History And Overview History And Overview Of Of

Microbial Identification MethodsMicrobial Identification Methods

Use of tubed and plated media - Basis of microbial Use of tubed and plated media - Basis of microbial identification till 1960sidentification till 1960s

Organisms were identified by “conventional methods”Organisms were identified by “conventional methods” Next step in evolution of identification methods- Next step in evolution of identification methods-

miniaturized commonly used biochemical reactionsminiaturized commonly used biochemical reactions Nowadays “system-dependent” approach is the industry Nowadays “system-dependent” approach is the industry

standardstandard Set of substrates that allow positive and negative Set of substrates that allow positive and negative

reaction patterns to emerge is selectedreaction patterns to emerge is selected Compared with established database profileCompared with established database profile

IntroductionIntroduction

No “FORMAL” definition of “RAPID”No “FORMAL” definition of “RAPID”Rapid test may mean results the same Rapid test may mean results the same

dayday

OR OR Rapid provides usable results in 2-4 hr Rapid provides usable results in 2-4 hr Make a difference to patient outcomeMake a difference to patient outcome

Approaches to Rapid IdentificationApproaches to Rapid Identification

APPROACH ONE- Vary conventional testing APPROACH ONE- Vary conventional testing approach- decreasing test substrate medium approach- decreasing test substrate medium volume and increasing concentration of bacteria volume and increasing concentration of bacteria

APPROACH TWO- Unique and novel substrates, APPROACH TWO- Unique and novel substrates, detect enzymatic activity without multiplication of detect enzymatic activity without multiplication of organism - NON GROWTH BASED TESTSorganism - NON GROWTH BASED TESTS

APPROACH THREE- Antigen-antibody reactionsAPPROACH THREE- Antigen-antibody reactions APPROACH FOUR-Molecular detection methods APPROACH FOUR-Molecular detection methods

Detection of Metabolic ActivityDetection of Metabolic Activity

Accuracy of identification of a systemAccuracy of identification of a system Reliable indication of utilization of substrateReliable indication of utilization of substrate Sensitivity and strength of detection signal Sensitivity and strength of detection signal Detection strategies for determining end productsDetection strategies for determining end products

COLORIMETRYCOLORIMETRY FLUORESCENCEFLUORESCENCE TURBIDITY TURBIDITY

COLORIMETRYCOLORIMETRY

ID systems detect colour change to detect ID systems detect colour change to detect presence of metabolic end productspresence of metabolic end products

Colour change indicatedColour change indicated pH indicators in mediapH indicators in media oxidation-reduction potential indicatorsoxidation-reduction potential indicators chromogenic substrateschromogenic substrates

Detected by Detected by unaided eyeunaided eye photoelectric cell photoelectric cell

FLUORESCENCEFLUORESCENCE

Fluorescence to measure metabolic activityFluorescence to measure metabolic activity Substrate-fluorophore complexSubstrate-fluorophore complex pH change due to metabolic activitypH change due to metabolic activity

Bacteria process substrate, fluorophore is Bacteria process substrate, fluorophore is released, fluorescence measuredreleased, fluorescence measured

Bacterial metabolic activity causes changes in Bacterial metabolic activity causes changes in pH which in turn causes changes in fluorophore pH which in turn causes changes in fluorophore fluoresce fluoresce quenched (lost) quenched (lost)

Contd..Contd..

Fluorogenic substrates are composed of Fluorogenic substrates are composed of carbohydrate or amino acid moities linked carbohydrate or amino acid moities linked through glycoside or peptide linkages to compds through glycoside or peptide linkages to compds like 4-methylumbelliferone and 4-like 4-methylumbelliferone and 4-methylcoumarin that produces fluorescent end-methylcoumarin that produces fluorescent end-products on hydrolysis.products on hydrolysis.

TURBIDITYTURBIDITY

Not used in identificationNot used in identification Determine growth in presence of inhibitorsDetermine growth in presence of inhibitors Optical density, measure of turbidity- Spectrophotometer Optical density, measure of turbidity- Spectrophotometer

LABELSLABELS

(REPORTER (REPORTER GROUPS)GROUPS)

B/F B/F

SEPARATIONSEPARATION SIGNAL SIGNAL DETECTIONDETECTION

Precipitation Precipitation immunoassaysimmunoassays

Not requiredNot required Not requiredNot required Naked eye, Naked eye, turbidity, turbidity, nephlometrynephlometry

Particle Particle immunoassaysimmunoassays

Blood cells, Blood cells, artificial particles artificial particles (gelatin, particles, (gelatin, particles, latex, etc)latex, etc)

Not requiredNot required Naked eye, pattern Naked eye, pattern analyzer, analyzer, spectrophotometry,spectrophotometry,particle countingparticle counting

RadioimmunoassaysRadioimmunoassays Radioisotopes Radioisotopes RequiredRequired Photon countingPhoton counting

Enzyme Enzyme immunoassaysimmunoassays

EnzymesEnzymes RequiredRequired Spectrophotometry, Spectrophotometry, fluorometry,photon fluorometry,photon countingcounting

Fluorescent Fluorescent immunoassaysimmunoassays

FluorophoresFluorophores RequiredRequired Photon countingPhoton counting

ChemiluminescentChemiluminescent

immunoassaysimmunoassaysChemiluminescentChemiluminescent

compoundscompounds RequiredRequired Photon countingPhoton counting

CLASSIFICATION OF VARIOUS IMMUNOASSAYSCLASSIFICATION OF VARIOUS IMMUNOASSAYS

ANTIBODY CAPTURE ANTIGEN CAPTURE

INDIRECT DIRECT

SOLID PHASE ASSAYS

BACTERIALBACTERIAL

STAPHYLOCOCCISTAPHYLOCOCCI

ALTERNATIVE COAGULASE ALTERNATIVE COAGULASE TEST PROCEDURESTEST PROCEDURES

Latex Agglutination Latex Agglutination Passive HamagglutinationPassive Hamagglutination

BBL™ Staphyloslide™ Latex BBL™ Staphyloslide™ Latex Test for Test for Staphylococcus aureusStaphylococcus aureus

PrinciplePrinciple

BBL Staphyloslide latex testBBL Staphyloslide latex test latex particles coated with human fibrinogen and latex particles coated with human fibrinogen and

