USMLE Biostats & Epi - A Complete Review - Azfar Basunia

USMLE
BIOSTATISTICS
A complete review
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Azfar Basunia, MD
2
Table of
Contents
Azfar Basunia, MD 3
1 Epidemiology & Population Health
Azfar Basunia, MD
Measures of
disease frequency
Prevalence:
╸ Disease frequency or burden of disease
# 𝑜𝑓 𝑐𝑎𝑠𝑒𝑠 𝑎𝑡 𝑎 𝑡𝑖𝑚𝑒 𝑝𝑜𝑖𝑛𝑡
𝑡𝑜𝑡𝑎𝑙 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑡ℎ𝑎𝑡 𝑡𝑖𝑚𝑒 𝑝𝑜𝑖𝑛𝑡
╸ Pretest probability of disease
╸ Directly related to PPV and inversely related to NPV
Azfar Basunia, MD 5
Measures of
disease frequency
Incidence:
╸ Measures risk of disease
╸ The proportion of at-risk, disease-free population that develops
the disease over a defined time period
# 𝑜𝑓 𝑛𝑒𝑤 𝑐𝑎𝑠𝑒𝑠 𝑜𝑣𝑒𝑟 𝑎 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑
𝑡𝑜𝑡𝑎𝑙 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑟𝑖𝑠𝑘 𝑡ℎ𝑒 𝑠𝑡𝑎𝑟𝑡 𝑜𝑓 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑
╸ Population at risk: SIDSà children > 1 year are not at risk
╸ Also known as cumulative incidence
Azfar Basunia, MD 6
Measures of
disease frequency
Incidence rate:
╸ Accounts for when disease occurs, dynamic population and
losses to follow-up
# 𝑜𝑓 𝑛𝑒𝑤 𝑐𝑎𝑠𝑒𝑠 𝑑𝑢𝑟𝑖𝑛𝑔 𝑜𝑏𝑠𝑒𝑟𝑣𝑎𝑡𝑖𝑜𝑛 𝑝𝑒𝑟𝑖𝑜𝑑
𝑡𝑜𝑡𝑎𝑙 𝑝𝑒𝑟𝑠𝑜𝑛 𝑡𝑖𝑚𝑒 𝑜𝑓 𝑜𝑏𝑠𝑒𝑟𝑣𝑎𝑡𝑖𝑜𝑛 𝑤ℎ𝑖𝑙𝑒 𝑎𝑡 𝑟𝑖𝑠𝑘 𝑑𝑢𝑟𝑖𝑛𝑔 𝑠𝑡𝑢𝑑𝑦
╸ Convention for person-time => per person-years or per
10/100/1000/ . . . person-years
Azfar Basunia, MD 7
Measures of
disease frequency
Relationship between incidence and prevalence
╸ Chronic diseases (DM, HTN): prevalence > incidence
╸ Increased survival and/or improved quality of care
╸ Acute/short lasting diseases (sepsis, flu): prevalence ≈ incidence
╸ Increased mortality or faster healing time
╸ Vaccination: decreases both incidence and prevalence
Azfar Basunia, MD 8
Measures of
disease frequency
Example
╸ Rural community with population of 10,000 people. 5,000 have T2DM at the
end of 2019. 100 new cases of T2DM arose between 2019 and 2020. In the
interval, 50 people passed away from complications of T2DM.
╸ Prevalence of T2DM at the end of 2019 = 5,000/10,000 = 0.5

╸ Prevalence of T2DM at the end of 2020 = (5,000+100-50)/(10,000-50) =
0.51
╸ Incidence of T2DM between 2019 and 2020 = 100/5,000 = 0.02
Azfar Basunia, MD 9
Measures of
Health Status
Mortality
╸ Number of deaths in a population within a time interval
Morbidity:
╸ Number of persons in a population with a disease at a specific
timepoint
Azfar Basunia, MD 10
Measures of
Health Status
Maternal mortality
╸ (Maternal deaths)/ (total number of live births in a year)
╸ Death of pregnant or w/i 42 days of birth or pregnancy termination
Children mortality
╸ Neonatal mortality: (# infant death w/i first 28 days of life) / (total
number of live births in a year)
╸ Infant mortality: (# infant death w/i first year of life) / (total number
of live births in a year)
╸ Under 5 mortality: (# children death w/i first 5 years of life) / (total
number of live births in a year)
Measures of
Health Status
Crude Rate
╸ Total number of cases at a given time point divide by the total
number of persons in the population
╸ Expressed as per 100/1000/10,000/. . .
╸ Does not account for/ adjust for confounding (e.g. age, sex)
Example:
╸ Town A: Population = 50,000; deaths = 3,000/year; crude mortality
rate = 60 per 1,000
╸ Town B: Population = 58,750; deaths = 4,100/year; crude mortality rate
= 70 per 1,000
╸ Is age distribution distorting the crude rates?
Measures of
Health Status
Adjusted Rate: Adjusts for possible confounding (e.g. age), allows for
better comparisons
Example: Age Specific mortality rates
Town Age # people % of total Deaths per Death rate per 1,000
group pop year
A ≤ 65 yr 25,000 50% 1,000 40
A > 65 yr 25,000 50% 2,000 80
B ≤ 65 yr 15,000 25.5% 600 40
B > 65 yr 43,750 74.5% 3,500 80
Measures of
Health Status
Standardization
╸ Adjustment of rates that take into account vital differences
between populations (typically age)
╶ Apply a standard age distribution (such as population
distribution of US by age group)
╶ Allow for directly comparison of health outcomes
╸ Commonly birth rates, death rates and unemployment rates
╸ Eg. Age-specific mortality rates among various US states
Measures of
Health Status
Case fatality rate (CFR)
╸ Proportion of patients with a particular disease/condition who dies
from the complications of the disease/condition
╸ Must be distinguished from mortality rate
╶ Analogous to risk of dying from a disease/condition in the
general population
╸ Eg. 10% of pts with diabetic nephropathy develop renal failure, but
75% pts with renal failure will die from its complications.
╶ CFR of renal failure among diabetic nephropathy pts = 75%
Measures of
Health Status
Life expectancy
╸ The average number of years an individual is expected to live
based on current death rates
╸ Account for social group, geographic region, sex and year of birth
etc.
Health-adjusted life expectancy

╸ The average number of years an individual can expect to live in
full health after adjusting for years lived in poor health due to
disease and/or injury
Measures of
Health Status
Risk factor
╸ an attribute that increases probability of disease
╸ Eg. Smoking is a risk factor for lung cancer
Population attributable risk (PAR) &

Population attributable risk percent (PAR%)
╸ Will be discussed in Measures of Association section
Measures of
Health Status
Latency
╸ Time between inciting pathologic events or exposure to disease
manifestation or development of clinical signs and symptoms
╸ Can apply to both risk factors and risk reducers
╸ Eg.
╶ Risk factors:
╶ Bacterial infections have short latency periods;
╶ Chronic diseases like HTN, DM, HIV have long latency periods;
╶ Risk reducers: Lung healing after smoking cessation in chronic
smokers may take years w/o full return to baseline functioning
Survival Analysis
Kaplan-Meier curve
╸ Measure disease prognosis,
time-to-event analysis
╸ Y axis, estimated probability
of event-free survival
╸ X axis, time to follow-up since
entry into study
╸ Each step/vertical drop denotes an event = death
╸ Small vertical lines in the graph represent censoring, those lost of
follow-up, dropped out etc.
Survival Analysis
Kaplan-Meier curve
╸ Simple inferences from graph
╸ Median survival = 50%
survival = 28 mo vs. 115 mo
╸ Survival at 36 mo = 46% vs.
75%
╸ Log-Rank test
╸ Compare survival curves
╸ p < 0.05, survival between groups is significantly different
╸ P > 0.05, survival between groups is not significantly different
20
Composite health status
indicators, measures of
population impact
Quality Adjusted Life Years (QALYs)
╸ Measure burden of disease
╸ Economic impact of interventions on population
╸ Time Trade Off (TTO) = a tool for QALY calculation
╶ 1 year in healthy life = 1 TTO; 1 year in disability, 0 < TTO < 1
╸ Interventions are aimed at maximizing QALYs
population impact
Example
╸ 40-year-old patient, previously healthy until age 30, history of
meningitis at age 30 and subsequent stroke and right sided
hemiparesis. Every 5 years of life in his current state = 1 year of life in
full health.
╸ His TTO after disability = 1/5 = 0.2. He lived 3o years in TTO = 1, and 10
years in TTO = 0.2.
╸ QALYs = (30 × 1) + (10 × 0.2) = 32
╸ NOTE: Formulas will differ for populations. Focus on understanding
when QALYs are utilized
population impact
Years of Potential Life Lost (YPLL)
╸ Measure of premature mortality based on standard, age-based tables
of life expectancy
╸ sum of the years of life lost annually by persons who suffered death
prior to life expectancy
population impact
Example
4 people A, B, C and D died at ages 70, 50, 10 and 90 years of age
respectively in a small rural community in 2019. Life expectancy for the
town is 75 years of age. Calculate YPLL
╸ YPLL of the community = YPLLA + YPLLB + YPLLC + YPLLD
╸ YPLLA = 75-70 = 5 years; YPLLB = 75-50=25 years; YPLLC = 75-10 = 65
years. YPLLD = 0 (since 90 > life expectancy of 75).
╸ YPLL of the rural community = 5+25+65+0=95 person-years
population impact
Disability Adjusted Life Years (DALYs)
╸ Compare overall health and life expectancy among countries
╸ Represent difference between current health situation and ideal
living in perfect health up-to the standard life expectancy
╸ Based on premature mortality (YPLL) and burden of living with a
disease or disability (year lived with disability, YLD)
╸ Interventions aimed at minimizing DALYs
population impact
Example
╸ A previously healthy male develops T2DM at 30 and passes away
from MI at 60. Standard life expectancy is 75 and disability weight for
T2DM is 0.5 (all hypothetical numbers).
╸ YPLL = 75-60 = 15 years
╸ YLD = years lived in disability × disability weight = 30 × 0.5 = 15 years
╸ DALYs = YPLL + YLD = 15 + 15 = 30 years
╸ NOTE: Formulas will differ for populations. Focus on understanding
when DALYS are utilized
population impact
Standardized Mortality Ratio (SMR)
╸ Used in occupational epidemiology
Observed deaths in the study group
Expected deaths in the study group
╸ Expected deaths based on age-specific mortality rates among
general US population
╸ SMR > 1 indicates greater deaths among the study group

compared to general US population
╶ E.g. SMR = 2 indicate 2× more deaths among study population
population impact
Standardized Mortality Ratio (SMR)
╸ SMR < 1 indicates fewer deaths among the study group compared
to general US population
╸ SMR = 1 indicated observed deaths = expected deaths

