Diagnostic Microbiology and Infectious Disease xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Diagnostic Microbiology and Infectious Disease

journal homepage: www.elsevier.com/locate/diagmicrobio

Detection of antibodies to both M. leprae PGL-I and MMP-II to recognize

leprosy patients at an early stage of disease progression
Hongsheng Wang a,1, Weijing Liu a,1, Yali Jin a, Meiwen Yu a, Haiqin Jiang a, Toshiki Tamura b,
Yumi Maeda b,, Masahiko Makino b
a
b

Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, 12 Jiangwangmiao Road, Nanjing, 210042, China
Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashimurayama, Tokyo, 189-0002, Japan

a r t i c l e

i n f o

Article history:
Received 13 April 2015
Received in revised form 15 July 2015
Accepted 18 July 2015
Available online xxxx
Keywords:
Leprosy
Phenolic glycolipid-I
Major membrane protein-II
Serology

a b s t r a c t
Antibodies to phenolic glycolipid (PGL)-I and major membrane protein (MMP)-II were evaluated for
serodiagnosis of leprosy in Southwest China, and the role in predicting the occurrence of the disease in household
contacts (HHCs) of leprosy was examined. Using PGL-I (natural disaccharide-octyl-bovine serum albumin)
antigenbased diagnosis (IgM antibodies), we could detect 94.9% of multibacillary (MB) leprosy and 38.9%
paucibacillary (PB) leprosy patients, whereas using MMP-II (IgG antibody), 88.1% of MB and 61.1% of PB patients
were positive. By combining the 2 tests and considering either test positive as positive, 100% of MB patients and
72.2% of PB patients were found to test positive. Of the HHCs of leprosy, 28.3% and 30% had positive levels of PGL-I
and MMP-II Abs, respectively. Seven out of 21 HHCs, who had high Ab titer to either antigen, developed leprosy
during the follow-up period of 3 years. These data suggest that the measurement of both anti-PGL-I as well as
anti-MMP-II antibodies could facilitate early detection of leprosy.
2015 Elsevier Inc. All rights reserved.

1. Introduction
Leprosy, caused by Mycobacterium leprae infection presenting progressive peripheral nerve injury and systematic deformity, remains an
important public health problem in many countries. During the year
2012, a global total of 232,857 new leprosy cases were detected, 6231
more cases than in 2011 (WHO, 2012). Although, China has already
achieved the elimination goal at the country level, in the recent
5 years, about 1200 new cases are detected every year. However, the
distribution of the occurrence of the disease is not uniform, so most
new cases of leprosy are concentrated in the Southwest China including
Yunnan, Guizhou, and Sichuan provinces, where it is often difcult to
reach due to the mountainous or hilly characteristics of the region.
With the introduction of multi-drug therapy (MDT), although leprosy
has been effectively controlled, signicant impairment of nerve function
and deformities and disabilities are found at greater frequency and severity in patients whose diagnosis was signicantly delayed; thus,
early diagnosis is recognized as key element in the prevention of the
spread of leprosy and minimizing the disabilities in patients. The diagnosis of leprosy is mainly based on clinical manifestations, microscopic
detection of acid-fast bacilli in skin biopsy, or slit-skin smear (WHO,
2009; WHO, 2010). However, these methods have low sensitivity because M. leprae bacilli cannot be detected easily, especially in
paucibacillary (PB) leprosy patients. Although PCR-based methods
Corresponding author. Tel.: +81-42-391-8211; fax: +81-42-394-9092.
E-mail address: yumi@niid.go.jp (Y. Maeda).
1
Contributed equally.

