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Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, January 2001, p. 138-142, Vol. 8, No. 1
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.1.138-142.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Phenolic Glycolipid I of
Mycobacterium leprae in Sera from Leprosy Patients
before and after Start of Multidrug Therapy
Sang-Nae
Cho,1,2,*
Roland V.
Cellona,3
Laarni G.
Villahermosa,3
Tranquilino T.
Fajardo Jr.,3
M. Victoria F.
Balagon,3
Rodolfo M.
Abalos,3
Esterlina V.
Tan,3
Gerald P.
Walsh,3
Joo-Deuk
Kim,1,2 and
Patrick J.
Brennan4
Department of
Microbiology1 and Institute for
Immunology and Immunological Diseases,2 Yonsei
University College of Medicine, Seoul 120-752, The Republic of Korea;
Leonard Wood Memorial Center, Cebu 6000, The
Philippines3; and Department of
Microbiology, Colorado State University, Fort Collins, Colorado
805234
Received 8 June 2000/Returned for modification 25 July
2000/Accepted 23 October 2000
 |
ABSTRACT |
A total of 100 untreated new leprosy patients were
recruited prospectively and examined for the presence of phenolic
glycolipid I (PGL-I) antigen in their serum specimens by dot
enzyme-linked immunosorbent assay (ELISA) using rabbit anti-PGL-I
antiserum. The presence of circulating PGL-I antigen was closely
related to the bacterial indices (BI) of the patients. The PGL-I
antigen was detectable in 27 (93.1%) of 29 patients with a BI of 4.0 or above and in 15 (68.2%) of 22 patients with a BI of 3.0 to 3.9. However, none of the 37 patients with a BI of less than 1.9 had detectable PGL-I antigen by the methods used in this study. The level
of PGL-I in serum declined rapidly by about 90% 1 month after the
start of multidrug therapy. This study showed clearly that anti-PGL-I
IgM antibodies and circulating PGL-I antigen levels reflect the
bacterial loads in untreated leprosy patients. The serological
parameters based on the PGL-I antigen may therefore be useful in the
assessment of leprosy patients at the time of diagnosis and possibly in
monitoring patients following chemotherapy.
| |
INTRODUCTION |
Despite the rapid reduction of
registered leprosy cases in the last decade, leprosy is still a major
public health problem in several countries 16. The finding
of no substantial decrease in the new-case detection rate (684,998 new
cases reported in 1997 [16] during the same period
undermines the successful multidrug therapy (MDT) programs directed by
the World Health Organization (WHO). Despite the WHO efforts to
eliminate leprosy by the year 2000, areas of hyperendemic infection
remain in many countries. In such areas, sensitive and specific
laboratory diagnostic tests will be of great value in detecting leprosy
patients at the early stages.
There have been tremendous efforts to develop sensitive and specific
serodiagnostic tests, and these have been reported in the literature.
Among the antigens evaluated for immunoassays, phenolic glycolipid I
(PGL-I) is still the only Mycobacterium leprae-specific
antigen 8, and it has been widely used for the
serodiagnosis of leprosy. The presence of the M. leprae-specific antigen(s) in clinical samples would be indicative
of current M. leprae infection. PGL-I was thus the target
antigen of choice because of its specificity to M. leprae
and its abundance. As well, there have been several studies of the
detection of PGL-I in various clinical specimens such as serum 1,
4, 12, 17, urine 4, 9, 10, 13, nasal washes
13, and biopsy specimens 14 for diagnosis and
determination of the prognosis following chemotherapy for leprosy.
In general, the PGL-I antigen is detectable in clinical specimens
mostly from multibacillary (MB) patients, mainly due to the limited
sensitivity of the current detection methods and to the rapid decline
of its level in sera soon after starting chemotherapy against leprosy
1, 10, 12. However, it has not been well established what
proportion of MB patients are positive for the PGL-I antigen in their
sera. The present study was therefore designed to compare the
bacterial indices with PGL-I detection in sera from untreated
leprosy patients. In addition, the PGL-I antigen level was measured
semiquantitatively in sera obtained serially from leprosy patients
after starting MDT. The results were then compared with bacterial
indices (BI) and immunoglobulin M (IgM) antibodies to the antigen to
determine which parameter was the better indicator to monitor the
effectiveness of chemotherapy against leprosy.
| |
MATERIALS AND METHODS |
Study patients and serum samples.