╶ Considered null value
population impact
Standardized Incidence Ratio (SIR)
╸ Used in cancer epidemiology, to compare the incidence of cancer in a
small population to a larger, control population
Observed cases in the target popupulation
Expected cases in a larger, control population
╸ Expected cases are based on age and sex-specific incidence rates
among the population of a state, the general US population etc.
╸ Interpretation similar to SMR:
╶ SIR = 1 => incidence similar
╶ SIR > 1 => incidence larger in the target population
╶ SIR < 1 => incidence smaller in the target population
Population pyramids &
impact of demographic
changes
Distribution of a population by sex
and age groups
Expansive pyramid:
╸ Higher percentage of youthful
population
╸ High birth rates + low life
expectancy
Source: Wikipedia
Population pyramids &
impact of demographic
changes
Constrictive pyramid:
╸ Higher percentage of elderly
population
╸ Low birth rate + high life
expectancy
Stationary pyramid:
╸ Age and sex distribution remain
stationary/constant over time
╸ Birth rate = death rate Source: health.pa.gov
Disease surveillance &
outbreak investigation
Disease surveillance:
╸ monitor spread of disease to establish patterns of progression
Disease Reporting
╸ Cornerstone of disease surveillance
╸ Formal reporting of notifiable infectious diseases required by healthcare
workers
╸ Around 80 reportable diseases in USA (COVID-19, TB, anthrax, rabies,
chlamydia, gonorrhea, HIV, measles, mumps, rubella etc.).
╶ Full list in CDC website: https://ndc.services.cdc.gov/
╸ Incidence of these diseases à indicators of overall health of population
Public health advisors (PHA)
╸ Public health workers doing field work or contact epidemiology
╶ Contacting, interviewing and locating people who have been
exposed to an infectious agent
╶ Offer treatment and follow-up options
╸ Called upon to respond to public health or humanitarian crisis
Health promotion
╸ Public policy to address health determinants such as income, housing,
food security, employment and working conditions
╸ Eg. Health literacy in schools, breastfeeding promotion in clinics
Recognition of clusters
╸ Cluster: An usually large
aggregate of a medical
condition, disease or event
within a particular geographic
location.
╸ If clusters of sufficient size or
importance are identified à
re-evaluate as outbreaks
Source: Johns Hopkins University
Communicable
disease transmission
Attack rate
╸ Cumulative incidence of disease, especially in the setting of outbreak
╶ Proportion of people with disease divided by total population at
risk
╸ Eg. In one year, 100 new cases of cholera in a rural village with
population of 8000.
╶ Attack rate of cholera = 100/8000 = 0.0125 = 1.25% per year
Communicable
disease transmission
Herd immunity
╸ Indirect protection from infectious diseases when certain percentage
of population becomes immune to an infection
╶ Either through vaccination or natural infection
╶ Reduces likelihood of transmission of infection to unvaccinated
individuals
╸ Threshold varies depending on infectivity of the pathogen
╶ Pertussis ~ 82%, Measles ~ 95%
Points of
intervention
Preventative Medicine
╸ Primordial prevention:
╶ Addressing risk factors (environmental, socioeconomic, behavioral).
╶ Eg. Encouraging healthy lifestyle and diet to prevent obesity, DM and
HTN (risk factors for cardiovascular disease)
╸ Primary prevention:
╶ Prevent disease occurrence
╶ Decreases both incidence and prevalence of disease
╶ Eg. Vaccinations, folate supplementation in pregnant women to
prevent neural tube defects in their children
Points of
intervention
╸ Secondary prevention:
╶ Target disease early in its course to promote early intervention,
prevent disease progression and irreversible damage
╶ Screening and follow-up management
╶ Eg. Cardiovascular screening (BP, lipid panel), cancer screening (eg.
Colonoscopy, mammograms)
╸ Tertiary prevention:
╶ Limit impairments and disability from a disease that has already
progressed to its advanced stages
╶ Decrease morbidity and mortality after disease onset
Points of
intervention
╸ Tertiary prevention:
╶ Eg. Stroke rehabilitation programs after acute stroke, tamoxifen
adjuvant therapy in breast cancer to reduce recurrence
╸ Quaternary prevention
╶ Limit unnecessary or excessive interventions by a health system that
could be harming more than benefiting the patients
╶ Eg. Avoid CT scan in infants unless absolutely necessary, avoid
prescribing antibiotics when viral disease is more likely
Points of
intervention
Community level
╸ Taxation (tobacco tax and soda taxes)
╶ Excising taxes on tobacco products and sugary drinks to
discourage purchase and reduce consumption
╶ Primordial and primary prevention
╸ Smoke-free cities, buildings, restaurants and public spaces
╶ Prevent harm from second-hand smoke exposure
╶ Risk of lung CA, emphysema, bronchitis and cardiovascular
diseases
╶ Primordial and primary prevention
Points of
intervention
School policies
╸ School based health and nutrition services promote early detection,
correction and prevention of disease and disability in children
╸ Eg. Safe-sex education, healthy lunches, presence of a school nurse
╸ Broader positive impacts in the children’s families and communities
Points of
intervention
Social determinants of health
╸ Economic and social factors that influence both individual and group
differences in health status, risk of disease or vulnerability to injury
╸ Eg. Education, income distribution, working conditions, healthy food
and clean water, housing, safe environments, gender, race, disability
╸ Possible Interventions
╶ Education: adequate teacher to student ratio, health curriculum
╶ Urban development: affordable housing, removal of lead paint,
parks, reliable public transportation
╶ Public policy: ADA, ACA, FMLA, smoking ban inside restaurants
2 Study design, types & selection
Azfar Basunia, MD
Descriptive
Studies
╸ (-) Cannot establish causality
Case report
╸ Describes an unusual disease presentation/outcome in a single
patient
╸ Can aid in hypothesis formulation
Case series
╸ Collect and analyze disease presentation, course or treatment
response of several patient cases
╸ (-) No control group, prone to selection bias since the researchers
are selecting the subjects
Analytical Studies:
Observational
Cross-sectional (individuals)
╸ Also known as prevalence study
╸ Examine associations between exposures (risk factors) and outcomes
(disease) at a particular time point
╶ Simultaneously measure frequency of exposure(s) and disease
╸ E.g. Investigators at a state hospital are studying links between
incarceration and development of active TB. They collected data on
all active TB cases and prior incarceration history from the hospital
charts.
Analytical Studies:
Observational
Cross-Sectional Surveys (Community Surveys)
╸ A form of cross-sectional study
╸ A group of subjects from a defined population are selected and
evaluated for exposure of interest at a particular time point
Analytical Studies:
Observational
Ecological studies
╸ Assess the relationship between outcome/disease and exposure
at a population level (country, state, city etc.)
╸ (-) Ecological Fallacy: Cannot make inferences on individual level
based on group characteristics
╸ E.g. Determining incidence of new COVID-19 cases among U.S.
counties and rates of vaccination
Analytical Studies:
Observational
Cohort Study
╸ Assess if an exposure/risk factor is associated with disease
╸ Method:
╶ Identify a group (cohort) with common characteristics (age,
background, geography, sex, etc)
╶ Divide subjects based on exposure status to risk factor
╶ Follow cohort over a period of time and determine if they
develop disease of interest
╸ Main outcome of interest: incidence.
╶ Risk of disease among exposed compared to risk of disease
among unexposed
Analytical Studies:
Observational
Prospective Cohort Study
╸ Study begins before outcome/disease has occurred
Retrospective Cohort Study
╸ Study begins after exposure + outcome/disease has occurred
╶ Patient charts are reviewed to gather data but the same
protocol as prospective cohort study is followed
Analytical Studies:
Observational
╸ Patients are followed over time
╶ (+) Can calculate incidence and risk of disease due to exposure
╶ (-) Need to determine exposure prior to study initiation
╶ (-) For rare diseases, large cohort needed, which drives up cost
Analytical Studies:
Observational
Example: Prospective design
╸ Clinicians want to investigate links between HTN development and heavy
smoking among males age 40-50
╸ A busy clinic had 900 new male pts w/o HTN between age 40-50.
55.56% of these pts are heavy smokers (>1 ppd).
╸ Pts’ are followed for next 5 years and new onset HTN is determined.
╸ 400 patients developed HTN among heavy smokers and 50 patients
developed HTN among non heavy smokers. RR = 6.4
╸ Conclusion: Risk of new onset HTN was 6.4 times greater among
heavy smokers for 5 years compared to non-heavy smokers
Analytical Studies:
Observational
Example: Retrospective design
╸ Clinicians want to investigate links between HTN development and heavy
smoking among males age 40-50
╸ 900 male pts w/o HTN between age 40-50 who were followed by the
clinic for the past 5 years were identified. 55.56% of these pts are heavy
smokers (>1 ppd) for the past 5 years.
╸ Pts’ charts were reviewed and new onset HTN w/i past 5 years is noted.
╸ 400 patients developed HTN among heavy smokers and 50 patients
developed HTN among non heavy smokers. RR = 6.4
╸ Conclusion: Risk of new onset HTN was 6.4 times greater among heavy
smokers for 5 years compared to non-heavy smokers
Analytical Studies:
Observational
Case-Control Study
╸ Assess if an exposure/risk factor is associated with disease
╸ Method:
╶ Identifying subjects with disease (cases) and subjects without
disease (controls)
╶ Matching Controls: patients w/o disease with similar features
(age, sex) from the same source population
╶ Goal is to limit confounding
╶ Look retrospectively and determine exposure/risk factor status
of cases and controls
Analytical Studies:
Observational
Case-control-study
╸ Main outcome measure: Odds ratio (OR).
╶ Compare odds of exposure among cases with odds of exposure
among controls
╸ Patients are not followed over time
╶ (+) Can be used in rare diseases (e.g. genetic diseases)
╶ (+) diseases with long latency period (e.g. cancer)
╶ (+) Cost efficient
╶ (-) Recall bias (especially in cases)
╶ (-) cannot calculate prevalence or incidence
Analytical Studies:
Observational
Example of Case Control Study
╸ Researchers are interested to investigate if hypertension (HTN) is associated
with heavy smoking (>1 pack/day) for 5 years in NYC.
╸ They identified 500 cases of HTN (disease) from 10 hospitals in NYC and
400 matched controls without HTN admitted in the same hospitals.
╸ Among pt with HTN, 400 were heavy smokers w/i past 5 years. Among pt
w/o HTN, 50 were heavy smokers w/i past 5 years
╸ Researchers then measured OR (28), which was statistically significant
(p<0.05). Odds of HTN were 28× greater among heavy smokers w/i past 5
years compared to non-heavy smokers.
Analytical Studies:
Observational
Nested Case-Control Study
╸ Cases and controls are drawn from participants of a prior, defined
cohort (i.e. cohort study)
╸ Cases have outcome of interest; controls do not
╸ Risk factors associated with outcome of interest can be investigated
╸ Eg. Examining effect of OCPs on VTE by drawing cases and controls
from nurse’s health study
Analytical Studies:
Interventional
Clinical Trial
╸ Prospective studies that assess whether new treatments are safe and
effective in patients with the disease of interest
╸ Exposures or treatments (drug, surgery, intervention) are assigned to
patients unlike observational studies
╸ End-points are defined prior to study initiation
╶ Primary: Outcome(s) the study is designed to evaluate. Number
of participants needed to detect meaningful differences of
intervention (i.e. power calculation/sample size determination) is
based on this. Eg. All-cause mortality, hospitalization
Analytical Studies:
Interventional
╶ Secondary: Outcomes of interest (e.g. effect of intervention on
co-morbidities, development of drug side effects )
╶ Combined: Combining multiple (categorical) endpoints (such
as all-cause mortality, MI, stroke, limb amputation). Can be
primary or secondary.
╶ Surrogate: Outcomes (lab test, imaging or physical finding)
that are predictive of future severe outcomes. E.g. rescue
inhalerà asthma control
╸ Outcomes reported: incidence, risk, survival analysis
Analytical Studies:
Interventional
Features of clinical trials
╸ Randomization: pts are randomized into treatment and control arm.
╶ Minimizes both obvious and hidden bias and confounding
╶ “Table 1” - baseline characteristics in treatment and control groups
╸ Blind: Not informing which pts are assigned to treatment or control
╶ Single blind: Only patients are unaware of their assignment
╶ Double blind: Both patients and researchers are unaware of
patient assignment. This is considered gold standard
╶ Triple blind: Patients, researchers and data analysts are unaware
of patient assignment.
Analytical Studies:
Interventional
Features of clinical trials
╸ Placebo controlled: Control subjects receive placebo
╸ Non-inferiority/ equivalence: Control subjects receive standard of
care or other treatment
╶ Goal: new drug is not worse than control drug by an acceptable
margin (the non-inferiority margin)
Analytical Studies: Non-inferiority study:
A. Not non-inferior and inferior
Interventional B. Non-inferior and inferior
D C. Non-inferior and not superior
D. Non-inferior and Superior
C
B ╸ Solid vertical line à superiority margin

