3_General Principles of Laboratory Diagnosis

HBV

the cause of infectious hepatitis and human immunodeficiency.

ignorance and carelessness.

two most common causes of laboratory infections are

spread of infections

Safety in the clinical laboratory prevents the ___ to laboratory workers

responsibility of laboratory personnel

safety in the workplace is the

BSL-4

Which BSL:

• Dangerous and exotic, posing a high risk of aerosol-transmitted infections. Infection caused by these microbes are frequently fatal without treatment or vaccines

• examples: Ebola virus, smallpox virus

BSL-3

Which BSL:

• Microbes there can either indigenous or exotic, and they can cause serious or potentially fatal disease through respiratory transmission

• Examples: Yersinia pestis (plague), Mycobacterium tuberculosis, SARS,

  rabies virus, West Nile Virus, hantaviruses

BSL-2

Which BSL:

• Moderate potential hazard to personnel and the environment. Includes bacteria and viruses that cause mild disease to humans, or are difficult to contract via aerosol in a lab setting.

• Examples: Hepatitis A virus, Streptococcus pyogenes, Borrelia

  burgdorferi (Lyme disease), Salmonella species

BSL-1

Which BSL:

• Not known to consistently cause disease healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment.

• Examples: Saccharomyces cerevisiae, E. coli K-12, and non-infectious bacteria, Bacillus subtilis

• Should be limited

• Barrier protection (coats, gloves) should be used.

• Teaching laboratory that does not work with known pathogens

HEPA filter

high-efficiency particulate air filter present in Biological safety cabinet

Diagnostic/Clinical Microbiology

A field of microbiology concerned with the etiological diagnosis of infection.

PHYSICIAN

• Ensures proper sample is collected for diagnostic methods

• Gives guidance by way of clinical diagnosis

MICROBIOLOGIST

• proper collection and transport;

• feedback for proper treatment

• Direct Detection of Pathogens

• Growth-Independent Diagnostic Methods

• Antimicrobial Drug Susceptibility Testing

Microbiological Identification of Pathogens:

Direct Detection of Pathogens

Microbiological Identification of Pathogens:

• Microscopy and related techniques

• Detection thru culture techniques (growth dependent techniques)

• Growth-independent methods

Growth-Independent Diagnostic Methods

Microbiological Identification of Pathogens:

• Immunoassays for Infectious Diseases

• Agglutination

• Immunofluorescence

• Enzyme Immunoassays, Rapid Tests, and Immunoblots

• Nucleic Acid Amplification

Antimicrobial Drug Susceptibility Testing

Microbiological Identification of Pathogens:

• Minimum Inhibitory Concentration

• Measuring Antimicrobial Susceptibility

Growth-independent methods

(ex. quantitative PCR (qPCR), Next-Generation Sequencing (NGS), Fluorescence In Situ Hybridization (FISH), etc

growth dependent techniques

(ex. dilution plating, colony morphology analysis, biochemical testing, and using selective/differential media)

• First, the specimen must be obtained from the actual site of the infection; the sample must be taken aseptically to avoid contamination with irrelevant microorganisms. Prior Requirements

• Next, the sample size must be large enough to ensure an inoculum sufficient for growth.

• Third, the metabolic requirements for organism survival must be maintained during sampling, storage, and transport.

• Finally, the sample must be processed as quickly as possible to avoid degradation.

Prior Requirements in Microbiological Identification of Pathogens:

Stuart’s, Cary-Blair, Amies Charcoal Medium

Availability of a Variety of Transport Media:

Stuart’s Medium

Availability of a Variety of Transport Media:

• (pH is maintained; no nutrients, prevents dehydration of secretions and enzymatic self-destruction of pathogens)

Cary-Blair Transport Medium

Availability of a Variety of Transport Media:

• ~ for fecal specimens; for recovery of Salmonella, Shigella, Vibrio, Campylobacter, etc.