A total of 100 untreated
patients were recruited prospectively among the leprosy patients who
presented at the Skin Clinics of the Leonard Wood Memorial Center for
Leprosy Research in Cebu City, Philippines. All patients were
classified based on clinical findings, histopathological examination,
and BI on the Ridley and Jopling scale 11. Of 100 patients
enrolled for the study, 28 patients were classified as lepromatous
(LL), 32 as borderline lepromatous (BL), 27 as borderline tuberculoid
(BT), 12 as tuberculoid (TT), and one as indeterminate; 89 patients
were bacteriologically positive (MB), and 11 were acid-fast
bacillus negative (paucibacillary). Serum samples were obtained from
the study patients before starting treatment and serially at regular
intervals of 1, 2, 4, 6, 9 and 12 months after starting WHO MDT.
Detection of PGL-I antigen in serum specimens.
The procedure
was a modification of that described previously 4, and it
was faster 3, 5. To facilitate the extraction of total
lipids from serum specimens, a single 100-µl specimen was added to a
filter paper disk (0.5 in. in diameter) (Schleicher & Schuell, Inc.,
Keene, N.H.) and dried completely. The lipids were then extracted using
2 to 3 ml of CHCl3-CH3OH (2:1) solution and
dried under N2. Serum lipids were dissolved in
CHCl3, applied to a Pasteur pipette packed with Florisil
60-100 mesh (Sigma Chemical Co., St. Louis, Mo.), and eluted with
CHCl3 followed by 5% CH3OH in
CHCl3. The lipid fraction eluted with 5% CH3OH
was saved and dried under N2 and examined for the presence
of PGL-I.
The dot enzyme-linked immunosorbent assay (ELISA) described by Hawkes
et al. 7 was used with minor modification as reported previously 3, 5. The purified lipid was dissolved in 100 µl of hexane, and a 5-µl portion was applied to a Tuffryn
(polysulfone) membrane (HT-200; Gelman Sciences, Inc., Ann Arbor,
Mich.), as originally used by Young et al. 17. High-titer
rabbit anti-PGL-I antiserum, prepared as described previously
4, was used as the primary antibody, and
peroxidase-conjugated goat anti-rabbit IgG (Cappel, Organon Teknika
Co., Durham, N.C.) was used as the secondary antibody. For color
development, 4-chloro-1-naphthol (Bio-Rad Laboratories, Inc., Richmond,
Calif.) was used and the results were read visually.
For comparison of the PGL-I antigen level in serum samples, purified
lipid from 100 µl of serum was twofold serially diluted to 1:256 in
hexane, and 5 µl of diluted lipid was applied to the Tuffryn membrane
and subjected to the dot ELISA procedures described above. The highest
dilution showing evidence of PGL-I was considered to be the titer of
PGL-I in this study in order to compare the PGL-I level between serum samples.
In each experiment, PGL-I was included at concentrations of 2.5, 1.0, 0.5, and 0.25 ng per spot as a positive control, and hexane was
included as a negative control. The detection limit was set at 0.5 to
1.0 ng of PGL-I, and any experiment giving a higher detection limit was
considered invalid and repeated.
Detection of antibodies to PGL-I.
An ELISA described by
Voller et al. 15 was used with minor modification as
reported previously 3, 6; however, instead of the
native glycolipid, the semisynthetic neoglycoprotein
O-(3,6-di-O-methyl-
-D-glucopyranosyl)-(1
4)-(2,3-di-O-methyl-
-1-rhamnopyranosyl)-(19)-oxynonanoyl-bovine serum albumin (natural disaccharide-octyl-BSA [ND-O-BSA])
2 was used. Briefly, 50 µl of diluted ND-O-BSA (20 ng of
sugar/ml) in carbonate buffer (pH 9.6) was added to the wells of
U-bottom microtiter plates (Dynatech Laboratories, Inc., Alexandria,
Va.), and incubated overnight at 37°C in a moist chamber. The wells were then washed with phosphate-buffered saline (PBS) solution (pH 7.4)
containing 0.05% Tween 20 (PBST) and blocked by the addition of 100 µl of PBST-0.05% BSA at 37°C for 1 h. After the wells were emptied, 50 µl of serum diluted 1:300 in PBST-5% normal goat serum (Gibco Laboratories, Grand Island, N.Y.) was added to the wells, which
were then incubated at 37°C for 90 min. After the wells were washed,
50 µl of affinity-purified peroxidase-conjugated goat anti-human IgM
(Behring Diagnostics, San Diego, Calif.) diluted 1:5,000 in PBST-5%
normal goat serum was added and incubation was continued at 37°C for
1 h. After another wash, 50 µl of substrate solution,
H2O2-o-phenylenediamine, was added
to the wells, which were then incubated at room temperature for about
15 min. The reaction was stopped with 50 µl of 2.5 N
H2SO4, and the absorbance was read at 490 nm.
