THE IMPORTANCE AND ADVANTAGES OF THE IMPORTANCE AND ADVANTAGES OF NUCLEIC ACID AMPLIFICATION TECHNIQUESNUCLEIC ACID AMPLIFICATION TECHNIQUES

ININDIAGNOSIS OFDIAGNOSIS OF

BACTERIAL INFECTIONSBACTERIAL INFECTIONS

FatihFatih KKÖÖKSALKSALUniversty Universty Of Of ÇÇukurovaukurovaFaculty Faculty of of MedicineMedicineDept Dept of of Clin MicrobiologyClin Microbiology

INFECTIOUS DISEASE INFECTIOUS DISEASE

High morbidityHigh morbidity

High mortalityHigh mortality

Health costHealth cost: > 5 : > 5 billionbillion USD USD

M.tuberculosis 8.0 .MillionDiarrheae 1.8 BillionC.trachomatis 88.0 Million

M.tuberculosis 8.0 .MillionDiarrheae 1.8 BillionC.trachomatis 88.0 Million

Tuberculosis 3.0 MillionDiarrhea 2.5 MillionAIDS 21.0 Million

Tuberculosis 3.0 MillionDiarrhea 2.5 MillionAIDS 21.0 Million

0

5

10

15

20

25

30

1. Çeyrek

Cardiovascular

İnfectious dis

malignantneoplasmsInj.,accid.,suic.

Chronic.lung dis

pregn.related dis.

Other

Digestive diseases

16.733.00016.733.0002929

2626

1212

Total death Total death 57.029.00057.029.000

996 5 44

14.867.00014.867.000

7.121.0007.121.000

Fauci Fauci et.al EID 11:519 2005et.al EID 11:519 2005

INFECTIOUS DISEASES ARE SECOND LEADING CAUSE OF MORTALITY

••The right drugThe right drug••The right patient The right patient ••The right The right timetime••The right costThe right cost

Rational Treatment of Infectious Diseases

••Identification of pathogenIdentification of pathogen••DetectionDetection of of resistanceresistance

••Hundered pathogensHundered pathogens••HowHow can can we solve we solve ??

DIAGNOSIS OF INFECTIOUS DISEASESDIAGNOSIS OF INFECTIOUS DISEASESIN THE MICROBIOLOGY LABORATORYIN THE MICROBIOLOGY LABORATORY

1.1. Direct methodsDirect methods::Microscopical evaluationMicroscopical evaluationIsolationIsolation of of microorganismsmicroorganismsIdentificationIdentification of of isolatesisolates

Microscopic identificationMicroscopic identificationBiochemical charactersBiochemical charactersProtein profileProtein profileAntibiotic susceptibilitiesAntibiotic susceptibilities

2. 2. Indirect methodsIndirect methodsELISAELISAIFA vbIFA vb

CULTURE IS GOLD STANDARTCULTURE IS GOLD STANDART

•• Slow growing Slow growing : M.: M.tuberculosistuberculosis, , Brucella sspBrucella ssp•• Fastidious Fastidious : H.: H.pyloripylori, , ChlamydiaChlamydia, , MycoplasmasMycoplasmas, N. , N. gonorrhoeagonorrhoea•• Unculturable Unculturable : : Tropheryma wippleii and other trephonemasTropheryma wippleii and other trephonemas•• Identification Identification : : BiotypingBiotyping, protein profile, , protein profile, serotypingserotyping, , phagetypingphagetyping•• Antibiotic resistanceAntibiotic resistance

1.1. Time Time consuming and Laboriousconsuming and Laborious

2. 2. Low reproducibiltyLow reproducibilty

3.3. Weak discrimnative powerWeak discrimnative power

4.4. Necessarly viabilityNecessarly viability

Could molecular techniques Could molecular techniques solve solve these problemsthese problems? ?

