laboratory diagnosis of infectious diseases prof dr gülden Çelik [email protected]
TRANSCRIPT
Learning ObjectivesAt the end of this lecture, the student should be
able to:
• list the main methods in diagnosis of infectious diseases caused by different type of microorganisms
• explain the importance of these methods in diagnosis• List the main advantages and disadvantages of each
type of test• Ensure the type, storage and transportation of
specimen in appropriate tests
Laboratory diagnosis
• Direct• Indirect
Laboratory diagnosis
• Direct: -Microscopy-Culture-Antigen-Nucleic acid
• Indirect:-Specific antibody (Serology)
Laboratory diagnosis
• Direct: -Microscopy-Culture-Antigen-Nucleic acid
• Indirect:-Specific antibody (IgG, IgM, IgA)
Boy with fever and rash
• In early June a 15-year old boy comes to your practice with his mother. He had been fine until about five days ago when he developed a fever. He has a stiff neck and a rash on his back. His mother reports that he was playing in the woods with some friends recently.
Which of the following bacteria may be the agent
• Pseudomonas aeruginosa
• Clostridium perfringens• Borrelia burgdorferi • Streptococcus pyogenes
• What do you see?• Which type of microscopy is this?
Tick-born disease
• Borrelia burgdorferi is the causative agent of Lyme disease.
• This bacterium, just like Treponema pallidum, is a member of the spirochetes, the family of spiral-shaped bacteria.
Boy with fever and rash
• After an incubation period of 3 to 30 days,
• develop at the site of the tick bite.
• The lesion (erythema migrans) begins as a small macule or papule and then enlarges over the next few weeks, ultimately covering an area ranging from 5 cm to more than 50 cm in diameter
Definition of Lyme Disease
• Lyme disease begins as an early localized infection, progresses to an early disseminated stage, and if untreated, can progress to a late manifestation stage.
• B. burgdorferi organisms are present in low numbers in the skin when erythema migrans develops. This has been shown by culture of the organism from skin lesions or detection of bacterial nucleic acids by polymerase chain reaction (PCR) amplification.
• Microscopic examination of blood or tissues from patients with Lyme disease is not recommended, because B. burgdorferi is rarely seen in clinical specimens.
Lyme Disease• Microscopy • Culture • Nucleic-Acid-Based Tests :65% to 75% with skin
biopsies, 50% to 85% with synovial fluid • Antibody Detection :• Spesific IgM: IgM antibodies appear 2 to 4 weeks
after the onset of erythema migrans in untreated patients; the levels peak after 6 to 8 weeks of illness and then decline to a normal range after 4 to 6 months.
• Spesific IgG
Microscopic Principles and Applications
• In general, microscopy is used in microbiology for two basic purposes:
1-the initial detection of microbes 2-the preliminary or definitive identification of
microbes.
Microscopic Principles and Applications
• The microscopic examination of clinical specimens is used to detect:
- bacterial cells, - fungal elements, - parasites (eggs, larvae, or adult forms), and - viral inclusions present in infected cells.
Microscopic Principles and Applications
• Characteristic morphologic properties can be used for the preliminary identification of
-most bacteria and -are used for the definitive identification of
many fungi and parasites.
Microscopic Methods
• Brightfield (light) microscopy • Darkfield microscopy • Phase-contrast microscopy • Fluorescent microscopy • Electron microscopy
Darkfield Microscopy
• Treponema pallidum (syphilis)• Leptospira spp. (leptospirosis)
Direct Examination
• The sample:• can be suspended in water or saline (wet mount),• mixed with alkali to dissolve background material (potassium hydroxide
[KOH] method) : fungal elements • mixed with a combination of alkali and a contrasting dye (e.g.,
lactophenol cotton blue: fungal elements Lugol iodine : Iodine is added to wet preparations of parasitology specimens to enhance contrast of internal structures. Facilitates differentiation of protozoa and host white blood cells.
• The dyes nonspecifically stain the cellular material, increasing the contrast with the background, and permit examination of the detailed structures.
Direct Examination
• A variation is the India ink method, • in which the ink darkens the background rather than the cell.• This method is used to detect capsules surrounding organisms, such as
the yeast Cryptococcus (the dye is excluded by the capsule, creating a clear halo around the yeast cell), and
• is a rapid method for the preliminary detection and identification of this important fungus.