Amies Charcoal Medium

Availability of a Variety of Transport Media:

• for the recovery of N. gonorrheae and other fastidious organisms.

Microscopy (direct examination) , Culture isolation and pathogen ID

Direct detection of pathogens:

Wet mount, 10% KOH, India ink, Lugol iodine

Direct microscopic examinations:

Wet mount

Direct microscopic examinations:

• Unstained preparation is examined by brightfield, darkfiled, or phase-contrast microscopy

10% KOH

Direct microscopic examinations:

• KOH is used to dissolve proteinaceous material and facilitate detection of fungal elements that are not affected by strong alkali solution. Dyes such as lactophenol cotton blue can be added to increase contrast between fungal elements and background.

India Ink

Direct microscopic examinations:

• Modification of KOH procedure in which ink is added as a contrast material. Dye is primarily used to detect Cryptococcus spp. in cerebrospinal fluid and other body fluids. Polysaccharide capsule of Cryptococcus spp. excludes ink, creating halo around yeast cell

Lugol iodine

Direct microscopic examinations:

• Iodine is added to wet preparations of parasitology specimens to enhance contrast of internal structures. This facilitates differntiation of amoeba and host white blood cells

Gram stain

Differential stain method:

• Most commonly used stain in microbiology laboratory, forming basis for separating major groups of bacteria (e.g., gram-positive, gram-negative). After fixation of specimen to glass slide (by heating or alcohol treatment), specimen is exposed to crystal violet and then iodine is added to form complex with primary dye. During decolorization with alcohol or acetone, complex is retained in gram-positive bacteria but lost in gram-negative organisms; counterstain safranin is retained by gram-negative organisms (hence their red color). The degree to which organism retains stain is function of organism, culture conditions, and staining skills of the microscopist.

Ziehl-Neelsen stain

Acid-fast stains:

• Used to stain mycobacteria and other acid-fast organisms. Organisms are stained with basic carbolfuchsin and resist decolorization with acid-alkali solutions. Background is counterstained with methylene blue. Organisms appear red against light blue background. Uptake of carbolfuchsin requires heating specimens (hot acid-fast stain)

24 hours

avoid gram variable, Specimen shall be useable up until

BAP, CAP, MHA, THB, SDA

Non-selective culture media:

MCA, MSA, XLDA, LJM, MA, CHROMa, IMA

Selective/Differential culture media:

BCYEa, CFa, Lim Broth, MCSa, RLA, TCBS a

Specialized Culture Media:

Ouchterlony immuno–double-diffusion

Selective Immunological techniques:

• Detect and compare antigen and antibody

• Fungal antigen and antibody

Immunofluorescence

Selective Immunological techniques:

• Detection and localization of antigen

• Viral antigen in biopsy (e.g., rabies, herpes simplex virus)

Enzyme immunoassay (EIA)

Selective Immunological techniques:

• Same as immunofluorescence

• Same as immunofluorescence

Immunofluorescence flow cytometry

Selective Immunological techniques:

• Population analysis of antigen-positive cells

• Immunophenotyping

ELISA and Radioimmunoassay (RIA)

Selective Immunological techniques:

• Quantitation of antigen or antibody

• Viral antigen (rotavirus); viral antibody (anti-HIV)

Western blot

Selective Immunological techniques:

• Detection of antigen-specific antibody or antigen

• Confirmation of anti-HIV seropositivity (antibody)

Complement fixation

Selective Immunological techniques:

• Quantitate specific antibody titer

• Fungal, viral antibody

Hemagglutination inhibition

Selective Immunological techniques:

• Antiviral antibody titer; serotype of virus strain

• Seroconversion to current influenza strain; identification of influenza

Latex agglutination

Selective Immunological techniques:

• Quantitation and detection of antigen and antibody

• Rheumatoid factor; fungal antigens; streptococcal antigens

Antibody Titers

• Measure of antibody quantity (titer) directed to antigens produced by the suspected pathogen.