have been developed, it is not practical to perform PCR in resourcepoor settings. In addition, leprosy is usually associated with low socioeconomic situation, patients often have limited access to medical care,
and early diagnosis becomes difcult. Proper clinical diagnosis has become difcult due to the scarcity of patients in big cities where the
leprologists undergo training, resulting in the delay of diagnosis and
treatment (Chen et al., 2000; Deps et al., 2006; Lockwood and Reid,
2001). In such situations, if leprosy-specic serological methods become available, it would be a boon to health workers and leprologists
to detect the disease easily.
Since the identication of phenolic glycolipid-I (PGL-I) specic for
M. leprae in 1981 by Hunter and Brennan (Hunter and Brennan,
1981), PGL-I is the most widely used antigen for serodiagnosis of leprosy, with the reports that it is highly antigenic and capable of inducing
high antibody titers against the unique sugar epitopes of this molecule
(Spencer and Brennan, 2011). Antibody response to the PGL-I antigen
is mainly studied under IgM class. Natural disaccharide-octyl-bovine
serum albumin (NDO-BSA) is a second-generation product of native
PGL-I; the terminal disaccharide haptens were synthesized to allow
chemical coupling to bovine serum albumin; and the polyvalent structure had the advantage of multiple hapten substitutions (up to 40) on
each polypeptide backbone, and, being water soluble, was amenable
to the development of assays more facile than conventional enzymelinked assays (Chatterjee et al., 1986).
One of the proteins identied from the cell membrane fraction of
M. leprae as an antigenic molecule capable of activating both antigenpresenting cells and T cells is major membrane protein-II (MMP-II)
encoded by the ML2038c gene (Maeda et al., 2005). Sera from

http://dx.doi.org/10.1016/j.diagmicrobio.2015.07.012
0732-8893/ 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Wang H, et al, Detection of antibodies to both M. leprae PGL-I and MMP-II to recognize leprosy patients at an early stage
of disease progression, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.07.012

H. Wang et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxxxxx

leprosy patients were reported to show higher IgG titer to MMP-II than
healthy subjects regardless of the clinical type of leprosy (Deshpande
et al., 1995).
We have previously evaluated IgM levels against NDO-BSA in Chinese leprosy patients and found that enzyme-linked immunosorbent
assay (ELISA) based on NDO-BSA is useful for screening early infection
with M. leprae (Wu et al., 2002). Here, we evaluated the use of NDOBSA and MMP-II for serodiagnosis of leprosy in Southwest China and
compared the capability of these 2 tests in diagnosis and predicting
the onset of leprosy.
2. Materials and methods
2.1. Subjects and samples
Sera were obtained under informed consent from healthy individuals, leprosy patients, household contacts (HHCs) of leprosy patients,
and tuberculosis (TB) patients from southwest regions of China. Sera
were frozen at 30 C until use. All leprosy patient sera used in this
study were obtained from recently diagnosed, previously untreated individuals, so there was no bias in the system, and double-blind test for
ELISA was conducted. The population studied included multibacillary
(MB, n = 59) and PB (n = 18) leprosy patients. Classication of leprosy
was carried out according to RidleyJopling's classication (Ridley and
Jopling, 1966) and WHO recommendations (http://www.who.int/lep/
classication/en/index.html). Bacterial index, clinical manifestation,
sensation involvement, nerve hypertrophy, and pathological ndings
were mainly observed. Patients diagnosed with TB (n = 60) had
either acid-fast bacilli in sputum smears (bacilli were stained with
ZiehlNeelsen stain) and/or M. tuberculosis grown in culture media.
HHCs of leprosy patients (n = 60) were dened as people living
under the same roof and sharing food with the patient for at least
6 months. HHCs were followed up at 1-year interval for a period of
3 years. When the HHCs develop clinical signs of leprosy, they are treated with MDT. Sera from Bacillus Calmette-Guerin (BCG)-vaccinated
healthy volunteers and blood donors (n = 53) were also collected
from southwest regions of China and used as negative controls (NCs)
in ELISA tests to determine the cut-off value of the assay. This study
was approved by the institutional ethics review board of the Institute
of Dermatology, Chinese Academy of Medical Sciences, and Peking
Union Medical College, Nanjing, Jiangsu, China (KYZR2009-016). All patients involved in the study provided written informed consent.
2.2. Determining antibody responses by ELISA
The ELISA for the detection of anti-MMP-II IgG Abs was performed as
presented previously (Hatta et al., 2009; Kai et al., 2008; Maeda et al.,
2007). Briey described as follows, MMP-II gene was expressed in
Escherichia coli as a fusion construct using a pMAL-c2X expression
vector (New England BioLabs, Ipswich, MA, USA) according to the instructions. Fifty microliters of the puried antigen was coated onto the
96-well plates (Immunosorb; Nalgene Nunc, Rochester, NY, USA) overnight at a concentration of 4 g/mL in 0.1 mol/L carbonate buffer
(pH 9.5). After blocking with 2% skim milk in phosphate-buffered saline