We purpose to/can take a small amount (several strands) of our interest/entire genome and cut it down

to make millions of copies of one small specific segment of target DNA

OUR PURPOSE WİTH PCR

In 1983, In 1983, KaryKary Mullis developed a revolutionary laboratory Mullis developed a revolutionary laboratory procedure: using the principles behind DNA replication, procedure: using the principles behind DNA replication,

he designed a way to make BILLIONS of copies of a he designed a way to make BILLIONS of copies of a specific DNA sequence in just a few hours!specific DNA sequence in just a few hours!

This process is called PCRThis process is called PCR

INDICATIONS of MOLECULAR METHODS INDICATIONS of MOLECULAR METHODS in CLINICAL MICROBIOLOGYin CLINICAL MICROBIOLOGY

1. Detection of pathogens

2. Identification of pathogens

3. Characterization of virulence factors

4. Determination of antimicrobial susceptibilities5. Surveillance of infectious diseases6. Host-pathogen interactions

Qualitative and quantitativeQualitative and quantitative

H.H.pylori or other helicobacterspylori or other helicobacters

H.H.pylori type pylori type I (I (CagA CagA + ) + ) oror H.H.pylori type pylori type II (II (CagA CagA --))

Glycolitic Glycolitic gen gen upregulation upregulation in in human gastric cell infected with human gastric cell infected with HPHP

BASIC PRINCIPLE OF AMPLIFICATION BASED BASIC PRINCIPLE OF AMPLIFICATION BASED MOLECULAR METHODSMOLECULAR METHODS

Amplification Amplification of of specific Nucleic Acid Fragmentspecific Nucleic Acid FragmentNAAT NAAT similar to similar to cultureculture

IdentificationIdentification of of ampliconsampliconsSpecific probsSpecific probs, RFLP, SSCP as , RFLP, SSCP as if if stainingstaining

Detection Detection of of specific mutationspecific mutationSpecific probsSpecific probs, RFLP, SSCP, RFLP, SSCP

CURRENT DEVELOPMENTS IN NUCLEIC ACID-BASEDTECHNIQUES

PCR :PCR : RTRT--PCR, PCR, HotHot--Start PCR, nStart PCR, n--PCR, PCR, Real Real Time PCRTime PCRTMA :TMA : Transcription Mediated AmplificationTranscription Mediated AmplificationNASBA :NASBA : Nucleic Acid SequenceNucleic Acid Sequence--Based Amplification Based Amplification SMART :SMART : Signal mediated Amplification Signal mediated Amplification of RNA of RNA TechnologyTechnologySDA :SDA : Strand Displacemend AmplificationStrand Displacemend AmplificationLAMP LAMP : : LoopLoop--mediated Isothermal Amplificationmediated Isothermal AmplificationLCR :LCR : Ligase Chain Reaction Ligase Chain Reaction RCA :RCA : Rolling Circle AmplificationRolling Circle AmplificationRAA :RAA : Ramification Amplification AssayRamification Amplification Assay

C.C.trachomatistrachomatis3 3 million infection per yearmillion infection per year70 % 70 % women asymptomaticwomen asymptomatic50 % man 50 % man asymptomaticasymptomatic

N.N.gonorrhoeagonorrhoea1 1 million infection per yearmillion infection per year

USAUSAMost common complicationsMost common complications::

PID, PID, Cervical ectopyCervical ectopy, , Tubal infertilityTubal infertility

NAAT IN INFECTIOUS DISEASESNAAT IN INFECTIOUS DISEASES

PCR, LCR, TMA, PCR, LCR, TMA, Hybrid CaptureHybrid Capture, , HybridizationHybridization, SDR, SDRFDAFDA--approved molecular diagnostic tests for approved molecular diagnostic tests for C/NC/N

STD is one of the major medical and social problems

Multiplex PCRMultiplex PCR in STDin STD

We can use of multiple primers to test for two or more target sequences at once in a clinicalsample

SyphilisSyphilisHerpesHerpes

ChancroidChancroid

Common NamesCommon Names

GonorrheaGonorrheaTrichTrich

GASTRODUODENAL COLONISATION OF H. GASTRODUODENAL COLONISATION OF H. pyloripylori

High morbidityHigh morbidityIncidenceIncidence prevalanceprevalance

DevelopedDeveloped countries countries % 0.5/ % 0.5/ year year % 10% 10--3030Developing countries Developing countries % 3% 3--10/ 10/ year year % 60% 60--8585