A ╸ Dashed vertical line à non-inferiority
margin
╸ Goal should be B, C, or D in non-
0 inferiority trial
Therapy under Therapy under
investigation is investigation is
inferior superior
Analytical Studies:
Interventional
Head et al. Eur Heart J. 2012
Analytical Studies:
Interventional
Types of clinical trials
╸ Phase 1:
╶ Evaluate drug safety: toxicity, maximum tolerated dose,
pharmacokinetics, pharmacodynamics
╶ Small number (<50) of healthy subjects
╸ Phase 2:
╶ Further evaluate drug safety: optimal dosing, efficacy and adverse
effects
╶ Small number (<100) of patients with disease of interest
Analytical Studies:
Interventional
Types of clinical trials
╸ Phase 3:
╶ Evaluate drug safety and efficacy for marketing: Compare drug to
current standard of care (if available) or placebo
╶ Randomized, blinded and controlled trial with large number (>100)
of patients with disease of interest
╶ Generally required for FDA approval
╸ Phase 4:
╶ Post marketing surveillance: safety studies ( rare and long-term
side effects)
Analytical Studies:
Interventional
Cluster, randomized controlled trials
╸ Participants are grouped into clusters, which are then randomized into
control or intervention.
╸ Individuals with similar background characteristics are usually
assigned to the same cluster
Factorial study (fully crossed design)
╸ (Randomized) study ≥ 2 interventions and all possible combinations.
╸ Eg. Exercise vs dietary modification for lowering BP. 4 possible
combinations: Exercise alone, diet alone, both, neither (control).
Analytical Studies: Abaluck et al. 2021.
Interventional
https://www.nber.org/system/files/
working_papers/w28734/w28734.
pdf
Community Intervention/ Community trials
╸ Trials involving entire communities instead of individuals
Example:
╸ Design: Cluster-randomized controlled trial with 600 communities in
Bangladesh with control arm (no special instructions), surgical masking
and cloth masking (both free mask distribution, how to mask, why its
important to mask).
╸ Monitor Sars-Cov-2 spread through symptoms + serological confirmation.
Results: 9% reduction in COVID transmission in surgical mask arm, cloth
masks arm transmission comparable to control arm.
Analytical Studies:
Interventional
Crossover study
╸ Participants serve as their own control after a brief washout period
╸ Design: Pts randomized to tx vs control à washout à switch
╸ (+) Controls for confounding, esp with fewer pts
╸ (-) Ineffective washout period and lingering effects of intervention
Systematic reviews
& meta-analysis
Meta-analysis
╸ Pooling data from several studies to increase statistical power
╶ detecting a difference in outcome of interest between groups
when one exists
╸ (+) More precise results
╶ good for rare diseases/outcomes
╶ Difference in outcomes between groups is small
╸ (-) Publication bias:
╶ Results are only as good as studies used
╶ pooling of biases and limitations of individual studies
╶ E.g. Using only studies with statistically significant results
Systematic reviews
& meta-analysis
Systematic reviews
╸ Compiling of primary studies and summarizing of evidence to
answer a defined question
╸ Does not involve statistical analysis
╸ (-) Publication bias
Systematic reviews
& meta-analysis
Funnel Plot:
╸ Assessment of publication bias
╸ Treatment effect (x-axis)
plotted against standard error
of treatment effect (y-axis)
╸ Look for symmetric distribution
of studies along the vertical line
╸ Asymmetric distribution
suggests publication bias
Source: Zhang et al. 2016
Systematic reviews
& meta-analysis
Study Heterogeneity:
╸ In an ideal world, studies pooled in meta-analysis would be undertaken
with the same experimental protocols à homogeneity
╶ Differences b/w outcomes only due to measurement error
╸ In real world, variability goes beyond what is expected from
measurement error
╶ Differences in investigated populations, treatment schedules,
endpoint definitions, etc
╸ Presence of some heterogeneity can be expected.
╶ Accounted by techniques such as random effects modeling,
stratified analysis
Systematic reviews
& meta-analysis
Risk of bias:
╸ Assess each study in a systematic review/ meta-analysis for bias in
domains of selection, performance, detection, attrition and report
╸ Classify as low risk, high risk or unclear
Risk of bias graph

from a single study
Source: Cochrane handbook
Systematic reviews
& meta-analysis
Risk of bias summary

from multiple studies in a
typical meta-analysis
Source: Cochrane
handbook
Systematic reviews
& meta-analysis
Effect size:
╸ Strength of relationship between 2 variables (correlation, mean
difference, risk ratio, odds ratio, regression coefficient etc)
╸ Purpose is to combine multiple effect sizes in meta-analysis
╸ Uncertainty in effect sizes used to calculate weights or importance
╶ Larger studies with smaller uncertainty à larger weights
╶ Smaller studies with larger uncertainty à smaller weights
Systematic reviews
& meta-analysis
Forest plot:
Graphical display of results
Individual studies (dark squares)
Effect size (size of dark squares)
Calculated combined effect (white
diamond, dashed vertical line)
Null value (solid line)
Source: Wikipedia
Obtaining &
Describing samples
Inclusion criteria:
Characteristics that patients
have to meet to participate
in the study
Exclusion criteria:
Characteristics that
disqualify patients from
participating in the study
From Pennell et al. NJEM 2021
Obtaining &
Describing samples
Lack of controls
- Cannot determine if patient outcomes/improvements are due to
intervention or inherent sample characteristics or bias
Selecting appropriate controls for studies
╸ Ideally control group will be similar to treatment group with the
exception of receiving the intervention
╸ Limits confounding (external variable affecting both
exposure/intervention and outcome)
Obtaining &
Describing samples
Matching
╸ Utilized in case-control studies
╸ Selecting controls from the same source population with matching
baseline characteristics (age, sex, comorbidities) who does not have
the disease of interest.
Obtaining &
Describing samples
Randomization
╸ Subjects are randomized to treatment and control groups
╸ Controls for both known and unknown confounders
╶ Effect of confounders averaged between groups
╶ Baseline characteristics approximately equal between groups
(table 1 in most trial studies)
Obtaining &
Describing samples
Concealed allocation
╸ Used in randomized trial to prevent selection bias
╸ Preventing investigators from gaining knowledge of research
participant assignment through techniques such as pharmacy
controlled randomization.
Obtaining &
Describing samples
Stratification
╸ Partitioning participants by confounding
╶ (-) requires prior knowledge of
confounding factors
╶ randomization is superior
╸ Stratified random sampling: Partitioning
possible participants into subgroups (such
as by age group, sex) and randomly
sampling from each group
╸ Stratified analysis: partitioning of results by
a possible confounder Source: Wikipedia
Methods to handle
noncompliance
Loss to follow-up
╸ Participants drop out of study prior to completion
╸ Can lead to attrition bias if one group selectively loses more subjects
╶ Remaining participants differ significantly
╶ E.g. in a placebo controlled trial, sicker patients from the
treatment arm drop out selectively, leading to overestimation of
of the study drug’s beneficial effects
╶ New bias=> loss of randomization advantage
Methods to handle
noncompliance
Per protocol treatment
╸ Assumes ideal scenario of no loss to follow-up
╶ Results calculated based on currently retained participants
╸ Overestimates effects of the study drug/intervention
Intention to treat (ITT) analysis
╸ Participants analyzed according to original assignment during
randomization regardless of study completion
╸ Preserve randomization during dropout and crossover studies
╸ More conservative estimate of the effect of intervention, which better
mirrors the expected effect in a practical clinical setting.
Methods to handle
noncompliance
Example
╸ New hypoglycemic diabetes drug X.
╸ New randomized placebo controlled trial for 1 year planned with 40
patients with T2DM planned with goal of A1c control between 6.5 – 7
at the end of study.
╸ 2o randomized in tx arm and 20 randomized in control
╸ At the end of trial,
╶ Tx arm: 4 pts had uncontrolled DM. 10 pt dropped out
╶ Control arm: 12 pts had uncontrolled DM. 7 pt dropped out
╸ Find ARR of uncontrolled DM
Methods to handle
noncompliance
Per protocol analysis
╸ ARR = (12/13) – (4/10) = 0.52 = 52%
╸ 52% reduction in risk of uncontrolled DM due to treatment with drug X
compared to placebo
Intention to treat analysis
╸ ARR = (12/20) – (4/20) = 0.15 = 40%
╸ 40% reduction in risk of uncontrolled DM due to treatment with drug X
compared to placebo
Methods to handle
noncompliance
Sensitivity analysis
╸ Determine robustness of the results or conclusions
╸ Repeat primary analysis by modifying methods, models, criteria or
variable ranges to see if such changes drastically affect the outcomes
or the results
╸ Some common scenarios for sensitivity analysis in clinical trials:
╶ Outliers or missing data (exclude or impute)
╶ Definition of outcomes: modify cutoffs
╶ Non-compliance: ITT or per protocol analysis
╶ Distribution of data: Eg. Normal vs. binomial vs. Poisson
Qualitative
Analysis
Qualitative Research
╸ Goal is to understand human behavior, the “why” and “how” of
decision making
╸ Research questions are discovery oriented, descriptive and
exploratory in nature
╸ Methods of gathering data: (1) participation in the setting, (2) direct
observation (3) interviews (4) focus groups (5) analysis of documents
╸ Categorize and report data for patterns that may arise
Qualitative
Analysis
Examples
╸ In a clinic, about half of all patients with HIV fail to adhere to HAART
╸ The researchers decide to interview patients, care givers, and
healthcare providers to gain insights into reasons for non-adherence
3 Measures of Association
Azfar Basunia, MD
Measures of
Association
2 ✕ 2 table: Organizing and representing data for calculations
╸ Disease as columns,
╸ Exposure or Test as rows
Disease + Disease -
Exposure/Test + True Positive (TP) False Positive (FP) Total exposure/test +

A B A+B
Exposure/Test – False Negative (FN) True Negative (TN) Total exposure/test –
C D C+D
Total disease + Total disease –
A+C B+D
Measures of
Association
Risk of outcome or disease
╸ Probability of the outcome/disease occurring over a certain period of time
╸ Calculated from cohort studies (subjects are followed over time)
╸ Cannot be calculated from case control studies since patients are not
tracked over time
Relative Risk or Risk Ratio (RR)

╸ Measures how strongly a risk factor is associated with an outcome or
disease among exposed individuals compared to unexposed individuals
Measures of Disease + Disease -
Association Exposure + A B Total exposure

+
Relative Risk calculation A+B
!. Exposure – C D Total exposure –

!"#$ "% &'( ()*+#(, !"#
RR = = $.
C+D
!"#$ "% &'( -%()*+#(, $"% Total disease + Total disease –
A+C B+D
╸ Range of RR [0, +∞)

╸ RR < 1: Exposure decreases risk of disease/outcome
╸ RR = 1: Exposure neither increases nor decreases risk of dz/outcome
╸ RR > 1: Exposure increases risk of disease/outcome
Measures of Stroke + Stroke –
Association Uncontrolled HTN