Differential Stains
• Differential stains:
• Gram stain : -bacteria-Yeasts (yeasts are gram-
Ipositive).• Iron hematoxylin and trichrome stains:protozoan parasites• Giemsa stain: blood parasites and other selected organisms
• Acid-Fast Stains• Ziehl-Neelsen stain: Used to stain mycobacteria
and other acid-fast organisms. • Kinyoun stain: Cold acid-fast stain (does not require
heating)
• Auramine-rhodamine: Same principle as other acid-fast stains, except that fluorescent dyes (auramine and rhodamine) are used for primary stain
• Modified acid-fast stain: Weak decolorizing agent is used with any of three acid-fast stains listed. Whereas mycobacteria are strongly acid-fast, other organisms stain weaker (e.g., Nocardia, Rhodococcus, Tsukamurella, Gordonia, Cryptosporidium, Isospora, Sarcocystis, and Cyclospora).
• These organisms can be stained more efficiently by using weak decolorizing agent. Organisms that retain this stain are referred to as partially acid-fast.
Microscopic Principles and Applications
• The microscopic detection of organisms stained with antibodies labeled with fluorescent dyes or other markers has proved to be very useful for the specific identification of many organisms.
• Fluorescent Stains• Acridine orange stain: Used for detection of
bacteria and fungi in clinical specimens. • Auramine-rhodamine stain: Same as acid-fast
stains.• Calcofluor white stain: Used to detect
fungal elements and Pneumocystis spp.
Direct fluorescent antibody stain
• Antibodies (monoclonal or polyclonal) are complexed with fluorescent molecules. Specific binding to an organism is detected by presence of microbial fluorescence. Technique has proved useful for detecting or identifying many organisms (e.g., Streptococcus pyogenes, Bordetella, Francisella, Legionella, Chlamydia, Pneumocystis, Cryptosporidium, Giardia, influenza virus, herpes simplex virus).
• Sensitivity and specificity of the test are determined by the number of organisms present in the test sample and quality of antibodies used in reagents.
Because most organisms are colorless and transparent, various dyes (stains) are used to see the individual cells
A variety of different types of stains are used in the microbiology lab, including:Contrast stains (e.g., methylene blue,
lactophenol cotton blue, India ink, iodine)Differential stains (e.g., Gram stain, spore
stains, acid-fast stains, Giemsa stain, silver stains, Trichrome stain)
Fluorescent stains (e.g., acridine orange, auramine-rhodamine, calcofluor white, antibody-conjugated fluorescent stains)
Stains
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Methylene Blue Stain
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Corynebacterium diphtheriae
Methylene Blue Stain
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Lactophenol Cotton Blue (LCB) Stain
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primarily for observing the morphology of fungal molds :
Aspergillus
Lactophenol Cotton Blue (LCB) Stain
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India Ink Stain
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The India ink stain: negative contrasting
stain Cryptococcus
neoformans. The ink is excluded by the fungal capsule so the fungi (arrows) are unstained and surrounded by a clear halo, while the ink particles provide a background contrast.
India Ink Stain
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Iodine Stain
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The iodine stain is a contrast stain used primarily for the detection of intestinal parasites (Entamoeba coli in this example).
Iodine Stain
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Gram Stain
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gram-positive (purple)
from gram-negative (red) bacteria.
Gram Stain
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Staphylococcus aureus and Candida albicans
S. aureus (black arrow) and
yeasts, in this case Candida albicans (red arrow).
Yeast can appear as gram-positive, although they tend to decolorize readily.
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Candida Species Candida albicans: yeast cells
and pseudohyphae.
Candida glabrata. These are smaller than other yeasts; they also do not form pseudohyphae. This is a common cause of urinary tract infections and is the second most common cause of fungemia.
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Acid-Fast Stains
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Mycobacteria
If a weak decolorizing solution is used to remove the primary stain, then partially acid-fast organisms such as Nocardia
Acid-Fast Stains
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differential stain used for detection of parasites in blood smears
Giemsa Stain
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Plasmodium
Giemsa Stain
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Silver stains are primarily used in anatomic pathology labs and not in microbiology labs.
Silver Stain
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Fungal elements (hyphae [photo] and cells) are stained with silver particles..
Silver Stain
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In Vitro Culture: Principles and Applications
• Anton van Leeuwenhoek : Microscobic observation (1676 )
• Pasteur: culture of bacteria almost 200 years later
• Over the years, microbiologists and cooks have returned to the kitchen to create hundreds of culture media that are now routinely used in all clinical microbiology laboratories.
In Vitro Culture: Principles and Applications
• Although tests that rapidly detect microbial antigens and nucleic-acid-based molecular assays have replaced culture methods for the detection of many organisms,
• the ability to grow microbes in the laboratory remains an important procedure in all clinical labs.
• For many diseases, the ability to grow a specific organism from the site of infection is the definitive method to identify the cause of the infection.
The success of culture methods is defined by:
• the biology of the organism• the site of the infection• the patient's immune response to the
infection • the quality of the culture media.