High mortalityHigh mortalityJapan Japan : : 23.123.1--43.7/100.000/43.7/100.000/year year

ZhouZhou W W FEMS FEMS ImmunImmun. . andand MedMed. . MicrobiologyMicrobiology 20032003

Wales Wales : : Gastric cancer Gastric cancer :12.000/:12.000/yearyearAllAll of of infections infections : 3.000/: 3.000/yearyear

H. pylori was recognised as Type I carcinogen

Acut gastritisAcut gastritis

Chronic Active GastritisChronic Active Gastritis

Antral gastritis Antral gastritis Multifocal atrofic gastritisMultifocal atrofic gastritis

Duodenal ulcer Duodenal ulcer GASTRIC ULCER GASTRIC CANCER

GASTRIC LYMPHOMA

NATURAL HISTORY NATURAL HISTORY ofof H.PYLORIH.PYLORI INFECTIONINFECTION

SampleSample

nn Culture

Culture 16s 16s rRNArRNA

gengeneeRandom Random sequencesequence

26 26 kDakDaSSA SSA gengenee

ureAureAgengenee

ureCureC ((glmMglmM))gengenee

7711112244111010221313

+ + + + + + + + + + +++ + + + + + + + ++++

++++++++++

++

++

++++++++

++

++++++++

JangJang--JihJih LuLu, , JournalJournal of of ClinicalClinical MicrobiologyMicrobiology 19991999

22--IDENTIFICATION OF H.IDENTIFICATION OF H.pylori pylori

ttyyppeespcspc

ggeennuussspcspc --

------

H.H.pylori pylori is is Type Type I I carcinogencarcinogen

cagPIcagPI: 40 : 40 kbp kbp 27 protein 27 protein codescodes ((cagAcagA--cagcagξξ))TypeType IV IV secretion systemsecretion systemType Type I I strainsstrainscagAcagA The most important The most important genegene in in cagPIcagPI

cagAcagA: 120: 120--140 140 kDakDa effectoreffector codes CagAcodes CagA proteinprotein--cytokinecytokineOncoprotein Oncoprotein found carsinogenic strainfound carsinogenic strainprovokes provokes inflammatoryinflammatory responseresponse

3-Characterization of virulence factors in H.pylori

YuriYuri ChurinChurin The Journal of Cell Biology The Journal of Cell Biology 20032003

••Repeated sequencesRepeated sequences on 3on 3’’ terminalterminalregionregion

••EPIYA gene EPIYA gene casettecasette is is responsible responsible from tyrosine phosphorilation from tyrosine phosphorilation whichwhich is a is a key enzymekey enzyme ininmalignant transformationmalignant transformation

••This sequence This sequence codescodes EPIYAEPIYA motivemotive

•• EPIYA gene EPIYA gene casettescasettes are found are found inindifferent number and different number and size in size in different different strainsstrains

3-Characterization of virulans factors in H.pylori

PatricePatrice BoquetBoquet TrendsTrends in in MicrobiolMicrobiologyogy 20032003

142 142 kDakDa

8888--95 95 kDakDa VacAVacA

37 37 kDakDa ––58 58 kDakDa

vagAvagA :3933 :3933 bpbp ORFORF

vacAvacA GENGEN

12871287--1296 1296 aa aa

vacAvacA and cagAand cagA gene profile in H.gene profile in H.pylori isolated frompylori isolated from PU PU patientspatients

Sample Sample : 156: 156ureCureC + : 142+ : 142

vacAvacA s1c : 41s1c : 41s1a : 101s1a : 101

m1a: 34m1a: 34m1b: 3m1b: 3m2 : m2 : --

cagA cagA : 144: 144BulentBulent K, K, Fatih KFatih K, , WorldWorld J J GastroenterolGastroenterol. 2003. 2003

PCR

4-MOLECULAR EPIDEMIOLOGY

Molecular epidemiology is essential tool in the outbreak setting to define ;Who isIs notA part of outberakWho/which/where is the possible source ?

