CHAGAS DISEASE Serological Diagnosis and the humoral immune response after specific treatment

CHAGAS DISEASESerological Diagnosis and the humoral

immune response after specific treatment

Alejandro O. Luquetti& Anis Rassi

Laboratório de Pesquisa em Doença de ChagasHospital das Clínicas e Instituto de Patologia Tropical

e Saúde Pública, Universidade Federal de Goiás

Brasil

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)


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


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



TO OBTAIN RELIABLE RESULTS1. 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 oneb)chech reactivity of lot

c)use several internal controls d)follow strictly technical instructions

3. Good laboratory practices4. Facilitate quiet environment, to avoid technical

errors



- 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



-Chronic, non treated, have usually high titers:IIF: 95% have 1/640 up to 1/10,240ELISA(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 lessELISA(crude): 95% < 0.8 (rec.) 95%<0.2

SEROLOGY IN NON-TREATED CHAGASIC PATIENTS

FOLLOW UP OF CHRONIC PHASE, CARDIAC FORM, NON TREATED PATIENT

MJS, female, 32 sera collected during 23 years

Date Age Ye ELISAOD/CO

IFI

titer

HAI

titer

BIOOD/CO

FIOOD/CO

WIEOD/CO

IgM RR

150878 23 0 2.4 640 32 3.3 3.0 8.2 <5 P

181082 27 4 3.1 1280 32 5.6 2.9 7.1 P

090596 41 17 3.6 1280 32 P

140797 42 19 2.6 1280 16 7.0 4.4 7.5 <5 P

101297 42 19 2.5 2560 32 8.0 4.6 8.3 P

231098 43 20 2.2 1280 32 7.9 5.0 8.5 P

080499 44 20 2.5 2560 32 5.7 3.7 7.0 P

221199 44 21 2.6 1280 16 8.2 5.3 7.8 P

280600 45 21 2.5 2560 64 8.1 5.6 7.4 P

030401 46 22 3.1 2560 32 8.4 5.1 7.3 P

020102 46 23 2.9 2560 32 6.4 4.3 7.5 P

SEROLOGY IN NON-TREATED CHAGASIC PATIENTS

FOLLOW UP OF CHRONIC PHASE, CARDIAC FORM, NON TREATED PATIENT

ICD, female, 32 sera collected during 13 years

Date Age Ye ELISA IFI HAI BIO FIO WIE IgM RR

181088 55 0 3.5 5120 256 7.6 8.0 6.9 40 P

040797 64 9 5.9 5120 512 8.2 5.2 20 P

040897 64 9 3.4 2560 512 8.4 4.5 80 P

030997 64 9 3.9 2560 256 9.4 7.3 160 P

090298 64 9 4.7 5120 512 8.5 7.4 80 P

170398 64 9 3.4 5120 256 8.1 7.5 40 P

241198 65 10 5.5 5120 512 9.9 6.2 7.2 20 P

221199 66 11 6.5 9999 1024 9.9 5.1 7.9 160 P

220500 66 11 4.3 5120 1024 9.9 7.4 7.6 80 P

070601 67 12 7.1 5120 256 8.8 3.9 7.3 5 P

141101 68 13 6.5 5120 512 9.9 7.5 6.6 160 P

SEROLOGICAL DIAGNOSIS OF T. cruzi INFECTION

CONVENTIONAL SEROLOGICAL TESTS

Values observed in infected and non infected

TEST RESULT NEGATIVE INCONCLUSIVE POSITIVE

ELISA O.D./Cut-off <0,9 0,9-1,1 >1,1

IIF TITER < 1/20 1/20-1/40 >1/40

IHA(2ME) TITER <1/8-1/10 1/8-1/10 >1/8-1/10

(Values differ according to trade of the kit and laboratory internal control)

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%.

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?


• 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


• 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)

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


• 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)

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.

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

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

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.

