Laboratory Diagnosis of Infectious Diseases

Prof Dr Gülden Çelikgulden.yilmaz@yeditepe.edu.tr

Laboratory Diagnosis of Infectious Diseases

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+997.800)] 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 I(urine!)Malaria

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): -sensitive

-specific-rapid-easy to use

-expensive

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: N.gonorrhoae in maleCulture:+antibiotic susceptibilityAntigen detection:rapid, less sensitive(Strep

A/RSV/Rota../in urine: L. pneumophila)NAAT: -real-time PCR(M.tuberculosis, Clostridium

difficile, MRSA,VRE..) -multiplex-PCR, quantitation, resistance mutation detection

Indirect:spesific IGM: viral

infections(measles,mumps,rubella..Quantitattive IgG: immunity:

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