Clinical Diagnosis, Clinical Study and Epidemiology

Laboratory Techniques for Clinical Diagnosis

Aim of clinical microbiology

• provide accurate and timely info about microbes that may be involved in patients’ disease process

• perform antibody testing: detect specific pathogens for diagnositing infection

• provide info on antimicrobial susceptibility of bacteria isolated

• ★ meaningful lab testing requires correct specimen typed collected correctly at the correct time and sent to the lab quickly using correct transport condition

Specimen collection and delivery

• take appropriate specimen

• collect specimen at appropriate time, during acute phase of disease

• collect beore patient receives antimicrobials

• avoid contamination from

  • normal flora

  • non-sterile equipment

• correct containers and appropriate transport media

Ways to diagnose infections

Direct visualisation of microorganism

• Gram stain

  • identify gram positive and negative bacteria

• acid-fast stain (Ziehl-Neelsen stain)

  • some bacteria (esp. mycobacteria) do not take up Gram stain

    • due to waxy mycolic cell wall

  • rely on the ability of such organism to retain the atain in the presence of ‘decolorising‘ agents

    • e.g. acid and alcohol

  • process

    • primary stain: Carbolfuchsin

    • mordant by heating

    • decoloriser: acid alcohol

    • counter stain: methylene blue

• Fluorescene staining

  • detected by fluorescene microscope

  • direct test

    • labeled antibodies bind on antigen of pathogen

    • emit fluorescent

  • indirect test

    • unlabeled antibodies bind on antigen of pathogen

    • labeled anti-immunoglobulin bind on surface of antibodies

    • emit fluorescent

• Electron microscope

  • direct identification of virus particles and detection of virus

  • fluid used for examinsation: dried on a copper grid → examine

    • disadvantage: require technical expertise and expensive

  • ~ 10^6 virus particles/mL to detect

  • suitable for stool, vesicular fluid spcimen

    • e.g. rotavirus

Culture microorganism

• Gold standard

  • enables antibiotics or antifungal sensitivty testing

  • labor-intensive

  • slow (days)

  • fastidious microbes cannot be cultured in artificial media

  • viable organisms: diff to recover from specimen of patients if received antimicrobial therapy

  • very tedious for virus

• culture condition

  • oxygen

  • carbon dioxide

  • temerature

• culture media

Identify specific microbial gene or product

• nucleic acid detection (PCR)

  • PCR

  • Real-time qPCR

    • ground state fluorophore: quencher inhibit the fluorescent reporter

    • → excited state fluorophore: polyerase cleavge → fluorescent signal is emited

  • Sequencing

    • Sanger sequencing

    • Next generation sequencing

• antigen detection: soluble carbohydrate antigens

  • e.g. Cryptococcus neoformans

• Viral proteins or bacterial toxin

  • western blotting

  • immunofluorescence staining

  • MALDI-TOF

Detect specific antibodies of pathogens

• antibodies to be detected

  • IgM: detected earlier in infection & indicative of active infection

  • IgG: indicative of exposure in recent past

  • divided into 2 parts: constant region and variable region

• Hemagglutination inhibition assay

  • usually for diagnosis of influenza

  • result intepretation

    • only RBCs: no reaction

    • virus + RBCs: hemagglutination

    • virus + RBCs + antibodies: hemagglutination inhibition

  • do not differentiate IgG and IgM

• Class switching reconbination (CSR)

• ELISA (enzyme-linked immunosorbent assay)

  • bind antigen to solid phase

  • addd unknown antibodies

  • detect bound antibodies with labeled anti-antibodies

  • adaptable for quantitation of antibodies

• Western blotting

  • proteins are separated by electrophoresis and transferred to a membrane

  • probing with antibodies and detect the target protein by enzyme reaction

• immunochromatographic test

  • convenient, quick, easy and ni power requirement

  • not as sensitive as conventional ELISA

  • adaptable to both antigen and antibody expression

Clinical Study

Case report or case series

Nature

• descriptive

• often used to new diseases with unclear cause

• more than one cases are analysed

  • symptoms

  • patients’ demographics

• purpose: investigate clues about etiology or course

Application

• emerging infectious disease

• rare disease

Cross-sectional study

Nature

• measure frequency of an outcome and/or exposure ina defined population at a given time frame

• single population only

  • e.g. all HK residents

• identify frequency of diease and risk factors

  • e.g. lung cancer and smoke

• ╳ follow-up required

Application

• prevalence of disease

Case-control study

Nature

• identify ppl with outcome (case) and a representative group of ppl w/o outcome (control)

• compare them regarding to their past exposure

• matching cases with control

  • reduce confounding

  • balance all potential confounders between cases and controls

Application

• odd ratio

  • odds of disease in the exposed/ ods of disease in the unexposed

Cohort study

Nature

• compare groups with or without exposure

• two types

  • prospective: monitor group over time

  • retrospective: look back time among groups

Application

• identify risk factors

• quantify odds ration between diseased and non-dieased

• not applicable for rare disease

  • likelihood of disease onset is too low

  • too few cases to draw conclusion

Epidemiology and Control of infectious disease

Definitions

• Epidemiology: study of distribution and determinants of health-related states or events in specified population and the application of the study to control of health problem

• Incidence: number od new cases occurring in a population during a specific period of time

• Prevalence

  • umber of existing cases ina population at a give point in time

  • can be influenced by

    • occurrence of new cases (incidence)

    • duration of each case

  • examples

    • disease with short duration: mainly influenced by incidence

    • chronic disease with low mortality: high prevalence even if incidence is low

• time periods

  • latent period: between infetion nad becoming infectious

  • infectious period: infected individual being able to transmit infectious agent

  • window period: bwteen infection and when diagnositic tests first become positive

  • incubation period: from infection time till the individual develops symptoms

Epidemiological Triangle

Susceptible host

• age, sex, race, gene, immune status,occupation…

Vehicles

• temperature, humidity, altitude

• crowding, housing, neighbourhood

• water, food

• radiation, pollution, noise

Sourse

• biological: bacteria, virus

• chemical: poison, smoke, alcohol

• physical: trauma, radiation, fire

• nutritional: lack, excess

Factors affecting infectiousness

• infectious agent

  • time of incubation period

  • time of infectious period

  • probability of transmission

• environment

  • type of contacts causing infection

  • number of contacts

• characteristics of individuals in the population

  • immunity

  • susecptibility

Reproduction number

Basic reproduction number (R0)

• the average number of infected secondary cases produced by each infectious case in a totally susceptible population

• R0=c*d*p

  • c: number of contacts per unit time

  • d: duration of infectiousness of the case

  • p: transmission probability

Effective reproduction number (Rt)

• the average number of sevondary cases ina population where not all individuals are susceptible

• net reproductive rate depends on R0 and proportion of susceptible individual

• Herd immunity

  • occur when a significant proportion of population are vaccinated (or other immune acquired methods)

  • → protection for susceptible individuals

• Interpretation of Rt

  • Rt >1: each infected patient will infect more than one person on average → number of cases wil increase exponentially over time

  • Rt =1: each infected patient will infect one person on average → stabilised outbreak and number of cases will remain stable

  • Rt <1: each infected patient will infect fewer thanone person on average → outbreak will slow down over time