1. Conducting flawless research – the right question, the right design Dr. Dick Menzies July 10 th, 2015

2. The starting point in Research – a Problem An unusual clinical observation –An unusual cluster (Outbreak of XDR in S Africa) –An unusual complication (Acquired Rifamycin resistance) An observation from reported data –Temporal trend (Increase in TB in Foreign-born) –Geographic clustering (TB in hospitals) Problems in diagnosis –Delayed diagnosis (cultures, DST) –Missed diagnosis (AFB smears in HIV infected) Problems in treatment: –Treatment too long (Standard “short-course” therapy) –Treatment too toxic (INH) –Treatment too long AND too toxic (MDR, 9INH)

3. An unusual clinical observation – example Extensively drug-resistant tuberculosis as a cause of death in patients with TB and HIV in rural South Africa Neel R Gandhi, Anthony Moll, A Willem Sturm, Robert Pawinski, Thiloshini Govender, Umesh Lalloo, Kimberly Zeller, Jason Andrews, Gerald Friedland Among 475 patients with culture-confirmed TB: –39% (185 patients) MDR-TB –6% (30 patients) XDR-TB. Of XDR-TB: –45% had been previously treated for TB ; –67% had a recent hospital admission. –100% of tested for HIV were co-infected. –98% died, with median survival of 16 days

4. From clinical problem (XDR outbreak) to Research question Questions arising: How (and why) did the patients acquire XDR? Why the association with HIV co-infection? Why the association with hospitalization? Why was mortality so high? Why was mortality so rapid? Each of these questions needs a separate study, and unique design - to be answered. WHICH question should be answered? IS this the right problem?

5. Choosing the right study question Step 1: Definition of the problem –Who gets the problem, –How important is it - does it affect death? Costs? Quality of life? Step 2: Understanding the biology? Manifestations - what organs are affected Pathogenesis - probable or know Transmission/hosts/reservoirs Step 3: make the question as precise as possible –(PICO or pseudo-PICO format)

6. Choosing the right study question “That year in the lab saved me a day in the library” Step 4. Review the literature (again) (and again) –This is often under-utilized - can avoid wasted time –Avoid repeating errors others have made –Do not study a problem that has been well described. RCT of FDCs (have been 15), Comparing positive TST vs IGRA (have been hundreds) –Know what has been described Effect of standard treatment Accuracy of standard diagnostics Step 5. Get the opinions of others –Speak to experts (or pseudo-experts) –Speak to providers –Speak to patients, community members, others affected

7. From Research qst to study design The biggest determinants of study design: 1) Time – investigators’ and patients’ 2) Money (“The biggest determinant of sample size is the budget”) 3) Resources – what do you have to work with? (Diagnostic Laboratory, Molecular Epi, Molecular Biology, Genetics) 4) Collaborators – who do you have to work with? What can they do? These 4 factors determine the kinds of studies you can do – which determines the question you can answer.

8. Hierarchy of study designs Descriptive studies Case reports, and case series Reported data, Prevalence surveys Analytic studies –Observational Cross-sectional Ecologic studies Case control studies Cohort studies –Experimental studies Uncontrolled trials Randomized controlled trials –Individual level –Field trials = group level Increasing costs and complexity

9. Reported Data Useful to: –Define incidence/prevalence –Identify geographic or temporal differences –Describe clinical characteristics –Describe outcomes. Implicit comparison with general population –Risk factors can be identified. –Useful if data is COMPLETE and ACCURATE

10. Coal use ( ) and TB incidence ( ). Temporal trends may indicate clues to causal exposure Coal use and TB in USA: 1953 – 2003

11. Total coal combustion ( ) Notified cases of TB ( ). Temporal trends may indicate clues to causal exposure Coal use and TB in China: 1978 - 2004

12. Advantages Usually very easy and quick studies Take advantage of already gathered data –Exposures –Diseases Disadvantages Relationship may be due to completely unmeasured factors VERY substantial potential for confounding Ecologic Studies

13. Directionality in research Retrospective: Start with persons with disease and ‘look backward’ in time to ascertain exposures. –Advantages: Biggest is convenience – do not have to wait a long time, because disease HAS happened. Makes these studies much quicker to complete, and much cheaper. Prospective: Start with a population and observe them ‘going forward’ in time. Exposures are measured first, and health events are measured afterward, as they occur. –Advantages: Biggest is accuracy of exposure, and certainty that exposure precedes disease.

