prof dr gülden Çelik [email protected] laboratory diagnosis of infectious diseases
TRANSCRIPT
Learning ObjectivesAt the end of this lecture, the student should be able to:
list the main methods in diagnosis of different types of infections caused by microorganisms
explain the importance of these methods in diagnosis
List the main advantages and disadvantages of each type of test
Methods in Laboratory diagnosis of infectious diseases
DirectIndirect
Laboratory diagnosisDirect: -Microscopy
-Culture-Antigen-Nucleic acid
Indirect:-Specific antibody (Serology)
Laboratory diagnosisDirect: -Microscopy
-Culture-Antigen detection -Nucleic acid detection
Indirect:-Specific antibody (IgG, IgM, IgA)
Laboratory diagnosisDirect: -Microscopy
-Culture-Antigen detection -Nucleic acid detection:
Nucleic acid amplification techniques
(NAT=NAAT)
Indirect:-Specific antibody (IgG, IgM, IgA)
Determinating the value of tests
SensitivitySpecificityPositive predictive valuesNegative predictive values
SensitivityAnalytical or epidemiologicAnalytical sensitivity refers to the ability
of a test to detect very small quantities of antibodies as occur during seroconversion
Epidemiologic sensitivity(clinical sensitivity): refers to the ability of a test to detect persons with established infection.
Sensitivity
True positivesSensitivity = X 100
True positives + False negatives
Specificity
True negativesSpecificity = X 100
True negatives+ False positives
Positive predictive value
True positives PPV = X 100
True positives + False positives
Negative predictive value
True negativesNPV = X 100
True negatives+ False negatives
Presence of
disease
Number of people with + test
Number of people with - test
Total
Sick
Healthy
TP FN TP+FN
FP TN FP+TN
Total TP+FP FN + TN TP + FP + FN + TN
Diagnostic Sensitivity= [TP/(TP+FN)] x 100 (Positivity in sick people)
Diagnostic spesifity= [TN/(TN+FP)] x 100 (Negativity in healty people)
Positive predictive value= [TP/(TP+FP)] x 100
Negative predictive value= [TN/(FN+TN)] x 100
PREVALANCE : % 0.02Presence of
disease
Number of people with + test
Number of people with - test
Total
Sick
Healthy
199 1 200
2000 997.800 999.800
Total 2199 997.801 1.000.000
Diagnostic Sensitivity= % 99.8 (Positivity in sick people)
Diagnostic spesifity= = % 99.8 (Negativity in healty people)
Positive predictive value= [199/(199+2000)] x 100 = % 9
Negative predictive value= [997.800/(1+997.801)] x 100 = %100
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 diseaseBorrelia 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 begins as an early localized infection, progresses to an early disseminated stage, and if untreated, can progress to a late
manifestation stage.
MicroscopyMicroscopic examination of blood or tissues
from patients with Lyme disease is not recommended, because B. burgdorferi is rarely seen in clinical specimens.
B. burgdorferipresent in low numbers in the skin So: culture of the organism from skin lesions detection of bacterial nucleic acids by
polymerase chain reaction (PCR) amplification
is not prefered
Lyme DiseaseMicroscopy 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
In every infectious diseaseThe tests to be usedThe clinical material
should be properly selected !
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- But lacks sensitivity !
Microscopic MethodsBrightfield (light) microscopy Darkfield microscopy Phase-contrast microscopy Fluorescent microscopy Electron microscopy
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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Direct Examination
The sample:can be 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 :parasitology specimen
?
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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Enterobius vermicularis
Pinworm eggs: deposited by adults at night
in the perianal area. Eggs are collected by
pressing tape on the anal surface
Eggs appear as an embryo surrounded by a colorless shell that is characteristically flattened on one side.
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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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Direct Examination
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.
?
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.
But now antigen detection is preferred
India Ink Stain
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Differential Stains
Gram stain : -bacteria-Yeasts (yeasts are
gram-positive).
Gram stainingNeisseria gonorrhoae detection in uretral
specimen from males
Differential StainsAcid-Fast StainsZiehl-Neelsen stain: Used to stain
mycobacteria and other acid-fast organisms.
Kinyoun stain: Cold acid-fast stain (does not require heating)
Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis
Conventional tests for laboratory confirmation of TB include• acid-fast bacilli (AFB) smear microscopy(24 hours)• culture Although rapid and inexpensive, AFB smear microscopy is limited by its poor sensitivity (45%–80% with culture-confirmed pulmonary TB cases)
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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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.
Panels A and B, Cryptosporidia. Panel C, Cyclospora. Panel D, Isospora.
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Differential Stains
Giemsa stain: blood parasites
differential stain used for detection of parasites in blood smears
Giemsa Stain
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Plasmodium
Giemsa Stain
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Fecal leucocyte - : viral?
Fecal leucocyte +:bacterial?
Darkfield Microscopy
Treponema pallidum (syphilis): !used in routine diagnosis
Leptospira spp. (leptospirosis) not used
Microscopic Principles and ApplicationsThe 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.
Treponema pallidum in the direct fluorescent antibody test for T. Pallidum:more sensitive
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.
Antibiotic susceptibility test
The success of culture methods is defined by:
the biology of the organism the site of the infectionthe patient's immune response to the infection
the quality of the culture media.
Certain bacteria need special conditions:Legionella is an important respiratory
pathogen; media should be supplemented with iron and l-cysteine.
Campylobacter, an important enteric pathogen, highly selective media should be 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 MediaCulture media can be subdivided into four
general categories: (1) enriched nonselective media, (2) selective media, (3) differential media, and(4) specialized media
Cell Culture: not routine ! Some bacteria and all viruses are strict
intracellular microbesThey 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.