IgGIgG Mixing latex reagent with Mixing latex reagent with Staphylococci Staphylococci having having

clumping factor or protein A – visible agglutinationclumping factor or protein A – visible agglutination Commercially available asCommercially available as StaphAurex (Murex)StaphAurex (Murex) Slidex Staph (bioMerieux) Slidex Staph (bioMerieux) Staphylatex (Microscan) Staphylatex (Microscan) Staph Rapid (Roche)Staph Rapid (Roche)

Passive HaemagglutinationPassive Haemagglutination

Tests use sheep red blood cells sensitized with Tests use sheep red blood cells sensitized with fibrinogen to detect clumping factor on surface fibrinogen to detect clumping factor on surface of of S.aureusS.aureus cells cells

Staphyloslide (BD Biosciences)Staphyloslide (BD Biosciences) Hemastaph (Remel)Hemastaph (Remel) Staphyslide (bioMerieux)Staphyslide (bioMerieux)

Advantage – Nonsensitized red cell suspension Advantage – Nonsensitized red cell suspension used as negative controlused as negative control

Rapid identifications systemsRapid identifications systems

RapiDEC StaphRapiDEC Staph API Staph-IDENT (bioMerieux)API Staph-IDENT (bioMerieux) API Staph (bioMerieux)API Staph (bioMerieux) APIID32 StaphAPIID32 Staph Vitek GPI card (bioMerieux)Vitek GPI card (bioMerieux) MicroScan Rapid Pos Combo Panel (Dade/Microscan)MicroScan Rapid Pos Combo Panel (Dade/Microscan) BBL Crystal Gram Positive(GP) Identification System BBL Crystal Gram Positive(GP) Identification System

(BD Biosciences)(BD Biosciences) Staph-Zym(ROSCO,Denmark)Staph-Zym(ROSCO,Denmark) Microbial Identification SystemMicrobial Identification System Biolog Microplate Identification SystemBiolog Microplate Identification System

RapiDEC StaphRapiDEC Staph (bioMerieux) (bioMerieux) Identifies isolate with “Aurease” in 2hrsIdentifies isolate with “Aurease” in 2hrs Four cupules Four cupules

controlcontrol fluorescent coagulase substratefluorescent coagulase substrate Alkaline phosphatase substrate Alkaline phosphatase substrate ββ--galactosidase substrategalactosidase substrate

After 2hrs incubation cupulae are observed under UVAfter 2hrs incubation cupulae are observed under UV Cupulae 1 - controlCupulae 1 - control Cupulae 2Cupulae 2 - S.aureus - S.aureus Cupulae 3- Cupulae 3- S.epidermidisS.epidermidis Cupulae 4- Cupulae 4- S.saprophyticusS.saprophyticus

Rapid tests for drug resistant Rapid tests for drug resistant StaphylococciStaphylococci

Rapid detection of drug resistanceRapid detection of drug resistance MRSA- Methicillin Resistance due to MRSA- Methicillin Resistance due to mecAmecA Tests availableTests available

Slide latex agglutination tests-Slide latex agglutination tests- MRSA Screen,(Denka Seiken,Japan)MRSA Screen,(Denka Seiken,Japan) Oxoid Penicillin Binding Protein latex agglutination test OxoidOxoid Penicillin Binding Protein latex agglutination test Oxoid

Fluorescence technology (Crystal MRSA ID System)Fluorescence technology (Crystal MRSA ID System) PCR PBP2A gene amplificationPCR PBP2A gene amplification CHROM AgarCHROM Agar

MRSA Latex KitMRSA Latex KitMRSA Screen detects PBP2A, antibodies ie monoclonal antiPBP2A are attached to latex beads, If isolate is MRSAmecA codes for PBP2A causes visible clumps

Media Containing Chromogenic Media Containing Chromogenic SubstratesSubstrates

CHROMagar formulations available as –CHROMagar formulations available as – CHROMagar Staph aureus (CHROMagar Microbiology, France)CHROMagar Staph aureus (CHROMagar Microbiology, France)

S.aureus colonies are MAUVES.aureus colonies are MAUVE S.aureusS.aureus ID agar (bioMerieux) ID agar (bioMerieux)

S.aureus colonies are GREEN S.aureus colonies are GREEN

CONS colonies are white, blue or beigeCONS colonies are white, blue or beige Production of Production of αα-glucosidase by organisms during growth-glucosidase by organisms during growth Rapid test yields results in 18-20 hr for identifying Rapid test yields results in 18-20 hr for identifying

S.aureusS.aureus, incorporation of oxacillin and methicillin , incorporation of oxacillin and methicillin determines methicillin resistance determines methicillin resistance

Molecular methods for detecting Molecular methods for detecting S.aureusS.aureus in clinical specimens in clinical specimens

DNA ProbesDNA Probes PCRPCR AccuProbe for AccuProbe for S.aureus S.aureus (Gene-Probe,San (Gene-Probe,San

Diego,CA)Diego,CA)

Multiplex PCR for MRSA

• mecA and fem B most common

• mec A identifies meth R

• fem B identifies Staph aureus

• mec A + fem B + – MRSA mec A - fem B + - MSSA mec A + fem B - - MR CNS mec A - fem B - - MS CNS

• Results in a few hours, may be performed

from patient’s samples without culture

MRSA

MSSA

MR-CNS

MULTPLEX PCR FOR RAPID DETECTION OF MRSA

310 mec

A651 bp fem B

Beta lactamase detectionBeta lactamase detection

STREPTOCOCCISTREPTOCOCCI

NOW® Strep A TestNOW® Strep A Test

Rapid immunochromatographic assay for detection of Rapid immunochromatographic assay for detection of Streptococcus pyogenesStreptococcus pyogenes Group A antigen Group A antigen

Sample type – Throat swabSample type – Throat swabTime for test results – 6 minutesTime for test results – 6 minutesSensitivity – 92%Sensitivity – 92%Specificity – 100%Specificity – 100%

CONJUGATE

CAPTURING Ab

SAMPLE DEVELOPMENT

Ag/Ab

Principle of ICTPrinciple of ICT

Streptex latex test kit for detection Streptex latex test kit for detection of group B of group B StreptococcalStreptococcal (GBS) (GBS)

antigen antigen

Strep OIA (Biostar))

