Serological Antibody Profile IgM and IgG of Mycobacterium leprae

PGL-1 and L-ESAT-6 in Patients and Household Contact from

Leprosy Endemic Area in East Java Indonesia

A. Dinar Adriaty

, M. Irfan Hadi

1,3

, Irmadita Citrashanty

, Iswahyudi

, Indropo Agusni

1,2

, Shinzo

Izumi

, Cita Rosita S. P.

Leprosy Study Group-Institute of Tropical Disease, Universitas Airlangga

Kampus C, Mulyorejo Surabaya 60115 East Java Indonesia

Dermatology and Venereology, Faculty of Medicine Universitas Airlangga, Surabaya 60286 Indonesia

Faculty of Psychology and Health Science, Islamic State University of Sunan Ampel, Surabaya 60237 Indonesia

Keywords: L-ESAT-6, PGL-1, Mycobacterium leprae, leprosy, patients, household contact

Abstract: Leprosy patients in Indonesia is the third largest in the world. The problem of high transmission is the

difficulty of early detection of leprosy. At present, diagnosis leprosy still based on clinical sign. Some of

supporting diagnostic tools are developed, such as serological test. The most widely used antigen for

diagnostics is Phenolic-glycolipid-1(PGL-1), but some limitations of the antigen, provide a challenge to find

a potential candidate antigen representing specific Mycobacterium leprae. Purpose of this research is for

studying Mycobacterium leprae L-ESAT-6 (epitope AA11-36) compare to PGL-1 to leprosy patients and

household contacts in leprosy endemic region. Analysis have been conducted to MB and PB leprosy patients,

as well as their families with total 173 respondents by testing Indirect ELISA of the L-ESAT-6 and PGL-1. In

general, the profile of ELISA test anti PGL-1 vs L-ESAT-6 in all the patients don’t have significant difference,

but in household contacts, with the Pearson correlation test, it can be concluded that there are significant

difference between PGL-1 and L-ESAT-6 (p-value = 0.049; p <α = 0.05). Healthy individuals who are exposed

to M.leprae found high titers of antibody anti L-ESAT-6 and lower profile of antibody anti PGL-1. It is

assumed that the individual is relatively immune to M.leprae. So it can be concluded that L-ESAT-6 (AA11-

36) can be used as candidate diagnostic test which is a predictor marker for people living in endemic leprosy

areas, for it still need a further research.

1 INTRODUCTION

Leprosy in Indonesia still become a health problem

with the discovery of new cases is still increasing

from year to year (WHO, 2016). The most important

cause of the high incidence rate is the difficulty of

early detection which play an important role in the

transmission process so that subclinical leprosy stage

continues to be manifest (Agusni, 2001). Diagnostic

tools to detect leprosy is still limited, based on clinical

sign. Laboratory testing based on a serological by the

method of ELISA (Eenzyme Linked Immunosorbent

Assay) and MLPA method (Mycobacterium leprae

particle agglutination) is used mostly using PGL-1

(Phenolic-glycolipid-1), but the lack of this antigen,

provide a challenge for finding a potential biomarker

used for early detection of leprosy (Spencer et al,

2012). Inspired from the successful use of

Mycobacterium tuberculosis ESAT-6 (T-ESAT-6)

for the detection of M. tuberculosis associated

specific responses (Parkash et al, 2007), we have

previously found that the use of ESAT-6 of M. leprae

was limited. Mycobacterium leprae ESAT-6 (L-

ESAT-6) is a protein secreted by the extracellular

M.leprae molecular weight 6kDa and an antigen

peptide representing specific epitopes of M. leprae

potent. L-ESAT-6 encoded by the genes of

pathogenic RD1 consists of 95 amino acids that has

multiple epitopes of which are located on the N-

Terminus (Geluk et al, 2002). In previous studies

conducted by Kurdi (Kundi et al, 2010) on epitope

tracking with B cell epitope scanning techniques, has

proven that serum leprosy patients type MB, PB and

subclinical leprosy reactive against L-ESAT-6 (AA

Adriaty, A., Hadi, M., Citrashanty, I., , I., Agusni, I., Izumi, S. and S. P., C.

Serological Antibody Proﬁle IgM and IgG of Mycobacterium leprae PGL-1 and L-ESAT-6 in Patients and Household Contact from Leprosy Endemic Area in East Java Indonesia.