+
A = 30 B = 100-30 = 70 A+B=100
Uncontrolled HTN
Example: –
C=5 D = 50-5 = 45 C+D=50
100 pts w/ uncontrolled HTN and 50 normotensive pt were followed for 5

years. 30 pt w/ uncontrolled HTN developed stroke while 5 normotensive pt
developed stroke. Calculate RR of stroke in pt w/ uncontrolled HTN.
!! &'⁄
!"# (''
RR = $! = )⁄ =3
$"% )'
Conclusion: Patients with uncontrolled HTN are 3x likely to develop stroke

over 5 years compared to normotensive patients
Measures of
Association
Odds
╸ Mathematically, probability of event occurring/probability of event not
occurring
╸ Preferred measure of association for case control studies
╶ Cannot calculate risk from case control study
╶ Odds of exposure for both pt w/ disease and pt w/o disease
Odds ratio (OR)
╸ The odds of exposure to risk factor among patients with disease or
outcome compared to the odds of exposure to risk factor among
patients without disease or outcome
Measures of
Association
Odds ratio calculation Disease + Disease –
/,,# +0 ()*+#-1( 23+%4 ,"#(#(5 !. 7×9 Exposure + A B

$
OR = = #. =
/,,# +0 ()*+#-1( 23+%4 ,"#(2#(6 % :×; Exposure – C D
╸ Range of OR [0, +∞)

╸ OR < 1: Exposure less likely among cases
╸ OR = 1: Exposure equally likely among cases and controls
╸ OR > 1: Exposure more likely among cases
Measures of Stroke + Stroke –
Association Uncontrolled HTN +

A = 60 B = 10
Uncontrolled HTN –
Example: C = 100-60 = 40 D = 50-10 = 40
100 50
100 pts who were admitted for stroke (cases) from a hospital are evaluated for
hx of uncontrolled HTN. 50 pts with similar baseline characteristics but no hx
of stroke (controls) were also evaluated for uncontrolled HTN. 60 cases and 10
controls had uncontrolled HTN. Calculate OR
#×% ()× *)
OR = &×' = +) × *) = 6
Conclusion: The odds of having uncontrolled HTN among patients with
stroke are 6x higher than the odds of having uncontrolled HTN among
patients without stroke
Measures of Disease + Disease –
Association Exposure + A B
Exposure – C D
Odds ratio of disease
╸ The odds of disease among patients w/ exposure or risk factor compared
to the odds of disease among patients w/o exposure or risk factor
,--. /0 -1.23.2 34/56 278/.2- !! #×% ,--. /0 278/.9:2 34/56 -1.23.2;
OR = ,--. /0 -1.23.2 34/56 95278/.2- = $!# = &×' = ,--. /0 278/.9:2 34/56 -1.23.2<
%
Exposure – C D
Rare disease assumption
╸ Mathematically, OR can approximate RR in disease prevalence is low (i.e.
disease is rare) => B >> A, D >> D
╸ Cut off for rare disease is arbitrary
!
=1.> /0 -1.23.2 34/56 278/.2- !! #×%
!"# #
RR = = $ ≈ $! = = OR
=1.> /0 -1.23.2 34/56 95278/.2- % &×'
$"%
╸ Use case control studies to approximate RR under rare disease assumption
Measures of
Association
Hazard Ratio
╸ Measure of effect in survival or time-to-effect studies
╸ Interpretation similar to RR
╶ RR only takes into account the occurrence of an event and can only
be calculated at the end of the study
╶ HR takes into account the timing & can be calculated at any time
╶ HR < 1: Outcome more likely among unexposed (protective effect);
╶ HR = 1: Outcome equally likely among exposed + unexposed (no
benefit or harm);
╶ HR > 1: Outcome more likely among exposed (detrimental effect).
╸ Calculated using Cox Proportional Hazards model
Measures of Source: Andre Thierry, et al. NJEM 2020
Association
Example: Phase 3 randomized clinical trial comparing pembrolizumab vs
conventional ctx for MSI-H–dMMR or metastatic colorectal cancer

Exposure – C D
Absolute Risk
╸ probability an individual develops an outcome or disease in the study
period => approximately equal to incidence rate
╸ Risk => calculated from cohort studies
╸ Absolute Risk =
?@?AB CDEFAEF;
?@?AB GA?DFH?E
I;J
= I;K;J;L

Attributable risk (AR) Exposure – C D
╸ How much an outcome may be attributable to the exposure/ risk factor

╸ Aka Risk Difference between exposed and unexposed individuals
╸ AR =
I
I;K
-
J;L
J
Absolute Risk Reduction (ARR)

╸ Measure of therapeutic effect in clinical trials
╸ How much risk of disease is reduced due to therapeutic intervention in the
treatment group compared to control group
╸ ARR =
J;L
J
-
I;K
I

Measures of
Association
Number needed to harm (NNH)
╸ No. of pts needed to be exposed before 1 pt develops the outcome
╸ Directly correlates with safety of the exposure
╸ NNH=
+
IMM:1N9M3NO2 =1.>
=
+
I=
Number needed to screen (NNS) or Number needed to treat (NNT)
╸ NNT: No. of pts needed to be treated to prevent negative outcome in 1 pt
╸ NNS: No. of pts needed to be screened to detect 1 case of the disease
╸ NNT or NNS =
+
IN./9M2 =1.> =2-9PM1/5
=
+
I==

Exposure – C D
Attributable risk percent (ARP)
╸ % of disease incidence among exposed patients that can be attributed to
the risk factor, which can be eliminated if exposure is avoided
╸ ARP =
==<+
==
× 100%
╸ Also, ARP =
I=
=1.> 15 278/.2-
×100%, where risk in exposed =
I
I;K

Association Treatment/
exposure +
A B
Treatment/ C D
Relative risk reduction (RRR) exposure –
╸ Determine how much tx/intervention reduces risk of negative outcomes

╸ % of risk reduced due to tx/intervention in the tx group compared to the
control group
╸ RRR =
+<==
+
× 100% = (1-RR) × 100%
╸ I==
Also, RRR = =1.> 15 P/5M:/O 6:/98 ×100%, where risk in control = J;L
J

Exposure – C D
Population attributable risk (PAR)
╸ The proportion of all cases of an outcome/disease in the total population
that could be attributed to the risk factor
╸ PAR = absolute risk – risk in unexposed =
I;J
I;K;J;L J;L
-
J
╸ Also, PAR = AR ×
M/M3O 278/.9:2 ;
M/M3O 83M125M.
=(
I
I;K
-
J
J;L
)×
I;K
I;K;J;L

Exposure – C D
Population attributable risk percent (PAR%)
╸ The percentage of disease in the observed population that is attributable
to the risk factor
╸ What % of an outcome could possibly be prevented if a risk factor were to
be removed from the population
╸ PAR% =
QI=
3N./O9M2 :1.>
×100% =
3N./O9M2 :1.> <:1.> 15 95278/.2-
3N./O9M2 :1.>
×100%,
╸ Also,
8:2R3O25P2 /0 278/.9:2 × (==<+)
PAR% = +;(8:2R3O25P2 /0 278/.9:2 × ==<+ )

Measures of
Association
Example 1
A large longitudinal 10-year cohort study is planned with 2400 patients
with similar baseline characteristics to investigate association between
smoking > 1 pack per day (ppd) and esophageal cancer. The prevalence
of smoking > 1ppd is 40% in the study population. Among patients who
smoked > 1ppd, 300 patients developed esophageal cancer. Among
patients who smoked <1ppd, 50 patients developed esophageal cancer.

Measures of Esophageal Esophageal
Association > 1 ppd
CA +
A = 300
CA –
B = 660 960
< 1 ppd C = 50 D = 1390 1440
Step 1: Complete 2 ✕ 2 table
╸ A = 300, C = 50
╸ A + B = (Prevalence of smoking > 1ppd) ✕ total study population
= 0.4 ✕ 2400 = 960
╸ B = 960 – 300 = 660
╸ C + D = (Prevalence of smoking < 1ppd) ✕ total study population
= 0.6 ✕ 2400 = 1440 (also 2400-960)
╸ D = 1440 – 50 = 1390

Measures of
Esophageal CA + Esophageal CA –
Association
> 1 ppd A = 300 B = 660
< 1 ppd C = 50 D = 1390
What is the incidence rate of esophageal CA over 10 year period?

I;J U));V)
Absolute Risk of esophageal CA= = = 0.146 = 14.6%
I;K;J;L W*))
How much more likely are individuals who smoke > 1 ppd to get
Esophageal CA compared to individuals who smoke < 1ppd
*
*"+ U))/Y()
Relative Risk (RR) = , = V)/+**) = 9.
,"-
What is the added risk of esophageal CA in individuals smoking > 1ppd

compared to individuals smoking < 1ppd
I J U)) V)
Attributable Risk (AR) = I;K - J;L = Y() - +**) = 0.278 = 27.8%
Measures of
Association
> 1 ppd A = 300 B = 660
< 1 ppd C = 50 D = 1390
What percentage of esophageal cancer in the individuals smoking > 1ppd

can be attributed to their exposure (smoking > 1ppd)
Attributable risk % (ARP)
I= ).W\]
= = = 0.889 = 88.9%
=1.> 15 Z+88- U))/Y()
==<+ Y<+ ]
= ==
= Y
= Y = 0.889 = 88.9%
How many individuals need to smoke > 1ppd before one develops
esophageal CA?
+ +
Number needed to harm (NNH) = = = 3.6 ≈ 4
I= 0.278

Measures of
Association
> 1 ppd A = 300 B = 660
< 1 ppd C = 50 D = 1390
What percentage of esophageal cancer observed in the population can be

attributed to smoking > 1 ppd
Population attributable risk (PAR)
I;J J UV) V) +
= absolute risk – risk in <1ppd = I;K;J;L - J;L = W*)) - +**) = Y = 0.111
Z+88- Y()
= AR × = 0.278 × = 0.111
M/M3O 83M125M. W*))
Population attributable risk % (PAR%)
QI= ).+++
= = = 0.762 ✕ 100% = 76.2%
3N./O9M2 :1.> ).+*(
8:2R3O25P2 /0 Z+88- × (==<+) ).* ×(Y<+)
= +;(8:2R3O25P2 /0 Z+88- × ==<+ )
= +;( ).* ×(Y<+))
= 0.762 ✕ 100% = 76.2%
Measures of
Association T2DM No DM
Example 2
> 30 min 300 cases/ 5000 cases/
A large prospective cohort study to daily 21,000 22,000
evaluate effect of daily > 30 min exercise person-years person-years
exercise on the incidence of T2DM
in men with BMI >30. 10,000 obese
men with no hx of DM by A1c < 30 min 3000 cases/ 1700 cases/
measurements were enrolled in the daily 21,700 20,000
study and followed up for 10 years. exercise person-years person-years
The following data are reported.
Calculate ARR, RRR

Measures of
Association T2DM No DM
&''' &''
J I .(/'' .('''
ARR= - = &''' (/'' - &'' )''' > 30 min 300 cases/ 5000 cases/
J;L I;K ; ;
.(/'' .'''' .(''' ..''' daily 21,000 22,000
())) ++ exercise person-years person-years
= - = 0.56 = 56%
Y(]Y +](
I== ).V(
RRR = , = 0''' = 0.9 = 90%
,"- 1021
< 30 min 3000 cases/ 1700 cases/
daily 21,700 20,000
exercise person-years person-years

Measures of
Association
Alternate wording for Interpretations
(1) In a case control study, OR = 1.7 for GI bleed for patients taking > 1.2g of
ibuprofen daily compared to those not taking ibuprofen daily.
à The odds of GI bleed are about 70% greater among patients taking
>1.2g of ibuprofen daily compared to those not taking ibuprofen daily.
(2) In a prospective cohort study, RR = 0.65 for subsequent MI for pts with
prior MI on statins compared to those with prior MI and not on statins.
à The risk of subsequent MI is 35% lower in pts with prior MI on statins
compared to those with prior MI and not on statins