4-MOLECULAR EPIDEMIOLOGY

Major Pathogens : P.aeruginosa, A.baumanii, MRSA

Clinical importance : High morbitity, mortality, cost effect2 million patiens4.7-5.4 Billion Dolar

Source of problems : Epidemiological knowledgeSolution of problem: Molecular methods

PFGERFLPrep-PCRAP-PCRDNA Sequencing

Who isIs notA part of outberakWho/which/where is the possible source ?

HOSPITAL INFECTIONSHOSPITAL INFECTIONS

55-- DeterminationDetermination of of antimicrobial susceptibility with antimicrobial susceptibility with traditionaltraditional methodsmethods

••FlexibleFlexible••Detect susceptibilityDetect susceptibility——better better criterioncriterion for treatment optionsfor treatment options••CheapCheap

••Many variables affect results: Many variables affect results: standardisationstandardisation required required

••Sometimes difficult to interpretSometimes difficult to interpret

••Microbiological breakpoint definition of resistance is impreciseMicrobiological breakpoint definition of resistance is imprecise

••Correlation between phenotypic methods and Correlation between phenotypic methods and in vivoin vivo results?results?

ADVANTAGESADVANTAGES

DISADVANTAGESDISADVANTAGES

•Can result faster

•Can detect the genetic mechanisms of resistance

55--AdvantageAdvantage of NAAT in of NAAT in Detection of Detection of Antibiotic ResistanceAntibiotic Resistance

••Detect only known mechanismsDetect only known mechanisms : novel mechanisms will : novel mechanisms will be missedbe missed

••Some resistance genes may be unSome resistance genes may be un--expressed: false positivesexpressed: false positives

ADVANTAGESADVANTAGES

DISADVANTAGESDISADVANTAGES

0 24 48 72 96 hrs

Direct plating on agarEnrichment broth Subculture onblood agar

Subcult BA + cefox disk diff

suspicious:

Slidex+

cefoxitinR

:

MRSA screenMRSA screen

Swab test

+72 – 96 hrs

-

Pre - enrichment broth

DNA extractPCR

+confirmation with conventional test

Confirmation with conventional test

I

IIDNA extract PCR

+1-3 hours

+24(I)-48(II) hours

FASTER RESULTSFASTER RESULTSHow many days we need to diagnosis of MRSA ?

AN INVESTIGATIONOF ANTIBIOTICSAN INVESTIGATIONOF ANTIBIOTICSRESISTANCE OF H.RESISTANCE OF H.pyloripylori SPECIESSPECIES

N : 386CLR : 65 19 %Methods :PCR-RFLP

Periods2002 - 2003 2003 - 2004 2004 - 2005 Total

n % n % n % n %

21422143others

A-G 12 70.5 21 80.8 20 86.9 53 81A-G 3 17.6 3 11.5 3 13.1 9 14

2 12.2 2 7.8 - - 4 5

17 14.9 26 17.9 23 18.1 65 100

Koksal F. Helicobacter 2006

55--Potential Clinical Benefits of Potential Clinical Benefits of NAATNAATDetection of Antibiotic Resistance for Patient CareDetection of Antibiotic Resistance for Patient Care

Early adequate treatmentReduction of empiric use of broad-spectrum antibioticsImproved Clinical outcomeReduction of hospital lenght of stayReduction in spread of resistance into community

ConclusionConclusionMolecular methods are required:

For the diagnosis of slow growing, fastidious and unculturable bacteria.

Subtyping and quantitation of bacteria in clinical samples.

Detection of drug resistance

Evaluation of epidemics and nosocomial infections

Response to therapy and prognosis.

Most important limitation of molecular approaches is that: They can detect only known target sequences and point mutations.

In spite of recent developments in molecular techniques, conventional methods will be in use for the routine clinical practice .NAATs are the complementary methods for traditional approaches