1. The utility of anti-trypomastigote lytic antibodies for determining cure of T. cruzi infections in treated patients: an overview and perspectives Antoniana Ursine Krettli CNPq Fellow Belo Horizonte, MG – BRAZIL akrettli@cpqrr.fiocruz.br Instituto René Rachou - FIOCRUZ

2. Chagas Disease and Professor Zigman Brener UFMG, FIOCRUZ

3. Problems with treatment and control of cure (i)Less than 1% of 510 chronic cases had a history of acute infection in a 30 years study (Coura et al 1983) (ii) In 544 children exposed 16 months to natural transmission2.6% became infected, 1/3 had symptoms compatible with an acute infection (Teixeira, MG Thesis MS UFRJ 1997) (iii)Most patients are treated in the chronic phase when parasitemia is subpatent and CS the main diagnosis test (iii)Our approach - other tests indicative of ongoing infections

4. (i) Sera or IgG from the chronic phase neutralize trypomastigote infectivity to mice (ii) Human sera agglutinates the Y parasites (iii) Some sera neutralize parasite infectivity (iv) The immunossuppressed host develops high parasitemia Functional antibodies in chronic T. cruzi infections (Krettli & Brener, J. Immun 1976)

5. Antibodies from chronic infections + live Y trypomastigotes

6. Antibody induced complement-mediated lysis (CoML) Victor & Ruth Nussenzweig (1976- BH) AK - NUY – 1977 Krettli et al Clin Exp Immuno 1979

7. Functional antibodies in T. cruzi infections Krettli & Brener, J Immunol 1982

8. The targget for lytic antibodies A glycoprotein of 160kDa the target for lytic antibodies (Martins et al 1985; Norris et al 1991) Tc160 - a DAF like protein, or the complement regulatory protein (CRP), cloned, sequenced and expressed as a recombinant seem ideal in ELISA tests (Kipnis et al 1985; Norris et al 1991)

9. Proposed mechanism of complement activation Reviewed in Krautz et al (2000). Norris et al. (1991)

10. (i)conventional serology - CS (CFR; IIF) (ii) complement mediated lysis (CoML) Krettli et al Trans R Soc trop Med 1983 (iii) hemocultures* *Up to 120 days cultures -two tests 54% positive Galvão et al Braz J Med Biol Research, 1989 T. cruzi human infections: treated cases Colaboration with Profs.J. R. Cançado & Brener, 1979

11. Patient, Age (Years) Last treatment in Years of follow up after treatment IIF titer Sera with positive lysis / total tested Hemocultures tests* AFO, 441981 12 1:800/65 AAA, 491982 14 1:800/55 ASS, 29 1983 10 1:1600/1619 LPC, 541986 6 1:3200/58 MCS, 57 1984 6 1:1600/68 MLP, 371985 7 1:1600/97 MPS, 511983 11 1:1600/1512 PMPM, 501982 8 1:320 0/73 APP, 57 1983 12 1:1600/97 ANC, 471979 8 1:800/1019 GV, 53**1986 16 1:3200/59 SCPL, 331977 17 1:40 0/68 T. cruzi drug-treated presumably cured patients

12. Chagasic patients presumably cured, i.e. the dissociated group: negative lysis & positive serology Most patients treated twice Some treated 3 (ex PM) Other patients treated 5 times (ex GV) Patients treated by Prof. JR Cançado, BH MG Drugs used: Nx, Bz mostly

13. Treated infections: a 15 year follow up study

14. % lysis with sera from non-treated CHA patients: hemocultures positive (A) or (B) negative. Lucia Galvão Tese Dr. UFMG Galvão et al Trans Roy Soc Trop Med & Hyg 1993 (C) Sera from non-chagasic (D) Sera from chagasic dissociated (Positive CS)

16. How long one has to wait? Follow ups An acute acidental case in USA treated with nifurtimox CoML was negative in a month (Israelski et al Am J Trop Med Hyg 1985) An acute case in treated with Bz CoML became negative in a month ( Krettli et al Trans Roy S.T. Med 1983) In one chronic case treated with Bz CoML reverted to negative years before CS became negative (six years later) (Galvão et al Trans Roy STMed 1993 )

17. Treatment at Months after treatment Sera samples tested Number (%) of positive sera CFRIIFCoML Chronic Phase JFS 3-74 2 (50%) 4 (100%) 4 (100%) 8-126 2 (33%) 6 (100%) 1 (17%) 13-241 1 (100%) 1 (100%) 0 (0%) 25-723 0 (0%) 2 (67%) 0 (0%) 73-845 0 (0%) 0 (0%) 0 (0%) 85-1084 0 (0%) 0 (0%) 0 (0%) Adapted from Galvão et al. Trans. R. Soc trop Med Hyg 1993 Treated cured patient: negative lysis in one year; CS negative in 6 years

18. Criteria of T. cruzi cure based on negative conventional serology, Prof. Cançado and others Patients treated / time for an expected negative serology < one year infection- one year after parasite removal 10-15 years infection 5-10 years after parasite removal >15 years infection over 10 years after parasite removal Reviewed by Villa et al Memorias IOC 2007 Cites Rassi & Luqueti 2002

19. Replacing the lytic antibodies test By flow cytometry excelent results (Martins. Cançado, Brener et al ); requires live parasites thus is not practical By ELISA Tc160 - a DAF like protein (Kipnis et al 1985) Tc160- a complement regulatory protein (CRP), cloned, sequenced and expressed as a recombinant protein, ideal in ELISA tests (Norris et al 1991, 1994; Meira et al 2004) Other recombinant proteins

20. CoML replaced by Immunoenzimatic assays (ELISA) with T. cruzi antigens Correlation CoML x ELISA Which antigens are reliable to control cure?

21. Antigen MW (kDA) Correlation with CoML References Gp160*160100%Martins et al 1985. Cruzipaine (Cisteine protease)** 57/5170% Gazzinelli et al 1993. F2 - Mucine GPI anchored 74/96100% Almeida et al 1993 Antigen excreted/secreted by trypomastigotes 70 to 220100% Krautz et al 1994. Complement regulatory protein (GP160 or the CRP) 160100% Norris et al; 1991; 1994. Tc24 – calcium binding protein 2486% Krautz et al 1995 Heat shock protein 7094% Krautz et al 1998 CRA/FRA antigens 4567% Silva et al 2002 Recombinant CRP r160100% Meira et al 2004

22. 1- Tests with live trypomastigotes although ideal to determine cure are not practical for use in clinical routine to control cure of treated patients 2- CoML test can be replaced by ELISA with adequate antigens; control trials need to be undertaken 3- Based on negative CoML revertion 40% of treated patients treated are cured, only 10% with neg CS 4- The ultimate goal to control cure should be the absence of parasitemia, use PCR? Conclusions

23. The future Opinions vary considerably in terms of: which regimens and length of therapy is curative? which serological test is ideal? which functional test is feasible to control cure? “More partnership is needed...to increase access to adequate and better adapted diagnosis and treatment” reviewed by the DNDI group (Ribeiro et al 2009 Plos Neglected Trop Diseases) A protocol ideal for PCR to detect T. cruzi DNA