Certain bacteria need special conditions:
Legionella is an important respiratory pathogen; however, it was never grown in culture until it was recognized that recovery of the organism required using media supplemented with iron and l-cysteine.
Campylobacter, an important enteric pathogen, was not recovered in stool specimens until highly selective media were incubated at 42° C in a microaerophilic atmosphere.
Chlamydia, an important bacterium responsible for sexually transmitted diseases, is an obligate intracellular pathogen that must be grown in living cells.
Types of Culture Media
Culture media can be subdivided into four general categories:
(1) enriched nonselective media, (2) selective media, (3) differential media, and(4) specialized media
Cell Culture
• Some bacteria and all viruses are strict intracellular microbes
• They can only grow in living cells.• In 1949, Enders described a technique for
cultivating mammalian cells for the isolation of poliovirus.
• This technique has been expanded for the growth of most strict intracellular organisms.
Cell Culture
• The cell cultures can either be cells that grow and divide on a surface (i.e., cell monolayer) or grow suspended in broth.
• Some cell cultures are well established and can be maintained indefinitely. These cultures are commonly commercially available. Other cell cultures must be prepared immediately before they are infected with the bacteria or viruses and cannot be maintained in the laboratory for more than a few cycles of division (primary cell cultures).
Molecular Diagnosis
• Like the evidence left at the scene of a crime, the DNA (deoxyribonucleic acid), RNA (ribonucleic acid), or proteins of an infectious agent in a clinical sample can be used to help identify the agent.
• In many cases the agent can be detected and identified in this way, even if it cannot be isolated or detected by immunologic means. New techniques and adaptations of older techniques are being developed for the analysis of infectious agents.
Molecular methods in infectious diseases
• Target molecule
– DNA
– RNA
Molecular Diagnosis
• The advantages of molecular techniques:• their sensitivity• Specificity• safety..
Genetic Probes
• DNA probes can be used like antibodies as sensitive and specific tools to detect, locate, and quantitate specific nucleic acid sequences in clinical specimens . Because of the specificity and sensitivity of DNA probe techniques, individual species or strains of an infectious agent can be detected, even if they are not growing or replicating.
Hybridization
• Nucleic acid probes labelled with enzymes,chemiluminescent reporter molecules
• Combine to the complementary nucleic acid sequences with high degree of specificity
HYBRIDIZATION
• More specific compared to serologic methods
• Nucleic acids are more stable• The same procedure () for every
microorganism• Sensitivity??
PPCCRR
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hainhain
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PCR• The polymerase chain reaction (PCR):• amplifies single copies of viral DNA millions of
times over• one of the newest techniques of genetic analysis• a sample is incubated with - two short DNA oligomers, termed primers, that
are complementary to the ends of a known genetic sequence within the total DNA- a heat-stable DNA polymerase (Taq or other polymerase obtained from thermophilic bacteria)- nucleotides, and buffers.
PCR• The oligomers hybridize to the appropriate sequence of DNA and act as
primers for the polymerase, which copies that segment of the DNA. • The sample is then heated to denature the DNA (separating the strands of
the double helix) and cooled to allow hybridization of the primers to the new DNA.
• Each copy of DNA becomes a new template. The process is repeated many (20 to 40) times to amplify the original DNA sequence in an exponential manner.
• A target sequence can be amplified 1,000,000-fold in a few hours using this method.
• This technique is especially useful for detecting latent and integrated virus sequences, such as in retroviruses, herpesviruses, papillomaviruses, and other DNA viruses.
What is PCR?
• PCR uses the DNA replication ‘machinery’ of a cell to make multiple copies of a specific DNA sequence.
• PCR is perhaps the most successful technique in Biology
• PCR can take a trace amount of DNA and make enough copies of it for testing
What is it used for?
• PCR is useful in any situation where a small amount of DNA is insufficient for analysis.
• PCR is used to establish blood relationships, to identify remains, and to help convict criminals or exonerate the falsely accused.
• PCR is an essential procedure in any genetics laboratory.