• is low during the acute stage of the infection and rises during convalescence

titer

• defined as the highest dilution (lowest concentration) of serum at which an antigen–antibody reaction is observed

• is the inverse of the greatest dilution (lowest concentration [e.g., dilution of 1:64 = titer of 64]) of a patient’s serum that retains activity in one of the immunoassays

acute stage of the infection

antibody titer is low during the ___ and rises during convalescence.

convalescence

antibody titer is low during the acute stage of the infection and rises during ___

Agglutination

• reaction between antibody and particle-bound antigen resulting in visible clumping of the particles.

• a commercially available suspension of latex beads coated with antibodies to protein A and clumping factor, two proteins found exclusively on the surface of Staphylococcus aureus cells, is specific for identification of clinical isolates of S. aureus.

visible clumping of particles

Positive result for agglutination:

Immunoflourescence

• Antibodies containing conjugated fluorescent dyes (fluorescent antibodies) can be used to detect antigens on intact cells.

• Direct – antibody with fluorescent dye links with target antigen,

• Indirect – non-fluorescent antibody links with target antigen; fluorescent antibody links with non-fluorescent antibody

• Viewed through fluorescence microscope

fluorescence microscope

Immunoflourescence is Viewed through

Direct Immunoflourescence

antibody with fluorescent dye links with target antigen

Indirect Immunoflourescence

non-fluorescent antibody links with target antigen; fluorescent antibody links with non-fluorescent antibody

ELISA (Enzyme-Linked Immunosorbent Assay)

• enzyme is covalently attached to an antigen or antibody molecule, creating an immunological tool with high specificity and high sensitivity.

• enzymes typically bound to antigen or antibody include peroxidase, alkaline phosphatase, and β galactosidase, all of which interact with enzyme specific substrates to form colored reaction products that can be detected in very low amounts.

Direct ELISA

Type of ELISA:

• Detects - pathogen antigen

• Uses - enzyme-labeled pathogen specific antibody (supplied)

Indirect ELISA

Type of ELISA:

• detects - pathogen-specific antibodies

• Uses - enzyme-labeled antibody directed (supplied) to pathogen specific antibodies in patient samples

Sandwich ELISA

Type of ELISA:

• Detects - pathogen-specific antibodies

• Uses - enzyme-labeled pathogen antigen (supplied)

Combination ELISA

Type of ELISA:

• detects - pathogen-specific antibodies and antigen

• Uses:

  • enzyme-labeled pathogen antigen (supplied)

  • Enzyme-labeled pathogen-specific antibody

no enzyme reaction = No antigen-antibody complex

In ELISA, if the test kit does not turn into blue, there is __

Nucleic Acid Amplification

• examines DNA derived from suspected infected • do not depend on pathogen isolation or growth, or on the detection of an immune response to the pathogen.

• species-specific nucleic acid sequences that are detected in the assays. • used for identification of a number of individual pathogens; particularly useful for identifying viral and intracellular infections, where culturing the responsible agents may be very difficult or even impossible.

• Can diagnose genetic disease and detect low levels of viral infection.

• In forensic medicine it is used to analyze minute traces of blood and other tissues in order to identify the donor by his genetic “fingerprint.”

• Three basic components:

  • (Make cDNA copy of RNA Sample – RTPCR)

  • Extraction of subject DNA or RNA

  • Amplification of the nucleic acid using gene-specific primer

  • amplified nucleic acid product (the amplicon) is visualized through gel electrophoresis or other methods.

Oxoid’s MIC Evaluator (MICE) strips: agar diffusion method

• Antibiotic susceptibility determined by the Etest (AB BIODISK, Solna, Sweden) for different antibiotics.

• Each strip, laid on an inoculated plate before incubation, is calibrated in μg/ml starting with the lowest concentration from the center of the plate.

• The lowest concentration of antibiotic that inhibits bacterial growth is the MIC value.

Minimum Inhibitory Concentration (MIC) value

The lowest concentration of antibiotic that inhibits bacterial growth is the