1. CHAGAS DISEASE Serological Diagnosis and the humoral immune response after specific treatment Alejandro O. Luquetti & Anis Rassi Laboratório de Pesquisa em Doença de Chagas Hospital das Clínicas e Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás Brasil

2. DIAGNOSIS OF CHAGAS DISEASE SEROLOGICAL DIAGNOSIS (chronic phase) - antibody titers in chronic patients (have parasites = Ag) - Kinetics of decrease of Ab titers in passive transmission (no parasites, no immunological memory) -Ab titers in specifically treated and cured -(no parasites, but memory, that finally forgets)

3. DIAGNOSIS OF CHAGAS DISEASE LABORATORIAL DIAGNOSIS OF THE CRHONIC PHASE - Antibodies anti-T.cruzi present in > 98% - Parasites present in no more than 50%, even with PCR - The parasitemia is usually low or absent, inconstant, variable, erratic, and even if there, not necessarily present at the sample (# from Ab) - Solutions versus suspensions

4. DIAGNOSIS OF CHAGAS DISEASE SEROLOGICAL DIAGNOSIS - Presence of Ab in constant concentration in a given individual - Conventional tests (ELISA, IIF, IHA) in use since 1975 - Great cumulated experience in all endemic countries - Performance depends of good quality kits and good Laboratory practice - Most with no purified antigen - Problems (few) of specificity mainly with leishmaniosis

5. DIAGNOSIS OF CHAGAS DISEASE SEROLOGICAL DIAGNOSIS TO OBTAIN RELIABLE RESULTS 1.Serum in good conditions (glycerol 50%). (bad storage (4º.C) low titers) 2.Comercial kits: ought to use (no in house) a)select a good performance one b)chech reactivity of lot c)use several internal controls d)follow strictly technical instructions 3. Good laboratory practices 4. Facilitate quiet environment, to avoid technical errors

6. DIAGNOSIS OF CHAGAS DISEASE SEROLOGICAL DIAGNOSIS - If properly done, allowed differences are of one titer, with same serum, different days - Very precise tool (if comercially acquired) - All in duplicate in different zone of plate - If duplicates do not fit, repeat (i.e. 1.5- 2.7) - Once system is working, go ahead

7. DIAGNOSIS OF CHAGAS DISEASE SEROLOGICAL DIAGNOSIS -Chronic, non treated, have usually high titers: IIF: 95% have 1/640 up to 1/10,240 ELISA(crude): 95% higher than 2 times OD/CO up to 6 times (i.e. 3.5) ELISA(rec.): 95% higher than 5 (i.e. 7.0) -Non infected: IIF: 95% titers 1/10 or less ELISA(crude): 95% < 0.8 (rec.) 95%<0.2

8. SEROLOGY IN NON-TREATED CHAGASIC PATIENTS FOLLOW UP OF CHRONIC PHASE, CARDIAC FORM, NON TREATED PATIENT MJS, female, 32 sera collected during 23 years DateAgeYeELISA OD/CO IFI titer HAI titer BIO OD/CO FIO OD/CO WIE OD/CO IgMRR 1508782302.4640323.33.08.2<5P 1810822743.11280325.62.97.1P 09059641173.6128032P 14079742192.61280167.04.47.5<5P 10129742192.52560328.04.68.3P 23109843202.21280327.95.08.5P 08049944202.52560325.73.77.0P 22119944212.61280168.25.37.8P 28060045212.52560648.15.67.4P 03040146223.12560328.45.17.3P 02010246232.92560326.44.37.5P

9. SEROLOGY IN NON-TREATED CHAGASIC PATIENTS FOLLOW UP OF CHRONIC PHASE, CARDIAC FORM, NON TREATED PATIENT ICD, female, 32 sera collected during 13 years DateAgeYeELISAIFIHAIBIOFIOWIEIgMRR 1810885503.551202567.68.06.940P 0407976495.951205128.25.220P 0408976493.425605128.44.580P 0309976493.925602569.47.3160P 0902986494.751205128.57.480P 1703986493.451202568.17.540P 24119865105.551205129.96.27.220P 22119966116.5999910249.95.17.9160P 22050066114.3512010249.97.47.680P 07060167127.151202568.83.97.35P 14110168136.551205129.97.56.6160P

10. SEROLOGICAL DIAGNOSIS OF T. cruzi INFECTION CONVENTIONAL SEROLOGICAL TESTS Values observed in infected and non infected TEST RESULT NEGATIVE INCONCLUSIVEPOSITIVE ELISA O.D. /Cut-off 1,1 IIF TITER 1/40 IHA(2ME) TITER 1/8-1/10 (Values differ according to trade of the kit and laboratory internal control)

