testing to confirm clinical cases of BU has been
reported (3,9), and the cumulative delayed-type hypersensitivity
(DTH) findings for this study have been described (Table 1) (3).
Sera were also tested for reactivity to MUCF antigen by Western
blot (Table 2).


Preparation of MUCF Antigen for Serologic Analysis

M. ulcerans strain S-WT (from the Centers for Disease Control and
Prevention culture collection; Atlanta) was recovered from a 1-ml
lyophilisate in 10% glycerol by incubation in 5 ml Middlebrook
7H9 with OADC supplement (Remel, Lenexa, KS) at 32.5°C for 7
days. This 5-ml inoculum was passaged into 25 ml Middlebrook
7H9 broth supplemented with tryptose and glucose (7H9TG; 0.1%
wt:vol and 2% wt:vol, respectively) and incubated at 32.5°C for 10
days, followed by passage into 100 ml of 7H9TG and growth at
32.5°C for 10 days. Cultivation in this protein- and serum-free
media allowed isolation and analysis of specific secreted
mycobacterial proteins. The 100-ml culture was passaged into 1L
of 7H9TG, incubated at 32.5°C in 850-cm 2 roller bottles, and
rotated at 90 rotations per hour. Bacilli were harvested during mid-
log phase growth by centrifugation. The 1L of MUCF, containing
mycobacterial proteins of interest, was concentrated at 4°C under
N 2 by using a stirred-cell apparatus (Amicon Inc., Beverly, MA)
with a 10-kDa molecular weight membrane. MUCF was
exchanged into 0.1 M NH 4 HCO 3 by dialysis, lyophilized, and
purified by separation of residual small molecular weight
contaminants from antigens of interest by size exclusion
chromatography. MUCF was loaded onto a fast-performance liquid
chromatography system (FPLC) (Pharmacia, Piscataway, NJ)
equipped with a G-25 superfine Sephadex desalting column
(Pharmacia, Piscataway, NJ), UV monitor, and conductivity
monitor for separation and detection of proteins and salts,
respectively. MUCF was eluted with an isocratic gradient of 0.1 M
NH 4 HCO 3 . MUCF protein was quantitated by bicinchoninic
acid assay (Pierce Chemical Co., Rockford, IL) and lyophilized
until needed. Approximately 5 mg of MUCF protein was obtained
per liter of culture.

Aliquots of 8 g of MUCF protein were resolved by discontinuous
sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-
PAGE) (10) by using 10% to 20% gradient 10-well gels (Novex,
San Diego, CA). MUCF proteins were visualized by staining with
silver (11), and this profile was reexamined throughout these
experiments to ensure that the MUCF proteins did not degrade
upon storage; no antigen variability was seen during our studies.
7H9TG media alone processed in the same way contained no
proteins by silver stain analysis after SDS-PAGE and bicinchoninic
acid assay.

Amino Acid Sequence Analyses

The N-terminal amino acid sequences of MUCF proteins were
determined and compared with known mycobacterial sequences.
One hundred micrograms of MUCF protein was resolved by two-
dimensional gel electrophoresis (2D-GE) by the method of
O'Farrell (12) with the modifications of Sonnenberg and Belisle
for resolution of mycobacterial proteins (13). 2D-GE resolved
proteins were transferred to 0.1 mm polyvinyl difluoride (PVDF)
membrane (14) and visualized by staining with 0.5% Coomassie
blue R250 in 40% methanol/10% acetic acid for 2 minutes. The N-
terminal amino acid sequence was obtained by loading membranes
containing the excised spot of interest onto a Procise-cLC
automated Edman sequencer (Applied Biosystems, Foster City,
CA) and reading the generated chromatographs against amino acid
standards.