4 Distributions of data
Azfar Basunia, MD
Distribution
of data
Data type
╸ Categorical/nominal variable
╶ Finite number of categories w/o any discrete order.
╶ Eg. Sex (male, female, other), States in USA
╸ Quantitative variable
╶ Discrete: only whole numbers/values; may have a logical order.
Eg. Diabetic category (no DM, pre-DM, DM) based on A1c
╶ Continuous: any real number values. Eg. SBP, temperature

Distribution
of data
Measures of Central tendency
╸ Mean
╶ arithmetic average.
╶ most affected by outliers / skewed data
╸ Median
╶ middle value.
╶ for even number of data points, avg of 2 middle values
╶ more resistant to outliers compared to mean
╸ Mode
╶ most common value in dataset.
╶ most resistant to outliers / skewed data
Distribution
of data
Skewness:
╸ + skew =>
long tail on
positive end.
╸ - skew =>
long tail on
negative end
Source: Wikipedia

Distribution
of data
Example
╸ Consider SBP readings of a patient over the period of 1 week (89, 98,
100, 121, 93, 93). Find mean, median, mode
╸ Data arranged in ascending order = 89, 93, 93, 98, 100, 121
Mean = (89+93+93+98+100+121)/6 = 99
Median = (93+98)/2 = 95.5
Mode = 93

Distribution
of data
Which measure of central tendency to use
╸ Continuous data, normal distribution à mean
╸ Continuous data or ordinal data (categorical data w/ natural order,
such as age groups ), skewed distribution à median
╶ Many useful statistical and analytical techniques have been
described with the median rather than mode
╸ Nominal data (categorical data w/o natural order like sex, race) à
mode, proportions

Distribution
of data
Measure of variability/ dispersion
╸ Standard deviation:
╶ dispersion of study data around
around its sample mean
╶ SD = 𝜎 =
∑( _ < _̅ ).
𝒏<+
╶ n ↑ => σ ↓
╶ Like mean, influenced by outliers
Source: Wikipedia

Distribution
of data
╸ Variance
╶ Another measure of dispersion around the mean
╶ Square of standard deviation
╶ 𝜎W =
∑( _ < _̅ ).
H<+
╸ Standard error around the mean
╶ Deviation of the study sample mean from the true population mean,
i.e. how closely the sample mean represents the population mean
╶ SEM = bH

Distribution
of data
╸ Range: (maximum value – minimum value) in a dataset
╸ Percentile: A value below which % of data falls.
╶ Eg. 10th percentile => 10% data falls below the value
╸ Quartile: 25% or quarter of a dataset.
╶ Arrange data in ascending order (min to max value)
╶ 1st quartile = 0-25% of dataset, 2nd quartile = 25-50% of dataset, etc
╸ Interquartile range: range from 2nd to 3rd quartile or 25th to 75th percentile
╶ Assess dispersion of data when median is used for central tendency
╶ More resistant to outliers like median => good for skewed distribution
Distribution
of data
Normal distribution
╸ Symmetric bell shaped curve
╸ Mean = median = mode
╸ 68-95-99.7 rule
╶ 1 SD => ~68% data
╶ 2 SD => ~95% data
╶ 3 SD => ~99.7% data
Source: Wikipedia

Distribution
of data
Standard normal distribution (Z distribution)
╸ Normal distribution with mean = 0, sd = 1
╸ Allows comparison of populations with different means and sd
╸ Z score: how does a single datapoint compare to the mean
╶ 𝐙 = _<cb
╶ Z + 1, +2, +3: 1 sd, 2 sd and 3 sd above mean respectively
╶ Z -1, -2, -3: 1 sd, 2 sd and 3 sd below mean respectively

Distribution
of data
Example
Blood glucose (BG) was measured in 400 patients with normal distribution and
mean = 100. BG= 120 was greater than 2 standard deviations of the sample.
(1) Standard deviation
_<c
𝐙= => 2σ = 120-100 => σ = 20/2 = 10.
b
(2) How many patients will have BG <80?

Z = (80-100)/10 = -2 => 80 is 2 sd lower than mean
ÞUsing 68-95-99.7 rule, 2.5% of pt will have BG < 80 = 2.5/100 x 400 = 10 pt

Distribution
of data
Regression to the mean
╸ Repeated observations tend to return to their mean value
╸ Prevents overdiagnosis from single extreme observation
╸ Eg. Diagnosis of HTN => elevated BP with repeated measurements in at
least 2 readings on 2 separate occasions.

5 Correlation & Regression
Azfar Basunia, MD
Correlation & Correlation coefficients, r
regression ╸ Linear association between 2 variables
╶ Strength of association
╸ DOES NOT IMPLY CAUSATION
╸ Range (-1, 1)
╶ Closer to |1| => stronger correlation
╶ (+) : direction, both ↑ or ↓ together
╶ (-) : direction, one ↑ while the other ↓

Correlation &
regression
╸ r = 0.8
╸ Plot 1: Possibly true
linear relationship
╸ Plot 2: parametric
(non-linear)
relationship
╸ Plots 3,4 : effect of
extreme values

Correlation &
regression
╸ Independent: x
╶ The exposure or baseline characteristic variables
╶ Eg. age, sex, drug dosing
╸ Dependent: y
╶ Effect or outcome variable
╸ Regression
╶ Mathematical relationship between dependent and independent
variables

Correlation &
regression
╸ Simple linear Regression
╶ 1 x and 1 y variable
╶ x can be categorical or continuous; y must be continuous
╶ y = β0 + β1x1 ; β1 = slope, β0 = y intercept
╶ Eg. Effect of age (x1) on blood pressure (y)
╶ (-) confounders are not accounted for (eg. BMI, smoking status)
╸ Multiple linear regression
╶ Multiple x and 1 y variable
╶ (+) Adjust for possible confounders
╶ y = β0 + β1x1 + β2x2 + β3x3; β1 ,β2 ,β3 = slopes, β0 = y intercept
╶ Eg. Effect of age (x1), BMI (x2) and smoking status (x3) on BP (y)
Correlation &
regression
╸ Logistic regression
╶ x can be categorical or continuous; y must be categorical/
dichotomous
╶ Simple logistic: 1 x, 1 y
╶ Multiple logistic: multiple x, 1 y
╶ Eg. Effect of age, sex, BMI, blood sugar (x variables) on presence
of diabetes (y variable)

6 Principles of testing & screening
Azfar Basunia, MD
Sensitivity, NPV Disease + Disease –
specificity, PPV Test + True Positive (TP) False Positive (FP)
Test – False Negative (FN) True Negative (TN)

Diagnostic tests
╸ Identify individuals with or without disease
╸ Often compared to the gold standard = best available test
Sensitivity = True positive rate
╶ The proportion of individuals with disease who test positive
╶ Sensitivity = dQdQ;ef
Specificity = True negative rate
╶ The proportion of individuals without disease who test negative
╶ Sensitivity = dfdf;eQ


Positive predictive value (PPV)
╸ The proportion of individuals with positive test results who actually
have the disease
╸ Disease prevalence ↑ => PPV ↑ and vice versa
╶ directly correlates w/ pre-test probability
╸ PPV =
dQ
dQ;eQ


Negative predictive value (PPV)
╸ The proportion of individuals with negative test results who actually do
not have the disease
╸ Disease prevalence ↑ => NPV ↓ and vice versa
╶ inversely correlates w/ pre-test prob
╸ NPV =
df
df;ef

Sensitivity, NPV
specificity, PPV
Disease + Disease –
Test + True Positive False Positive

(TP) (FP)
Test – False Negative True Negative
(FN) (TN)
UP “Trues” at the “top”

╸ U => Sensitivity, Specificity
╸ P => PPV, NPV
Sensitivity, NPV
specificity, PPV
Disease + Disease –
Test + True Positive False Positive

(TP) (FP)
Test – False Negative True Negative
(FN) (TN)
UP “Trues” at the “top”

╸ U => Sensitivity, Specificity
╸ P => PPV, NPV
Sensitivity, NPV
specificity, PPV
Example
The prevalence of Lung CA in a small rural town is 10%. Using a new
screening test, 90% people with Lung CA tested positive while 50% people
without Lung CA tested negative. Find Sensitivity, Specificity, PPV, NPV
Assume a population = 1,000 Lung CA + Lung CA –

Since prevalence = 10% Test + True Positive (TP) False Positive (FP)
Total Lung CA+ = 100 Test – False Negative (FN) True Negative (TN)
Total Lung CA- = 900 100 900

specificity, PPV Test + 90 450

Test – 10 450
Example continued
The prevalence of Lung CA in a small rural town is 10%. Using a new screening
test, 90% people with Lung CA tested positive while 50% people without Lung
CA tested negative. Find Sensitivity, Specificity, PPV, NPV
Sensitivity = 90%, specificity = 50%

TP = 0.9✕100 = 90; FN = 100–90 = 10; TN = 0.5✕900 = 450; FP = 900–450 = 450;
PPV = 90/(90+450) = 0.167 = 16.7%
NPV = 450/(10+450) = 0.978 = 97.8%

Sensitivity, NPV Healthy Disease
specificity, PPV TN TP
pts
Suppose graphs represent A1c FN FP
A: A1c = 6.5; B: A1c = 5

By moving diabetes dx A1c cutoff A à B
╸ Sensitivity: ↑ Healthy DM
╸ Specificity: ↓
╸ PPV: ↓
╸ NPV: ↑
B A

Sensitivity, NPV
specificity, PPV
Suppose we move cut point from C à D
What happens to sensitivity, specificity, PPV, NPV?
Imagine again Disease = DM and C: A1c = 6.5, D: A1c = 8
╸ Sensitivity: ↓
Healthy Disease
╸ Specificity: ↑
╸ PPV: ↑
╸ NPV: ↓
C D

Sensitivity, NPV
specificity, PPV Healthy Disease
To maximize both sensitivity and

specificity, ideal cutoff point =
intersection of the the graphs

ROC Curves
Receiver-Operator Characteristic Curve
╸ Graphical representation of true positive rate (sensitivity, y axis) and
false positive rate (1-specificity, x axis) of diagnostic test(s) for various
cut-off points/thresholds
╸ Goal is to maximize sensitivity (closer to 1 on y axis) and specificity
(closer to 0 in x axis)
╸ ↑ Area under the curve => ↑ diagnostic accuracy
╶ Best theoretical test => area = 1
╶ Useless test would follow the diagonal line with area = 0.5

ROC Curves Source: Wikipedia
╸ Each line denotes a

possible test
╸ Red dashed line : useless
test
╸ Comparing tests: Blue line
> Orange line > green line
╸ Blue arrow point to the
ideal cutoff in the best test
╶ Leftmost corner or
inflection point

Properties of a
screening test
Precision / reliability
╸ Reproducibility, how closely repeated
measurements match each other
╸ High precision = minimal random error
╶ Smaller standard deviation
Accuracy / validity
╸ “trueness” of the test results, how
closely measurements match reality
╸ High validity = minimal systematic error
(non-random error) and bias Source: St. Olaf college

Properties of a
screening test
╸ Screening test Healthy Disease
╶ Initial test
╶ Negative result rules out the
disease => low FN
╶ High sensitivity, low
specificity => high FP
╸ Confirmatory test
╶ Confirm disease presence Confirmatory
Screening
after screening test=> low FP test test
╶ High specificity, low
sensitivity => high FN

specificity, PPV
Test + True Positive (TP) False Positive (FP)
Criteria for screening and confirmatory tests

