BASIC MOLECULAR TECHNIQUES IN

INFECTIOUS DISEASES

Dr.Sarookhani

Clinical laboratory

• 1)bacteriology & mycology

• 2)parasitology & protozoalogy

• 3)virology

• 4)hematology

• 5)biochemistry&hormon&metabolism

• 6)immunology&serology

• 7)cytology&histo-pathology & genetics Dr.Sarookhani

Types of laboratory methods(for infectious diseases)

• Direct methods– look for/detect the agent

• Indirect methods– detect host response to the agent

Dr.Sarookhani

Ag Ab Reactions

• PRIMARY• IF٬ RIA ٬ ELISA ,CLIA

• SECONDARY• Percipitation• Agglutination• Fulccolation

Dr.Sarookhani

Direct methods(Bacteriology&mycology& Parasitology&Virology)

1. Macroscopic evaluation

2. Staining

3. Direct microscopy

4. Electron microscopy

5. Rapid tests

6. Molecular methods

7. Propagate the agent (culture&sensitivity)

No propagation required

Dr.Sarookhani

MOLECULAR TECHNIQUES ADVANTAGES

• High speed

• high analytical sensitivity

• high clinical sensitivity

• conceptually simple

• highly specific

• Amenable to full automationDr.Sarookhani

BASIC CATEGORIES OF ANALYSIS USED TO

CHARACTERIZE DNA&RNA

• 1)electrophoretic seperations(total,RE,PFGE)

• 2)hybridization assays

• 3)amplification techniques (NAAT)

• 4)restriction fragment length polymorphism(RFLP)

• 5)sequencing

Dr.Sarookhani

LABORATORY SPECIMENS FOR MOLECULAR TECHNIQUES

• 1)whole blood& PBMC• 2)serum• 3)body fluids (urine, semen,

CSF,ameniotic fluid,...) • 4)biopsies• 5) placenta & CVS• 6)blastomer cells of embryo• 7)others(hair,stool,smears,..)

Dr.Sarookhani

ELECTROPHORETIC SEPERATION OF

NUCLEIC ACIDS

Dr.Sarookhani

RFLP CONCEPT

Dr.Sarookhani

HYBRIDIZATION ASSAY FORMATS

1)liquid or solution phase hybridization

2)solid support hybridization

a)DOT/blot(&inverse DOT/blot)hybridization

b)southern&northern blot hybridization

c)in situ hybridization(tissue,cells,chromosomes )

d)NA chip technology Dr.Sarookhani

HYBRIDIZATION CONCEPT

Dr.Sarookhani

SOUTHERN&NORTHERN BLOT HYBRIDIZATION

Dr.Sarookhani

FLOURESCENT IN SITU HYBRIDIZATION (FISH)

– Whole cells or tissue section affixed to glass slides.

– Clinical applications in formalin-fixed paraffin embedded tissues.

tissueDr.Sarookhani

NA CHIP TECHNOLOGY

Dr.Sarookhani

MICRO ARRAY TECHNOLOGY

Dr.Sarookhani

Application of microarray for pathogen detection

Dr.Sarookhani

DNA SEQUENCING

Dr.Sarookhani

Nucleic Acid Amplification Technologies (NAAT)(NAAT)

• 1)TARGET AMPLIFICATION METHODS a)PCR & modifications b)NASBA c)TMA d)SDA

• 2)PRIMER(PROB) AMPLIFICATION METHODS a)LCR b)Q-beta replicase c)cleavase / invader technology

• 3)SIGNAL AMPLIFICATION METHODS a)b DNA & b)HCA

Dr.Sarookhani

principles

Dr.Sarookhani

Dr.Sarookhani

PCR-based modification techniques• RT-PCR• nested PCR• hot start PCR• PCR-LiPA• PCR-SSP• PCR-ARMS• PCR-RFLP• multiplex PCR• PCR-SSCP• RACE-PCR • Real time PCR

Dr.Sarookhani

Schematic of Multiplex PCR

In multiplex PCR more than one target sequence can be amplified by including more than one

pair of primers in the reaction. ( Amplifying various genes simultaneously)

Locus A

Locus C

Locus B

A

C

B

small large

Dr.Sarookhani

Multiplex PCR

Dr.Sarookhani

In the field of infectious diseases

the technique has been shown to be a valuable method for identification of:

• viruses• bacteria • fungi • parasites• All

Dr.Sarookhani

REAL TIME PCR

Dr.Sarookhani

Dr.Sarookhani

APPLICATIONS OF MOLECULAR MOLECULAR TECHNIQUESTECHNIQUES IN MEDICAL

MICROBIOLOGY

Dr.Sarookhani

Microbiology Laboratory• Clinical Microbiology comprises essentially 5 sections.