Immunoassay for direct detection of gp A StreptococciImmunoassay for direct detection of gp A Streptococci in throat swab specimensin throat swab specimens Swab extracted with acetic acid Swab extracted with acetic acid Mixed with anti-groupA AbMixed with anti-groupA Ab bound to HRP, drop placed bound to HRP, drop placed on slide coated with on slide coated with anti-groupA Ab anti-groupA Ab HRP substrate HRP substrate colour examinedcolour examined Purple spot if antigen presentPurple spot if antigen present Gold or tiny dot if negative Gold or tiny dot if negative

Meridian Meritec*-Strep Beta-Meridian Meritec*-Strep Beta-Hemolytic Grouping SetHemolytic Grouping Set

Standardized coagglutination procedure Standardized coagglutination procedure Results within one minute following culture Results within one minute following culture For the identification of For the identification of Beta-hemolytic streptococciBeta-hemolytic streptococci

based on serogroups described by Lancefield based on serogroups described by Lancefield

Fisher Sure-Vue* Strep A Lateral Fisher Sure-Vue* Strep A Lateral Flow Test KitFlow Test Kit

One-step lateral-flow immunoassay for the qualitative One-step lateral-flow immunoassay for the qualitative detection of Group A Streptococcal antigen from throat detection of Group A Streptococcal antigen from throat swab specimens swab specimens

Results in five minutes Results in five minutes Sensitivity: 90.2%; specificity: 98.1% Sensitivity: 90.2%; specificity: 98.1% Membrane strip coated with rabbit anti-Strep A antibody Membrane strip coated with rabbit anti-Strep A antibody

on the test band region and goat anti-rabbit antibody on on the test band region and goat anti-rabbit antibody on the control band region the control band region

Control band verifies sufficient volume has been added Control band verifies sufficient volume has been added and proper flow is obtained and proper flow is obtained

Other rapid tests for identification of Other rapid tests for identification of StreptococciStreptococci

API RAPID STREP(Biomerieux)API RAPID STREP(Biomerieux) BBL Crystal Gram-Positive Identification SystemBBL Crystal Gram-Positive Identification System Rapid ID 32 STREP (Biomerieux)Rapid ID 32 STREP (Biomerieux) RapID STR(Remel)RapID STR(Remel) Vitek Gram-Positive Card(Biomerieux)Vitek Gram-Positive Card(Biomerieux)

PNEUMOCOCCIPNEUMOCOCCI

BBL Pneumoslide testBBL Pneumoslide test

Rapid latex agglutination procedure for the Rapid latex agglutination procedure for the identification of identification of S. pneumoniaeS. pneumoniae

Specific antibody against all capsular Specific antibody against all capsular serotypes absorbed onto latex beadsserotypes absorbed onto latex beads

When mixed with a suspension of When mixed with a suspension of unknown organism, strong agglutination of unknown organism, strong agglutination of the latex beads indicates the presence of the latex beads indicates the presence of S. pneumoniaeS. pneumoniae

Latex agglutination for MeningitisLatex agglutination for Meningitis

Latex agglutination testLatex agglutination testCommercially available kits contain Commercially available kits contain

reagents for detecting following antigens: reagents for detecting following antigens: Neisseria meningitidisNeisseria meningitidis serogroup A serogroup A Neisseria meningitidisNeisseria meningitidis serogroup B serogroup B Neisseria meningitidisNeisseria meningitidis serogroup C serogroup C Streptococcus pneumoniaeStreptococcus pneumoniae Haemophilus influenzae b Haemophilus influenzae b

Latex slide agglutination test for detection of Latex slide agglutination test for detection of antigens toantigens to Neisseria meningitidis Neisseria meningitidis Groups A, C, Groups A, C, Y and W135 in cerebrospinal fluid (CSF), serum Y and W135 in cerebrospinal fluid (CSF), serum or urine or urine

Visible agglutination occurs as bacterial Visible agglutination occurs as bacterial antigens react with respective antibody-coated antigens react with respective antibody-coated latex beadslatex beads

Test kit provides confirmation and serogrouping Test kit provides confirmation and serogrouping capabilities from suspected colonies of capabilities from suspected colonies of N. N. meningitidis meningitidis Groups A/Y or C/W135Groups A/Y or C/W135

Directigen™ Meningitis Test System Directigen™ Meningitis Test System

The initial enthusiasm for the rapid diagnosis of bacterial The initial enthusiasm for the rapid diagnosis of bacterial meningitis by antigen detection has been tempered by meningitis by antigen detection has been tempered by more recent experiencesmore recent experiences

When latex agglutination tests for H.influenzae, When latex agglutination tests for H.influenzae, Streptococcus agalactiae, Neisseria meningitidis and Streptococcus agalactiae, Neisseria meningitidis and S.pneumoniae were performed on 1,540 CSF samples S.pneumoniae were performed on 1,540 CSF samples from patients with suspected acute bacterial meningitis from patients with suspected acute bacterial meningitis antigen was detected in only 27 antigen was detected in only 27

Use of these tests has declined and cannot be Use of these tests has declined and cannot be recommended recommended

Koneman’s colour Atlas & Textbook of Diagnostic Microbiology 6 Edn

Commercial systems available for Commercial systems available for NeisseriaNeisseria

RapID NH panel (Remel)RapID NH panel (Remel) Vitek Vitek Neisseria-HaemophilusNeisseria-Haemophilus Identification(NHI) Identification(NHI)

card (bioMerieux)card (bioMerieux) Haemophilus-NeisseriaHaemophilus-Neisseria Identification(HNID) Identification(HNID)

Panel (Dade/Microscan)Panel (Dade/Microscan) API NH Strip (bioMerieux)API NH Strip (bioMerieux)

BactiCard Neisseria FLUORESCENT ANTIBODY TEST

RapID NH system(Remel) MicroScan HNID panel

API NH panel API NH N.meningitidis

GONOCHEK IIGONOCHEK II

N.meningitidisN.meningitidis- - γγ--glutamyl-glutamyl-pp-nitroanilide-nitroanilideN.gonorrhoeae- N.gonorrhoeae- propyl-propyl-ββ-naphthylamide-naphthylamide

NM

NG

(EY Laboratories,CA)

ENTEROBACTERICAEENTEROBACTERICAE

EnterobactericaeEnterobactericae and coliforms on and coliforms on chromagarchromagar

