Early Diagnosis of leprosy bas

ed on Multiple Assays

Beijing Tropical Medicine Research Institute

Email: weny8@163.com

2013-9-16

Introduction Early diagnosis of Leprosy is a big challenge in endemic countries. At the pres

ent, no single assay can predict the persons who have infected M. Leprae (indicated by PGL-1 positive in sera) may development leprosy or what time they may development leprosy.

KaiYuan city, in Honghe Profecture, Yunnan Province has been a higher lepro

sy-endemic area in China. During 1996-2006, the twenty cases were newly detected in the 618 villagers from the three villages, Kaiyuan. Moreover, 2002-2006,the average detective rate is as high as 6.5/100000 in the city. Therefore, the three villages were selected as the field site for early diagnosis.

The multiple assays are consist of ELISA, PCR and T cell assays, as well as Acid-Fast staining, histopathology. Those assays have been applied to the following up or monitoring in the villagers and household contacts since 2007. The aim of the study is to demonstrate whether the assays may be useful for early detection of leprosy.

The surveillance program in the villagers and HHCs

New and treated Patients, HHCs and villagers in 3 endemic villages

were surveyed by clinical examination and ELISA (ND-O-BSA IgM

and LID-1 IgG).

If suspected cases are found, the PCR would be used for diagnosis

The three times or more following –up have been conducted since

2007.

Assays performed in the study Serologic tests:

ELISA on PGL-1 (Providing by Prof. P. J. Brennan ) and LID-1( Providing by Dr. M. S. Duthie ).

Nested-PCR and Real time PCR:

Amplify RLEP fragment from skin biopsy, paraffin tissue and whole blood.

T cell assay :

IFN-γ by Whole blood and PBMC.

Routine examinations: AF staining and Histopathology

FIG. 1: Amplification of M. Ieprae DNA from patient blood using nested-PCR.First round: Lane1 to 3 MB (+) ; 4 , 5 PB ( - ) ; 2nd round : Lane(4) (5) PB ( + ) .

372bp—

—200bp

1 2 3 4 5 + — (4) (5) + — M

FIG. 2: Serial ten-fold dilutions of M. leprae in negative blood

Nested PCR : It was successfully amplified from one bacterium (from Dr. Thomas P. Gillis)

—400bp372bp—

109 108 107 106 104105 103 102 10 1 — M

—400bp372bp— —400bp372bp—

109 108 107 106 104105 103 102 10 1 — M

AB C

DE

F

G

Amplification Plot ： A: 107 copies /μlplasmid ； B: 106 copies /μlplasmid ； C: 105 copies /μlplasmid ； D: 104 copies /μlplasmid ； E: 103 copies /μlplasmid ； F: 102 copies /μlplasmid G: 10 copies /μlplasmid 。

TaqMan real time PCR

Select unique proteins from M. leprae database based on comparative genomic analyses

Synthesize peptides to be used in vitro PBMC/whole blood stim

ulation assays

21 peptides chosen from 14 different M. lepraeproteins were synthesized for in vitro testing

Synthesize peptides

Synthetic peptides: the stimulate antigens of the 21 polypeptides

Code Peptide location Code Peptide location

P1 ML0044 P12 ML0497-1

P2 ML0049-1 P13 ML0497-2

P3 ML0049-2 P14 ML0574

P4 ML0049-3 P15 ML1001

P5 ML0050 P16 ML1989

P6 ML0073-1 P17 ML1990

P7 ML0073-2 P18 ML2283

P8 ML0410-1 P19 ML2346-1

P9 ML0410-2 P20 ML2346-2

P10 ML0411-1 P21 ML2567

P11 ML0411-2

Newly diagnosed case

during the following up in Kaiyuan City

A girl at age 12, HHC. Her chest, back with multiple size differin lesions of the ring, touch drops were found when the third following-up. Three consecutive ELISA antibody was rising in the over one year. pathological examination, AFB 3+ and S-100 staining showed the nerve infiltration and no granuloma can beseen in HE staining.

time

ELISA results

ND-0-BSA LID-1

2008.06 0.338 0.298

2009.02 0.381 0.746

2009.10 0.948 1.406

Back lesions S-100 dyeing(X 400)

A-F staining （ X 1000 ）

Case 1 , early BL

H&E X 200

邓早期病例的PCR产物测序结果

Case 2 and Case 3, who is villager and his lesion looks like as the case 2. However, diagnosed as TT by histopathology.Both of cases AF (-), but PCR +, DNA sequence are totally matched with RLEP fragment on M. leprae.