Each test was performed in duplicate, and the mean absorbance of
BSA-only wells was subtracted from that of wells with ND-O-BSA before analysis.
| |
RESULTS |
Detection of PGL-I in untreated patients.
Crude lipids were
extracted from serum samples obtained from leprosy patients before
starting chemotherapy, and the lipid fractions containing the PGL-I
antigen were purified using the Florisil column. The purified lipids
were then applied to a polysulfone membrane and subjected to dot ELISA.
The results were read visually, and some of the dot ELISA results are
shown in Fig. 1. In general, the size and
color intensity of dots varied markedly from 1+ (Fig. 1 dot 5E) to 2+
(dot 4B) to 3+ (dot 6A) between serum samples. This thus indicated that
the circulating PGL-I level in blood is markedly different between
patients.
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FIG. 1.
Dot ELISA for the detection of the PGL-I-containing
lipid fraction from serum samples of leprosy patients. A 5-µl portion
of serum lipid dissolved in hexane was applied to each square, and the
rest of the ELISA steps were then carried out. Any visible dot was
considered PGL-I antigen positive. Note the difference in the sizes and
intensities of dots because of different quantities of PGL-I in
serum.
|
|
To determine the range of BI in leprosy patients whose serum samples
were PGL-I positive, the leprosy patients were grouped based on PGL-I
detection results, and the BI of leprosy patients were plotted in each
group. As shown in Fig. 2, PGL-I was
detectable in patients with BI as low as 2.2, although the majority of
PGL-I-positive patients had BI over 3.0. On the other hand, a portion
of leprosy patients with BI over 3.0 had no circulating PGL-I antigen
in sera detectable by the method employed in this study. When the prevalence of PGL-I antigenemia was analyzed based on the average BI,
the PGL-I antigen was detectable in sera from 27 (93.1%) of 29 patients with BI of 4.0 to 5.0, from 15 (68.2%) of 22 patients with BI
of 3.0 to 3.9, and from 2 (16.7%) of 12 patients with BI of 2.0 to
2.9, respectively (Table 1). However,
none of the 37 patients with BI of less than 2.0 had circulating PGL-I
antigen in their sera. When the results were analyzed based on the
clinical spectrum of leprosy, PGL-I was detectable in 25 (89.3%) of 28 LL patients, 18 (56.3%) of 32 BL patients, and 1 (3.7%) of 27 BT
patients (data not shown). None of 13 TT and 1 indeterminate patients
had PGL-I antigenemia.
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FIG. 2.
Distribution of BI of patients with or without
detectable PGL-I antigen in their serum specimens.
|
|
Monitoring the PGL-I level after chemotherapy.
A
semiquantitative assay was used to compare PGL-I antigen levels before
and after starting chemotherapy. The purified lipid fraction containing
PGL-I was serially diluted twofold in hexane, and each diluted lipid
was assayed for the presence of PGL-I. Figure
3 shows some of the results of PGL-I
titer determinations in sera serially obtained from a leprosy patient.
The PGL-I titer in serum obtained before treatment was 1:128, and it
decreased rapidly to 1:8 2 months after starting chemotherapy and was
barely detectable at 6 months. Likewise, eight patients with BI of 4.5 or over were examined for their PGL-I antigen levels weekly during the
first month of chemotherapy and after 2, 4, and 6 months. In addition,
anti-PGL-I IgM antibodies were assayed in the serum samples and the
results were compared with the PGL-I antigen level. As expected, the
mean titer of PGL-I antigen decreased rapidly soon after starting
chemotherapy: by about 50% within 2 weeks of chemotherapy, by about
90% after 1 month, and by more than 95% after 2 months (Fig.
