International Symposium Centenary of Chagas Disease 1909 . 2009

1. International Symposium Centenary of Chagas Disease 1909 . 2009 July, 2009 – Rio de Janeiro Use of Polymerase Chain Reaction (PCR) to Establish Drug Efficacy - Value and Limitations Constança Britto cbritto@ioc.fiocruz.br Oswaldo Cruz Foundation, FIOCRUZ

2. I- Etiological Treatment should focus on Parasite Clearance Chemotherapy during the acute stage of Chagas disease leads to parasitological cure in up to 80% of the cases, but its effectiveness in the long-lasting chronic phase remains unclear. • The main limitations are: • long-term follow-up (persistence of specific antibodies); • continuing administration of toxic nitroimidazolederivatives; • differences in drug susceptibility among distinct T. cruzi strains; • lack of reliable tests to ensure parasite elimination. • Conventional Serology may remain positive for several years despite repeated negative parasite detection methods. • Rather poor sensitivity of Xenodiagnosis and Hemocultive

3. PCR to assess therapy in Chagas disease SPECIFICITY IMPROVEMENT (DNA sequences with the ability to distinguish between organisms) Target DNA sequence DNA in vitro amplification Chromosome The use of PCR to detect minimal amounts of T. cruzi DNA in the blood of chagasic patients opened new possibilities for the diagnosis of chronic infections and evaluation of chemotherapeutic treatment (Moser et al. 1989; Sturm et al. 1989; Avila et al. 1991; 1993;Britto et al. 1993; Wincker et al, 1994; Britto et al, 1995). HIGHER SENSITIVITY (Multiple genome targets) II- PCR contributions for the diagnosis of Chagas disease

4. PCR performance for the analysis of clinical specimens has revealed discordant results concerning sensitivity, depending on: • epidemiological characteristics of the studied populations (geographic areas, high genetic heterogeneity of T. cruzi strains, etc); • volume of blood collected (intermittent presence and quantity of circulating parasites at the time of blood collection); • method used to isolate DNA; • selection of parasite target-sequences, reagents used, as well as thermo-cycling conditions. Several therapeutic studies confirm the usefulness of PCR to evaluate treatment outcome in Chagas disease, as a complement to serological tests. III- Is PCR a valid technique to address cure?

5. PCR appears remarkably useful for the early detection of treatment failure, in cases of resistance or reactivation of T. cruzi infection. • If the persistence of positive PCR is considered therapeutic failure, assessment of parasite load by quantitative real-time PCR could be correlated with the impact of trypanocidal treatment on the disease evolution. • Clearance of parasitaemia and disappearance of antibodies are taken as cure criteria. There is no guarantee that a single “flash” of negative PCR means parasitological cure. The value of PCR tests lies mainly in the positive results they yield. 330 bp fragment Detection of a single parasite in 20mL of blood lysate IV- The value of PCR to establish drug efficacy

6. Parasite search by microhematocrit, hemoculture and PCR-based on satellite nuclear DNA (100 µL plasma) • PCR (during treatment & at the end of follow-up) confirmed 100% of Bz treatment efficacy in association with conventional serology negativation (Russomando et al, 1998). Parasite clearance more rapidly assessed by PCR [15 days after treatment] than serology [3-8 months] during a 4 years follow-up (Russomando et al, 1998). • Schijman et al (2003) evaluated treatment outcome (Nf and Bz) in 40 infected infants monitored for 2-3 years by PCR (satellite DNA) and conventional methods. Two mL of blood were collected for PCR. Cure achieved by negativation of PCR + serology in 100% of infants with <3 months, in 66.7% between 7 months-2 years old and in 12.5% with >3 years old. In those infants who started therapy in their first months of life (<3 months), cure was achieved early after treatment. V- Treatment of congenital infections