11. LABORATORY DIAGNOSIS OF T. cruzi INFECTION INCONCLUSIVE RESULTS - By simultaneous use of three conventional tests(ELISA, IFI, HAI) - From 7.849 sera, 4.800 (61,2%) positive and 2.194 negative - Only 133 (1,7%) inconclusive: - This means: 1 test on cut-off (+/-10%), 1 negative and 1 positive or 2 – 3 on gray zone - Possible cause detected in 63 (47,5%) with aid of IIF leishmania, IgM, Reumatoid factor, Montenegro test - Leishmaniosis in 26%, treated in 14%, acute phase in 3%, passive transference of Ab in 3% and hepatitis in 1,5%.

12. LABORATOY DIAGNOSIS OF T. cruzi INFECTION SOME RESULTS OBTAINED IN NOT CLEARLY DEFINED SERA (n = 205/16.150 sera), WITH NEW TOOLS - Agreement among recombinant antigens (at least three kits): All negative: 35 sera (11/35 do not agree with 4# ELISA) All positive: 61 sera (14 do not agree with 4# ELISA) ----------- Agreements: 96/205 (47 %) - Lack of agreement (some P, other N): 64 sera - Partial agreement (majority P or N): 45 sera - Those 109 sera, are P or N? What to do? Believe in which?

13. LABORATOY DIAGNOSIS OF T. cruzi INFECTION RELIABLE RESULTS IN SEROLOGY –Matherials: approved kit, retested for lot at the lab (internal panel with low positives and high negatives) Good laboratory practices: temperatures, pH, etc. POP : description of each procedure, in detail –Methods: Programs of technical training (Telelab) –Quality: Lab ought to participate in an External Quality Control Programme, provided that: This programm send at least 2 panels/year The Lab should be approved

14. LABORATOY DIAGNOSIS OF T. cruzi INFECTION PROGRAMS of EXTERNAL QUALITY CONTROL –Initial difficulty to mount serum panels (AR/CH/BR/PY) Meeting OPS-BH 1994) –Difficult to obtain panels in non endemic countries –Initiative of OPS/PANEL São Paulo (BR) 1995 –Programm operative in >18 countries –Priority in reccommendations of South Cone (1999) –Programms of Hemotherapy Societies (AR, BR) –Programm of MPH, BR, COSAH>ANVISA (2001)

15. LABORATOY DIAGNOSIS OF T. cruzi INFECTION CONTROL QUALITY PROGRAM OF THE MINISTRY OF HEALTH, BR FOR EXTERNAL CONTROL IN BLOOD BANKS –Joint venture National Agency of Sanitary Surveill./Fiocruz –Both belong to Ministery of Health, coordination, execution –Technical Committee, 1 by area (syphilis, HIV, etc) –Several meetings/year, evaluation of results by marker –Three panels/year (6x3) – by post –Started on 2000, processed 5.718 plasma bags (2007) –From 2001 to 2008, 18 evaluations, of 135 services (90% public) –Increase in results, from 3,6 % to 0,9% discordances.) –Indirectly detected problems with different lots of used kits

16. LABORATOY DIAGNOSIS OF T. cruzi INFECTION EVALUATION OF ELISA KITS AVAILABLE IN BRAZIL –Study performed by Lab Coordination, Min.Health –Bought all certified kits available in Brazil (n=12) –Selection of 152 sera (half negative) –Blind tests by 4 labs: MG, PE, MS, GO –Kappa index of 0,71 to 0,98. Sensitivity 0,97 a 1,0 –6 kits sensitivity = 1,0. 5 kits = 0,99. 1 kit =0,97. –Trades: Adaltis, Bioma, Biomerieux, Bioschil, Biozima, –Ebram, Hemagen, Omega, REM, Wama, Wiener –This study allows to exclude some trades, based on published data (available at the site of the M.Health)

17. CHAGAS DISEASE DIAGNOSIS OF INFECTION BY T. cruzi IEIN EACH SITUATION 3)DIAGNOSIS OF CONGENITAL TRANSMISSION: - Send by pediatritian, to exclude transmission from the mother - Reassure that mother is really infected: serology previously or, serology of blood from umbilical cord: if negative, no transm. - Look for parasites in the new-born (micro-hematocrit) - Positive serological tests, only indicate maternal transference - IgM (IIF not recomended). Anti-SAPA not available - Better advise physician for new sample at 6-8 months: - If IgG present, transmission is proved and child ought to be treated.

