South Africa

July 2016

Valeria Rolla, Afranio Kritski, Bruno Andrade,

Marcelo Cordeiro, Betina Durovni,

Solange Calvacante, Jose Roberto Lapa e Silva, Catherine McGowan, Timothy

Sterling

Protocol

900 adults with culture-positive pulmonary TB

Cohort A

Drug-susceptible or drug-resistant

Followed for response to therapy, TB recurrence

2,700 close contacts of the TB patients in Cohort A

Cohort B

TST or QuantiFERON positive or negative

Followed for development of TB

Duration of follow-up: 24 months

Specimen Collection

TB Patients

Baseline, 2 months, end of therapy, TB recurrence

Close Contacts

Baseline, end of therapy (if any, or at 6 months), TB

disease

Biorepository

Study sites

Sites and Specimen flow

Rio sites and Specimen flow

Duque de Caxias

Fiocruz - INI

Rocinha

UFRJ

Laboratory Quality ControlCurrent Status

Mycobacteriology labs (SMILE)

Evaluations: twice a year

All Labs approved:

Fiocruz: approved since August 2013

Salvador: approved since March 2015

Manaus: approved since March 2015

+UFRJ: developing new lab (space, equipment, personnel)

Repeat SMILE visit to all labs in October 2016

Laboratory Quality ControlCurrent Status

Immunology labs (Duke for PBMCs, Qiagen for QuantiFERON)

Evaluations: every quarter

Rio de Janeiro

Fiocruz: approved since June 2015

UFRJ: approved since June 2015

Salvador: approved since March 2016 (new lab)

Manaus: approved since December 2015

Specimen processing, flow, storage: SOPs finalized at allsites

Data/Specimen Quality

Shipping of specimens:

From Rio de Janeiro’s study sites to the immunology and

microbiology labs at Fiocruz and UFRJ

From labs to the Biorepository:

Temperature control, time to receive samples and storage

Protocol / data collection / specimen monitoring:

Case Report Forms (CRFs)

Label system

REDCap system to collect data from study participants, and track

specimen transportation from the study sites to local labs, then to

Biorepository

Biorepository

Biorepository: Salvador, Bahia

Fundação José Silveira: Instituto Bahiano de Reabilitação

All equipment installed, personnel trained

Freezers, N2 containers, computer/inventory system, back-up generators

Monitoring systems in place

FreezerPro system working very well

Institutional infrastructure, SOPs in place

Started from a brand new location and now will be expanded

More than 12,500 samples!

Load: Freezers (7,5%), N tank (9%)

Biorepository

Biorepository Expansion:

- Fundação José Silveira investment

Biorepository

Distribution (+12,500):

Plasma: 4,099 vials

PBMCs: 931 vials

Whole blood: 972 vials

Urine: 4,131 vials

Sputum: 810 vials

TB isolates: 436 vials

QuantiFERON: 1,200 vials (400 each)

Paxgene tubes: 343 tubes

Receiving area

Inventory/Management system - office

Budget Received:

$1.5M from NIH

$1.0M from Brazilian Ministry of Health

$500K received in 2016

Extra costs not originally planned:

Equipment for labs, lab QC programs, extra personnel and

suplies for immunology labs, IATA training, etc.

Special labels (long duration)

FreezerPro

Specimen transportation

BSC and HEPA filters for Biorepository

Generators, alarms...

EnrollmentStatus as of July 8 2016

Protocol approved at all study sites; CONEP approval notrequired

All 5 sites enrolling:

Cohort A: 184 TB cases

20% treatment completed

10% MDR

12% Diabetes

Cohort B: 162 close contacts

EnrollmentStatus as of July 8 2016

Rio de Janeiro:

INI-Fiocruz – 89 participants (started JUN 2015)

Rocinha – 62 participants (started SEP 2015)

Duque de Caxias – 121 participants (started SEP 2015)

Salvador – 23 participants (started JUN 2016)

Manaus – 51 participants (started JAN 2016)

Overall Areas of Scientific FocusTranslational studies

M. tuberculosis transmission and infection

Biomarkers to identify patients at risk for:

Progression from M. tuberculosis infection to TB disease

Poor response to TB treatment

Risk factors for TB recurrence (relapse, reinfection)

Role of mixed M. tuberculosis infection

Epidemiologic studies

MDR-TB and XDR-TB

TB/HIV

TB and other co-morbid conditions: diabetes, alcohol,

tobacco, etc.

Scientific projects affiliated with RePORT

Biomarker discovery for tuberculosis infection and disease

Co-funded by CNPq (Brazil) and NIH (U.S.)