BactiCard® E. coliBactiCard® E. coli

For presumptive rapid identification of For presumptive rapid identification of Escherichia coliEscherichia coli using enzyme technology using enzyme technology Tests: Indole, MUG Tests: Indole, MUG

Biolog GN Microplate system with 96 well microtitre plate Biolog GN Microplate system with 96 well microtitre plate containing 95 carbon substrates and redox indicator containing 95 carbon substrates and redox indicator tetrazolium. If carbon substrate utilized then purple tetrazolium. If carbon substrate utilized then purple colour develops and “metabolic fingerprint” is matchedcolour develops and “metabolic fingerprint” is matched

REMEL RapID ONE panel has 19 sustrates for identification of 70 organisms belonging to Enterobacteriaceae Each test interpreted visually after 4hours of incubationResults are scored and compared with database

MicroScan gram-negative ID Panel

-Microtitre wells inoculated with heavy suspension of organism -Incubated at 35C for 15-18 hr -Interpreted visually-Biochemical tests converted into seven or eight-digit biotype number which is looked up in manufacturers codebook-Same panels compatible with WalkAway System which processes both rapid and conventional overnight panels. (Courtesy Dade Behring)

PSEUDOMONASPSEUDOMONAS

SALMONELLASALMONELLA

RAPID TESTSRAPID TESTS FOR DIAGNOSING FOR DIAGNOSING TYPHOIDTYPHOID

TyphidotTyphidot test detects presence of IgM and IgG in one test detects presence of IgM and IgG in one hour (sensitivity>95%, Specificity 75%) hour (sensitivity>95%, Specificity 75%)

TyphidotTyphidot-M, that detects IgM only (sensitivity 90% and -M, that detects IgM only (sensitivity 90% and specificity 93%) specificity 93%)

TyphidotTyphidot rapidrapid (sensitivity 85% and Specificity 99%) is (sensitivity 85% and Specificity 99%) is a a rapidrapid 15 minute immunochromatographic test to detect 15 minute immunochromatographic test to detect IgM IgM

IgM dipstick testIgM dipstick test

Typhoid fever dipstick assayTyphoid fever dipstick assayKITKIT

Dipstick contains two Dipstick contains two horizontal bands: an antigen horizontal bands: an antigen band (lower band) and an band (lower band) and an internal control (upper band)internal control (upper band)

Assay is based on binding of Assay is based on binding of specific IgM antibodies to the specific IgM antibodies to the antigenantigen

Bound IgM antibodies are Bound IgM antibodies are specifically detected with an specifically detected with an anti-human IgM dye conjugateanti-human IgM dye conjugate

Minitek system (BD) uses substrate-impregnated filter Minitek system (BD) uses substrate-impregnated filter discs distributed to each test well before inoculation with discs distributed to each test well before inoculation with bacterial suspension bacterial suspension

Rapid detection of Clostridial toxinsRapid detection of Clostridial toxins

SYPHILISSYPHILIS

RAPID PLASMA REAGIN(RPR) CARD TESTCARBOGEN reagent : Particulate carbon suspension coated with lipid complexesAntilipoidal antibodies are detectedSerum sample need not be heatedAfter mixing serum with test reagent observe for flocculation macroscopically in 8 min

BRUCELLOSISBRUCELLOSIS

Immunochromatographic Immunochromatographic Brucella-Brucella-Specific Specific Immunoglobulin M and G Lateral Flow Assays for Immunoglobulin M and G Lateral Flow Assays for

Rapid Serodiagnosis of HumanRapid Serodiagnosis of Human BrucellosisBrucellosis

Brucella dipstick assayBrucella dipstick assay

Dipstick assay : Rapid Dipstick assay : Rapid detection of detection of Brucella-Brucella-specific specific IgM antibodies in human IgM antibodies in human serum or whole blood samples serum or whole blood samples in early stage of disease in early stage of disease

Based on the binding of Based on the binding of specific IgM antibodies to a specific IgM antibodies to a lipopolysaccharide fraction lipopolysaccharide fraction prepared from prepared from B. abortusB. abortus

Assay utilizes stabilized non-Assay utilizes stabilized non-enzymatic detection reagents enzymatic detection reagents

Time to detection- three hours Time to detection- three hours

LEPTOSPIRALEPTOSPIRA

LEPTOTEK DRI DOTLEPTOTEK DRI DOT Standard method for diagnosis of leptospirosis is the Standard method for diagnosis of leptospirosis is the

microscopic agglutination test (MAT)microscopic agglutination test (MAT) MAT is laborious and time-consuming, interpretation of MAT is laborious and time-consuming, interpretation of

results is subjectiveresults is subjective

PrinciplePrinciple

LeptoTek Dri Dot assay : Coloured latex particles LeptoTek Dri Dot assay : Coloured latex particles activated with broadly reactive activated with broadly reactive LeptospiraLeptospira antigen dried antigen dried onto an agglutination cardonto an agglutination card

Assay based on binding of Assay based on binding of LeptospiraLeptospira-specific -specific antibodies, present in serum sample, to antibodies, present in serum sample, to LeptospiraLeptospira antigen causing a fine granular agglutination at edge of antigen causing a fine granular agglutination at edge of droplet droplet

LEPTO Dipstick assayLEPTO Dipstick assay

PrinciplePrincipleDipstick assay rapid detection of Dipstick assay rapid detection of Leptospira-Leptospira- specific IgM specific IgM antibodies in human serum or whole blood samplesantibodies in human serum or whole blood samples

Based on binding of specific IgM antibodies to a broadly Based on binding of specific IgM antibodies to a broadly reactive antigen prepared from non-pathogenic strain reactive antigen prepared from non-pathogenic strain ensuring detection of wide range of serovarsensuring detection of wide range of serovars

Time to detection: three hours Time to detection: three hours Interpretation depends on intensity of staining of antigen Interpretation depends on intensity of staining of antigen

band: determined by comparison with a coloured band: determined by comparison with a coloured reference stripreference strip

LeptoTek Lateral Flow assayLeptoTek Lateral Flow assay

FCM for leptospirosisFCM for leptospirosis

FCM detects the size and shape of the bacteria by FCM detects the size and shape of the bacteria by measurement of light scatter parameters: forward scatter measurement of light scatter parameters: forward scatter (FSC) and side scatter (SSC) (FSC) and side scatter (SSC)