containing 0.1% Tween 20 (PBST), the plates were washed with PBST,
and human sera (normal, patients, or HHCs) diluted 200 times in
blocking solution were added and incubated at room temperature for
2 hours. After washing with PBST, biotinylated anti-human IgG (Vector
Laboratories, Burlingame, CA, USA) in blocking solution was added at a
dilution of 1:1500 and incubated for 1 hour. The plates were incubated
with reagents from the ABC Kit (Vector Laboratories) in PBST for 30 min.
After washing with PBST, substrate solution consisting of 0.2 mg/mL of
OPD (o-phenylene diamine) and 0.02% H2O2 in 0.1 mol/L citrate buffer
(pH 5) was added, after which the reaction was stopped with 0.5 mol/L
sulfuric acid. Optical density (OD) was measured using a Vmax spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
Detection of anti-NDO-BSA Abs in sera by ELISA is described previously (Wu et al., 2002). Briey, 96-well plates (Immunosorb; Nunc)
were coated with NDO-BSA at a concentration of 100 ng/mL; then the
plates were placed at 37 C thermostat for more than 18 hours to dry.
The plates were soaked for 20 min with PBS before blocking. After
blocking with 5% skim milk in PBS (pH 7.2), the plates were washed
with PBS, and human sera diluted 100 times in 2.5% skim milk in PBS
were added and kept at 37 C for 2 hours. After washing with PBS,
biotinylated goat anti-human IgM (Vector Laboratories) in blocking
solution was added at a dilution of 1:1500 and incubated at 37 C for
1 hour. After washing with PBS, substrate solution consisting of
0.1 mg/mL of OPD was added as described for anti-MMP-II antibodies.
The OD of each well was read at 490 nm, for all assays.
2.3. Statistical analyses
For statistical analysis, MEDCALC software (MedCalc, Ostend,
Belgium) was used. A receiver operating characteristic (ROC) curve
was drawn to calculate the cut-off levels using the OD values of MB leprosy patients' sera and healthy controls. Pearson correlation coefcient
was also calculated by MEDCALC software. Student's t test was used to
calculate the P value. The P value of b0.05 was considered to be statistically signicant.
3. Results and discussion
3.1. Evaluation of NDO-BSA ELISA and MMP-II for serodiagnosis of leprosy
It is well documented that the detection of antibodies to M. leprae
antigens can be used for diagnosis of MB patients but that of PB patients
is limited (Oskam et al., 2003). To determine the serum antibody responses to both NDO-BSA and MMP-II antigens, we collected sera
from leprosy patients, TB patients, and HHCs of leprosy from Yunnan,
Guizhou, and Sichuan provinces of China. When anti-NDO-BSA IgM
Abs were measured, by taking the cut-off value of OD 0.087 as dened
using a ROC curve, 94.9% (n = 59) of MB patients were positive and
38.9% (n = 18) of PB patients had positive antibody levels. The sensitivity and specicity of the test were 94.9% and 86.8%, respectively, as calculated using ROC statistics. When the IgG antibody levels against
MMP-II antigen were examined with the same patients' sera, 88.1% of
MB patients and 61.1% of PB patients were determined to have positive
antibody titers. The cut-off value was 0.059 as determined using a ROC

Table 1
Evaluation of NDO-BSA and MMP-IIbased serodiagnosis in various groups of patients.
Group (n)

MB (59)
PB (18)
HHC (60)
TB (60)
NC (53)

NDO-BSA

MMP-II

NDO-BSA or MMP-II

Positive (%)

Positive (%)

Positive (%)

56 (94.9)
07 (38.9)
17 (28.3)
11 (18.3)
07 (13.2)

52 (88.1)
11 (61.1)
18 (30.0)
14 (23.3)
06 (11.3)

59 (100)
13 (72.2)
23 (38.3)
22 (36.9)
11 (20.7)