14. General approach: Measure disease(s) and exposure(s) all at once in a population. Estimate effect: Prevalence odds ratio Advantages Good for common/chronic diseases Good for common exposures Allows one to measure multiple disease or conditions and multiple exposures Disadvantages Measurement of exposure may be difficult –Recall problems if long latency –May change over time (Alcohol, smoking, blood pressure) Can not distinguish cause and effect –(Tobacco Industry defense) Cross-sectional or Prevalence Studies

15. Tuberculin (or IGRA) Surveys A special type of cross-sectional survey Once TST or IGRA convert to positive with TB infection – they remain positive lifelong (some exceptions) Cross-sectional survey – detects all with positive tests – from recent or remote From prevalence of positive test at a given age – can calculate average annual risk of TB infection. Can compare prevalence in different populations If different ages, or different exposure periods can estimate trends in infection

16. University students in Brazil (All BCG vaccinated in infancy) Silva et.al. IJTLD2000; 4:420-426 Average ARI: Preclinical0.2% Clinical2.9% Incidence of TB in Brazil:75/100 000

17. Example of Kaplan-Meier analysis: General Hospital Ventilation and time to TST conversion

18. General Approach: Measure exposures retrospectively in persons with disease (cases) and without disease (controls) Estimate odds of exposure for disease (Odds ratio) Advantages Relatively cheap and quick Particularly useful for studying rare conditions –Or conditions with long latency Disadvantages Controls, Controls, Controls –Very difficult to select proper controls –This is the source of most problems in case control studies –And is why they are generally considered weak evidence. Difficulties of retrospective exposure assessment –particularly if long latency Case Control Studies

19. Fitness costs of drug-resistance mutations in MDRTB: a household-based case-control study. Salvatore…. and Cohen T, JID 2015 Cases: MDR index patients with at least 1 other HH member with same strain Controls: MDR but no-one in HH with same strain Matching: –Same number of contacts in House-Hold –Same extent of Drug Resistance –Same time to detection Not matched: Extent of disease, Cavitation, smear, duration of symptoms, prior episodes, Size of house Findings: katG Ser315Thr mutation associated (OR: 2.39; 95% CI: 1.21- 4.70). BUT combination of katG Ser315Thr & rpsL-Lys43Arg mutations was protective.

20. Nested Case Control study – example TB outbreak in Inuit village Behr, Fox, Khan, Lee, Menzies & many others Major ‘outbreak’ of TB in small village in N Quebec 695/940 residents investigated as contacts during outbreak –Newly infected –50 Confirmed disease, 19 probable disease Case control study of housing, nutrition, lifestyle determinants –“Nested” within all 695 investigated as contacts Case-control1: New Infection –Cases=Newly infected, Controls = Not infected Case-control2: Disease –Cases=Confirmed or Probable, Controls = Infected but no disease

21. General approach: Start with a population free of disease. Measure exposures, and follow them all. Estimate of effect: Risk ratio (Risk disease in Exposed/unexposed) Advantages: Can measure many exposures, and many diseases Temporal relationship clearer (cause before disease) Disadvantages Long and expensive (often very $$$) Good for common diseases (some cancers, cardiovascular). But not for rare diseases or long latency Also what if you fail to measure key determinants –(Solution = freezer) Cohort Studies

22. Average Annual Incidence of Tuberculosis Among Navy Recruits By History of Household Contact Comstock, Edwards, and Livesay; Am Rev Respir Dis 1974; 110:576