DOI: 10.5220/0008156703230327

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 323-327

ISBN: 978-989-758-494-7

323

11-36), while in healthy people in endemic areas no

such reactivity. Those studies also have found three

types of epitopes of the N-Terminus region of the L-

ESAT -6 as follows: epitope markers leprosy of type

LL and BB leprosy (MB) is LEQCQES (28-34),

epitope VNELQG (14-19) which is an epitope

markers of type TT leprosy (PB), epitope IDALLE

(24 -29) are only reactive with antibodies contained

in the sera of healthy household contact (HHC) group.

What is interesting in this case, that the three epitopes

found is not in the primary structure of the T-ESAT-

6 (Mycobacterium tuberculosis) so this epitopes are

highly specific to M.leprae (Spencer et al, 2002).

M.leprae is uncultivable mycobacteria, production of

synthetic proteins was conducted on L-ESAT-6

(AA11-36). The purpose of this study was to analyzed

the profile of specific antibodies reaction of L-ESAT-

6 (AA11-36) compare to PGL-1 against leprosy

patients and household contact. The development of

tools is important to facilitate diagnosis and provide

a more thorough understanding of transmission and

the incidence of M.leprae infection in high endemic

regions, even throughout the country.

2 METHODS

This study was approved by national ministry of

health and local ethic commission from Dr.Sutomo

Hospital Surabaya and paticipants were included only

after signing written informed consent forms. Patients

groups has the following inclusion criteria: newly

diagnosed and previously untreated or recently

diagnosed and within the first 3 months treatment

with WHO-MDT. Household contacts of both MB

and PB leprosy patients were recruited as a group at

elevated risk of subclinical leprosy. Samples

consisted of 3 groups, leprosy patients Multibacillary

(MB) type, Paucibacillary (PB) type and household

contact (HHC), each of the groups were 42, 36 and 95

respondents respectively and they were taken from a

district in Lamongan, one of the endemic areas in East

Java Indonesia. The number of samples is calculated

based on the formula stratified random sampling.

2.1 ELISA (Enzyme Linked

Immunosorbent Assay

A total of 3 mL of blood serum isolated to then

proceed with the analysis of indirect ELISA. The

antigen is a synthetic antigen : PGL-1 (NT-P-BSA)

and synthetic L-ESAT-6 (epitop AA 11-36) include 3

epitope markers for MB, PB and HHC, consists of

LEQCQES (28-34), VNELQG (14-19), IDALLE

(24-29)-N-terminus labeled with Biotin. Antigen

diluted 1 mg / ml with carbonate buffer, all

components coated into 96-wells microtiter plates

(Nunc, Maxisorp) for L-ESAT-6, microplate coated

with streptavidin. Blocking buffer consisting of 1%

skimmed milk / PBS and serum total diluted 1/300 in

0.1% skimmed milk / PBS / Tween-20. Samples were

analyzed in duplicate and incubated for 1 hour at

room temperature. The wells were washed with PBS-

Tween20, and then incubated with horseradish

peroxidase (- HRP-) conjugated antibodies (Dako,

Denmark) and then diluted to 0.1% skimmed

milk/PBS/Tween-20.

After washing, the plates stained with substrate

ortho-phenilen-diamine (OPD) and peroxidase 30%

(MERCK) in phosphate-citrate buffer and incubated

until developed a yellow color and stopped with

1.25M H

. Both antibody IgM anti-NT-P-BSA

and antibody IgG anti-L-ESAT-6 were measured.

The antibody titer was measured by optical density

(OD) of all the wells that have been read at a

wavelength of 450 nm and a reference wavelength at

492nm. Real OD obtained from reduction of OD in

both wavelengths (delta-OD) and converted

automatically by the BIOLISE software to unit/mL.

2.2 Evaluation of ELISA Test Results

Interpretation of the ELISA test result is to see the

yellow signal above background values, it is

recommended to use the plot algorithm. The results

of diagnostic tests in the form of quantitative data and

for the statistical analysis used Pearson correlation

test by SPSS (Statistical Package for the Social

Sciences version 16.0).

3 RESULTS

3.1 Distribution of Samples

There were totally 173 inhabitants who participated

in this study, consisting of 69 respondents (39.88%)

were male and 104 (60.12%) were female. In terms of

age, respondents are divided into 3 groups: children

(0-21 years), adults (22-45 years) and elderly (more

than 45 years) and the adults group is the most

frequent 56.7% (98/173).