4). Six of eight patients had no PGL-I detectable 6 months after starting MDT. In contrast, there was no
significant decrease in mean anti-PGL-I IgM antibody levels during the
first 6 months, particularly in the patient group. Thus, PGL-I antigen
seems to be a good marker for monitoring the effectiveness of
chemotherapy during the early phase of MDT.
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FIG. 3.
Titer determination of PGL-I-containing lipids purified
from serum samples that were obtained serially from a leprosy patient
following chemotherapy. Rows: A, before starting chemotherapy; B, 1 week after starting chemotherapy; C, 2 weeks; D, 3 weeks; E, 1 month;
F, 2 months; G, 4 months; H, 6 months; I, 9 months.
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FIG. 4.
Comparison between the PGL-I antigen level and
anti-PGL-I serum antibody levels following chemotherapy of lepromatous
leprosy patients. Each value represents the mean and standard deviation
for eight patients.
|
|
To determine the duration of PGL-I antigenemia in patients undergoing
MDT, serum samples obtained at predetermined intervals from leprosy
patients were examined for the presence of the PGL-I antigen. In
general, within 2 to 6 months of starting MDT, the PGL-I antigen level
declined rapidly to undetectable in serum samples from patients who had
a low (1+; group I) or moderate (2+; group II) level of PGL-I at the
time of diagnosis (Table 2). Only one
(6.3%) of 16 patients with a PGL-I level of 2+ had the antigen
detectable in this study after 6 months of therapy. Although patients
with a high level (3+; group III) of PGL-I at the time of diagnosis
showed a longer duration of PGL-I antigenemia, 14 (77.8%) of 18 patients were PGL-I antigen negative after 6 months of MDT and only 1 patient had circulating PGL-I antigen after 12 months of MDT. In
contrast, there was no substantial decrease in BI in the majority of
patients after 12 months of MDT. The mean BI declined from 4.3 ± 0.5 at the time of diagnosis to 3.4 ± 0.8 after 12 months of MDT
in group III. This was in comparison to the other groups, in which the
BI declined from 4.2 ± 0.7 to 2.9 ± 0.9 after 12 months for
group II patients and from 3.4 ± 0.7 to 1.5 ± 0.5 for group
I patients. The results clearly indicate that PGL-I antigen is a better
indicator for monitoring the effects of chemotherapy on leprosy.
| |
DISCUSSION |
This study showed that the majority of MB leprosy patients with BI
over 3.0 had PGL-I antigenemia and that the PGL-I antigen disappeared
from the circulation within 6 months of starting MDT. This thus
indicated that the PGL-I antigen is a useful marker for monitoring the
effect of MDT in leprosy patients with high bacterial loads. Since
PGL-I is the M. leprae-specific antigen, the presence of the
antigen in serum and other clinical samples may indicate an active
M. leprae infection in the body. Previous studies showed
that the PGL-I antigen was detectable in serum, urine, and biopsy
samples from certain groups of leprosy patients, primarily MB patients.
However, it has not been fully evaluated what proportion of MB patients
would have PGL-I antigenemia and how long the antigen would be present
in blood after starting MDT in leprosy patients.
One of the objectives of this study was therefore to determine the
proportion of patients with PGL-I antigenemia in comparison to the
average BI of leprosy patients. As expected, PGL-I was detectable in
most serum samples from untreated leprosy patients with an average BI
over 4.0. Its prevalence declined markedly among patients with a BI of
3.0 to 4.0, and it was hardly detectable in sera from patients with a
BI less than 3.0. These results supported reports by Roche et al.
12 and Chanteau et al. 1, in which the
majority of leprosy patients with a BI over 4.0 or LL patients had
PGL-I antigenemia before starting MDT. The presence of PGL-I in sera
thus clearly reflects the higher bacterial loads. It was also of
interest that several patients whose BI was over 3.0 had no detectable
PGL-I in sera, despite having a relatively high BI, up to 4.0 to 4.2. The patients with a BI over 4.0 had a relatively high level of
anti-PGL-I antibodies despite the absence of the antigen in serum (data
not shown), indicating production of PGL-I by M. leprae.