Correlation coefcient
(NDO-BSA versus MMP-II), R
0.196
0.523
0.279
0.117
0.282

P value NDO-BSA
versus NC

P value MMP-II
versus NC

b0.0001
0.039
0.323
0.514

b0.0001
0.005
0.041
0.167

Antibodies to NDO-BSA (IgM) and MMP-II (IgG) were evaluated by ELISA in the sera of MB and PB patients as well as HHCs of leprosy patients. Antibodies in the sera of TB patients were
also analyzed. As healthy controls, sera of noncontacts (NC) of leprosy were examined. Correlation coefcient (NDO-BSA versus MMP-II) and p values against healthy controls are shown

Please cite this article as: Wang H, et al, Detection of antibodies to both M. leprae PGL-I and MMP-II to recognize leprosy patients at an early stage
of disease progression, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.07.012

H. Wang et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxxxxx

curve, where the sensitivity of the test was 88.1% and the specicity was
88.7% (Table 1). NDO-BSA IgM and MMP-II IgG antibody levels were signicantly higher in MB patients than the healthy controls, HHCs, TB patients, and PB patients (P b 0.05 for NDO-BSA and MMP-II). Further, by
combining both tests and taking either test positive as positive, it was
found that 100% of MB patients were positive, and 72.2% of PB patients
were positive. It was also shown that 36.9% of TB patients and 20.7% of
healthy controls were positive. The data indicate that there are limitations to taking either positive as positive. Nevertheless, when seropositive individuals are detected, it is still necessary to search for other
clinical signs of leprosy. The dot plot was plotted by taking NDO-BSA
OD values of MB or PB patients on X-axis and that of MMP-II values on
Y-axis (Fig. 1D and E) to determine the correlation between the 2 antibody titers. Calculation of Pearson correlation coefcient of MB patients
indicated that the relationship between the variables was weak (r =
0.196); OD values of PB patients indicated that there is a moderate positive correlation (r = 0.523). HHC and TB patients also showed a weak
correlation between the variables (Table 1). One of the reasons for a
weak correlation between the variables in all groups may be due to the

detection of separate classes of antibody, IgM versus IgG, for glycolipid

and protein, respectively. When the P values in each group were calculated against the healthy controls (NC) by taking the OD values, the MB and
PB patients showed signicantly high results. HHC showed signicantly
low value (P = 0.32) for NDO-BSA detection, whereas those for MMPII detection showed signicance (P = 0.04) (Table 1).
Thus, serological data using both antigens NDO-BSA and MMP-II are
still not a sure test of leprosy detection, but it could be probably used to
conrm difcult cases such as pure neural leprosy and MB cases that do
not present the signs and symptoms of leprosy.
3.2. Prognostic value of NDO-BSA-ELISA and MMP-II-ELISA in
HHCs of leprosy
We evaluated the antibody levels of both NDO-BSA and MMP-II in 60
HHCs of leprosy. Seventeen HHCs (28.3%) were positive by NDO-BSA
ELISA, and 18 (30.0%) HHCs were positive for MMP-II ELISA (Table 1).
Among these positive HHCs, 13 (21.7%) individuals were positive for
both antigens. We could follow-up only 21 of HHCs because, in these

Fig. 1. Distribution of anti-MMP-II and anti-NDO-BSA antibody levels (OD values). Antibody response of leprosy patients and HHCs of leprosy patients in southwest provinces of China.
Sera from healthy individuals, having no history of contact with leprosy patients (NC), HHC, MB, and PB leprosy patients, were assessed by ELISA for antibodies to NDO-BSA (A) and
MMP-II (B). OD of each sample is plotted by individual markers. The thick horizontal line indicates the average OD values in each group. Errors bars are shown (95% condence interval
for mean). (CE) OD values of NDO-BSA are plotted on X-axis, and that of MMP-II is plotted on Y-axis. Sera from HHCs (C), sera from PB leprosy (D), and sera from MB leprosy (E) are taken
for the evaluation of the antibodies. Thick lines show cut-off values of each analysis (NDO-BSA, 0.087; MMP-II, 0.059).