Statistical Analyses

Statistical analyses were done by the Mantel-Haenszel chi-square
method. Statistical significance for the chi-square p values and
Fisher's exact p values was evaluated at alpha = 0.05. Fisher's
exact p value was used when the expected cell count was <5. Odds
ratios (OR) were determined by calculating the ratio of the odds of
exposure among cases to that among controls. The test for
specificity was determined by dividing the true negatives (n = 24;
disease-negative and 70 kDa-negative) by the total number of
controls (n = 27). All analyses were done on BU patients and
healthy controls from the Daloa region of Côte d'Ivoire, where the
disease is endemic, unless otherwise specified. All data were
analyzed by using Exact 2.0b software (D. Martin and H. Austin,
Atlanta, GA).

Results

Seventeen (43.6%) of the 39 skin-tested BU patients had
induration of > 10 mm within 72 hours of Burulin administration,
with PPD induration at least 3 mm smaller than that with Burulin
(Table 1). Only 3 (14.3%) of the 21 skin-tested healthy controls
from the area had a similar DTH pattern (Table 1). In addition, 11
(28.2%) of the 39 BU patients had an equally strong response to
Burulin and PPD, which suggests that Burulin skin testing may not
be reliable in regions where BU and TB are endemic and BCG
vaccination is widely used (Table 1). Dual-positive responses
included, 28 (71.8%) of the 39 BU patients were Burulin positive.
However, when this total Burulin-positive population was
delineated by disease stage, positive responses were observed in
most of the patients with healed (93.8%) and ulcerative disease
(64.7%), but in few (33.4%) patients with early ulcerative disease
(Table 2). When healed and active Burulin responses were
compared with the DTH response seen in early ulcerative BU
patients, a significant difference between the disease stages was
found (Fisher's exact p = 0.009). These skin-tested BU patient and
control populations from the Daloa region were then tested for
reactivity of their sera to MUCF by Western blot, and Burulin
induration was compared with serum antibody response (Table 2).
Twenty-six (66.7%) of the 39 skin-tested BU patients had an
antibody response to the MUCF, versus 7 (33.3%) of the 21 skin-
tested controls (chi-square p = 0.014). BU patient populations had
approximately the same positive antibody responses to the MUCF,
regardless of disease stage. Specifically, 68.8% of the healed
patients were antibody positive, with active ulcerative disease at
64.7% and early disease at 66.7% (Table 2). When the antibody
responses of healed and active patients were compared with those
seen in early BU patients, the serologic response did not differ
significantly by disease stage (Fisher's exact p = 0.999). Therefore,
serologic testing may be useful in the early diagnosis and
surveillance of BU.

Figure. Western blot reactivity to and Silver stain analysis of
Mycobacterium ulcerans culture filtrates (MUCF). A)
Representative antibody responses to MUCF. M; molecular weight
markers with annotations corresponding to molecular weight on the
left; lanes 1-6, representative BU patient sera with reactivity to
MUCF; lanes 7-10, representative antibody reactivity in healthy
persons from the disease-endemic area; lanes 11-12, serologic
reactivities to MUCF of two representative tuberculosis (TB)
patients. TB patient sera that were serologically reactive to MUCF
were included as a control for cross-reactivity. Regions of antibody
reactivity corresponding to M. ulcerans antigens of 70, 38/36, and
5 kDa are noted (arrows). B) Silver-stained SDS-PAGE gel of
MUCF. The identification of putative proteins corresponding to the
serologically reactive 70, 38/36, and 5 kDa MUCF antigens are
noted (stars). For Western blot analyses, aliquots (50 ˜g protein) of
MUCF were resolved by discontinuous SDS-PAGE (10) with
preparative 10% to 20% gradient 1-well gels (Novex, San Diego,
CA) and then transferred to nitrocellulose (14). Nitrocellulose
sheets were cut into 2-mm strips and stored in 5% skim milk in
Tris-buffered saline, pH 7.6, until used. Antibody to MUCF was
detected by probing nitrocellulose strips with sera at a 1:50 (vol:vol
dilution). Bound serum antibodies were detected with alkaline
phosphatase-conjugated anti-human antibody (Sigma Chemical
Co., St. Louis, MO), and the substrate 4-Bromo-3- Chloro-2-
Indoyl-1-Phosphate/Nitro blue tetrazoleum (Sigma Chemical Co.,
St. Louis, MO). All sera were tested in duplicate for confirmation
of the antibody responses.

