MOLECULAR METHODS IN DIAGNOSIS AND MONITORING OF INFECTIOUS

DISEASES

Fera Ibrahim Dept. Of Microbiology, Medical Faculty,

University of Indonesia

LABORATORY DIAGNOSIS OF INFECTIOUS DISEASES

• DIRECT – Isolation of the agents– Detection of Antigens

–Detection of nucleic acids• INDIRECT

– Detection of Antibodies– Histopathology

NUCLEIC ACIDSThe Basics of DNA and RNA

NUCLEIC ACIDSThe Basics of DNA and RNA

NUCLEIC ACIDSThe Basics of DNA and RNA

3’

5’

5’

3’

Transcription and Translation in Prokaryotes

Ribosome

Ribosome5’

mRNA

RNAPol.

Transcription and Translation in Viruses

Indications for molecular testing

• To detect slow growing, fastidious or uncultivable microorganisms

• To determine the cause of significant outbreaks

• To detect and charaterize more common microorganisms

- Rapid- Easier to use- Less expensive

Development of molecular assays for infectious diseases

• SIGNAL AMPLIFICATION METHODS– Nucleic acid Probes– Hybrid Capture– Branched DNA– In Situ Hybridization

• NUCLEIC ACID AMPLIFICATION– Basics of the Polymerase Chain reaction (PCR)– Other methods of Nucleic Acid Amplification– Modifications of PCR

• POSTAMPLIFICATION ANALYSIS– Traditional Methods of Detection– Reverse Hybridization– DNA Sequencing

• REAL TIME NUCLEIC ACID AMPLIFICATION – Methods of Detecting the Products of Real Amplification

• STRAIN TYPING– Non amplification Based Typing– Amplification Based Typing

Development of molecular assays for infectious diseases

SIGNAL AMPLIFICATION METHODS– Nucleic acid Probes

• DNA Probes that contain a chemiluminescent label• Target the rRNA (rRNA > rDNA genes)• Clinical Applications :

– Direct detection of bacteria in clinical specimens– A rapid and specific method of identifying microorganisms in culture

(sensitivities and specificities 90s to 100%)– The advantage is time savings, the disadvantage is higher cost– N.gonorrhoeae, C.trachomatis, Group A and B Streptococcus, M.tbc,

M. Kansasii, Mgordonae, M.avium intracellulare complex, Campylobacter, Haemophilus influenzae, streptococcus pneumoniae, Staphylococcus aureus, Listeria monocytogenes

– Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis

Hybridization

Hybridization

SIGNAL AMPLIFICATION METHODS– Hybrid Capture

• The RNA probes used are complementary to the DNA molecule from the microorganism of interest in a tube or microtiter plate

• A monoclonal antibody recognizes and immobilizes a DNA-RNA hybrid, another antibody labeled with reporter molecule recognizes that complex. A reporter generate a signal in the form of light.

• The amount of signal generated in the assay is proporsional to the amount of target present

• Clinical Applications :– The detection of high risk HPV subtypes, Cytomegalovirus, HBV– N. Gonorhoeae, C. Trachomatis (more sensitive than culture)– The hybrid capture is less sensitive and specific than PCR

Hybrid Capture

SIGNAL AMPLIFICATION METHODS• Branched DNA (bDNA)

• The organism-specific bDNA oligonucleotides probes hybridize to the target nucleic acid molecule and this complex is captured onto a solid substrate. Branched DNA oligonucleotide reporter molecules which are conjugated to reporter enzymes are added and generate a chemiluminescent signal after the addition of appropriate substrate.

• Clinical Applications• The detection and quantification of pathogens such as

HIV, HBV, and HCV• It has become the standard of care to monitor the viral

loads of patients infected with HIV (75-500 copies of HIV1 RNA)

• Although the sensitivity of the bDNA assays may be slightly less than that for PCR, there is overall a good correlation between viral loads generated by these different assays.