23. Incidence of active TB after a positive IGRA The 5 largest studies from high burden countries (Rangaka, Pai, et al. Lancet Inf Dis, 2012) CountryNTestIncidence of active TB in IGRA+ groups The Gambia [Hill et al. 2008] 2348ELISPOT (in- house) 9/1000 person-yr Turkey [Bakir et al. 2008] 908ELISPOT (T-SPOT.TB) 21/1000 person-yr S Africa [Mahomed et al. 2009 (abstract)] 5248QFT 6/1000 person-yr Colombia [del Corral et al. 2009] 2060In-house whole- blood CFP-10 assay 11/1000 person-yr Senegal [Lienhardt et al. PLoS One 2010] 2679ELISPOT (in- house) 14/1000 person-yr

24. Advantages Best method to evaluate an intervention Best control of bias and confounding Disadvantages Not easy or feasible for all interventions Very Expensive and long Not for studies of risk factors or natural history Substantial refusal or drop-out rates can restrict generalizability Population selected may not be representative –Younger adults with only one condition –Often exclude pregnant woman, kids, elderly! Solution – Biomarker? –2 month culture conversion instead of fail/relapse Randomized Trials

25. Five Phase 2 trials: 2 month culture conversion with FQN (all patients received 4 months HR in continuation) Author Year Patients (N) FQN regimen Control Regimen 2 Month Culture Conversion FQNControl Burman 2006 277HRZMHRZE71% Rustomjee 2008 205HRZG/MHRZE79%/42%64%/36% Conde 2009 146HRZMHRZE80%63% Dorman 2009 328MRZEHRZE60%55% Wang 2010 123HRZEMHRZE90%78%

26. Three Phase 3 trials of 4 month regimens with FQN StudyPts. (N) FQN – 4 monthsStandard - 6 months CureDeathFail/ Relapse CureDeathFail/ Relapse OFLUTB1585 79%1%16%83%1%10% Remox 1900 83%1%11%92%1%3% Rifaquin 592 82%018%95%1%4%

27. Experimental Community or Field Trials General Design Pick an intervention to be applied at a community level –Fluoride in water, public education, vaccination Find several communities or population groups Apply intervention to some and not others –Randomly again Measure outcomes at population or group level

28. Cluster randomized Trials – example Effect of improved tuberculosis screening and isoniazid preventive therapy on incidence of tuberculosis and death in patients with HIV in clinics in Rio de Janeiro, Brazil: a stepped wedge, cluster-randomised trial Betina Durovni, Valeria Saraceni, Lawrence H Moulton, Antonio G Pacheco, Solange C Cavalcante, Bonnie S King, Silvia Cohn, Anne Efron, Richard E Chaisson, Jonathan E Golub 29 HIV clinics in Rio de Janeiro. Staff trained in TB screening, TST and INH. Clinics randomly allocated to date of starting the intervention. 2 clinics started every 2 months starting from Sept 2005, until Aug 2009 Outcome: TB incidence +/- death

29. A randomized trial to compare 4 months Rifampin vs 9 months INH for the treatment of LTBI Phase 1: Compliance and completion Completed in 2003 Phase 2 – Adverse events and costs Completed in 2007 Phase 3: Efficacy and effectiveness Enrolment completed in 2014: 840 children, 6020 adults Follow-up will finish in 2016 Publication in ???

30. Thanks

31. Randomized Trials Intensified regimen containing RIF and Mfx for TB meningitis: an open-label, randomised controlled phase 2 trial Rovina Ruslami*, A Rizal Ganiem*, Sofiati Dian, Lika Apriani, Tri Hanggono Achmad, Andre J van der Ven, George Borm, Rob E Aarnoutse, Reinout van Crevel Open-label, phase 2 trial. Factorial design Patients aged >14 with TB meningitis –All received INH & PZA & Steroids Randomly assigned for 14 days – by computer-generated schedule: –RIF: 450 mg vs 600 mg, intravenously, and –Mfx 400 mg, or Mfx 800 mg, or EMB 750 mg daily. 14 days of treatment all patients, then routine treatment. Endpoints: PK analyses (blood and CSF) adverse events attributable to tuberculosis treatment, and survival.