18. CHAGAS DISEASE VERTICAL TRANSMISSION SEROLOGY: NEWWBORNS FROM INFECTED MOTHERS -RESULTS OBTAINED ON 56 NEWBORNS -First month age: elevated titers, similar to those of the mother - (n=17) IFI 1.280 Elisa: 2,6 HAI 256 PaGIA pos - Second month sera: IFI 320 Elisa: 1,5 HAI 16 PaGIA variab - Third month: IFI 80 Elisa: 1,2 HAI 8 PaGIA variab - Fourth month: IFI 40 Elisa: 0,6 HAI 4 PaGIA neg - Sixth month (n=13): IFI 10-40 Elisa: 0,6 HAI <4 PaGia neg - Seventh month (n=9): IFI<1/10 Elisa: 0,4 HAI <2 PaGIA neg - Eight month (n=8): IFI<1/10 Elisa: 0,4 HAI<2 PaGIA neg -No antibodies a-T. cruzi, as non infected = passive transference

19. CHAGAS DISEASE DIAGNOSIS OF INFECTION BY T. cruzi IN EACH CONTEXT 4) FOLLOW UP OF A SPECIFICALLY TREATED: -Send by physician, looking for cure -- For Tc2b: - Cure is possible in all newborns, 60% of acute phase and children and up to 25% of adults, after benznidazol x 60 days - Cure criteria is abscence of antibodies, formerly present - Time to attain: months in congenital, years in acute/children and decades in adults - For these, need to preserve previous sera with glicerol - To procede in paralel with sera before and after treatment

20. SEROLOGY IN TREATED CHAGASIC PATIENTS FOLLOW UP OF ACUTE PHASE (50 DAYS EVOLUTION) IN A SUCCESFULLY TREATED PATIENT DPS, female, fever by 07/03/81, Romaña sign. Flagellates on wet smear. Benznidazol x 60 days. DateAgeTTELISAIFIHAIBIOFIOWIEIgMRR 2704811001.4160641.26.27.0640P 11058110151.71280323.54.27.4P 090981105m1.020161.93.42.3N 280482111y1.21041.12.30.5N 221183122y1.210<21.44.10.1N 090584133y0.91041.21.80.1N 250785144y0.9<10<40.91.10.1N 310787166y0.8<1041.01.20.3N 280590199y1.1<10<40.81.10.4N 19049422130.8<1040.70.50.1N 07059625150.92040.40.60.1N

21. SEROLOGY IN TREATED CHAGASIC PATIENTS FOLLOW UP OF CHRONIC PHASE IN A SUCCESFULLY TREATED PATIENT TRS, treated at 31 years old, benznidazol x 60 days, on march 1982 DateAgeTTELISAIFIHAIBIOFIOWIEIgMRR 2603823102.0640324.93.87.8 1503833212.1640164.93.18.0 0108853431.78084.22.97.2 1202873551.08042.71.42.7 0408883761.24082.01.02.2 2504914091.48043.51.77.2 16079342111.34043.73.44.6 09059645140.98042.61.27.5 15059746151.38043.01.57.0 04029947170.84082.61.36.7 28109948170.82082.31.06.8

22. SEROLOGY IN TREATED CHAGASIC PATIENTS FOLLOW UP OF RECENT CHRONIC PHASE, THERAPEUTIC FAILURE ARS, male, treated with 8 years old (1988), benznidazol x 60 days. Positive xenodiagnosis after treatment DateAgeYeELISAIFIHAIBIOFIOWIEIgMRR 220987702.4640646.46.08.4P 020988703.71280645.36.4P 19888treatedP 20038982.2640644.72.5P 231289912.01280323.33.5P 110190922.81280321.76.2P 3004911032.5640163.83.77.3P 25119818102.91280166.13.3P 26110323153.51280645.63.48.2P 15060423163.11280326.41.77.9P 07110626182.82560645.68.1P

23. SEROLOGY IN TREATED CHAGASIC PATIENTS FOLLOW UP OF CHRONIC PHASE IN A SUCCESFULLY TREATED PATIENT MDS, treated at 47 years old, in 1980, with benznidazol x 60 days. DateAgeELISAIIFIHA ELISA Biomer. ELISA Biomang. ELISA Wiener 201178453.61/25601/5128.37.08.2 280979463.01/12801/1287.55.38.0 198047treated 100998651.040<1/41.01.14.5 271198650.910<1/41.11.25.0