Branched DNA Signal Amplification Assay

SIGNAL AMPLIFICATION METHODS– In Situ Hybridization

• This method has been used in the molecular pathology for the detection of chromosomal translocation, gene amplification and for the identification of infectious agents

• FISH oligonucleotide probe is labeled with a fluorophore, direct fluorescence microscopy

• Chromogenic in situ hybridization (CISH) oligonucleotide is labeled with an enzymes, traditional light microscopy

• The microbial rRNA may be used as the target for hybridization probe rRNA >rDNA (more sensitive); rRNA ~ a viable organism

• PNA ISH a peptide nucleic acid probe (better than DNA probe with regard to penetration hybridization on intact organism

• A battery probes bacterial meningitis, community acquired pneumonia

– In Situ Hybridization• Clinical Applications :

– Direct detection of bacteria, mycobacterium, fungi and parasites : bacteria in water samples and biofilm (Legionella, E coli); Helicobacter pylori in gastric biopsies; Legionella pneumophila in fixed respiratory specimens; Chlamydia, M tbc, M leprae (cannot be cultivated on artificial media); S. aureus, P. Aeruginosa, S. maltophilia, B.cepacia and H. Influenza in specimen patient with cystic fibrosis

– To differentiate viruses that produce identical cytophatic effects (HSV and VZV) and the high and low risk HPV subtypes, to detect viruses with neoplasia : HPV, EBV, HHV8

– Fungi and parasites : Aspergillus Vs Fusarium/Pseudallescheriapathogen and different antifungal susceptibility profile ; Pneumocystis jiroveci; to rapid identify and differentiate Candida albicans, C. Glabrata, C. Krusei and C. Parapsilosis in blood culture

SIGNAL AMPLIFICATION METHODS

In Situ Hybridization

In situ RNA hybridization (ISH) for FISH, CISH applications

NUCLEIC ACID AMPLIFICATION– Basics of the Polymerase Chain reaction (PCR)

• First described by Kary Mullis in 1983• Exploits the basic biochemistry of DNA replication with the

end goal of amplification of a particular portion of DNA usually contains diagnostically useful information

• PCR reaction consist of the target DNA, oligonucleotide DNA primers, the four nucleotide triphosphate, thermostable DNA polymerase, MgCl2 and water

• Three phase : DNA denaturation, primers annealing, primers extention DNA synthesis

– Clinical Applications• To detect organisms that could not be cultured,were

fastidious or slow growing• Almost every microorganism of clinically interest has been

detected and studied by PCR commercial assays, FDA approved

NUCLEIC ACID AMPLIFICATION– Other methods of Nucleic Acid Amplification

• The nucleic acid sequence based amplification (NASBA)– Isothermal assays use 3 enzymes : Reverse Transcriptase (RT),

Rnase H and T7 DNA dependent RNA polymerase– RT make a cDNA copy of target molecule (usually RNA), The primer

contains the T7RNA polymerase binding sequence on the 5’ end of the molecule, DNA-RNA hybrid is hydrolized by Rnase H, 2nd primer then binds and creates the complementary strand of the DNA this complete cDNA serves as the template for T7RNA polymerase which transcribes numerous copies of RNA to be detected by a variety of methods

• Transcription mediated amplification (TMA) sda• Strand-displacement amplification (SDA)

– An isothermal reaction that relies on the ability of the DNA polymerase to displace one strand of DNA at the site of a single strand nick and proceed with DNA replication or amplification in this assays

– Require special conditions and endonucleases that will generate a nick in only one of the strands of the double stranded DNA molecule

NUCLEIC ACID AMPLIFICATION– Other methods of Nucleic Acid Amplification

• Ligase Chain Reaction (LCR)– The ligation or connection of two probes that anneal contiguously on

the template DNA strand– N. Gonnorhoeae, Chlamydia trachomatis

• Clinical Applications– FDA approved commercial assays available for the detection of N.