Shift of light scatter parameter to a different location with Shift of light scatter parameter to a different location with higher FSC and SSC values, indicates formation of higher FSC and SSC values, indicates formation of leptospiral aggregatesleptospiral aggregates

Rapid less than 1.5 hRapid less than 1.5 h FCM analysis is objective FCM analysis is objective Can be automated Can be automated

H.pyloriH.pylori

Rapid Urease test (CLO Test) Rapid Urease test (CLO Test) Biopsy sample is incubated on an agar based medium Biopsy sample is incubated on an agar based medium

containing ureacontaining urea If If H.PyloriH.Pylori is present in sample, bacterial urease converts is present in sample, bacterial urease converts

urea to ammonia, colour change indicator detects the urea to ammonia, colour change indicator detects the subsequent pH rise in the medium subsequent pH rise in the medium

Time to detection one hourTime to detection one hour

Helicobacter pylori Stool Antigen Helicobacter pylori Stool Antigen Test (HpSA)Test (HpSA)

Test utilises polyclonal anti-H. pylori capture Test utilises polyclonal anti-H. pylori capture antibody adsorbed to wellsantibody adsorbed to wells

Diluted patient samples and a peroxidase Diluted patient samples and a peroxidase conjugated polyclonal antibody added to conjugated polyclonal antibody added to wells and incubated for one hour at RTwells and incubated for one hour at RT

Wash to remove unbound materialWash to remove unbound material Substrate is added and incubated for ten Substrate is added and incubated for ten

minutes at RT. Colour develops in the minutes at RT. Colour develops in the presence of bound enzymepresence of bound enzyme

Stop solution is added and results are Stop solution is added and results are interpreted visually or spectrophotometricallyinterpreted visually or spectrophotometrically

Test can be performed in less than 90 Test can be performed in less than 90 minutes by any laboratory, since no special minutes by any laboratory, since no special equipment is neededequipment is needed

ImmunoCard STAT! HpSAImmunoCard STAT! HpSA(Meridian Bioscience)(Meridian Bioscience)

Rapid 5 minutes immunoassay, based on a lateral flow Rapid 5 minutes immunoassay, based on a lateral flow chromatography technique using a monoclonal antibody, chromatography technique using a monoclonal antibody, for the qualitative detection of for the qualitative detection of Helicobacter pyloriHelicobacter pylori Antigens in human stoolAntigens in human stool

Interpretation of ResultsNegative: one BLUE line (control)

Positive: one BLUE line (control) and one PINK-RED line (test)

CHLAMYDIACHLAMYDIA

Direct fluorescent antibody testDirect fluorescent antibody test Optical immunoassaysOptical immunoassays

MYCOBACTERIAMYCOBACTERIA

TUBERCULOSISTUBERCULOSIS

RAPID CULTURE METHODSRAPID CULTURE METHODS

BACTEC 460 AFB SYSTEMBACTEC 460 AFB SYSTEM MB/BacT MYCOBACTERIA DETECTION SYSTEMMB/BacT MYCOBACTERIA DETECTION SYSTEM ESP MYCO SYSTEMESP MYCO SYSTEM SEPTI-CHEK AFB SYSTEMSEPTI-CHEK AFB SYSTEM MYCOBACTERIA GROWTH INDICATOR TUBEMYCOBACTERIA GROWTH INDICATOR TUBE ALL REQUIRE PANTA / EQUIV ADDED Abs AND ALL REQUIRE PANTA / EQUIV ADDED Abs AND

OTHER NUTRIENTS (OADC)OTHER NUTRIENTS (OADC)

ADVANTAGES – RAPID, EASY TO USE, FAIRLY ADVANTAGES – RAPID, EASY TO USE, FAIRLY SENSITIVE & SPECIFICSENSITIVE & SPECIFIC

DISADV – HIGH COST, TECHNOLOGY ORIENTEDDISADV – HIGH COST, TECHNOLOGY ORIENTED

BACTEC 460 TB BACTEC 460 TB

BACTEC 460TB SystemBACTEC 460TB System

[[1414C] palmitic acid as a carbon source in medium is C] palmitic acid as a carbon source in medium is metabolized by microorganisms to metabolized by microorganisms to 1414COCO22 which is which is monitored by the instrumentmonitored by the instrument

Amount of Amount of 1414COCO2 2 and rate of production are directly and rate of production are directly proportional to growth rate of organism proportional to growth rate of organism

Rapid and accurate testing of Rapid and accurate testing of mycobacteria mycobacteria Detection — 4- 8 days Detection — 4- 8 days Differentiation —3- 6 days Differentiation —3- 6 days Susceptibility — 4-12 days Susceptibility — 4-12 days

BACTEC 9000 SYSTEMBACTEC 9000 SYSTEM

BACTEC 9000 systemBACTEC 9000 system

Automated continuously monitored systemsAutomated continuously monitored systems Fluorescence sensing method to detect growth Fluorescence sensing method to detect growth

instead of light (BacT/ALERT)instead of light (BacT/ALERT) Not based on radioisotopesNot based on radioisotopes BACTEC 9000MB System uses fluorescence BACTEC 9000MB System uses fluorescence

quenching-based oxygen sensor as MGITquenching-based oxygen sensor as MGIT

MYCOBACTERIA GROWTH MYCOBACTERIA GROWTH INDICATOR TUBEINDICATOR TUBE

MGIT(BD) contains Middlebrook 7H9 broth MGIT(BD) contains Middlebrook 7H9 broth supplemented with PANTA and ODACsupplemented with PANTA and ODAC

Fluoresence quenching-based oxygen sensor (silicon Fluoresence quenching-based oxygen sensor (silicon rubber impregnated with ruthenium pentahydrate)rubber impregnated with ruthenium pentahydrate)

Large amount of oxygen initially present in the medium Large amount of oxygen initially present in the medium quenches fluorescence of the sensorquenches fluorescence of the sensor

Growth of Growth of mycobacteria mycobacteria in broth depletes oxygenin broth depletes oxygen Indicator fluoresces brightly when tubes are illuminated Indicator fluoresces brightly when tubes are illuminated

with Wood’s lamp / long-wave UV light with Wood’s lamp / long-wave UV light Time to detection similar to BACTEC 460 TB SystemsTime to detection similar to BACTEC 460 TB Systems Positive tests - orange fluorescent glow at the tube basePositive tests - orange fluorescent glow at the tube base Negative tests - no glow Negative tests - no glow