7. Infants infected during the early years of life represent those patients amenable to specific treatment with great possibility of cure (Andrade et al. 1996; Sosa Estani et al. 1998). Galvão et al, 2003: J Clin Microbiol 41: 5066-70. • Cohort of T.cruzi seropositive schoolchildren from an endemic Brazilian area; • Blood samples (5 mL) were taken at baseline and 36 months after treatment with Bz (n=58) or placebo (n=53); • Bz group: 39.6%positive PCR (for kDNA) Placebo: 64.2% positive PCR (P=0.01) • Untreated patients had 1.6-fold-higher chance of remaining PCR positive than the Bz group (P<0.05); • Adequate correlation between high proportion of negative PCR and decrease in antibody titers in the Bz group; • PCR positivity occurred in patients without reductions in antibody titers; • PCR is a useful tool for revealing therapeutic failure on a short-term basis. VI- Treatment in early life

8. The efficacy of available drugs is limited for chronic patients (Urbina, 2002). The drugs are able to reduce the parasite population in blood and the risk for disease progression (Viotti et al, 1994; 2006). Up to 80% of chronic treated patients do not achieve parasitological cure as assessed by the persistence of positive serology (very slow decrease in antibody titers), now confirmed using PCR-based methods. (Britto C et al, 1995) Polymerase chain reaction detection of Trypanosoma cruzi in human blood samples as a tool for diagnosis and treatment evaluation. Parasitology 110: 241-247. 32 Bz-treated seropositive chronic patients attending the Hospital of Infectious Diseases (Fiocruz). Positive PCR (kDNA) observed in only 9 out of 32 treated patients who remained reactive with conventional serology (5 years post-treatment). VII- Treatment of chronic infections

9. Selection of patients(1979) Department of Tropical Medicine, University of Brasilia Vector transmission has been interrupted for more than 20 years 100 Xeno+ patients asymptomatic 85 – treated (Nf or Bz) (37 in the acute phase & 48 in the chronic phase) 15 – control group BA GO

10. Therapy evaluation in a 20 years follow-up XENO (pre & post-treatment) PCR (post-treatment) PCR was more sensitive than Xenodiagnosis in determining therapeutic failure, 20 years after chemotherapy Total PCR + 10 (27%) 17 (35%) 8 (53%) XENO + 5 (13.5%) 8 (16.7%) 4 (26.7%) PCR - 27 (73%) 31 (65%) 7 (47%) XENO - 32 (86.5%) 40 (83.3%) 11 (73.3%) Acute N=37 Chronic N=48 Placebo N=15

11. The agreement of negative results between serology and PCR is probably indicative of cure PCR + PCR - Total Acute Phase 10 (27%) 27 (73%) N=37 31 (65%) Chronic Phase 17 (35%) N=48 7 (47%) Placebo Group 8 (53%) N=15 PCR - Sero + Sero - Sero ? 15 (56%) 7 (26%) 5 (18%) Acute Phase 16 (52%) 3 (10%) 12 (38%) Chronic Phase Herein, as only one point in time was analyzed by PCR, a negative result could not be assumed as definitive cure. 7 (100%) 0 (0%) 0 (0%) Placebo Group

12. Treatment of patients with Chronic Chagas Cardiomyopathy (CCC) Multicenter, randomized, placebo controlled clinical trial of 3,000 patients with CCC (excludes severe disease). Full-scale trial follow-up will be 5 years (ending in 2011). Am Heart J 2008; 156: 37-43 BENEFIT includes a pilot study (600 patients) to evaluate whether 60 days of Benznidazole therapy will reduce parasite activity determined by PCR negativation and parasite load measure. PCR performed at randomization, at the end of therapy (60 days) and after 2 years of follow-up. Participation of 8 countries and 60 centers, conducted by the Canadian Institute of Health Research and WHO/TDR, with the Latin American coordinating center at Dante Pazzanese Institute (SP, Brazil). PCR Core Lab in Brazil (Oswaldo Cruz Institute/FIOCRUZ, RJ) = responsible for PCR analyses from 16 Brazilian centers. This is the largest trial yet conducted in Chagas disease. The study will clarify the role of trypanocidal therapy in preventing cardiac disease progression and death.