Social determinants of TB and HIV in Brazil (Rocinha)

Funded by Vanderbilt University

Pharmacogenomic predictors of treatment toxicity, failure, and relapse in HIV-related tuberculosis

R01 funding pending

Mixed M. tuberculosis infection and whole genome sequencing (Rio)

Afranio Kritski, Sebastien Gagneux. Submitted to FAPERJ

Mechanisms of emergence of drug resistance in MDR-TB

Bob Horsburgh (RePORT-India + Brazil) R01 to be resubmitted September 2016

Other areas of interest:

Biomarkers of TB infection/disease

TB/Diabetes

Gene ontologies differentially expressed in TST +

household contacts compared to active TB cases

Gene Ontologies %1Zinc Regulation 6,1

2Interferon Regulation 14,3

3Chemokines 2,0

4Cytokines 16,3

5Receptors & intracellular receptors 20,4

6Complement 6,1

7Neuronal precursors 6,1

8Grow Factors 4,1

9Mithocondrial Regulators 4,1

10Retinoic Acids 2,0

11Intracelluar mediators 18,4

Biomarker discovery for TB infection and

disease - CNPq and NIH: Preliminary Data

Metabolism profile for anti-TB drugs and

efavirenz in HIV/AIDS patients in Rio de

Janeiro

Samples from a randomized clinical trial to treat TB-HIV

patients with EFV 600 or 800mg and TB drugs in fixed-

dose combination

Study drug metabolism and ancestry markers in Rio de

Janeiro’s TB-HIV patients

53%45%

2%

Acetylation profile based only on theknown functional genotypes

Intermediate

Slow

Fast

Perfil de acetilação na população brasileira. Osgráficos mostram a distribuição dos status deacetilação nas 5 macrorregiões do Brasil. Os valoresestão expressos em percentagem.

Acetylation profile according to region of Brazil

Viral load <1000 and>1000 Fisher's exact test

NAT2

1 - 1 (1.8) 0 (0)

2 - 29 (51.8) 8 (66.7) 0.609

3 - 26 (46.4) 4 (33.3)

CYP2E1

1 - 69 (95.8) 12 (92.3)

2 - 3 (4.2) 1 (7.7) 0.492

CYP2B6

1 - 33 (46.5) 3 (25) 0.023

2 - 36 (50.7) 6 (50)

3 - 2 (2.8) 3 (25)

GSTM1

1 - 27 (37.5) 4 (30.8) 0.761

2 - 45 (62.5) 9 (69.2)

DOSE EFV 600 or 800mg

600mg - 36 (50) 5 (38.5) 0.642

800mg – 36 (50) 8 (61)

Adverse reactions during therapy

G1 and 2 - 35 (48.6) 10 (76.9) 0.114

G3 and 4 - 37 (51.4) 3 (23.1)

SNPs profile of TB-HIV patients treated with

efavirenz based regimen 600X800mg

SNP profile of TB-HIV patients treated with efavirenz

based regimen 600X800mgAdverse reactions to EFV (G1 and2XG3and4) Fisher's exact test

NAT2

1 - 0 (0) 1 (1.7)

2 - 8 (72.7) 30 (51.7) 0.431

3 - 3 (27.3) 27 (46.6)

CYP2E1

1 - 13 (92.9) 69 (95.8)

2 - 1 (7.1) 3 (4.2) 0.516

CYP2B6

1 - 7 (53.8) 30 (42.3) 0.54

2 - 5 (38.5) 37 (52.1)

3 - 1 (7.7) 4 (5.6)

GSTM1

1 - 7 (50) 24 (33.3 0.377

2 - 7 (50) 48 (66.7)

DOSE EFV 600 or 800mg

600mg - 3 (21.4) 39 (54.2) 0.051

800mg 11 (78.6) 33 (45.8)

Age

Mean 36.8 (9.6) 39.5 (10.2) 0.114

AFR EUR NAMSample Size

AFR 0.996 0.002 0.002 105

EUR 0.002 0.993 0.005 158

NAM 0.002 0.023 0.976 64

TB HIV 0.361 0.473 0.166 96

Genetic ancestry at the individual level using

autosomal ancestry informative marker (AIM)-indels

Recent published data reflecting RePORT-Brazil’s

other research interests related to TB

Conclusions

Outstanding progress has been made:

Protocol implementation

Laboratory infrastructure

Establishment of Biorepository

Participant enrollment

Focus over the next 6-12 months:

Continue enrollment and retention

Increase funding through grants and other sources