Such an absence of PGL-I antigenemia in MB patients with BI over 4.0 was also reported by Roche et al. 12. One possible explanation would be previous medication with anti-leprosy drugs despite denial by the patients at the time the clinical history was obtained.
The other objective of this study was to closely monitor the PGL-I
level immediately after the start of MDT in leprosy patients. When the
PGL-I level in sera were measured semiquantitatively in serial serum
samples, PGL-I declined rapidly. Even 1 week after the start of MDT,
there was an indication of a decrease in PGL-I level, and the mean
PGL-I titer was reduced by one-half after 2 weeks of MDT in this study.
Previously, it was shown that the PGL-I titer apparently declined in
serum samples obtained at least 1 or 2 months after starting MDT
1, 4, 12, 17. The results suggested that PGL-I was
actively secreted from live M. leprae into surrounding
tissues, and as soon as the bacilli were killed by chemotherapy, the
PGL-I antigen was no longer produced and only the residual antigens in
tissues and in blood disappeared from the circulation with a half-life
of about 2 weeks.
The duration of PGL-I antigenemia was apparently well correlated with
the initial level of PGL-I antigen in sera, which also reflected the
bacterial loads in leprosy patients. PGL-I was barely detectable in
sera from patients after 6 months of MDT. Only about 13% of patients
who had PGL-I antigenemia before chemotherapy had PGL-I antigen in sera
detectable by the method used in this study. Chanteau et al.
1 also showed that 3 of 10 patients had residual
PGL-I in sera 6 months after the start of MDT and that PGL-I level
decreased by 97% 1 year after MDT in MB leprosy patients. Therefore,
the PGL-I level in serum or other clinical samples may be a useful
marker for monitoring the effectiveness of MDT among MB patients,
particularly during the early stage of chemotherapy.
Interestingly, Roche et al. 12 reported that PGL-I levels
decreased very rapidly in patients infected with drug-sensitive M. leprae but decreased slowly in patients infected with
drug-resistant bacilli or with the erythema nodosum
leprosum (ENL) reaction. In our study, however, no patients
relapsed from WHO MDT even 3 to 4 years after their release from
treatment. Therefore, it was not possible to correlate the antigen
clearance rate with the outcome of relapse of drug-resistant M. leprae infection. In addition, although there were 10 ENL patients
among the study patients, we did not note any significant delay in
PGL-I antigen clearance compared to those in patients with the same BI
at the time of diagnosis. The slow clearance of PGL-I antigen in sera from ENL patients may reflect a high BI at the beginning of treatment.
As indicated in the literature, therefore, the PGL-I antigen detection
assay can be added to the existing methods for laboratory assessment of
leprosy patients after starting MDT. To date, the morphological index
has been used for early assessment of MDT, but it has not been
reliable, mainly due to variation in the results. BI has thus been most
widely used for monitoring the effectiveness of MDT in leprosy
patients. However, at least 1 year is required to see any measurable
decrease in BI. Anti-PGL-I antibody levels also decreased
following MDT, but the reduction rate was measured at 50% after 2 years. PGL-I antigen detection thus seems to have an advantage over
morphological index, BI, and anti-PGL-I antibody assay for monitoring
the effects of chemotherapy of leprosy.
| |
ACKNOWLEDGMENTS |
This study was supported in part by grants from the Korean
Science and Engineering Foundation (951-0705-026-2), Seoul, Korea; the
UNDP/World Bank/WHO Special Programme for Research and Training in
Tropical Diseases (TDR); and the Leonard Wood Memorial (American Leprosy Foundation), Rockville, Md. The native PGL-I antigen was provided through funds from the National Institute of Allergy and
Infectious Diseases, National Institutes of Health, contract N01-AI-05074.
We thank the technical staffs at the Leonard Wood Memorial Center in
Cebu, Philippines, for their help in collection and processing of
clinical samples.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Yonsei University College of Medicine, 134 Shinchon-dong, Seoul 120-752, Republic of Korea. Phone: 822-361-5282. Fax:
82-2-313-9028. E-mail: raycho{at}yumc.yonsei.ac.kr.
| |
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Clinical and Diagnostic Laboratory Immunology, January 2001, p. 138-142, Vol. 8, No. 1
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.1.138-142.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