Becton DickinsonPRINCIPLE

MGITMGIT

•Rapid, easy-to-use system Rapid, easy-to-use system •High accuracy High accuracy •Fluorometric technology Fluorometric technology •No RadioisotopesNo Radioisotopes•No special system requiredNo special system required

•Higher contamination ratesHigher contamination rates•Masking of fluorescence by bloodMasking of fluorescence by blood•lack of compatibility with lack of compatibility with decontamination proceduresdecontamination procedures

Advantages and Disadvantages

BACTEC MGIT 960BACTEC MGIT 960

Tubes are continuously monitored by the system, fully automated

BacT/MB ALERT systemBacT/MB ALERT system

BacT/ ALERT systemBacT/ ALERT system

At the base of each bottle is a colorimetric At the base of each bottle is a colorimetric COCO2 2 sensor separated from the broth by a sensor separated from the broth by a

COCO2 2 – semipermeable membrane that – semipermeable membrane that

monitors amount of COmonitors amount of CO2 2 in the bottlein the bottle

With microbial growth the sensor changes With microbial growth the sensor changes colour, altering the amount of light colour, altering the amount of light reflectedreflected

ESP Culture System IIESP Culture System II

Detects pressure Detects pressure changes in head space changes in head space above broth medium in a above broth medium in a sealed bottle as a result sealed bottle as a result of gas production or of gas production or consumption due to consumption due to growth of growth of mycobacteriamycobacteria

(Trek Diagnostic Systems)

0622 ANALYZERTrek Diagnostic System ESP Culture System II automated microbial detection system

Septi-Chek SystemsSepti-Chek Systems

Biphasic mediumBiphasic medium Bottle with 20ml 7H9 broth in Bottle with 20ml 7H9 broth in

enhanced CO2(20%) and enhanced CO2(20%) and paddle containing three types paddle containing three types of solid media: LJ, Middlebrook of solid media: LJ, Middlebrook 7H11 agar, Chocolate agar 7H11 agar, Chocolate agar

Advantageous for detection of Advantageous for detection of strains which do not grow in strains which do not grow in liquid medium liquid medium

Average time to detection is Average time to detection is longer than BACTEC460 but longer than BACTEC460 but shorter than conventional shorter than conventional methodsmethods

Becton Dickinson

MycoDot™ serological testMycoDot™ serological test

MycoDot™ serological test for diagnosis of MycoDot™ serological test for diagnosis of tuberculosis employs liparabinomannan (LAM) tuberculosis employs liparabinomannan (LAM) antigen bound to plastic combsantigen bound to plastic combs

When the combs are incubated in diluted serum, When the combs are incubated in diluted serum, specific anti-LAM antibodies from the sample, if specific anti-LAM antibodies from the sample, if present, bind to the antigenpresent, bind to the antigen

Combs are then washed to remove non-specific Combs are then washed to remove non-specific antibody, and incubated in a suspension of antibody, and incubated in a suspension of colored particles which bind to bound anti-LAM colored particles which bind to bound anti-LAM antibodies antibodies

If enough of the specific antibodies are present in If enough of the specific antibodies are present in the serum sample, a colored spot will form where the serum sample, a colored spot will form where the antigen is attached to the plastic comb the antigen is attached to the plastic comb

Sensitivity of test is calibrated so that only cases Sensitivity of test is calibrated so that only cases of active mycobacterial disease such as of active mycobacterial disease such as tuberculosis will provide a positive reaction by tuberculosis will provide a positive reaction by MycoDot™ MycoDot™

Luciferase reporter assayLuciferase reporter assay

Luciferase reporter mycobacteriophagesLuciferase reporter mycobacteriophages Phages are constructed as recombinant Phages are constructed as recombinant

derivatives of mycobacteriophages such as L5, derivatives of mycobacteriophages such as L5, D29, or TM4, carrying luciferase gene from D29, or TM4, carrying luciferase gene from fireflies (FFlux)fireflies (FFlux)

When these reporter phages are used to infect When these reporter phages are used to infect cultures of cultures of mycobacteriamycobacteria, luciferase gene is , luciferase gene is expressed and (when a luciferin substrate is expressed and (when a luciferin substrate is added) the infected cells emit lightadded) the infected cells emit light

Light is detected using luminometer Light is detected using luminometer

Luciferase reporter assayLuciferase reporter assay

MOLECULAR TECHNIQUESMOLECULAR TECHNIQUES

Molecular techniques for rapid identification of Molecular techniques for rapid identification of mycobacteriamycobacteria Confirmation of isolates recovered from clinical specimens using Confirmation of isolates recovered from clinical specimens using

DNA probesDNA probes Identification by DNA sequencingIdentification by DNA sequencing Direct detection in sputum by nucleic-acid amplification assaysDirect detection in sputum by nucleic-acid amplification assays DNA Fingerprinting and strain-typing DNA Fingerprinting and strain-typing

Commercially Available DNA ProbesCommercially Available DNA Probes

AccuProbeAccuProbe Identifies the isolate within 2hrIdentifies the isolate within 2hr Target 16S rRNA is released from the organism by Target 16S rRNA is released from the organism by

sonication sonication Labeled DNA probe combines with organisms rRNA to Labeled DNA probe combines with organisms rRNA to

form DNA-rRNA hybrid form DNA-rRNA hybrid Labeled product is detected by a luminometerLabeled product is detected by a luminometer

Direct Amplification testsDirect Amplification tests

Rapid rRNA amplification assay for detection of MTB Rapid rRNA amplification assay for detection of MTB from clinical samples from clinical samples

Target amplified probe is used Target amplified probe is used First available PCR kit for detection of MTB in clinical First available PCR kit for detection of MTB in clinical

specimens by Roche specimens by Roche AMPLICOR MTB amplifies 584bp region of 16S rRNAAMPLICOR MTB amplifies 584bp region of 16S rRNA Amplified Mycobacterium tuberculosis Direct test(AMTD)Amplified Mycobacterium tuberculosis Direct test(AMTD)

Gas-Liquid & High –Performance Liquid Gas-Liquid & High –Performance Liquid Chromatography Chromatography