RCD 2018 - The 23rd Regional Conference of Dermatology 2018

324

Table 1. Distribution of the respondents

Table 2. Antibody reaction of IgM anti PGL-1and IgG anti L-ESAT-6

AntibodyTiter

(unit/mL)

MB patients

PB patients

Household Contacts

(HHC)

Mean ± SD (range) Mean ± SD (range) Mean ± SD (range)

M anti PGL-1

32136.07±184706.60 (43-

126870

)

1508.36±1955.23 (219-

8240

)

774.93±752.79 (31-

4332

)

IgG anti L-

ESAT-6

990.28±1232.37

(

48-6300

)

575.5±349.38 (234-

1650

)

404.21±268.43 (108-

1512

)

3.2 Antibody-specific Responses in

PGL-1 and L-ESAT-6

Pearson correlation test, shows the levels of IgM anti

PGL-1 with IgG anti L-ESAT-6 epitope LEQCQES,

epitope marker for MB patients showed the value of

p = 0.598, p > 0.05. It concluded that there was no

correlation between them. Pearson correlation test

shows the levels of IgM anti PGL-1 with IgG anti L-

ESAT-6-epitope VNELQG, specific epitope for PB

patients is obtained p = 0.962, p> 0.05. It concluded

that there was no correlation between the levels of

IgM anti PGL-1.

The levels of IgM anti PGL-1 and IgG ESAT-6

epitope IDALLE, specific epitope for healthy

household contacts showed the value of p = 0.049

(p<0.05). It can be concluded that there is a

significant correlation between the levels of IgM anti

PGL-1 with IgG anti L-ESAT-6- epitope IDALLE.

The strength of the correlation is 0.584 it can be

seen from the Fig.2 that there is a pattern of negative

correlation between the levels of IgM anti PGL-1

with IgG antiL-ESAT-6-IDALLE.

4 DISCUSSION

The most extensively evaluated serologic test for

leprosy is still based on the detection of IgM anti-

PGL-1, because highly titer of anti-PGL-1 serology

reflects the bacillary load and has limited application

for diagnosis of all clinical forms of leprosy (8,9,10),

but according to the profile of IgM anti-PGL-1

compare to IgG ESAT-6, it seems this antigen still

recomended as an important adjunct tool for the

differentiation of PB and MB leprosy. For IgG anti L-

ESAT-6 profile, similar to anti-PGL-1 serology, the

presence of IgG antibodies that react to protein

antigens reflect to the bacillary load as well. Most MB

patients have high IgG titres but few PB patients are

responsive. The serological profile antibody anti- L-

ESAT-6 with specific epitope of MB and

PB patients

has not been able to support the lack of PGL-1.

Interesting result shows at the comparison

between IgM anti-PGL-1 and IgG anti-L-ESAT-6

with epitope IDALLE specific for healthy household

contacts. It has a negative significant correlation with

PGL-1 so that it can be said that healthy individuals

exposed to M.leprae if antibodies are found with high

Serological Antibody Proﬁle IgM and IgG of Mycobacterium leprae PGL-1 and L-ESAT-6 in Patients and Household Contact from Leprosy

Endemic Area in East Java Indonesia

325

titers of L-ESAT-6 epitope IDALLE and could

suppressed the development of bacterial load in the

body which indicated from the titer of IgM anti-PGL-

1, then it is certain that the individual is relatively

immune against M.leprae. This correlation means

that household contacts with the greater risk of

becoming leprosy has the greater protective antibody

against leprosy.

5 CONCLUSION

In the future it might said that antigen L-ESAT-6 can

be a candidate for predictor marker in order to

develop an alternative tools for detecting leprosy in

the early stage. For these purpose still needed further

research. The result of IgG anti-L-ESAT-6 in leprosy

patients might have a different profile if possible

tested in L-ESAT-6 epitope IDALLE that specific for

healthy household contact. This can be also as a

suggestion for further research.

Figure 1. Antibody responses of leprosy patients and household contacts. Multibacillary (MB; n=42), Paucibacillary (PB;

n=36), healthy household contacts of confirmed patients (HHC; n=95) were assesed. Results from each individual serum

sample against the mean titer in units/mL both from antibodies anti PGL-1 (IgM) (A) and anti ESAT-6 (IgG) (B)

Figure 2. Antibody responses of IgM anti-PGL-1 and IgG anti- Mycobacterium leprae protein antigen ESAT-6 epitope

IDALLE from household contacts (HHC)

ACKNOWLEDGEMENT

The authors are extremely grateful to all the

volunteers for the cooperation and to all the field and

laboratory staff who made this work possible for their

assistance. Also for the leprosy patients in Puskesmas

Brondong, Lamongan, East Java Province and all

their family who willingly supported in this research.

This work was funded by a grant RISBIN

IPTEKDOK from National Ministry of Health.

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Serological Antibody Proﬁle IgM and IgG of Mycobacterium leprae PGL-1 and L-ESAT-6 in Patients and Household Contact from Leprosy

Endemic Area in East Java Indonesia

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