• Aerobic and anerobic bacteriology

• Mycology

• Mycobacteriology (also called Acid-fast Bacteriology, AFB)

• Parasitology

• Virology

Dr.Sarookhani

IMPACT OF MOLECULAR METHODS ON CLINICAL

BACTERIOLOGY• 1)DIAGNOSIS & PATHOGEN IDENTIFICATION

a)for slow growing or difficult-to- culture organisms b)further examination & identification of agar-grown pure cultures c)simultaneous isolation of pathogens from specimens

• 2)THERAPY

• 3)EPIDEMIOLOGY & CONTROL MEASURES Dr.Sarookhani

MOLECULAR METHODS IN CLINICAL BACTERIOLOGY LAB.

• PCR & other amplification techniques

• nucleic acid hybridization techniques

• use of RE,s

• DNA sequencing analysis

• gene chip technology

Dr.Sarookhani

MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(1)

Streptococci group 16S rRNA RFLPGroup A streptococci Emm gene Hybridizat&PCRPneumococci Lyt A gene DNAprobe&PCRN.gonorrhea rRna DNA probeN.gonorrhea CPPB gene PCRN.gonorrhea Pillin gene LCRN.meningitidis rrn gene PCREntrococci rRNA ProbeStaphylococci HSP60 Colony DOT blotStaphylococci Nuc&femA PCR

Dr.Sarookhani

MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(2)

H.influenza PBP& rRNA Probe

H.influenza BexA&ompP6 PCR

Legionella rRNA Probe

Legionella mip PCR

H.ducreyi 16 S rRNA Probe

H.ducreyi groEL PCRMycoplasma&Ureaplasma MP & MCC ProbeMycoplasma&Ureaplasma Tuf&urease PCR

Dr.Sarookhani

MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA(3)

H.pylori Ure A,C&rRNA Probe&PCR

Y.entrocolytica ail PCR

Cholera toxin Elt&ctx&hlyA PCR

C.diphteria toxA Probe

M.tuberculosis IS probes

M.tuberculosis IS6110 PCR&RFLP&LCR

Dr.Sarookhani

PCR of M.tuberculosis

Dr.Sarookhani

Molecular detection of mycoplasma

Dr.Sarookhani

PCR DETECTION OF BRUCELLA

Dr.Sarookhani

PCR-based detection of H.pylori (cag A gene)

Dr.Sarookhani

PCR-based detection of T.pallidum

Dr.Sarookhani

PCR-based detection of Mycobacterium lepre in

skin biopsy

Dr.Sarookhani

PCR-based detection of Yersinia entrocolitica (chromosomal ail gene)

Dr.Sarookhani

APPLICATIONS OF MOLECULAR EPIDEMIOLOGY

• Detection of identityidentity of strains

• detection of genotypesgenotypes

• detection of emergence & spread of strains of an organism with unusualunusual resistance patterns or pathogenicity

• determining the efficiency efficiency of infection control procedures

• identification of sourcesource in outbreaksDr.Sarookhani

APPLICATION OF MOLECULAR METHODS IN VIROLOGY

• Hepatitis viruses(HBV,HCV,HDV):PCR&RT-PCR

• herpesviridae(CMV,HSV,EBV,VZV,...):PCR

• HTLV1 & HIV1,2, : nested RT-PCR

• ENTOVIRUSES :RT-PCR

• PARVOVIRUS B19 : HB & PCR

• HPV : FISH

• mumps,adenovridae,LCM,measles : PCR&RT-PCR

• rubella : RT-PCRDr.Sarookhani

QUANTITATIVE AMPLIFICATION RESULTS

MAY USEFUL FOR:

• Viral load

• prognosis

• monitoring response to therapy

Dr.Sarookhani

HCV RNA ( RT-PCR)

Dr.Sarookhani

QUANTITATIVE AMPLIFICATION

FOR HIV DETECTION•

• branched DNA assay

Dr.Sarookhani

Laboratory Diagnosis of Influenza

Comparison of Test Methods forComparison of Test Methods for InfluenzaInfluenza

Test MethodTest MethodTime to Time to ResultsResultsCommentsComments

SerologySerology>>22 wkswksRetrospective, requires paired seraRetrospective, requires paired sera

CultureCulture1-101-10 daysdaysStill gold standard(?), requires Still gold standard(?), requires expertise, provides virus for studiesexpertise, provides virus for studies