The antibody response to the MUCF was then determined for all
61 clinically diagnosed BU patients and 27 healthy controls, and
antibody reactivity to individual antigens was recorded. Three M.
ulcerans antigens of 70, 38/36, and 5 kDa were commonly
recognized by BU patient sera antibodies (Figure). The 36-kDa
antigen was found to be a degradation product of the 38-kDa
antigen (unpub. results). Forty-three (70.5%) of 61 BU patient sera
had antibodies to at least 1 of 3 antigens, compared with 10
(37.0%) of 27 sera from healthy controls from the area and 4
(30.8%) of 13 sera from TB patients (Table 3). The antibody
response to the 70-kDa M. ulcerans antigen was associated with
BU disease (OR = 12.33; chi-square p = 0.00002), and the
response to this protein was consistent throughout early to late
disease stage (OR = 12.4; chi-square p = 0.0000001; Table 3).
Results were similar for the 38/36-kDa antigen (OR = 5.94; chi-
square p = 0.0016), regardless of disease stage (OR = 3.00; chi-
square p = 0.0026).

Table 3. Antibody responses to specific Mycobacterium ulcerans
culture filtrate proteins in all Buruli ulcer patients and controls


N-terminal amino acid sequence of the 38 kDa protein was
DPAPAPAPRD, and had mycobacterial sequences that precede
glycosylation sites (15). The serologic response to the 5-kDa
antigen, although common in the seroreactive specimens tested, did
not differ significantly between BU patients and healthy controls
from disease-endemic areas or TB patients (chi-square p = 0.764
and Fisher's exact p = 0.999, respectively); nor were antibodies to
this antigen observed in BU patients at the early ulcerative stage
(Table 3). A cross-reactive serologic response to MUCF was
observed in some TB patients who had not been exposed to M.
ulcerans and in some healthy controls from disease-endemic areas
(Figure; representative sample and Table 3). These positive
responses may be due to exposure to other mycobacterial
pathogens with similar antigens as M. ulcerans or BU disease
undetectable by clinical diagnosis, respectively.

Conclusions

Diagnosis of BU currently relies on the clinical presentation of the
ulcerative disease stage, by which time the infection has already
caused damage, therapeutic options are limited, and outcome is
associated with severe disease (3,7,8). Preulcerative nodules and
edemous lesions, when identified, frequently contain numerous
extracellular mycobacteria (1,2). Thus, diagnosis of BU disease at
this early stage may lead to more effective treatment. Indeed,
diagnosis of preulcerative BU disease is associated with a better
therapeutic outcome and reduced illness (8), and accurate
surveillance at the preulcerative stage would enable assessment of
the prevalence and global impact of BU disease. Surveillance may
also identify risk factors as well as prevention strategies for BU.
Consistent with previous reports (3,9), DTH to Burulin, a crude
preparation of M. ulcerans lysate (9), was observed in many
(71.8%) of the clinically diagnosed BU patients. This overall
response was similar to the well-defined cell-mediated DTH
responses reported for other mycobacterial infections (16-18),
including M. tuberculosis (19,20). In contrast, in BU patients, the
Burulin response was associated with ulcerative and healed
disease, but not with early ulcerative disease. Thus, unlike other
DTH-based monitoring programs that indicate mycobacterial
infection before and during disease in immunocompetent persons,
Burulin testing may be more useful for confirming ulcerative
disease or monitoring past disease with M. ulcerans in conjunction
with PPD results. The marked shared reactivity to PPD and Burulin
antigens by BU patients, as measured by similar induration upon
intradermal injection, was expected to some degree, considering
the commonality of antigenic lipids on the mycobacterial surface,
genetic relatedness of mycobacterial pathogens, opportunity of
exposure to both mycobacterial pathogens and environmental
mycobacteria, and rate of BCG vaccination in the region. When
Burulin-positive results were analyzed in conjunction with either
the presence of a BCG vaccination scar or recollection of BCG
vaccination, 12 of 14 BCG-vaccinated BU patients were Burulin
positive, and 6 of these 12 patients were also PPD positive.
Additionally, all the Burulin- and PPD-positive controls either
recalled BCG vaccination or had a BCG scar (data not shown).