╸ SnNout & SpPIN
╶ High sensitivity à Negative result rules out disease => ideal
screening test
╶ High specificity à Positive result rules in disease => ideal
confirmatory test
╸ Sensitivity and specificity DOES NOT depend on disease prevalence
╸ Trade off between sensitivity and specificity for a diagnostic test

Properties of a
screening test
Lead time bias
╸ Early detection of disease leads to increase in perceived survival time
╶ Lead time: time between detection by screening and its usual
clinical presentation and dx (eg by sx, or traditional dx criteria)
╶ No apparent effect on prognosis or mortality rates => seen in
terminal diseases with lower cure potential (e.g. adult cancers)
╸ Solutions
╶ Adjust for severity of disease when detected by screening
╶ Use mortality rate instead of survival time

Properties of a
screening test Source: Wikipedia
Example: cancer screening

Note that death occurs at the same time regardless of earlier detection by screening

Properties of a
screening test
Length time bias
╸ A form of selection bias
╸ Screening test preferentially detects less aggressive and slowly
progressive forms of a disease
╶ More aggressive forms of the disease die off and/or are not detected
╶ Increase in apparent survival times
╸ Solution
╶ Adjust for severity of disease when detected by screening

Properties of a
screening test
Example: cancer screening
Screening appears to lead to
better survival
Source: Wikipedia
Probability
Pre-test probability
╸ Probability a patient has a specific disease
╸ Usually equal to prevalence of disease
╸ Affects PPV, NPV; does not affect sensitivity, specificity, likelihood ratio
╶ ↑ pre-test probability => ↑ PPV, ↓ NPV
╶ ↓ pre-test probability => ↓ PPV, ↑ NPV
Post-test probability
╸ Probability a pt has a disease after diagnostic testing
╸ Post-test prob = pre-test prob ✕ likelihood ratio

Probability
Likelihood Ratio (LR)
╸ Assess value of performing a diagnostic test
╸ How likely that an individual has a disease after diagnostic testing, i.e.
helps calculate post-test probability
╸ Does not depend on prevalence/ pre-test probability
╶ Calculated from sensitivity and specificity
╸ Interpretation
╶ LR < 1 => decrease in probability of presence of disease
╶ LR = 1 => pre-test and post-test prob are similar; no effect of testing
╶ LR > 1 => increase in probability of presence of disease
Probability
╸ Positive Likelihood Ratio (LR+)
╶ Probability an individual with dz tests positive compared to an
individual w/o dz testing positive
╶ LR+ =
𝐒𝐞𝐧𝐬𝐢𝐭𝐢𝐯𝐢𝐭𝐲
+<𝐒𝐩𝐞𝐜𝐢𝐟𝐢𝐜𝐢𝐭𝐲
╶ LR > 10 => large increase in probability of dz => high test specificity
╸ Negative Likelihood Ratio (LR-)
╶ Probability an individual with dz tests negative compared to an
individual w/o dz testing negative
╶ LR- = +< 𝐒𝐞𝐧𝐬𝐢𝐭𝐢𝐯𝐢𝐭𝐲
𝐒𝐩𝐞𝐜𝐢𝐟𝐢𝐜𝐢𝐭𝐲
╶ LR < 0.1 => large decrease in probability of dz => high test sensitivity
Probability Source: Wikipedia

Probability
Example: Likelihood ratio
A study investigating various diagnostic
Biom Sensi Speci LR + LR -
biomarkers for glioblastoma (GBM) arker tivity ficity
resulted in table to the right. A 98% 40% 1.63 0.05
╸ A pt is most likely to be free of GBM
B 70 % 95 % 14 0.32
when which biomarker is negative?
╶ Biomarker A (Lowest LR-) C 75 % 70 % 2.5 0.36
╸ A pt is most likely to have GBM when

which biomarker is positive?
╶ Biomarker B (Highest LR+)
Probability
Biom Sensi Speci LR + LR -
arker tivity ficity
A 98% 40% 1.63 0.05
B 70 % 95 % 14 0.32
Example: Likelihood ratio C 75 % 70 % 2.5 0.36
╸ Assuming a pt has GBM, how likely are they to test positive for biomarker
C compared to a patient without GBM testing positive for biomarker C?
╶ LR+ for biomarker C = 2.5
╸ Assuming a pt has GBM, how likely are they to test negative for
biomarker B compared to a patient without GBM testing negative for
biomarker B?
╶ LR- for biomarker B = 0.32

Probability
Probability properties
╸ Independent events:
╶ probabilities of events are not contingent upon one another.
╶ Eg. P(having a girl w/ autosomal dominant dz)
╸ Multiplication rule:
╶ for independent events, combined probability is calculated by
multiplying individual probabilities.
╶ Eg. P(having a boy w/ autosomal dominant dz) = P(having a boy) ✕
P(child has autosomal dominant dz)

Probability
Probability properties
╸ Addition rule:
╶ P(event 1 or event 2) = P(event 1) + P(event 2) – P(event 1 and 2)
╶ For mutually exclusive events:
╶ Eg. presence or absence of Neurofibromatosis 1)
╶ P(event 1 and 2) = 0 => since they never occur together
╶ P(event 1 or event 2) = P(event 1) + P(event 2)

Probability
Conditional probabilities
╸ P(event 1| event 2) = prob. of event 1 given that event 2 has occurred
╸ Eg. Sensitivity = P(Test + | Disease +), PPV = P(Disease + | Test +),
Bayes theorem
╸ Can be used to calculate conditional probabilities
╸ P(event 1|event 2) = P(event 2|event 1) × P(event 1)

P(event 2)
Law of total probability: P(A) = P(A|B)×P(B) + P(A| not B)×P(not B)

Sensitivity, NPV
specificity, PPV
The prevalence of Lung CA in a small rural town is 10%. Using a new
screening test, 90% people with Lung CA tested positive while 50% people
without Lung CA tested negative. Find Sensitivity, Specificity, PPV
Prevalence of disease = P(D+) = 0.1; => P(D-) = 1-0.1 = 0.9

Sensitivity = P(T+|D+) = 0.9; Specificity = P(T-|D-) = 0.5;
=> P(T+|D-) = 1-specificity = 0.5
Q(d;|L;)×Q(L;) Q(d;|L;)Q(L;)
PPV = P(D+|T+) = Q(d;)
=Q T+D+ Q L; ; Q T + D− Q(L<)
).Y × ).+
= = 0.167 = 16.7%
().Y×).+);().V×).Y)
Probability
Decision tree
╸ Visual representation of all outcomes sometimes with their probabilities
╸ Classic example: pedigree
Source: Wikipedia
7 Study interpretations &
drawing conclusions from data
Azfar Basunia, MD
Causation
Hypothesis-generating testing
╸ Investigating patterns in data to generate testable hypothesis using
qualitative research & descriptive statistics (mean, median, IQR, correlation)
╸ Eg. Investigating rising teenage obesity in a small town by analyzing
demographics, eating habits, and activity level of students
Hypothesis-driven testing
╸ Addressing a specific question using analytical studies and statistical
methods
╸ Eg. Are increased carbohydrate content in school lunches is linked to rising
obesity among school children in a small town
Causation
Causal criteria (Bradford-Hill Criteria): Causality in epidemiological studies
╸ Temporality:
╶ outcome occurs after exposure within an expected amount of time.
╶ Eg. Poison ivy causing type IV hypersensitivity reaction
╸ Temporal Sequence:
╶ The cause/exposure must happen before effect/outcome.
╶ There can be strong correlation between the cause and effect, but it
may be difficult to distinguish.
╶ Eg. Connection between poor performance and marijuana smoking.
Does poor performance cause more marijuana smoking or do
students with poor performance smoke more marijuana
Causation
Causal criteria (Bradford-Hill Criteria)
╸ Dose response relationship:
╶ Exposure to higher doses causes higher incidence of effect
╶ Eg. Patients with higher pack years of smoking have higher incidence
of lung and bladder cancer
╸ Reproducibility:
╶ Findings have been observed in studies with different persons,
different places, different sample sizes etc

Causation
Causal criteria (Bradford-Hill Criteria)
╸ Biological plausibility:
╶ Current biological or medical knowledge consistent with relationship
between exposure and effect.
╶ Eg. Relationship between carcinogens in cigarettes and lung CA
Reverse causality
╸ The risk factor/exposure and the outcome are strongly associated but
the relationship between exposure and disease process are reversed.
╸ Eg. Schizophrenia leads to low SES, not the other way around (Source:
Gerstman 2003)
Chance
Null Hypothesis (H0)
╸ Assumption that there is no relationship between the exposure and
outcome or no difference between the two variables
Alternate Hypothesis (H1)

╸ Assumption that there is a relationship between the exposure and
outcome or a difference between the two variables

Chance
Outcomes after hypothesis testing
╸ The null hypothesis was incorrectly rejected
╶ Type 1 error, α
╸ The null hypothesis incorrectly could not be rejected and alternate
hypothesis could not be accepted
╶ Type 2 error, β
╸ Null hypothesis was correctly rejected & alternate hypothesis is accepted
╶ Power, 1-β, the chance of detecting a difference when one exists
╸ The null hypothesis correctly could not be rejected
╶ 1-α
Chance
H0 is true H1 is true
Testing does not 1-α Type 2 error (β)

reject H0
Testing rejected H0 Type 1 error (α) Power (1-β)

Chance
Type 1 error rate, α
╸ False positive results, i.e. finding a difference when none exists in reality
╸ α significance level set by researchers prior to study
╶ Commonly 0.05, but 0.01 or 0.1 also possible
╶ If p value < α, H0 is rejected in favor of H1
╸ Random error or chance alone is the most common cause for type 1 error
occurrence

Chance
Multiple testing problem
╸ When multiple secondary endpoints are
evaluated, there is higher probability of
erroneously finding a statistically
significant result due random error
╶ If there are 10 secondary endpoints,
40% chance of rejecting H0 due to
chance alone!!
╸ Adjustment is needed to α level, p-value
(q values)
Source: Chen et al. 2017

Chance
Type 2 error rate, β
╸ False negative results, i.e. finding no difference when one exists in reality
╸ Common cause: Too few patients (small sample size)

Chance
Power (1-β):
╸ Finding a difference when one exists
╸ Conventionally set at 80%
╸ Positively correlates with:
╶ Large magnitude of association of interest (such as difference
between the means)
╶ Less overlap between data points (i.e. less scatter)
╶ Larger sample size
╶ Researchers can only control this.
╶ Strategy: Large trial or meta analysis from several smaller
studies
Chance
P-value
╸ The probability of observing test result at least as extreme when the null
hypothesis is correct
╸ If p<α (typically 0.05), the H0 is rejected in favor of H1
╸ P value is calculated by statistical tests of significance

Chance
Confidence interval (CI)
╸ Most commonly used is 95% CI, but 90% and 99% CI also possible
╶ Larger confidence level => wider confidence intervals
╶ 99% > 95% > 90%
╸ Definition: Given repeated sampling, the % of confidence intervals that
contain the true population parameter
╸ Calculated by test statistic (t-statistic, F-statistic, Z-score etc)
╸ Affected by sample size
╶ Larger sample size => smaller/tighter CI

Chance
Confidence interval (CI)
╸ Can used to interpret statistical significance in the absence of p-value
╶ Confidence intervals do not contain null value
╶ Null values:
╶ Ratios: OR, RR, HR = 1;
╶ Differences: AR, ARR = 0
╶ Confidence intervals do not overlap