Gonorrhoeae and C trachomatis that use TMA and SDA; HIV viral load that use NASBA technology; CMV pp67 assay for monitoring CMV level in a bone marrow transplant recipient (replace antigenemia assays); for the rapid detection of enterovirus from the cerebrospinal fluid (CSF)

NUCLEIC ACID AMPLIFICATION– Modifications of PCR

• RT-PCR (Reverse Transcription –PCR)– Make DNA copy from RNA – To detect RNA viruses, mRNA, rRNA (~viable organisms)– Clinical Applications

» To detect dengue virus , HIV, Hantavirus, SARS» To detect rRNA bacteria, parasites and fungi

• Broad range PCR– The primers of assay are designed to detect all the organisms in the

group of interest and exclude as many organisms that are not in this group

– The members of a larger group may be detected in a single reaction– Clinical Applications

» To detect the enteroviruses capable of causing aseptic meningitis

» To charaterize mycobacteria by rpoB gene provide information regarding the resistance of mycobacteria to rifampin

NUCLEIC ACID AMPLIFICATION– Modifications of PCR

• Multiplex PCR– An alternative method to broad range PCR for the detection of

multiple pathogens in one reaction– Clinical Applications

» To detect different microorganisms that cause the same types of diseases ex To detect S. pneumoniae, H influenzae and N. Meningitiditis the most common causes of bacterial menigitis

» To detect the agents of atypical and typical bacterial pneumonia• Nested / Seminested PCR

– Modification of PCR designed to increase the sensitivity of the assay reaction

– This modification consist of two primers sets directed against the same target

– Clinical Applications» To detect microorganisms that may be in low quantity in the

blood and tissue such as Rikettsia, Bartonella

POLYMERASE CHAIN REACTION (PCR)

Molecular detection of HTLV 1

Agarose gel electrophoresis and Southern blot of samples amplified by SYBR Green real time PCR assay. In the upper part of the figure: the two positive HTLV-I samples (lanes 1 and 11), the samples with HTLV-I indeterminate or positive serological assays (lanes 2–10), MT-2 representative cellsscalar dilutions (from 102 to 104; lanes 12–14) and molecular weight markers (lane 15) are shown. The bottom of the figure shows the Southern blot assay. The non-specific bands exhibited in the HTLV-I negative samples (lane 7–9) represent non-specific products that both did not hybridize withthe HTLV-I specific internal probe and showed an unrelated melting temperature in SYBR Green real time PCR.

M 1 2 3

CMV detection by PCR technique

Line 1 = positve DNA CMVLine 2 = H2O, PCR reaction controlLine 3 = H2OM = Marker phiX 174 DNA/haeIII

1353bp1078bp

872bp603bp

310bp

CMV detection by PCR technique

0.3

K+ K- S M Kbp

0.4

1.0

0.5

0.1

0.2 M = DNA LadderS = SampleK- = Negative controlK+ = Positive controlKbp = Kilo base pair

k+ k- S M

M: DNA LadderS: Plasma sample k+: Positive control k-: Negative control

HIV 1 detection by RT-PCR technique

– Traditional Methods of Detection• Gel Electrophoresis/Southern Blot Analysis

– The separation of the DNA in gel electrophoresis is largely due to size – The negatively charged DNA migrate toward the anode, – Visual comparison of the migration of the amplicon and the migration

of the DNA molecules of varying sizes in a ladder the user can estimate of the size of the DNA molecule amplified

– The amplified product was transferred to nitrocellulose paper and a radiolabelled oligonucleotide probe added exposed to X-ray film

• Enzymatic detection of Amplified products– The enzymatic reaction for the detection of the amplified product is

performed in a microtiter plate– The amount of signal generated is proportional to the amount of

amplicon present, when used with calibrated standards, quantitative information may be obtained

• Clinical Applications– Many of the commercially available systems use a colorimetric

reaction for the detection of the amplified products (Southern Blot labor intensive and time consuming)