Analysis of cellular long-chain fatty acids Analysis of cellular long-chain fatty acids Acid methanolysis technique used for isolating mycolic Acid methanolysis technique used for isolating mycolic

acid methyl esters from bacterial cellsacid methyl esters from bacterial cells FDA cleared MIDI Sherlock Microbial System consists of FDA cleared MIDI Sherlock Microbial System consists of

1100HPLC and computer is rapid & reliable method1100HPLC and computer is rapid & reliable method HPLC utilizing fluorescence detection (HPLC-FL) of HPLC utilizing fluorescence detection (HPLC-FL) of

mycolic acid directly from fluorochrome stained smearsmycolic acid directly from fluorochrome stained smears These systems are in direct competition with nucleic acid These systems are in direct competition with nucleic acid

based methods for rapid identificationbased methods for rapid identification

M LepraeM Leprae Dipstick assay is aimed at the rapid Dipstick assay is aimed at the rapid

detection of detection of M.lepraeM.leprae-specific IgM -specific IgM antibodies in human serum or whole antibodies in human serum or whole blood samplesblood samples

Assay is based on the binding of Assay is based on the binding of specific IgM antibodies to a semi-specific IgM antibodies to a semi-synthetic antigen consisting of the synthetic antigen consisting of the terminal sugar groups of the phenolic terminal sugar groups of the phenolic glycolipid-I (PGL-I) antigen of glycolipid-I (PGL-I) antigen of M. lepraeM. leprae attached to bovine serum albuminattached to bovine serum albumin

Use of this antigen ensures that the ML Use of this antigen ensures that the ML Dipstick has a sensitivity and Dipstick has a sensitivity and specificity which is comparable to specificity which is comparable to ELISA.ELISA.

Result of dipstick assay is obtained Result of dipstick assay is obtained after three hours and no special after three hours and no special equipment required equipment required

ANAEROBIC INFECTIONSANAEROBIC INFECTIONS

Rapid IdentificationRapid Identification Rapid (Rapid (2-4hr2-4hr) identification systems ) identification systems Detection of preformed (constitutive) enzymes by use Detection of preformed (constitutive) enzymes by use

of chromogenic or fluorogenic substrates of chromogenic or fluorogenic substrates

Rapid ID32A system (BioMerieux,France)Rapid ID32A system (BioMerieux,France) ANI cardANI card AN-I DentAN-I Dent API ZYM systemsAPI ZYM systems Microscan systemMicroscan system BBL crystal Anaerobe (ANR)BBL crystal Anaerobe (ANR) RapID ANAII System (Remel)RapID ANAII System (Remel)

Overall performance of these systems- mod to goodOverall performance of these systems- mod to good

NON SPECIFIC TESTS NON SPECIFIC TESTS C REACTIVE PROTEINC REACTIVE PROTEIN

Membrane solid phaseMembrane solid phase Immunometric assayImmunometric assay Porous matrix with anti-human Porous matrix with anti-human

CRP immunoglobulinsCRP immunoglobulins (CAPTURE ANTIBODIES)(CAPTURE ANTIBODIES) CRP if present binds to capture CRP if present binds to capture

antibodiesantibodies HRP-conjugateHRP-conjugate Chromogen:Tetramethyl Chromogen:Tetramethyl

benzidinebenzidine Intensity of blue colour is Intensity of blue colour is

proportional to concentrationproportional to concentration of CRP of CRP

1. Special device used to take blood and calibrated loop used to pick up blood

2. Sample diluted with the dilution buffer

3. Sample added till completely soaked

4. One droplet of conjugate added

5. One droplet of TMB solution added

6. One droplet of stop solution added

7. Interpretation of results by comparison with color chart provided with the kit

ENDOTOXIN ASSAYENDOTOXIN ASSAY

Reagent prepared from Reagent prepared from amebocytes of horseshoe amebocytes of horseshoe crab (crab (Limulus polyphemusLimulus polyphemus))

Lysate undergoes gelation Lysate undergoes gelation when in contact with traces when in contact with traces of endotoxinof endotoxin

Used with varying success Used with varying success in diagnosis of Gram in diagnosis of Gram negative sepsis negative sepsis

APPLICATIONS OF FLOW APPLICATIONS OF FLOW CYTOMETRY: isolation of CYTOMETRY: isolation of microbes and identificationmicrobes and identification

Isolation of microbes and their identificationIsolation of microbes and their identification Direct detection of microbial components (nucleic acids Direct detection of microbial components (nucleic acids

and proteins) in clinical samples is possible and proteins) in clinical samples is possible Bacterial detection: fluorochrome-labeled antibodies to Bacterial detection: fluorochrome-labeled antibodies to

specific antigens are a powerful tool specific antigens are a powerful tool Advantages of using antibodies cells not need not be Advantages of using antibodies cells not need not be

cultivable, the method is simple and fast, providing rapid cultivable, the method is simple and fast, providing rapid diagnosisdiagnosis

Disadvantage is still limited availability of antibodies Disadvantage is still limited availability of antibodies directed against particular microbes directed against particular microbes

PRINCIPLE- Use of different-sized fluorescent PRINCIPLE- Use of different-sized fluorescent microspheres coated with antibodies against microbes - microspheres coated with antibodies against microbes - a new application for direct diagnosis a new application for direct diagnosis

FCM in conjunction with fluorescent antibodies used to FCM in conjunction with fluorescent antibodies used to detect surface antigens in detect surface antigens in HaemophilusHaemophilus , , SalmonellaSalmonella , , MycobacteriumMycobacterium , , BrucellaBrucella , , Branhamella catarrhalisBranhamella catarrhalis , , Mycoplasma fermentansMycoplasma fermentans , , Pseudomonas aeruginosaPseudomonas aeruginosa , , Bacteroides fragilisBacteroides fragilis and and LegionellaLegionella

Detects by measuring decrease in fluorescence Detects by measuring decrease in fluorescence emission of microspheres due to shading effect of emission of microspheres due to shading effect of microbes on both the exciting and emitting lightmicrobes on both the exciting and emitting light

Use of different-sized fluorescent microspheres - Use of different-sized fluorescent microspheres - pathogens can be detected simultaneously in the same pathogens can be detected simultaneously in the same sample sample