Chance
Examples Confidence interval (CI)
In a randomized trial studying effect of BB on SBP, carvedilol caused SBP
change by -15 (95% CI: -20, -10), metoprolol by -30 (95% CI: -45, -15) and
placebo by +2 (95% CI: -4, +8).
╸ Is carvedilol and metoprolol effective in reducing BP compared to placebo?
╶ Yes! The 95% CI between placebo and metoprolol and between
placebo and carvedilol does not overlap.
╸ Are metoprolol and carvedilol different from each other in effectiveness?
╶ No! The 95% CI between carvedilol and metoprolol overlap, and thus
their efficacies are similar

Chance
Examples Confidence interval (CI)
In a randomized trial studying efficacy of drug X among pt with end stage
pulmonary fibrosis. Final outcome is death. Compared to placebo, HR at 6
months = 1.5 (95% CI: 1.1, 1.9) and at 1 year = 1.3 (95% CI = 0.5, 2.1).
╸ Is Drug X efficacious in preventing death at 6 months?
╶ Yes! The 95% CI for HR at 6 months does not contain null value ( = 1)
╸ Is Drug X efficacious in preventing death at 1 year?
╶ No! The 95% CI for HR at 1 year does contains null value ( = 1)

Chance
Statistical tests of significance
╸ T-test
╶ For continuous data
╶ Compares mean values from 2 samples
╶ Uses the T-distribution (which approaches normal distribution as n↑ )
╶ Compute a t statistic, which is then compared a t-table to get a p-
value

Chance
╸ Unpaired two sample T-test
╶ Independent samples T-test
╶ Difference between 2 sample means coming from 2 different groups
╶ Eg. Comparing morning mean BP b/w smokers vs. non-smokers?
╸ Paired two sample T-test
╶ Dependent samples T-test
╶ Same group, i.e. same patient, sampled 2 different times and serve as
their own control
╶ E.g. Crossover study to investigate BP after caffeine consumption in
resident doctors
Chance
╸ Analysis of Variance (ANOVA)
╶ For continuous data
╶ Compares mean values from 3 or more samples
╶ Computes an F statistic, which is used to obtain a p-value
╶ Eg. Randomized trial comparing mean blood pressure changes
between 4 BB: metoprolol, carvedilol, bisoprolol and propanolol

Chance
╸ Pearson Chi-square test
╶ For categorical data (no mean values)
╶ Compares proportions in 2 or more independent samples
╶ Data often represented in 2 × 2 contingency table for 2 samples
╶ Does not work for small samples (<10 in any cell 0f 2 × 2 table)
╶ Compute a 𝟀2 statistic, which is then used to obtain a p-value
╶ E.g. Trial studying the effect of nimodipine in preventing cerebral
vasospasm after stroke
╸ Fisher’s exact test
╶ For categorical data (no mean values) with small samples
Chance
╸ Mann-Whitney U test (Wilcoxon rank sum test)
╶ For t test or ANOVA, underlying assumption is approximate normal
distribution for both populations => compare means
╶ Here, we can compare any population distribution, including non-
normal or skewed distributions => compare medians
╶ Can also be used with small sample sizes
╶ H0: Population medians are equal;
╶ H1: population medians are not equal.
╶ Rank-sum test statistic can be used to compute a p-value

Chance
╸
Summary of statistical approach
Categorical data for dependent variable (y)

╶ Statistical tests: Chi-square, fisher’s exact (if small sample size)
╶ Regression: logistic regression
╸ Quantitative data for dependent variable (y)
╶ Statistical tests (comparing means):
╶ Independent samples (different individuals in each group)
╶ 2 groups: two sample T-test
╶ > 2 groups: ANOVA
╶ Dependent samples (same pt serve as control): paired T-test
╶ Statistical tests (comparing medians/ small sample size):
╶ Mann-Whitney U test (Wilcoxon rank sum test)
╶ Regression: linear regression
Chance
A priori analysis
╸ Comparisons are defined prior to seeing/collecting the data
╸ Eg. Defining primary and secondary endpoints of clinical trials prior to
running data analysis
Post-hoc analysis
╸ Comparisons are made after data is collected and evaluated
╸ Must account for multiple comparisons

Chance
Sub group analysis
╸ Analyzing data from a study after stratifying by a factor (e.g. diabetics vs.
non-diabetics, smoking status, sex, etc)
╸ May be defined apriori or performed post-hoc
╸ Multiple testing problem arises and thus adjustment of α or p-value is
necessary

Interpretation of
graphs/tables & text

Interpretation of

Interpretation of

Interpretation of

Interpretation of

Interpretation of
Fig: forest plots of the hazard
ratios for the primary outcome
according to prespecified
baseline subgroups

Bias, confounding &
threats to validity
Error
╸ Systematic error is caused by bias and can only be eliminated by
eliminating bias
╸ Random error is decreased by increasing sample size or taking multiple
measurements
Bias
╸ Errors in study design and execution that causes deviation of findings
from their true value

Bias, confounding &
threats to validity
Selection bias
╸ Subjects selected for study are not representative of the study
population because of improper/ loss of randomization.
╸ Sampling bias (ascertainment bias)
╶ Certain individuals are more likely to be selected for clinical studies
╶ Affects generalizability of study
╶ Non response bias: A form on sampling bias (from questionnaires or
surveys) where non-responders differ significantly from responders
=> non-random selection of candidates
╶ Volunteer bias: Individuals who volunteer for a study are different
from the general population
Bias, confounding &
threats to validity
Selection bias
╸ Berkson’s bias/fallacy
╶ Samples selected from hospitalized patients are not representative
of the general population (likely due to severity of illness)
╸ Attrition bias
╶ Commonly in prospective studies
╶ Participants lost to follow-up differ significantly from participants
retained in the study.
╶ Eg. Participants in the treatment arm of a trial are lost more
selectively due to more severe side effects
╶ Solution: Intention to treat analysis
Bias, confounding &
threats to validity
Selection bias
╸ Prevalence bias (Neyman bias/ survival bias)
╶ Commonly in case-control and cross-sectional studies
╶ Exposure happens long before disease assessment
╶ Those with more severe disease may die off early
╶ Those will less severe disease may recover prior to assessment
╶ The prevalence of disease during assessment is affected by those
who continue to have the disease at the time of assessment

Bias, confounding &
threats to validity
Information bias
╸ Inaccurate classification of exposure and/or outcome due to incorrect
data collection or interpretation
╸ Measurement bias
╶ Error in collecting or measuring the outcome.
╶ Solution: Use standardized data collection methods
╸ Recall bias
╶ Common in retrospective studies and case-control studies
╶ Inaccurate recall of risk factors or past exposures in select
participants, likely related to disease status or awareness
╶ Solution: Limit follow-up time between exposure and interview
Bias, confounding &
threats to validity
Information bias
╸ Observer bias (ascertainment bias)
╶ Also a form of cognitive bias, where researcher misclassifies data
due to preconceived expectations or individual differences in
interpretation regarding patient’s disease status or treatment
╶ Eg. A pathologists’ awareness of diabetes status in pts may influence
how frequently they call diabetic nephropathy from renal biopsies
╶ Observer-expectance effect (Pygmalion effect)
╶ A researcher subconsciously influences the participants due to
their beliefs in the treatment efficacy

Bias, confounding &
threats to validity
Information bias
╸ Observer bias (ascertainment bias)
╶ Hawthorne effect
╶ Study participants and researchers modify their behavior in
response to awareness of being studied.
╶ Eg. Physicians in antibiotic stewardship program may be less
likely to prescribe antibiotics
╶ Solution: blinding

Bias, confounding &
threats to validity
Placebo effect
╶ A form of cognitive bias
╶ Participants on placebo report outcomes based on preconceived
notions about the treatment’s efficacy
╶ Solution: blinding
Ecological fallacy
╶ Inferences about individuals are made from inferences about the
group to which individuals belong to based on ecological studies
╶ Eg. Countries with large fat consumption have high rates of breast
cancer => cannot deduce individual woman’s risk of breast CA based
on their fat consumption

Bias, confounding &
threats to validity
Confounding
╸ An extraneous variable directly associated with the exposure and
outcome that explains the observed relationship between exposure and
outcome
╸ Eg. In a study, smokers were found to have high rates of liver cirrhosis.
Alcohol consumption is a possible confounder
╶ Heavy alcohol users are more likely to be smokers and have cirrhosis

Bias, confounding &
threats to validity
Solutions to Confounding
╸ Randomization
╶ Controls for known and unknown confounders
╶ Successful randomization => averaging of all confounding variables
between the treatment and control arm
╸ Matching
╶ In case control studies, selecting controls who closely match cases
in other attributes except for disease/outcome
╶ (-) May not eliminate unknown confounders

Bias, confounding &
threats to validity
╸ Cross-over study design
╶ Each patient serve as their own control after a washout period.
╶ Initial treatment may be randomized
╶ (-) Ineffective washout may cause lingering tx effects.
╸ Restriction
╶ Selection criteria enforced to eliminate potential confounders.
╶ Eg. Effect of age minimized by enrolling only patients from certain
age groups
╶ (-) Loss of generalizability of results

Bias, confounding &
threats to validity
╸ Stratified Analysis
╶ Data in the sample are divided into subgroups by a strata or variable
and analyzed separately
╶ Can be used to differentiate between confounders and effect
modifiers
╶ Eg. Stratifying by hx of heavy alcohol consumption in the study and
then looking into association between smoking and liver cirrhosis
╶ (-) Must have collected data on the confounder/stratum

Bias, confounding &
threats to validity
Effect modification
╸ Effect of exposure on the outcome is modified by another variable
╸ NOT A SOURCE OF BIAS
╸ Often a natural/biological phenomenon
╶ exposure has different impact based on different circumstances
╶ cannot be eliminated but worth discussing in a study
╸ Eg. A new drug of asthma is only efficacious in adults but not children

Bias, confounding &
threats to validity
Effect modification vs confounding
╸ Bias
╶ Confounding is a source of bias and should be eliminated
╶ Effect modification is not bias and does not need to be eliminated
╸ Stratified analysis
╶ For confounding, the relationship between effect of exposure
disappears/ is not statistically significant in all strata
╶ Eg. Smoking status à cirrhosis and stratified by hx of heavy
alcohol consumption.
╶ RR in heavy alcohol users = 1.05 (95% CI: 0.75, 1.35); RR in non-
alcohol users = 1.2 ( 95% CI: 0.8, 1.4)
Bias, confounding &
threats to validity
Effect modification vs confounding
╸ Stratified analysis
╶ For effect modification, the relationship between effect of exposure
is preserved/ statistically significant in at least 1 stratum.
╶ Eg.
╶ Efficacy of new drug X on asthma stratified by adults and
children.
╶ In adults, HR of asthma exacerbation = 0.7 (95% CI: 0.5,0.9), In
children, HR of asthma exacerbation = 0.7 (95% CI: 0.55, 1.05)

Internal vs.
External validity
Internal validity
╸ Relates to conclusions regarding cause and effect
╸ Needed to properly demonstrate causal relationship between exposure
and outcome
╸ Increased when research environment is tightly controlled
╶ Generalizability may compromise internal validity
╸ Threatened by systematic error and bias (confounding, measurement,
selection), regression to the mean etc

Internal vs.
External validity
External validity
╸ Refers to generalizability
╶ How well the observed relationships between exposure & outcome
apply to the real world, i.e. individuals outside the study population
╸ Can be biased due to
╶ artificial (such as tightly controlled) research environments
╶ non-representative samples
╸ E.g. Trial of drug conducted among patients in China used to extrapolate
its beneficial effects among populations in Sub-Saharan Africa

