Profile of IgM Anti PGL-1 and IgM Anti LID1-NDO on Leprosy

Patients of Dr. Soetomo General Hospital, Surabaya

Iswahyudi

, D. Adriaty

, Prita P. K.

, I. Agusni

1,2

, S. Izumi

, C. R. S. Prakoeswa

1,2

Leprosy Study Group, Institute of Tropical Disease Universitas Airlangga Surabaya,

Dermatology and Venereology, Medical Faculty Universitas Airlangga, Surabaya,Indonesia

Keywords: leprosy, PGL-1, LID1-NDO

Abstract: Serological tests have been used to assist leprosy disease management. The latest serological test towards anti

LID1-NDO ((Leprosy IDRI Diagnostic 1- Natural Disaccharide Octyl) challenges the leprosy antigen already

in use, PGL-1, to provide a better, more sensitive leprosy serological test. This research is to study the profile

of IgM anti PGL-1 compared to IgM anti LID1-NDO in leprosy patients from Surabaya. Sera sample 91

leprosy patients from Dr. Soetomo Hospital, Surabaya (PB : 30, MB: 61) and tested against IgM anti PGL-1

and IgM anti LID1-NDO using the ELISA technique. The result of anti PGL-1 is stated in u/mL and in optical

density (OD) unit for IgM anti LID1-NDO. The average IgM anti PGL-1 titer in PB patients are 1577.1 u/ml,

whereas for anti LID1-NDO antibody the average OD is 0.166. In MB patients IgM anti PGL-1 has the

average of 3629.2 u/ml. The absorbance of IgM anti LID1-NDO was 0.228 on average. The majority of MB

patients presented with positive anti LID1-NDO and anti PGL-I responses (77.05 % and 68.85 %). For PB

patients presented with positive anti LID1-NDO and anti PGL-I responses (70% and 60%). strong correlation

was found (P=0.003) between IgM anti LID1-NDO and anti PGL-1. PGL-1 originates from the

polysaccharide of M. leprae, and LID1-NDO fuses this polysaccharide with another set of protein LID1-

NDO is potential to be used in serological tests for leprosy diagnosis and monitoring, as it performs to a

similar manner to PGL-1.

1 INTRODUCTION

Leprosy is a chronic infectious disease caused

Mycobacterium leprae. Although the disease affects

the skin and peripheral nerves, it can present with a

wide array of pathologies and clinical manifest to

cause leprosy present as a bacteriologic, clinical,

immunologic, and pathological spectrum ranging

from the extremes observed in paucibacillary (PB)

and multibacillary (MB) patients depending upon the

patient’s immune response. (Bührer-Sékula et al.,

2008; Spencer and Brennan, 2011; Amorim et al.,

2016) The diagnosis of leprosy is not simple and, not

surprisingly, many professionals have neither the

experience to recognize the various signs and

symptoms of the disease nor the ability to

differentiate them from other diseases. (Duthie et al.,

2014, 2007; da Conceição Oliveira Coelho Fabri et

al., 2015) Thus, leprosy patients often receive

incorrect diagnoses and appropriate treatment is

delayed. Antibody responses to specific M. leprae

antigens can be evaluated by several tests. (Duthie et

al., 2016) The detection of IgM antibodies against

phenolic glycolipid I (PGL-I) represents the most

evaluated serologic assay for leprosy, with levels

correlating with bacillary loads such that levels rise

across the TT to LL spectrum. Among these are

serologic tests that measure the levels of

immunoglobulin M (IgM) against phenolic

glycolipid-1 (PGL-1) the synthetic mimetope natural

disaccharide octyl – leprosy IDRI diagnostic 1 (LID1-

NDO). The aim of this study was to evaluate the

antibody responses against phenolic glycolipid-1

(PGL-1), Leprosy IDRI Diagnostic 1 Natural

Disaccharide Octyl (LID1- NDO) in leprosy patients

(Hunter et al., 1982)

2 METHODS

Blood (3cc) from 91 leprosy patients from Dr.

Soetomo General Hospital, Surabaya (PB : 30, MB:

61) were collected from the cubituous vena of each

patient, to be centrifuged and have the serum tested

320

Iswahyudi, ., Adriaty, D., K., P., Agusni, I., Izumi, S. and Prakoeswa, C.

Proﬁle of IgM Anti PGL-1 and IgM Anti LID1-NDO on Leprosy Patients of Dr. Soetomo General Hospital, Surabaya.

DOI: 10.5220/0008156603200322

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 320-322

ISBN: 978-989-758-494-7

against IgM anti PGL-1 and IgM anti LID1-NDO

using the Enzyme Linked Immunosorbent Assay

(ELISA) technique. Briefly, polysorp 96-well plates

(Nunc Maxisorp) were coated with 0.01 μg/mL

natural trisaccharide-phenyl conjugated to bovine

serum albumin (NT-P-BSA), the trisaccharide NTP-

BSA ride synthetic analog of PGL-I kindly provided

by Dr Fujiwara, Nara University, Japan, incubation

overnight at 4ºC and blocked with phosphate-

buffered saline tween (PBST)-2% skim

milk.Incubation for 1 h at 37ºC. Serum samples

diluted 1/300 in PBS 1% skim milk were added to

duplicate wells of either NT-P-BSA coated plates.

