TV4001 - Epidemiology 7 - Seroepidemiology

What is serological epidemiology?

One of the main constituents of serum that is frequently measured, is?

Investigation of disease and infection in populations, by the measurement of variables present in serum.

specific antibody activity of globulins

Methods of expressing amounts of antibody

The concentration of antibody is expressed as? What is that?

A titre - the highest dilution of serum that produces a test reaction

Animals with detectable antibody titres are?

nimals with no detectable antibodies are?

What is the third thing?

SEROPOSITIVE

SERONEGATIVE

Animals previously seronegative, and now seropositive, have SEROCONVERTED

Serology can be successfully used as an epidemiological tool in many ways

INTERPRETATION OF SEROLOGICAL TEST RESULTS

It is, generally, impossible to identify?

Why?

Probable origin or cause of a titre in a single test for antibody

Does not, ordinarily, differentiate between ACTIVE and PASSIVE IMMUNE RESPONSE or RECENT or PAST EXPOSURE

Methods of expressing amounts of antibody
If the highest dilution that produces a test reaction is 1 in 32, then the titre is?

How can we use the reciprocal in this example?

1/32

The reciprocal, 32, can be quoted, indicating that the undiluted serum contains 32

times the antibody for reaction

A true positive result derives from?

False positive results occur for a variety of reasons, example includes?

Actual infection

Cross-reaction can occur between an infectious agent and antibodies to different organisms with similar antigens,

A true negative result indicates?

False negative results can occur for several reasons, those being?

Absence of infection

• Some animals have induced/natural antigen tolerance = don’t produce ABs when exposed to agent

• Poor timing of test fails to detect infection - ABs aren’t made in that time

• Unsuitable tests for detecting infection

DETECTING DISEASE WITH SCREENING TESTS

Screening is the application of a test to?

Apparently healthy animals in order to detect infection or sub-clinical disease

• It is not diagnostic and requires apt follow up

• Can be done on a large scale

• Oft used in conjunction with more specific and expenny DX tests

DETECTING DISEASE WITH SCREENING TESTS

What is the “Gold Standard”?

The diagnostic test evaluates? How?

Describe the test results

A definitive DX done by biopsy, surgery, long-term followup or another acknowledged standard of diagnosis

Two categories of animals.

Firstly, animals found to have the disease of interest when tested by an accepted "Gold Standard" of diagnosis, such as biopsy or microbiological culture.

Secondly, disease-free animals tested by the same "Gold Standard"

There are four possible interpretations of test results. Two of which are correct and two of which are wrong

DETECTING DISEASE WITH SCREENING TESTS - Sensitivity and Specificity

The Gold Standard has ID’d which animals having the disease?

The sensitivity of a test is its ability to? How is this expressed?

Has identified (a + c) animals as having the disease of interest, and the "a" animals as having POSITIVE diagnostic results.

Detect diseased animals or its ability to detect the disease when it is present.

This is expressed as a/(a + c) (often as a %)

DETECTING DISEASE WITH SCREENING TESTS - Sensitivity and Specificity

Similarly, as shown in the vertical column of the contingency Table 3, the absence of disease is expressed as?

What does this represent?

d/(b+d).

The specificity of a test, which is the ability of a test to detect nondiseased animals or the ability of a test to identify the absence of disease correctl

DETECTING DISEASE WITH SCREENING TESTS - Sensitivity and Specificity

A sensitive test should rarely?

A specific test will rarely?

miss an animal with the disease.

misclassify animals without the disease as diseased

DETECTING DISEASE WITH SCREENING TESTS - Uses of sensitive tests?

• Helpful during early stages of DX workup to RULE OUT diseases that are unlikely possibilities

• Sensitive tests that are positive usually in disease presence should be used if there is penance for missing a disease

• Also useful when the probability of disease is somewhat low and the purpose of this test is to discover disease

DETECTING DISEASE WITH SCREENING TESTS - Uses of specific tests?

• Specific tests are useful to "RULE IN" or confirm a diagnosis suggested by other data, a highly specific test is rarely POSITIVE in the absence of disease

• Test is apt if a FALSE POSITIVE can harm the animal i.e. test and slaughter policy or cause financial distress or prevalence of disease in pop. is low

PREDICTIVE VALUE OF SCREENING TEST RESULTS

The predictive value of a POSITIVE test is defined as?

How to calculate?

Proportion of true diseased animals among those that test POSITIVE - the probability the animal has the disease when test is +ve

Calculated as a/(a+b)

PREDICTIVE VALUE OF SCREENING TEST RESULTS

The predictive value of a NEGATIVE test is?

How to calculate?

The proportion of true non-diseased animals among those that test NEGATIVE - the chance the animal doesn’t have the disease when the test is negative

It is calculated as d/(c+d)

Went up to 15

Methods of improving predictive value

How can we do this?