AUTOMATED SYSTEMSAUTOMATED SYSTEMS

IDENTIFICATION DATABASESIDENTIFICATION DATABASES

Principle : Generate a database for each Principle : Generate a database for each species that contains percentage probability for species that contains percentage probability for a positive result with each test in the batterya positive result with each test in the battery

To identify unknown isolate, metabolic profile is To identify unknown isolate, metabolic profile is obtained and converted to a numeric code which obtained and converted to a numeric code which enables comparison with databaseenables comparison with database

Binary code conversion systemBinary code conversion system Octal code conversion scheme- octal profile Octal code conversion scheme- octal profile Identity provided by computer databaseIdentity provided by computer database

PRODUCTPRODUCT ORGANISMORGANISM FORMATFORMAT TIME TIME RESULTRESULT

METHODMETHOD

API System API System bioMerieuxbioMerieux

GNB,GNB,StaphStaph,,StrepStrep GPC,CD GPC,CD

Cupules, Cupules, microtubes in microtubes in stripstrip

2-72hr2-72hr Colorimetric, Colorimetric, fluorescence fluorescence turbidityturbidity

BBL Crystal BBL Crystal BDBD

Enterobacteria Enterobacteria GNB,GPC,GNB,GPC,

MicroplateMicroplate 3-20hr3-20hr Colorimetric Colorimetric fluorescencefluorescence

BBL Minitek BBL Minitek BDBD

Enterobacteria Enterobacteria GNB,GPCGNB,GPC

MicroplateMicroplate 4-48hr4-48hr ColorimetricColorimetric

BBL BBL EnterotubeEnterotube

EnterobacteriaEnterobacteria Substrate Substrate chamberschambers

18-48hr18-48hr ColorimetricColorimetric

GN-GN-GPMicroplateGPMicroplate

GNB,GPCGNB,GPC MicroplateMicroplate 4-24hr4-24hr ColorimetricColorimetric

Summary of commercial bacterial identification Summary of commercial bacterial identification systemssystems

PRODUCTPRODUCT ORGANISMORGANISM FORMAT FORMAT RESULTRESULT METHODMETHOD

Micro-ID Micro-ID (Remel)(Remel)

EnterobacteriaEnterobacteria Substrate Substrate chambers in chambers in plastic stripsplastic strips

Non growthNon growth

2hr2hrColorimetricColorimetric

Quad-FERM Quad-FERM (bioMerieux)(bioMerieux)

NeisseriaNeisseria CupulesCupules Non growthNon growth

2hr2hrColorimetricColorimetric

Rapid IDRapid ID EnterobacteriaEnterobacteria Tray with Tray with substratesubstrate

Non growthNon growth

2hr2hrColorimetricColorimetric

Vitek Vitek (bioMerieux)(bioMerieux)

EnterobacteriaEnterobacteria

GNB,StaphGNB,StaphCards with Cards with wellswells

Growth Growth 2-18hr2-18hr

ColorimetricColorimetric

MicroScan MicroScan (Dade)(Dade)

EnterobacteriaEnterobacteria

GNB,StaphGNB,StaphMicrotitre plateMicrotitre plate 2-42hr2-42hr Colorimetric Colorimetric

fluorescencefluorescence

Flow cytometerFlow cytometer

MICROSCAN WALKAWAYMICROSCAN WALKAWAY

MICROSCAN WALKAWAYMICROSCAN WALKAWAYPLATESPLATES

VITEK 2VITEK 2

Automates almost entire processAutomates almost entire process Bacterial suspension of defined density of Bacterial suspension of defined density of

isolated organism inoculatedisolated organism inoculated Comp asstd growth analysis to calc MICComp asstd growth analysis to calc MIC turbidimetrically determined kinetic turbidimetrically determined kinetic

measurements of growth for linear regression measurements of growth for linear regression analysisanalysis

VITEK GPS SA- 86% accuracyVITEK GPS SA- 86% accuracy

VITEK 2 SYSTEMVITEK 2 SYSTEM

VITEKVITEK

5 VITEK models available—all of which provide the 5 VITEK models available—all of which provide the same analysis with rapid results and only differ in the same analysis with rapid results and only differ in the number of cards they process: 32, 60, 120, 240 or 480 number of cards they process: 32, 60, 120, 240 or 480

VITEK Test Card is the size of a playing card and is VITEK Test Card is the size of a playing card and is made up of 30 or 45 microwells containing either made up of 30 or 45 microwells containing either identification substrates or antimicrobics identification substrates or antimicrobics

VITEK 2 TEST STRIPSVITEK 2 TEST STRIPS

GNI+ Card (Gram-GNI+ Card (Gram-Negative Plus) V1311Negative Plus) V1311

GPI Card (Gram-Positive) GPI Card (Gram-Positive) ANI Card ANI Card (Anaerobes) V1309 (Anaerobes) V1309

THE FUTURE OF THE FUTURE OF DIAGNOSTICSDIAGNOSTICS

LAB-ON-A-CHIP!

A typical on chip system would be capable of

Controlling fluid transport (injection, pumping, valves)

Separation (diffusion based, size, field flow)

Purification and analysis (chromatography, electrophoresis, isoelectric focusing)

Sensing (chemical, biological, flow, viscosity)

Detection (fluorescence, electrochemical, etc.)

Capture of analyte from patient sample

E

Discrete Test Regions (DTRs) Y Y

E

Antibodies covalently bound to biochip surfaceRandox Biochip surface containing

up to 100 DTRs

Generation of light output which is quantified to determine the level of analyte in patient sample

Secondary enzyme-labelled antibody which also binds to the analyte from the patient sample

SCHEMATICS OF A BIOCHIP

REFERENCESREFERENCES

Koneman’s Color Atlas & Textbook of Diagnostic Koneman’s Color Atlas & Textbook of Diagnostic Microbiology 6 EdnMicrobiology 6 Edn

Clinical Diagnosis & Management by Laboratory Clinical Diagnosis & Management by Laboratory Methods,John Henry,20EdnMethods,John Henry,20Edn

Bailey & Scott’s Diagnostic Microbiology,11 EdnBailey & Scott’s Diagnostic Microbiology,11 Edn Applications of Flow Cytometry to Clinical MicrobiologyApplications of Flow Cytometry to Clinical Microbiology

Clin Microbiol Rev. 2000 April; 13(2): 167–195 Clin Microbiol Rev. 2000 April; 13(2): 167–195