24. SEROLOGY IN TREATED CHAGASIC PATIENTS FOLLOW UP OF CHRONIC PHASE IN TREATED PATIENTS, ON THE WAY FOR CURE DEMONSTRATION Two patients, treated at 37 and 15 years old, with benznidazol x 60 days. Date PAM Age (Time) ELISAIIFIHA ELISA Biomer. ELISA Biomang. ELISA Wiener 05108836 (0)3.31/25601/1285.96.08.2 25100554 (17 y)1.41/6401/162.41.35.6 31030654 (18 y)1.51/3201/82.11.03.0 ARS 15038814 (0)1.81/12801/325.64.0 02088815 (0)2.41/25601/644.52.57.5 11040632 (17 y)0.61/1601/81.40.8

25. SPECIFIC TREATMENT IN CHAGAS DISEASE FOLLOW UP OF AN UNSUCCESFULL CASE, TREATED AT THE ACUTE PHASE - VRSA, female, 36 y, parasites in fresh preparation DataIdadeELISAIFIHAI HAI+2ME ELISA Biolab ELISA Fiocruz 280683360.710<4-1.10.8 010783361.5808<80.61.2 150983360.71608<81.2- 110785382.264064165.1- 210786392.516064323.4- 140788413.164032-4.75.1 090903563.3256064-8.56.4

26. RECOMBINANT ANTIGENS YEAR sponsor Nr CENTERS / Antigens Nr SERA Country (Sera) 1989 WHO 1990 CYTED 1998 IMTSP 10 4 5 Nr Rec AssayNr aproved Reference, Year 50 215 541 17 10 6 Several phage dot blot ELISA 10 4 Moncayo & Luquetti, 1990 Levin et al, 1991 Umezawa et al. 1999. EVALUATION IN THREE MULTICENTRIC STUDIES Brazil BR, AR, VE BR, AR, VE, BO, CH, CO, AS, GU, HO

27. DIAGNOSIS & TREATMENT OF T. cruzi INFECTION SOME OF THE GREAT DREAMS that latin- americans transformed in facts 1)CONSENSUS ON THE BETTER NEW REAGENTS FOR SEROLOGICAL DIAGNOSIS: multicentric study WHO 1989- 1990, Moncayo & Luquetti, 1990. 2)ELIMINATION OF TRANSMISSION BY Triatoma infestans AT THE SOUTH CONE, 1991: 3 certified countries(UR,CH,BR)and 2 in advanced stage (PY, BO) 3)CONSENSUS Tc1/Tc2, Rio, 1999. 4)CONSENSUS ON PCR PROCEDURES AND OPTIMIZA- TION OF SPECIFICITY, TDR, Buenos Aires, 2008 5)WHO reference Serum: Prototypes Tc1 & Tc2 lyophilization (2L) scheduled, 2009.

28. DIAGNOSIS & TREATMENT OF T. cruzi INFECTION CONCLUSION 1) Most diagnostic situations are easily solved by serology at the chronic phase 2) Serological diagnosis nowadays is precise and reliable 3) Cure is return to non infected status: i.e. no parasites, nor antibodies (Cancado) 3) In succesfully treated patients, serological tests became negative after a period of time 4) It is expected to have same curve than passive transmission: no Ag, Ab should come down 5) Immunological memory, made the difference: parasites by weeks (acute), Ab dissapear in months. parasites < 10 y (recent chronic): Ab clear in few years parasites > 10 y: Ab start to decline after 2 decades, negative after 3-4 decades 6) Individual differences. Recombinants not better than crude. Purified good for individual cases. Single tool not enough.

29. COLLABORATORS / PARTNERS OF RESEARCH, CHAGAS DISEASE Goiânia:Dr. Joffre M. de Rezende (Gastroenterology) Dr. Anis Rassi (Cardiology) Dr. Helio Moreira (Proctology) Dr. Ênio Chaves de Oliveira (Surgery esophagus and colon) Dra. Dayse Elizabeth Campos (Cardiology) Dra. Maria da Glória Merheb Vaz (Gastroenterology) Dra. Rita Francis G. y R. Branco (Cardiopediatry) Dra. Neusa G. Leal Marra (Cardiology in obstetrics) Psic. Auta Mendes (Psicology) Dr. Ionizete Garcia da Silva (xenodiagnosis) (IPTSP, UFG) Dra. Ana Maria de Castro (hemoculture) (IPTSP, UFG) São PauloDra. Nobuko Yoshida (Unifesp) Dr. José Franco da Silveira (Unifesp) Dra. Eufrozina S. Umezawa (USP) Rio Jan.Dr. Octávio Fernandes (Fiocruz) UberabaDr. Aluisio R. Prata (FM Triângulo Mineiro) Argentina Dr. Mariano Levin and Alejandro Schijman (markers, PCR) U. S. A.Dr. Miercio Pereira (markers) U. K. Dr. Michael A. Miles (strains)