MolecularMolecular((RT-PCRRT-PCR))2-42-4 hrshrsBecoming gold standard(?), requires Becoming gold standard(?), requires

expertise & expensive equipmentexpertise & expensive equipment

Antigen Detection Antigen Detection )IF()IF(2-42-4 hrshrsRequires reading expertise & IF Requires reading expertise & IF

microscopemicroscope

Antigen DetectionAntigen Detection ((Rapid EIA-likeRapid EIA-like))15-3015-30 minminWidely available, requires little Widely available, requires little

expertiseexpertiseDr.Sarookhani

Specimen Types• Upper respiratory tract

– Nasal or naso-pharyngeal (NP) swabs

– Throat swabs

– NP aspirates or washes

• Lower respiratory tract– Tracheal aspirates

– Bronchoalveolar lavages

• Store at 2-8°C < 72 hours or freeze at < -70°C.– Transport with cool-pack

Dr.Sarookhani

Possible contamination due to the throat- wash sampling method

Dr.Sarookhani

Dr.Sarookhani

Dr.Sarookhani

MOLECULAR DETECTION OF PARASITES &PROTOZOA

•

Hydatidosis(protoscolex)

r-DNAITS 1

PCR-RFLP

Plasmodium sp. NestedPCR-RFLP

A.culcifacies Mt-DNA PCR-RFLP

toxoplasmosis B 1 DOT/BLOT&PCR

E.histolytica &(dispar var.)

r RNA Hybridization& PCR

leishmaniosis PCR

Dr.Sarookhani

Nested PCR for Leishmania diagnosis

Dr.Sarookhani

PCR-BASED DETECTION OF TOXOPLASMA GONDII

Dr.Sarookhani

MOLECULAR DETECTIONOF FUNGI

• T.verrocosum :HSP 70 :PCR

• Candida sp. :PCR

• Cryptococcus neoformans:nested PCR(CSF)

• Histoplasma capsulatum: probe

• Coccidioides immitis :probe

• Blastomyces dermatidis :probe

• Acanthamoeba :PCRDr.Sarookhani

Detection of Cryptococcus neoformans by nested PCR

Dr.Sarookhani

MOLECULAR METHODS FOR CO-IDENTIFICATION OF MULTIPLE AGENTS

Dr.Sarookhani

Infectious agentInfectious agent Pathogens targetedPathogens targeted Clinical manifestation(s) and/or Clinical manifestation(s) and/or

specimenspecimen

CombinationCombination

((All agentsAll agents))

HSV, HSV, H. ducreyiH. ducreyi, and , and T. pallidumT. pallidum

Genital ulcer disease Genital ulcer disease

HPVs, HPVs, HSV, and HSV, and C. trachomatisC. trachomatis

Genital swabsGenital swabs

Adenovirus, Adenovirus, HSV, and HSV, and C. trachomatisC. trachomatis

Keratoconjunctivitis Keratoconjunctivitis

EV, influenza viruses A EV, influenza viruses A and B, RSV, PIV types 1 and B, RSV, PIV types 1 and 3, adenovirus, and 3, adenovirus, M. M. pneumoniaepneumoniae, and , and C. C. pneumoniaepneumoniae

Acute respiratory Acute respiratory tract infectiontract infection

Dr.Sarookhani

Application of multiplex PCR for diagnosis of viral infections

(viruses in CNS)

•

Clinical manifestation(s)Clinical manifestation(s) Specimen(s)Specimen(s) Viruses and/or other Viruses and/or other

agent(s) targetedagent(s) targeted

Meningitis, encephalitis, Meningitis, encephalitis, and/or and/or

meningoencephalitismeningoencephalitis CSFCSFHSV-1, HSV-2, and CMV HSV-1, HSV-2, and CMV

HSV and VZV; EBV and HHV-6 HSV and VZV; EBV and HHV-6 HSV-1, HSV-2, VZV, CMV, HSV-1, HSV-2, VZV, CMV,

HHV-6, and EBVHHV-6, and EBV

six herpesvirusessix herpesviruses

that maythat may infect the CNS infect the CNS

HSV-1, HSV-2, VZV, CMV, HSV-1, HSV-2, VZV, CMV, HHV-6, EBV, and Ent.V HHV-6, EBV, and Ent.V

CMV, EBV, HHV-6, HHV-7, CMV, EBV, HHV-6, HHV-7,

and HHV-8and HHV-8

Dr.Sarookhani

16 S rDNA

Dr.Sarookhani

Advantages of Molecular techniques in Infectious Diseases

• increased sensitivity and specificity of identification

• faster report turnaround time• Confirmation of culture• Identification of organisms that are non-viable or

cannot be cultured• Identification of fastidious, slow growing

organisms• Identification of organisms that are dangerous to

culture• Identification of organisms in small numbers or

in small volume specimensDr.Sarookhani