Serologic Methods(Immunologic techniques)
DetectIdentifyQuantitate antigen or antibodyDisadvantage: Cross reaction: False
positivity-similar or common epitope
Serologic, Serodiagnosis,Serology
Detection of antigen or antibody in serumThe term serologic is used also for
searching antigen or antibody in mediums other than serum(saliva,urine)
Serologic assay=immunoassay
ImmunoassaysAntigen or antibody is detectedIn a variety of clinical specimens:
Mostly seraBody fluids(cerebrospinal fluid)TissuesEnvironmental substances
AntibodiesPolyclonal:Heterogeneous antibody preparationsRecognizes many epitopes on a single
antigenMonoclonal:Recognize individual epitoses on an
antigen
Methods of detectionAntibody-antigen complexes can be
detected:DirectlyLabelling the antibody or the antigen:-enzyme-radioactive-fluorescent dye
Classical serologic methodsPrecipitationImmunodiffusion techniquesAgglutinationOther serologic methodsComplement fixationHemagglutination inhibitionNeutralization
Agglutination testsClumping of antigen with its antibodyFlocculation: 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 testsAntigens passively absorbed on
carriers:passive agglutination-Red blood cells: passive hemagglutination-gelatin particles: particle agglutinationClassical agglutination in test tubes:-Salmonella:Gruber Widal-Brucella:Wright-Rickettsiae:Weil-Felix reaction
Agglutination negative
Agglutination positive
PrecipitationTubes:solutionsGels
ImmunoassaysImmunofluorescence (IFA)Enzyme-linked immunosorbant assay
(ELISA)-Western blot
Radioimmunoassay (RIA)
Serologycan be used to identify the infecting agentevaluate the course of an infection, or
determine the nature of the infection-whether it is a primary infection or a reinfection, and whether it is acute or chronic.
Serologic testing is used to identify viruses and other agents that are difficult to isolate and grow in the laboratory or that cause diseases that progress slowly
In the diagnosis of infectious diseases by immunoassays
Either spesific antigen:Directly from specimenFrom the culture for identification
Specific antibodies are detected:IgGIgMIgA
Specific antibody detection
Seroconversion occurs when antibody is produced in response to a primary infection.
IgM: early in infection (2-3 weeks)transient (3-6 months)
*sometimes persists longer IgG: forms later (immunity)
highest in 4-6 monthsusually persists during the whole life
IgG avidity: High: past infectionLow: new infection
Western blot (WB)
Examples of Viruses Diagnosed by Serology
Epstein-Barr virus Rubella virus, Measles,Mumps Hepatitis A, B, C, D, and E viruses Human immunodeficiency virus Human T-cell leukemia virus Arboviruses (encephalitis viruses)
Diagnosis of acute infection By specific IgM detection by ELISA:HAVMeaslesRubellaMumpsParvovirus B19Varicella zoster…
Quantitative antibody detection:Anti-HBs: 10mIU/ml(immune to HBV
infection)Rubella IgG: 10-15 IU/ml(immune to
Rubella infection)
Antigen detectionMembrane ELISAImmunochromatograhic methodsLatex agglutinationRapidLess sensitiveLess specific
Rapid antigen assaySensitivity !Specificity !
Antigen detectionStrep ARSVInfluenzaRotaNorovirusLegionella pneumophila serogroup IMalaria
Nucleic acid amplification techniques
(NAT=NAAT)
Target molecule
DNA
RNA
Molecular Diagnosis
The advantages of molecular techniques:their sensitivitySpecificitysafety..
Hybridization
Nucleic acid probes labelled with enzymes,chemiluminescent reporter molecules
Combine to the complementary nucleic acid sequences with high degree of specificity
HYBRIDIZATIONMore specific compared to serologic methods
Nucleic acids are more stableThe same procedure () for every microorganism
Sensitivity??
PPCCRR
olymeraseolymerase
hainhain
eactioneaction
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
HistoryDiscovered 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 amplifiedTwo types of synthetic primers,
complementary to the ends of the target sequence
Large amounts of the four DNA nucleotidesTaq1, a heat-resistant form of DNA
Polymerase
How it works…
Number of amplified pieces = 2n (n = # of cycles)
The Thermocycler
PCRThe polymerase chain reaction (PCR):amplifies single copies of viral DNA millions of
times over
Very sensitiveDanger !:ContaminationFalse positive
Postamplification detectionGel analysisColorimetric microtitre plate systemTarget amplification and detection systems
occur simultaneously in the same tube (Real- Time PCR)
RV12
İnfluenza A
Rapid molecular techniques!!!
Rapid real –time PCR
Direct sequencingCombination of PCR with dideoxynucleotide
chain termination methods can be used to determine sequence of DNA.
Genotyping of virusesIdentification of bacteria and fungiAntimicrobial susceptibilty testing to detect
mutations
DNA microarraysThousands of oligonucleotides are on a
solid supportA labelled amplification product is
hybridized to the probes
Microarray
Multiplex PCR:
Now more often: !!!! -antigen detection -rapid real-time PCR -multiplex PCR
-quantitative detection for follow-up treatment
-antimicrobial resistance
Laboratory diagnosisDirect:
Microscopy: Culture:+antibiotic susceptibilityAntigen detection:rapid, less sensitive(Strep
A/RSV/Rota..)NAAT: real-time PCR(M.tuberculosis,
MRSA,VRE..) multiplex-PCR, quantitation FOR FOLLOW-UP OF TREATMENT, resistance mutation detection
Indirect:spesific IGM: viral
infections(measles,mumps,rubella..Quantitative IgG: immunity: exception:?
oAnti-Rubella IgGoAnti-HBs
The success of the Microbiology laboratory
Quality of the specimenThe way its sentThe method usedThe interpretation:Do not hesitate to have contact with your
microbiology laboratory!
Reference:
7th ed 2013