Internal vs.
External validity
Efficacy trials/studies
╸ Also called explanatory trials,
╸ Goal: determine if an intervention produces the expected result under
ideal circumstances
Effectiveness trials/studies
╸ Also called pragmatic trials
╸ Goal: measure the degree of beneficial effect under clinical settings
closely resembling the real world
Note: Trials/studies are not either-or and can exists in a continuum

Internal vs.
External validity Source: Singal et al. 2014

Statistical and
Clinical Significance
Statistical significance
╸ Probability that the relationship between exposure and outcome does
not occur by chance alone.
╸ Evaluated by tests of significance, compared by p-value (<0.05)
Clinical significance
╸ Observed (statistically significant) differences from a study are
important for the prognosis or management in the real world
╸ Magnitude of effect is important
╸ Eg. New BP med + dietary modification has statistically significant drop
of BP by 2 mmHg compared to dietary modification alone
Endpoints
Clinical endpoints/outcomes
╸ An objectively measurable outcome or endpoint to measure whether an
intervention in a clinical trial was beneficial
╸ Continuous (eg. BP, blood glucose),
╸ Categorical (eg. stroke, MI, death),
╸ Soft = not directly harmful (eg. pain)
╸ Hard = directly harmful (eg. death)

Endpoints
Surrogate endpoints/outcomes
╸ An endpoint used closely related to a primary outcome when the primary
clinical endpoint is not available for measure
╸ Generally lab tests, imaging or physical findings that are predictive of
future severe primary outcomes.
╸ E.g. rescue inhalerà asthma control

8 Clinical decision making, interpretation
and evidence-based data use
Azfar Basunia, MD
Clinical decision making
USPSTF guidelines on assessing level of evidence

╸ Level I: Evidence obtained from at least 1 randomized controlled trial
╸ Level II-1: Evidence obtained from several well designed controlled trials
w/o randomization
╸ Level II-2: Evidence obtained from several well designed cohort or case
control studies
╸ Level II-3: Evidence obtained from several time series experiments w/ or
w/o intervention.
╸ Level III: Expert opinion based on descriptive studies

USPSTF categories of recommendations

╸ Level A: Good scientific evidence, benefits outweigh risks. Clinicians
should discuss/offer to patients
╸ Level B: Fair scientific evidence, benefits outweigh risks. Clinicians should
discuss/offer to patients
╸ Level C: Fair scientific evidence, benefits may outweigh risks. Clinicians
should weigh individual pt considerations prior to offering
╸ Level D: Fair scientific evidence, risks outweigh benefits. Clinicians should
not offer to asymptomatic pt
╸ Level I: Scientific evidence is lacking or of poor quality

Evidence based medicine

╸ Integrating individual clinical expertise with best scientific evidence
╸ Individual patient factors (beliefs, values, risk profile) must be taken into
account and respected in most cases despite EBM guidelines
╶ Notable exception: communicable disease, child and elder abuse
╸ Evidence and recommendations judged by USPSTF guidelines and recs

Synthesis of concepts from real data

╸ Answerable clinical question formulated à PICO model
╶ Patient, intervention, comparison, outcome
╸ Systematic retrieval of best evidence from literature
╸ Critical appraisal of literature à internal validity and bias, precision,
clinical importance and external validity
╸ Application of results to clinical practice
╸ Evaluation of performance

Limitations
╸ Patient values are not commonly emphasized
╸ Large lag between study conductance, result publications, and
application of recommendations
╸ Cognitive biases, anecdotal and/or experience may cause clinicians to
reject EBM
╶ Defensive medicine: practice of over-prescribing tests, Rx,
procedures to reduce threat of lawsuits
╶ Overtreat b/c of experience with rare, shocking outcome or pt’s
emotional needs

9 Research Ethics
Azfar Basunia, MD
Informed consent
for research
Principles of Informed consent
╸ Patient was provided sufficient information regarding diagnosis, options,
benefits and risks of treatment options.
╸ Patient must have decision making capacity (no altered mental status,
acute psychiatric conditions like acute mania, psychosis)
╸ Patient is making the decision voluntarily (no coercion)

Informed consent
for research
Required disclosures to patient
╸ Purpose of study and all aspects of the study’s protocols and design
╸ Any potential risks and or benefits to the patient from the study
╸ Alternative treatment options
╸ Potential conflicts of interest, financial disclosures, industry sponsorship
Patients must provide informed consent prior to participating in the study
╸ Participants have the right to withdraw at any time and are not obligated
to provide reasons for withdrawing

Informed consent
for research
Informed consent from vulnerable populations
╸ Vulnerable populations:
╶ pregnant, fetuses, children (age < 18 yrs), prisoners
╸ Regulated by Office for Human Research Protection (OHRP)
╸ Pregnant individuals
╶ Preclinical studies in animals and clinical studies in non-pregnant
patients have already been conducted
╶ Data regarding potential harm to pregnant mothers and fetuses are
available

Informed consent
for research
╸ Children
╶ Provide direct benefit and is not more harmful to children compared
to adults. Eg. COVID-19 vaccine
╶ Require informed consent from parents and assent from children
╶ Unless emancipated, children cannot provide informed consent
╶ Assent: agreement to participate. No assent => no participation
╸ Prisoners
╶ Research investigates health issues that directly benefit prisoners
╶ Risk cannot be greater in prisoners compared to general population
╶ Cannot be coerced into participation with benefits such as sentence
mitigation as compensation

HIPAA
Health Insurance Portability and Accountability Act of 1996 (HIPAA)
╸ Identifies protected health information and sets standards for disclosure
╸ Only minimum necessary information should be disclosed
╸ No consent necessary for disclosure for the following:
╶ Health care providers directly involved in pt care (eg. consultants)
╶ Payment processing agencies (pt’s insurance company)
╶ Health care operations (legal services, auditing services)
╶ Special public health interests (transmissible disease, threat of harm
to pt or others, elder/child abuse)

IRB
Institutional Review Boards (IRBs)
╸ Reviews and approves studies involving human subjects in the US
╸ Responsible for ensuring rights and welfare of research participants
╸ Group consists of at least one scientist and one non-scientist
╸ Applies research ethics by reviewing research methods
╶ May approve, deny or ask for modifications to study design
╶ Ensures adequate consent has been obtained
╶ Ensures studies are following current institutional, ethical and
regulatory guidelines

Intervention
analysis
Interim analysis
╸ Analysis of data prior to completion of study
╸ Reasons for stopping trial prior to completion
╶ Intervention clearly beneficial
╶ Intervention clearly harmful
╶ Intervention clearly futile and continuing the study will not lead to
clinically meaningful effect
╶ Eg. MADIT II trial studying ICD vs antiarrhythmics in patients with prior
ventricular arrhythmias was stopped when ICD arm showed
significant clear benefit à 31% reduction in all-cause mortality
compared to antiarrhythmic arm (Source: Moss et al. NJEM 2002)
Intervention
analysis
Safety monitoring
╸ Monitoring of adverse event immediately after occurrence by an
independent physician with relevant expertise
╸ Independent safety officer (ISO)
╶ A clinician or researcher who is independent of the study team
╶ Carries out monitoring for patient safety, adverse events, trial
progress and data quality

Regulatory
issues
Drug development
╸ Process of bringing a new drug to the market once a biologically active
drug with likely therapeutic usefulness has been identified
Phases of approval:
╸ Phase 1:
╶ Evaluate drug safety: toxicity, maximum tolerated dose,
pharmacokinetics, pharmacodynamics
╶ Participants: Small number (<50) of healthy subjects

Regulatory
issues
Phases of approval:
╸ Phase 2:
╶ Further evaluate drug safety: optimal dosing, efficacy and adverse
effects
╶ Participants: Small number (<100) of patients with disease of interest
╸ Phase 3:
╶ Evaluate drug safety and efficacy for marketing: Compare drug to
current standard of care (if available) or placebo
╶ Randomized, blinded and controlled trial with large number (>100) of
patients with disease of interest
╶ FDA approval for general use granted after this stage
Regulatory
issues
Phases of approval:
╸ Phase 4:
╶ Post marketing surveillance: safety studies ( rare and long-term side
effects)

Regulatory
issues
Appropriateness of placebo
╸ Existing treatment with demonstrated efficacy exists => placebo-
controlled trial for new drug is unethical
╸ New intervention/treatment compared to current treatment (e.g. in non-
inferiority trial)

Regulatory
issues
Randomized controlled trials may not be appropriate when
╸ Interventions to prevent rare diseases (like SIDS) or uncommon adverse
effects of drugs may require very large sample sizes for adequate power
╶ Better assessed by observational studies (eg. Case-control studies)
╸ New intervention clearly beneficial and control subjects with either
placebo or standard regimen will clearly have poor outcomes

Regulatory
issues
Scheduling
╸ Federal policies and regulations determining manufacture, import, use,
possession and distribution of controlled substances.
╸ FDA and DEA determine addition and removal of drugs from schedules
╸ Schedule I: High abuse potential, no acceptable medical use. Eg. Heroin
╸ Schedule II: High abuse potential, restricted medical use. Eg. Morphine
╸ Schedule III: Moderate abuse potential, medical use permitted. Eg.
Ketamine, anabolic steroids
╸ Schedule IV: Lowest abuse potential, medical use permitted. Eg.
Benzodiazepines, tramadol
Regulatory
issues
Off label use
╸ Using a prescription or OTC drug in for an FDA unapproved clinical
indication or in an FDA unapproved age group, dosage or route of
administration.
╸ Generally legal unless it grossly violates ethical guidelines or safety
regulations.

Thanks!
You can find me at:
@AzfarBasunia
Azfar Basunia, MD
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USMLE Biostats &amp; Epi - A Complete Review - Azfar Basunia

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USMLE

╸ Prevalence of T2DM at the end of 2019 = 5,000/10,000 = 0.5

A > 65 yr 25,000 50% 2,000 80

B ≤ 65 yr 15,000 25.5% 600 40

B > 65 yr 43,750 74.5% 3,500 80

Health-adjusted life expectancy

Population attributable risk (PAR) &

╸ Will be discussed in Measures of Association section

╸ SMR > 1 indicates greater deaths among the study group

╸ SMR = 1 indicated observed deaths = expected deaths

B ╸ Solid vertical line à superiority margin

Head et al. Eur Heart J. 2012

Risk of bias graph

Source: Cochrane handbook

Risk of bias summary

Exposure/Test + True Positive (TP) False Positive (FP) Total exposure/test +

Relative Risk or Risk Ratio (RR)

Association Exposure + A B Total exposure

!. Exposure – C D Total exposure –

╸ Range of RR [0, +∞)

Association Uncontrolled HTN

100 pts w/ uncontrolled HTN and 50 normotensive pt were followed for 5

Conclusion: Patients with uncontrolled HTN are 3x likely to develop stroke

/,,# +0 ()*+#-1( 23+%4 ,"#(#(5 !. 7×9 Exposure + A B

╸ Range of OR [0, +∞)

Association Uncontrolled HTN +

╸ Use case control studies to approximate RR under rare disease assumption

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Attributable risk (AR) Exposure – C D

╸ How much an outcome may be attributable to the exposure/ risk factor

Absolute Risk Reduction (ARR)

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╸ Determine how much tx/intervention reduces risk of negative outcomes

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< 1 ppd C = 50 D = 1390

What is the incidence rate of esophageal CA over 10 year period?

What is the added risk of esophageal CA in individuals smoking > 1ppd

< 1 ppd C = 50 D = 1390

What percentage of esophageal cancer in the individuals smoking > 1ppd

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< 1 ppd C = 50 D = 1390

What percentage of esophageal cancer observed in the population can be

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(2) How many patients will have BG <80?

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USMLE Biostats & Epi - A Complete Review - Azfar Basunia

USMLE Biostats & Epi - A Complete Review - Azfar Basunia