13. VIII- Real-time PCR for evaluating therapy I- Rapid quantification of T. cruzi in host tissue by real-time PCR (Cummings & Tarleton, 2003) • Accurate quantification of tissue parasite burden in experimental infected mice using SYBR green withtwo distinct primer sets: satellite nuclear DNA and kDNA. kDNA nuclear A useful tool for monitoring parasite burden in hosts under treatment II- Development of a real-time PCR assay for T. cruzi detection in blood samples(Piron et al, 2007) • TaqMan probe directed to satellite DNA for parasite detection in blood of chronic individuals and acute congenital infection = qualitative assay (90% concordance with Nested PCR for the same parasite target). • The system was used to quantify T. cruzi epimastigotes DNA in reconstituted blood samples, reaching a detection limit of 10 parasites/mL.

14. III. Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients (Duffy et al, 2009) • SybrGreen targeted to T. cruzi repetitive satellite sequence. • To estimate parasite load in pediatric patients (n=43) and for the follow-up of those receiving treatment with Bz (n=38); aging 15 days to18 years old. • To monitor Chagas disease reactivation in heart-transplanted patients (n=3) who received immunosuppressive therapy. Melting curve analysis of satellite amplicons from reference stocks melting temperatures > 85ºC Dynamic range of T. cruzisatellite DNA based Q-PCR in reconstituted blood

15. IV. PIDC – Chagas Disease Integrated Program (FIOCRUZ) • 2008 Meeting: Thematic area of Diagnosis • Development and validation of reagents and calibrators for Real Time PCR (TaqManqPCR) – Oswaldo Cruz Institute (RJ), Carlos Chagas Institute (PR), BioManguinhos • DNA extraction standardization by an automated platform • Generating a recombinant plasmid (internal calibrator) A question to be addressed: Which target should be considered?? Standard curve generated from Dm28c (TcI) epimastigotes DNA in blood (10-fold dilutions) y = - 3,1455x + 42,07 2 = 0,9898 R 40 38,57 Cl Brener (TcIIe) Dm28c (TcI) satellite DNA 36,36 35 32,81 31,98 28,82 28,80 30 26,62 25,87 25 Cycle Number (Ct) 22,10 19,49 20 kDNA 15 y = - 3,1675x + 35,148 10 INPA 4167 (Z3) 2 = 0,9977 R Y (TcIIb) 5 0 0.1 0.01 1 10 100 Parasite equivalents/mL blood • Both assays yielded the same detection limit • kDNA was more effective if one considers the lower Ct values inferred from each point of the curve (higher number of sequence targets) • Satellite DNA gave more reproducible results inter-assays Dynamic range of T. cruzi kDNA based Real-Time PCR [n = 6]. Serial dilutions of epimastigote DNA representative of T. cruzi populations. Cl Brener (TCIIe) Cl Brener (TCIIe) Cl Brener (TCIIe) Dm28c (TCI) Dm28c (TCI) Dm28c (TCI) INPA 4167 (Z3) INPA 4167 (Z3) INPA 4167 (Z3) Y (TCIIb) Y (TCIIb) Y (TCIIb)

16. TDRnews March 2009 Held on Buenos Aires (INGEBI-CONICET), coordinated by A. Schijman Support by TDR & PAHO, INGEBI-Conicet UBA and the United Nations University UNUBIOLAC Urgent need of an international multicentric initiative for PCR standardization and validation (different protocols yielding non uniform results) • Participants from 14 countries including Spain, France, Belgium, United Kingdom and USA -CDC Guanidine-EDTA Blood Samples Best sensitivity Best specificity Phenol DNA Extraction Silica Membrane Column K DNA SATELLITE DNA REAL TIME PCR K DNA SATELLITE DNA REAL TIME PCR 100% specificity SYBR green The best PCR practices for clinical setting led to the definition of a Standard Operating Procedure – SOP (to be published soon!)

17. THANK YOU ... AND ENJOY RIO !!!!