After and washing with PBS-Tween, horseradish

peroxidase-conjugated to anti- human IgM (Wako,

USA) was then added. After incubation for 1 h at

37ºC and further washes, Antibodies against PGL- 1

were detected by indirect ELISA with peroxidase-

conjugated anti-human IgM with 0-phenylene

diamine as substrate (Sigma) was added for each well.

The colour reactions of the entire plate were stopped

with 2.5 N-H

2SO4. The optical density (OD) was read

at 492 nm /620 nm using microplate reader Texan

Infinite f50. For final OD value PGL-1 (NTP-BSA)

of each serum sample was calculated by Biolise

program software (Titer). The Cut-off was defined as

Titer > 605 u/ml.

Serum IgM antibodies to the di-fusion protein

LID1-NDO were detected by serology ELISA. LID1-

NDO provided by Dr Malcom S Duthi, from

Infectious Disease Research Institute (IDRI) Seatle ,

USA. Polysorp 96-well plates (Nunc Maxisorp) were

coated with 2 μg/mL of LID1-NDO at 4ºC overnight

and blocked with PBST with 2% skim milk for 1 h at

37 ºC. Serum samples diluted 1/300 in 1% PBS-skim

milk were added in duplicates and incubated for 1h at

37 C. Plates were washed and incubated with added

50 μL of peroxidase-conjugated with anti-human IgM

(Sigma) diluted to 1/6,000 in PBS, 0.1% skim milk.

After washings, reactions were developed with anti-

human IgM with 0-phenylene diamine as substrate

(Sigma) was added for each well. The colour

reactions of the entire plate were stopped with 2.5 N-

2SO4. The optical density (OD) was read at 492 nm

/620 nm using microplate reader. The corrected OD

of each well at 492 nm / 620 nm was read using a

microplate reader. The results were expressed as

mean absorbance of the duplicates. The final OD

value LID1-NDO of each serum sample was

calculated by subtracting the OD value. The cut-off

was defined as OD > 0.053.

3 RESULTS

The study group was composed by 91 leprosy patients

(MB : 61, PB:30) with ages ranging from 18-64 years

(median = 41 years) of whom the majority was male

(Table). The IgM anti PGL-I ELISA positive rate

among the MB leprosy patients tested was determined

to be 68,85 % (42/61), the titers average is 3629,2

u/ml. Among these same MB leprosy patients, the

seropositive rate in IgM anti LID1- NDO test was

found to be slightly higher at 77,05% (47/61), the

average is OD 0.228. Cluster aread on the scatter

diagram for MB patients are OD 0.5 (anti LID1-

NDO) and 1500u/ml (anti PGL-1).

The IgM anti PGL-I ELISA positive rate among

the PB leprosy patients tested was determined to be

60 % (18/30), the titers average of 1577,1 u/ml. The

seropositive rate in IgM anti LID1- NDO test was

found to be slightly higher at 70% (21/30), the

average OD is 0.166. For PB patients, cluster area are

OD=0.25 and titer 350 u/mL for their respective

antigens. Statistical analysis a strong correlation

(p=003) between IgM anti LID1-NDO and IgM anti

PGL-1.

Table 1: Proportion of Seropositivity for antibody against

LID1-NDO and PGL-1 according to Group

Leprosy Type

No of sample

Gender

(M/F)

No (%) positive

LID1-NDO

PGL-1

46/21

20/10

(77.05)

(70.00)

(68.85)

(60.00)

MB : Multibacillary, PB: Pausibacillary, M : Male, F :

Female

4 DISCUSSION

Leprosy serology has been studied frequently and

many of the factors determining seropositivity are

well known, as has been reviewed by Oskam et al. We

examined the presence of antibodies against

particular M. leprae antigens among MB and PB

groups, Patients were classified based on the WHO

classification system,

all were undergoing treatment

for leprosy

treatment MDT-WHO. PGL-1 originates

from the polysaccharide of M. leprae, and LID1-NDO

fuses this polysaccharide with another set of protein,

ML0405 and ML2331.The majority of MB patients

Proﬁle of IgM Anti PGL-1 and IgM Anti LID1-NDO on Leprosy Patients of Dr. Soetomo General Hospital, Surabaya

321

presented with positive anti LID1-NDO and anti

PGL-I responses (77.05 % and 68.85 %,

respectively). For PB patients presented with positive

anti LID1-NDO and anti PGL-I responses (70% and

60%). In comparative analyses of anti LID1-NDO

and anti PGL-I responses in the statistically strong

correlation. Although serological tests appear to have

a limited ability to aid the diagnosis of PB patients,

our data also identified that a greater number of PB

patients were seropositive for antibodies against

PGL-1 and LID1-NDO. Our data indicate that tests

detecting antibodies to PGL-I and/or LID1-NDO

represent effective tools for the detection of MB and

PB patients.

5 CONCLUSIONS

LID1-NDO is potential to be used in serological tests

for leprosy diagnosis and monitoring, as it performs

to a similar manner to PGL-1.

ACKNOWLEDGEMENTS

To Dr Tsuyoshi Fujiwara, Nara University, Japan, for

generously providing the NT-P-BSA antigen. We

also thank American Leprosy Mission who provided

funding to the Infectious Disease Research Institute

(IDRI).

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