Date Age TT ELISA IFI HAI BIO FIO WIE IgM RR

270481 10 0 1.4 160 64 1.2 6.2 7.0 640 P

110581 10 15 1.7 1280 32 3.5 4.2 7.4 P

090981 10 5m 1.0 20 16 1.9 3.4 2.3 N

280482 11 1y 1.2 10 4 1.1 2.3 0.5 N

221183 12 2y 1.2 10 <2 1.4 4.1 0.1 N

090584 13 3y 0.9 10 4 1.2 1.8 0.1 N

250785 14 4y 0.9 <10 <4 0.9 1.1 0.1 N

310787 16 6y 0.8 <10 4 1.0 1.2 0.3 N

280590 19 9y 1.1 <10 <4 0.8 1.1 0.4 N

190494 22 13 0.8 <10 4 0.7 0.5 0.1 N

070596 25 15 0.9 20 4 0.4 0.6 0.1 N


FOLLOW UP OF CHRONIC PHASE IN A SUCCESFULLY TREATED PATIENT

TRS, treated at 31 years old, benznidazol x 60 days, on march 1982

Date Age TT ELISA IFI HAI BIO FIO WIE IgM RR

260382 31 0 2.0 640 32 4.9 3.8 7.8

150383 32 1 2.1 640 16 4.9 3.1 8.0

010885 34 3 1.7 80 8 4.2 2.9 7.2

120287 35 5 1.0 80 4 2.7 1.4 2.7

040888 37 6 1.2 40 8 2.0 1.0 2.2

250491 40 9 1.4 80 4 3.5 1.7 7.2

160793 42 11 1.3 40 4 3.7 3.4 4.6

090596 45 14 0.9 80 4 2.6 1.2 7.5

150597 46 15 1.3 80 4 3.0 1.5 7.0

040299 47 17 0.8 40 8 2.6 1.3 6.7

281099 48 17 0.8 20 8 2.3 1.0 6.8


FOLLOW UP OF RECENT CHRONIC PHASE, THERAPEUTIC FAILURE

ARS, male, treated with 8 years old (1988), benznidazol x 60 days. Positive xenodiagnosis after treatment

Date Age Ye ELISA IFI HAI BIO FIO WIE IgM RR

220987 7 0 2.4 640 64 6.4 6.0 8.4 P

020988 7 0 3.7 1280 64 5.3 6.4 P

1988 8 treated P

200389 8 2.2 640 64 4.7 2.5 P

231289 9 1 2.0 1280 32 3.3 3.5 P

110190 9 2 2.8 1280 32 1.7 6.2 P

300491 10 3 2.5 640 16 3.8 3.7 7.3 P

251198 18 10 2.9 1280 16 6.1 3.3 P

261103 23 15 3.5 1280 64 5.6 3.4 8.2 P

150604 23 16 3.1 1280 32 6.4 1.7 7.9 P

071106 26 18 2.8 2560 64 5.6 8.1 P


FOLLOW UP OF CHRONIC PHASE IN A SUCCESFULLY TREATED PATIENT

MDS, treated at 47 years old, in 1980, with benznidazol x 60 days.

Date Age ELISA IIF IHA ELISA

Biomer.ELISA

Biomang.

ELISA

Wiener

201178 45 3.6 1/2560 1/512 8.3 7.0 8.2

280979 46 3.0 1/1280 1/128 7.5 5.3 8.0

1980 47 treated

100998 65 1.0 40 <1/4 1.0 1.1 4.5

271198 65 0.9 10 <1/4 1.1 1.2 5.0


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)

ELISA IIF IHA ELISA

Biomer.ELISA

Biomang.

ELISA

Wiener

051088 36 (0) 3.3 1/2560 1/128 5.9 6.0 8.2

251005 54 (17 y) 1.4 1/640 1/16 2.4 1.3 5.6

310306 54 (18 y) 1.5 1/320 1/8 2.1 1.0 3.0

ARS

150388 14 (0) 1.8 1/1280 1/32 5.6 4.0

020888 15 (0) 2.4 1/2560 1/64 4.5 2.5 7.5

110406 32 (17 y) 0.6 1/160 1/8 1.4 0.8 0.8

SPECIFIC TREATMENT IN CHAGAS DISEASE

FOLLOW UP OF AN UNSUCCESFULL CASE, TREATED AT THE ACUTE PHASE

- VRSA, female, 36 y, parasites in fresh preparation

Data Idade ELISA IFI HAI HAI+2ME ELISA

Biolab

ELISA

Fiocruz

280683 36 0.7 10 <4 - 1.1 0.8

010783 36 1.5 80 8 <8 0.6 1.2

150983 36 0.7 160 8 <8 1.2 -

110785 38 2.2 640 64 16 5.1 -

210786 39 2.5 160 64 32 3.4 -

140788 41 3.1 640 32 - 4.7 5.1

090903 56 3.3 2560 64 - 8.5 6.4

RECOMBINANT ANTIGENS

YEAR

sponsor

Nr

CENTERS/Antigens

Nr

SERA

Country (Sera)

1989WHO

1990CYTED

1998

IMTSP

10

4

5

Nr Rec

Assay Nr aproved

Reference, Year

50

215

541

17

10

6

Several

phage dot blot

ELISA

10

4

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

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,

20085)WHO reference Serum: Prototypes Tc1 & Tc2 lyophilization (2L) scheduled, 2009.

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 yearsparasites > 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.

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 Paulo Dra. Nobuko Yoshida (Unifesp)

Dr. José Franco da Silveira (Unifesp)

Dra. Eufrozina S. Umezawa (USP)

Rio Jan. Dr. Octávio Fernandes (Fiocruz)

Uberaba Dr. 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)

Documents

CHAGAS DISEASE Serological Diagnosis and the humoral immune response after specific treatment