1. Early Diagnosis of leprosy based on Multiple Assays Beijing Tropical Medicine Research Institute Email: weny8@163.com 2013-9-16

2. Introduction  Early diagnosis of Leprosy is a big challenge in endemic countries. At the present, 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 leprosy-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.

4. 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.

5. 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

6. 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 ( + ). 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)

7. Amplification Plot ： A: 10 7 copies /μlplasmid ； B: 10 6 copies /μlplasmid ； C: 10 5 copies /μlplasmid ； D: 10 4 copies /μlplasmid ； E: 10 3 copies /μlplasmid ； F: 10 2 copies /μlplasmid G: 10 copies /μlplasmid 。 TaqMan real time PCR

8. Select unique proteins from M. leprae database based on comparative genomic analyses Synthesize peptides to be used in vitro PBMC/whole blood stimulation assays 21 peptides chosen from 14 different M. leprae proteins were synthesized for in vitro testing Synthesize peptides

9. Synthetic peptides: the stimulate antigens of the 21 polypeptides CodePeptide locationCodePeptide location P1ML0044P12ML0497-1 P2ML0049-1P13ML0497-2 P3ML0049-2P14ML0574 P4ML0049-3P15ML1001 P5ML0050P16ML1989 P6ML0073-1P17ML1990 P7ML0073-2P18ML2283 P8ML0410-1P19ML2346-1 P9ML0410-2P20ML2346-2 P10ML0411-1P21ML2567 P11ML0411-2

10. Newly diagnosed case during the following up in Kaiyuan City

11.  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-BSALID-1 2008.060.3380.298 2009.020.3810.746 2009.100.9481.406 Back lesions S-100 dyeing(X 400) A-F staining （ X 1000 ） Case 1, early BL H&E X 200

12. Case 2 IND age at 5, HHC. At the second time of following up, a single light spot was found on his back. Single lesion Histo-Path: No epidermal atrophy, no subepidermal clear zone and no definite granuloma seen; perivascular lymphocyte infiltration, some epitheliod cells and a single suspected AFB seen. ELISA: ND-O-BSA 0.0885; PCR for M.leprae (+) LID-1 0.1610. Acid-fast staining (-) Diagnosis: IND leprosy S-100 400X HE 400 X

13. 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.

14. 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.

16. Evaluation on T cell assay based on the peptides

17. 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

18. Preliminary summary on T cell assay based on the 21 peptides  Although we succeeded in the diagnosis one case and no cross reaction with TB and normal groups. However, the results 0f all 21 peptides for both PBMC or whole blood assay on MB:24, PB:6 and HHC:17 showed no peptide may induce IFN-γ in significant differences between the petients and control. In addition, the experimental repeatability is not good.  The design or selection of the peptides and proteins require to further study.

19. 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

20. To compare IFN- γ production between 37 ℃ and 37 ℃ + CO 2 incubation with nine antigens on whole blood from the 3 individuals.

21. 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.

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

23. Results (ML2044 and 229-88)

24. Results (ML2055 and 229-112)

25. Results (ML2028)

26. Results (P-ML2044 and P-ML0405)

27. Summary  The results of IFN- γ production between 37 ℃ and 37 ℃ +CO 2 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 CO 2.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.

28. 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 Profecture.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 Mycobacterium leprae DNA in BI=0 biopsy specimens or paraffin- embedded skin biopsy samples or whole blood for Early diagnosis of leprosy.

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