Please cite this article as: Wang H, et al, Detection of antibodies to both M. leprae PGL-I and MMP-II to recognize leprosy patients at an early stage
of disease progression, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.07.012

H. Wang et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxxxxx

Table 2
Measurement of antibodies to NDO-BSA and MMP-II in household contacts of leprosya
follow up study.

MMP-II (+)
MMP-II (+)
MMP-II ()
MMP-II ()

NDO-BSA (+)
NDO-BSA ()
NDO-BSA (+)
NDO-BSA ()

Positive (no.)

Developed leprosy (no.)

13
5
4
38

5
1
1
1

rural areas, it is rather difcult to track them at the same place due to
their unstable living incomes and jobs. Out of 21 HHCs, 8 (38.1%) developed leprosy during the 3-year follow-up period. This value was high
compared to that previously observed in the HHCs of Indonesian leprosy patients (22%) (Hatta et al., 2009). Among the 8 HHCs who developed
leprosy, 5 had positive antibody titers to both NDO-BSA and MMP-II,
and the 2 were positive to one of the antigens and 1 was negative for
both antigens (Table 2). Notably, 38.5% (5 out of 13 HHCs) of those
HHCs who were positive to both serological tests developed leprosy
during follow-up period. One of the HHCs who developed leprosy was
negative for both antigens at the beginning of the survey. However, on
the second visit, the antibody titers were high for both antigens and
also manifested clinical signs of leprosy. After drug treatment, the antibody titer showed negative values. So, HHCs who have high antibody
titer should be carefully monitored for any clinical signs of leprosy in
order to promptly initiate chemotherapy. It should also be noted that
6 of the HHCs who developed leprosy were contacts of PB leprosy patients, and therefore, a close examination of the HHCs of not only MB
but also PB leprosy patients is necessary. Overall, the data are indicative
of the prognostic value of serology with NDO-BSA and MMP-II.
Early detection of infection by M. leprae, followed by effective treatment, is critical to reduce disease progression. Sensitive and specic
tools for early detection of infection are critical for an effective leprosy
elimination campaign. However, the correct diagnosis of leprosy is
often delayed because the recognition of clinical signs and symptoms
is the only way the disease can be identied. Nowadays, few clinicians
are able to condently identify these clinical symptoms, and simple
tests to facilitate leprosy experts to identify patients are not widely available. To nd new patients, clinical examination aided by simple serological
tests would be helpful. One of the earlier studies showed that a combinatorial
approach using anti-PGL-I and anti-45 kDa antibodies for detection of
M. leprae infection seems to provide a method with higher specicity
(Parkash et al., 2006). However, limitations with serology still exist.
Here, we evaluated the performance of 2 leprosy serological tests
(detection of antibodies to NDO-BSA and MMP-II). As expected, the 2
tests readily detected clinically conrmed patients with MB leprosy.
Fig. 1 shows the distribution of the antibody titers in the patients and
HHCs of leprosy. Anti-MMP-II antibody titers in MB patients showed a
fairly diverse distribution, whereas anti-NDO-BSA showed more compact distribution, whether this difference in the distribution of titers is
due to difference in the class of antibody (either IgG or IgM) is unknown. It is interesting to note that a distinct group of anti-MMP-II
antibodypositive individuals could be distinguished from the
antibody-negative group in the HHC group.

In summary, the combination of the NDO-BSA and MMP-II test appears to have utility for detection and monitoring of leprosy patients
and HHCs of leprosy. We believe that it could enhance surveillance
and facilitate referrals and would be an important tool in trials of new
interventions and treatments.
Acknowledgments
The authors would like to thank the healthy volunteers for their cooperation and the eld and laboratory staff for their kind assistance in
collecting the sera. This work was supported by the National Natural Science Foundation of China (81371751), the fund for Natural Science Foundation of Jiangsu Province (2014020044) and 'Research Program on
Emerging and Re-emerging Infectious Diseases' from Japan Agency for
Medical Research and development, AMED in part by a US-Japan Cooperative Medical Science Program.
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Please cite this article as: Wang H, et al, Detection of antibodies to both M. leprae PGL-I and MMP-II to recognize leprosy patients at an early stage
of disease progression, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.07.012