M

552

558徐 +- 邓

RLEP-372bp

M552

558徐 +邓

Nested-PCR

RLEP-131bp

MM-有

-无

M:DNA 标准-：阴性对照邓：早期病例（皮损标本)徐：可疑病例(组织液）+：阳性对照

100bp

400bp

100bp

200bp

巢式-PCR方法扩增16S rRNA麻风菌特异片段

M:DNA 标准-：阴性对照，有：表示用第一轮PCR阴性产物作为模板；无：为水对照

邓：早期病例徐：可疑病例+：阳性对照

Case 4(out-patient in Beijing)

A 40 year-old woman, the 5 lesions on leg, foot and buttocks. Not lost of sense Pathological examination:, and epithelioid cells infiltration around blood vascular ,adnexal

and granuloma . The AF (-). Suspecting as sarcoidosis but PCR (+) . Now the lesions are significantly regressed after a half year of MDT. The patient was diagnosed with BT.

Evaluation on T cell assay based on the peptides

Case 5 A 30 year-old woman,in endemic area, the lesion on ankle, lost of sense,but not easy to get biopsy.T cell assay based on the 21 peptides for IFN- γ secretion >50 pg/ml was 15/21 peptides,MDT for 6 months.

Before MDT After MDT

Pepti des P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11I FN-γpg/ml（ ）

90.6 58 20.4 139.6 89.2 0 54.8 72 141.7 79.6 62.6

Pepti des P12 P13 P14 P15 P16 P17 P18 P19 P20 P21I FN-γpg/ml（ ）

179.2 52.5 0 289 350.4 0 15.7 71.1 110.7 34.5

Preliminary summary on T cell assay based on the 21 peptides

Although we succeeded in the diagnosis one case and no cross rea

ction with TB and normal groups. However, the results 0f all 21 pepti

des for both PBMC or whole blood assay on MB:24, PB:6 and HH

C:17 showed no peptide may induce IFN-γ in significant difference

s between the petients and control. In addition, the experimental re

peatability is not good.

The design or selection of the peptides and proteins require to furth

er study.

Evaluation on T cell assay based on the proteins

The objects ： MB ： 7 ； PB ： 3 ； HHC ： 5 ； TB ： 11 ； NC ( endemic area normal) ： 10.

Antigens ： Proteins(from Dr.Duthie) ： LID-1 ； ML89 ； ML2044 ； 229-88 ； ML2055 ； 229-112 ； ML2028 ；

Peptides(the synthesis by us) ： P-ML2044 ； P-ML0405.

Methods ： Follow the assay by I-AS-006 IDRI, Seattle, Washington

98104, Subject No. I-AS-006

To compare IFN- γ production between 37℃ and 37℃ + CO2 incubation with nine antigens on whole blood from the 3 individuals.

To compare IFN-γ production between co-culture with IL-12 and without IL-12 in 37℃ plus nine antigens respectively on whole blood from the 3 individuals.

To compare IFN-γ responses to nine antigens on whole blood from the patients and control. (LID-1 and ML89)

Results(ML2044 and 229-88)

Results(ML2055 and 229-112)

Results(ML2028)

Results(P-ML2044 and P-ML0405)

Summary The results of IFN- γ production between 37℃ and 37℃+CO2

incubation with nine antigens on whole blood from the 3 individuals showed no significant difference ， p=1.06 ；

The results of IFN- γ production between co-culture with IL-12 and without IL-12 in 37℃ plus nine antigens respectively on whole blood from the 3 individuals,it has a significant difference ， p=0.0001 ；

IFN-γ responses to nine antigens on whole blood from the patients and control at 37 in 24 hours incubation without ℃ CO2.The threshold is 50 pg/ml, nine antigens were no cross reaction with TB, NC. Better sensitivity antigen are: LID - 1, ML89, ML2044 and ML2028.

Future Plans

Further identify M.leprae specific peptides and protein antigens by the

whole blood assay in progress at the field of the endemic areas.

Follow-up of HHCs (PGL-I \LID-1 Ab/ responding to 0.2 in Honghe P

rofecture.Whether the surveillace is worth to apply to the field site

or not, the cost-effect should be furher considered.

Evaluation of Real-Time PCR Targeting RLEP for Detection of Mycoba

cterium leprae DNA in BI=0 biopsy specimens or paraffin-embedde

d skin biopsy samples or whole blood for Early diagnosis of leprosy.

Thank You

Lunar Year of Tiger Celebration with cured patients with their families, at Jiuhua Shanzhuang (hot spring resort), Jan 29, 2010.