Screen only high prevalence pop. i.e. pop. with likely high rate of infection

• Studies find subgroups with high risk of infection in these groups

• Screening can then be concentrated on those with high risk

2nd method - Use more than one screening test

Can be done several ways

• Use an inexpensive, relatively sensitive on all animals THEN use a more sensitive expenny test on the animals +ve to first test

• Ciuld also use 2+ tests simultaneously to all individuals

Methods of improving predictive value

2nd method - Use more than one screening test

• Using 2+ tests simultaneously to all individuals

When tests are used in this manner, the resultant sensitivity and specificity are dependent on the way the results are interpreted, in what ways can we do this?

Parallel interpretation - Animal D+ if it reacts +Ve to one or both tests

• Inc sensitivity but dec specificity of combined tests

Series interpretation - Animal D+ if BOTH tests are +ve

• Inc specificity but dec sensitivity

MOLECULAR EPIDEMIOLOGY

Molecular epidemiology of infectious disease looks at?

The practical goals of molecular epidemiology are to?

Distribution and determinants of infectious diseases by utilizing molecular biology methods

• ID agents responsible for infectious diseases

• Determine their physical sources, bio relationships, transmission route

• ID genes causing virulence

• Find vaccine relevant Ags and Drug resistance

Applications of molecular strain-typing methods to epidemiologic problems

Includes? Describe each

Determining dynamics of disease transmission in geographically widespread areas:

• Study intro and spread of agent into area,

• ID changes in prevalence of drug-resistance,

• find factors that aid transmission

Identifying risk and quantify attributable risk fractions in sporadic outbreaks of infectious diseases

• Distinguish epidemic from endemic occurrences of Dz

• ID new clones of infectious agents

• ID specific vehicles and RF for sporadic infections

Stratifying data and refining epidemiologic study designs

Distinguishing parhovars from nonparhovars

• ID new virulence factors and pathos

Addressing hospital and institutional infectious disease problems

• Studying nosocomial drug resistant pathos

• Distinguish btw nosocomia outbreaks from non-outbreak infection clusters

• ID RF for opportunistic infection

• Study polyclonal infections

Identifying genetic determinants of disease transmission

• ID reasons for enhanced spread of strain in a region

• ID gene diffs btw commensal and pathos

• ID reasons for emergence of new infectious agents

When to use molecular strain-typing methods

The decision to use a molecular technique as opposed to a particular conventional method should be based on the following considerations…

(i) simplicity, (ii) high throughput, (iii) cost, and (iv) appropriatenessIssues affecting the Sensitivity and Specificity of molecular tests

Issues affecting the Sensitivity and Specificity of molecular tests

Field perspective aspects include?

Collection techniques - Collect only tissue of interest and avoid contaminants amplifying the incorrect product (affects specificity)

Collection of correct tissue

• Some agents only found in specific tissues, PCR not specific if wrong one took

• Agent may not be uniformly distributed within single tissue type (need subsampling to inc specificity)

Storage / degradation of materials

• degradation of nucleic acids is expected and can dec spec and sens

Bacterial loads

• May be too low to be picked up by PCR - affects sensitivity

Evolution

• Primers good for genes of interest but genes may have mutated - affects specificity

Issues affecting the Sensitivity and Specificity of molecular tests

Lab perspective aspects include

Isolation?

Lysis?

RNA?

Size?

Contamination?

Quantity?

Primer design?

Ratio?

Concentration?

Isolate correct tissue - improper purification can lower the sensitivity test reactions

Shit lysis of cells - no DNA in solution can be amplified = loss of sens

Loss of DNA during purification - DNA not picked up = poor sens

RNA degradation - dec PCR specs&sens

Size of Template - PCR more specific for smaller base pair fragments

Contamination

• Buffers, primers, plastic, can skew results

• Incorrect DNA by user error/poor purification

DNA quantity

• Sample too small or too small quantities

• OR product is amplified but detection limit too low to be visible to human eye on gel

Primer design

• Poorly designed? = Won’t attach or attach to non-specific products

Primer:Template ratio

• Too much primer = reaction prone to gen non-specific products

• Too little primer = poor sens

MgCl2 concentration

• Mg2+ determines how well primers bind to template

• Mg also needed for polymerase and dNTP to work

• Higher Mg2+ = less spec binding

• Lower Mg2+ = nothing binds

Issues affecting the Sensitivity and Specificity of molecular tests

Lab perspective aspects include

Separation?

Cycles?

Inhibitors?

Mutations?

Conditions?

Temp?

Issues with sensitivity related to template separation

Inhibitos affect sens&specs

• PCR inhibitors inc: EDTA, Phenol, Proteinase K

  • Methods

    • Polymerase binding

    • DNA interaction

    • Interact with polymerase during extension

Cycles

• PCR continued beyond exponential amplification phase = non-specific product amplification OR desired product disappears

Polymerase-induced mutations

• Taq can’t proofread = shit specs

Reaction conditions

• Changed conditions = primers can’t anneal

• Changes inc: add glycerol, Dec pH, dec primers/dNTPs/MgC12

Temp

• Annealing temp KEY for specificity

• Higher temperature, and shorter annealing and extension periods = Higher specs