We found that a specific response to the MUCF was observed in
BU patient sera, but unlike the Burulin DTH response, the antibody
response did not correlate with disease stage  (Table 2). The
antibody reactivity in the sera from some of the controls from
disease-endemic areas may reflect preclinical infection with M.
ulcerans or exposure to other mycobacteria and cross-reactivity to
antigens common to mycobacterial species. Interestingly, 10
(55.6%) of the 18 antibody-negative BU patients, versus 15 (33%)
of the 43 antibody-positive BU patients, recalled or had a BCG
vaccination scar, which suggests that BCG vaccination did not
contribute to the antibody response of BU patients to MUCF (data
not shown).

Thirty percent of the TB patient sera were reactive to MUCF, with
reactivity principally towards lower molecular weight proteins and
not the 70- or 38/36-kDa proteins (Figure; Table 3). N-terminal
amino acid sequence of one of these lower molecular weight
proteins, a 31 kDa protein, was found to be FSRPGLPVEY and
demonstrated homology with the mycolyl transferases found in M.
tuberculosis and other mycobacteria (21). Thus, reactivity of the
TB patient sera based on antibodies generated against common
mycobacterial antigens is the likely explanation for the few TB
patient sera reactive to MUCF.

Our preliminary results indicate the usefulness of the MUCF in
detecting BU; specifically, individual antigens in the MUCF may
be used for the development of a sensitive and specific test for BU
(Table 3). Both the 70- and 38/36-kDa proteins were indicators of
BU disease, and antibody responses to both of these proteins were
similarly present in BU-reactive patient serum samples, regardless
of disease stage. In addition, reactivity to the 70-kDa protein was
specific for M. ulcerans infection (88.8%).

The 70- and 38/36-kDa proteins may be useful for the development
of a serologic test for BU in areas where TB is endemic. Based on
these results, we are isolating and characterizing these two M.
ulcerans antigens for additional testing. The N-terminal sequence
for the 38/36-kDa protein has already been determined as part of
the purification process for this protein, and identification and
purification of the 70-kDa protein are under way. If a serologic test
proves to be diagnostic, early excision of nodules and
antimycobacterial chemotherapy trials of patients could reduce the
public health and socioeconomic impact of this emerging disease.

Acknowledgments

We thank John Stanford for providing the Burulin reagent, Jennifer
Ekmark for discussion and critical reading of the manuscript,
Merywin Wigley for laboratory assistance, Carolyn Carter for N-
terminal sequence analysis, the Emory Medical Care Foundation
for collection and use of the TB patient and control sera (HIC #
455-96), and the National Center for Infectious Diseases for the
initial case study of BU disease in Côte d'Ivoire.

This study was supported in part by Centers for Disease Control
and Prevention Cooperative agreement No. U50/ CCU416560-01-
1, Novel Diagnostic Tests for Infections of Public Health
Significance, and by an Oak Ridge Institute for Science and
Education Fellowship awarded to Karen M. Dobos.

Dr. Dobos is a senior postdoctoral fellow in the laboratory of Dr.
C. Harold King at the Emory University School of Medicine. Her
current research interests focus on bacterial proteomic analysis.
She will continue studying the proteins of Mycobacterium ulcerans
for development of future vaccine and diagnostic candidates. 

Address for correspondence: C. Harold King, Emory University
School of Medicine, 69 Butler St. S.E., Atlanta, GA 30303, USA;
fax: 404-880-9305; e-mail: cking01@emory.edu.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

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