History
• Discovered in 1983 in California by Kary Mullis
• Published in a 1985 paper
• Sold by Cetus Corporation for $300 million
• Mullis won the 1993 Nobel Prize in Chemistry for his discovery
Requirements of PCR:
• Knowing parts of the target DNA sequence to be amplified
• Two types of synthetic primers, complementary to the ends of the target sequence
• Large amounts of the four DNA nucleotides• Taq1, a heat-resistant form of DNA Polymerase
How it works…
Number of amplified pieces = 2n (n = # of cycles)
The Thermocycler
Postamplification detection
• Gel analysis• Colorimetric microtitre plate system• Target amplification and detection systems
occur simultaneously in the same tube (Real- Time PCR)
RV12
İnfluenza A
Rapid real –time PCR
Other amplification methods
• TAS: transcription-based amplification system• 3SR: self sustained sequence replication• NASBA: nucleic acid sequence-based
amplification– (very similar)
Other amplification techniques (II)
• LCR : ligase chain reaction• bDNA: branched DNA• Qbeta replikase
Molecular Techniques • Technique Purpose Clinical Examples• RFLP: Comparison of DNA Molecular epidemiology, HSV-
1 strains• DNA electrophoresis: Comparison of DNA Viral strain
differences (up to 20,000 bases)• Pulsed-field gel electrophoresis: Comparison of DNA (large
pieces of DNA) Streptococcal strain comparisons• In situ hybridization: Detection and localization of DNA
sequences in tissue Detection of nonreplicating DNA virus (e.g., cytomegalovirus, human papillomavirus)
• Dot blot Detection of DNA sequences in solution Detection of viral DNA
• Southern blot: Detection and characterization of DNA sequences by size Identification of specific viral strains
• Northern blot: Detection and characterization of RNA sequences by size Identification of specific viral strains
• PCR: Amplification of very dilute DNA samples Detection of DNA viruses
• RT-PCR: Amplification of very dilute RNA samples Detection of RNA viruses
• Real-time PCR: Quantification of very dilute DNA and RNA samples Quantitation of HIV genome: virus load
• Branched-chain DNA: Amplification of very dilute DNA or RNA samples Quantitation of DNA and RNA viruses
• Antibody capture solution hybridization DNA assay: Amplification of very dilute DNA or RNA samples
Quantitation of DNA and RNA viruses
SDS-PAGE: Separation of proteins by molecular weight Molecular epidemiology of HSV
Direct sequencing
• Combination of PCR with dideoxynucleotide chain termination methods can be used to determine sequence of DNA.
• Genotyping of viruses• Identification of bacteria and fungi• Antimicrobial susceptibilty testing to detect
mutations
DNA microarrays
• Thousands of oligonucleotides are on a solid support
• A labelled amplification product is hybridized to the probes
Microarray
Serologic Methods(Immunologic techniques)
• Detect• Identify• Quantitate antigen or antibodyDisadvantage: Cross reaction-similar or common epitope
Antibodies
Polyclonal:• Heterogeneous antibody preparations• Recognizes many epitopes on a single antigenMonoclonal:• Recognize individual epitoses on an antigen
Antibodies
• The development of monoclonal antibody technology revolutionized the science of immunology.
• Monoclonal antibodies are the products of hybrid cells generated by the fusion and cloning of a spleen cell from an immunized mouse and a myeloma cell, which produces a hybridoma.
• The myeloma provides immortalization to the antibody-producing B cells of the spleen. Each hybridoma clone is a factory for one antibody molecule, yielding a monoclonal antibody that recognizes only one epitope. Monoclonal antibodies can also be prepared and manipulated through genetic engineering and "humanized" for therapeutic usage
The advantages of monoclonal antibodies
• (1) that their specificity can be confined to a single epitope on an antigen and
• (2) that they can be prepared in "industrial-sized" tissue culture preparations.
• A major disadvantage of monoclonal antibodies is that they are often too specific, such that a monoclonal antibody specific for one epitope on a viral antigen of one strain may not be able to detect different strains of the same virus.
Methods of detection
Antibody-antigen complexes can be detected:• Directly• Labelling the antibody or the antigen:-enzyme-radioactive-fluorescent dye
Serologic methods
• Detect either• Antigen using a known antibody• Antibody using a known antigen
Classical serologic methods
• Precipitation• Immunodiffusion techniques• AgglutinationOther serologic methods• Complement fixation• Hemagglutination inhibition• Neutralization
Agglutination tests
• Clumping of antigen with its antibody• Flocculation: similar to agglutination; except
that agglutinats float rather than sediment• Prozone reaction: high antibody causes false
negative. The sera should be diluted!!• Antigens passively absorbed on
carriers:passive agglutination
Agglutination tests
• Antigens passively absorbed on carriers:passive agglutination
-Red blood cells: passive hemagglutination-gelatin particles: particle agglutinationClassical agglutination in test tubes:-Salmonella:Gruber Widal-Rickettsiae:Weil-Felix raection
Agglutination negative
Agglutination positive
Immunoassays
• Immunofluorescence (IFA)• Enzyme-linked immunosorbant assay (ELISA)
-Western blot• Radioimmunoassay (RIA)
Western blot (WB)
The success of the Microbiology laboratory
• Quality of the specimen• The way its sent• The method used• The interpretation