POST AMPLIFICATION ANALYSIS

Gel Electrophoresis

HIV 1 detction by RT-PCR technique

Electrophoretic gel profile of RT-PCR products generated using the Applied Biosystems ViroSeq HIV-1 genotyping system. Fifteen members of the HIV-1 subtype panel at 5,000 RNA copies/ml were subjected to sequence analysis by the ViroSeq assay using the model 3100 genetic analyzer. The gel profile for purified amplified product is shown and is representative of all of the HIV-1 subtypes tested. Lanes 1 and 12 contain two amounts of the DNA mass ladder. Lanes 3 and 4 contain the two subtype D isolates (46 and 44 ng). Lanes 5, 6, and 7 contain subtypes F, H, and A (74, 70, and 53 ng). Lane 8 contains the positive control provided in the kit (84 ng). Lane 10 contains the negative control.

Cockerill FR III. Arch Pathol Lab Med. 2003;127:1112 (www)

HPV DETECTION BY PCR and SB HYBRIDIZATION TECHNIQUE

Southern blot hybridization of PCR products, amplified by SPF1/2 (top row), GP51/61 (middle row), and My11/09 (bottom row). PCR products from 10-fold dilutions of plasmids, containing genomic sequences of HPV genotypes 16, 35, and 45 were analyzed. Lanes 1 to 5 represent PCR products starting with 100 fg, 10 fg, 1 fg, 100 ag, and 10 ag of HPV DNA, respectively.

GP5/6 and GP5 +/6 + PCR on 1 ng of DNA of 22 cloned HPVs diluted in 100 ng of human placental DNA. PCR products areshown after gel electrophoresis (upper panels) or after Southern blotting and hybridization with the HPV cocktail probe under low stringency conditions (lower panels). The level of the 150 bp PCR products is indicated at the right.

A combination of the general primers GP5 and GP6,originally selected from the HPV L1 region on the basisof sequence information from HPV-6, - 11, - 16, - 18, -31and -33, was found to amplify target DNA of at least 27 mucosotropic HPV genotypes under conditions that allow mismatch acceptance

– Reverse Hybridization• To immobilize all the probes of interest on a nitrocellulose

strip and then apply the amplicon to the strip and determine which probe hybridized with the amplicon

• Opposite to how a southern blot is performed• The assay has the advantage of using a chromogenic

reaction , rather than radioactivity to detect hybridization• Easier to perform than DNA sequencing, to asses only the

sequence for which there are probes and does not afford the opportunity to evaluate new mutations

• Clinical applications– Line probe assays (LiPA, Innogenetics), Reverse Blot strips (Roche),

INNO-LiPA (Bayer)– To differentiate the various genetic subtypes HCV, HPV– To detect mutations associated with organisms resistence to antiviral

agents or antibiotic HIV, M. tbc

– DNA Sequencing

POST AMPLIFICATION ANALYSIS

HPV DETECTION BY HIBRIDIZATION TECHNIQUE

FIG. 1. HPV genotyping of PCR product by reverse line blot method. Schematic of the reverse line blot genotyping assay from L1 consensus primer-generated PCRproducts. The drawing represents the detection of a hypothetical mixed infection of HPV 16, 31, and 11.

Probe layout of the HPV genotyping strip. (a) HPV genotyping strips (n 5 28) hybridized with the HPV L1 consensus PCR product generated from the HPV targets indicated to the right. Fifty microliters of PCR product generated from amplification of 106 HPV plasmid targets (with the exception of HPV 51 and 68,which were amplified with 103 plasmid targets) in a background of human cellular DNA (12.5 ng/PCR) was hybridized to the HPV genotyping strips and detected by the previously described reverse line blot method. (b) Line blot genotyping hybridization results for 10 clinical specimens in the previously described study. Fifty microliters of denatured PCR product was hybridized to each strip. The genotyping results for the specimens are as follows: no. 333, HPV negative; no. 334, HPV negative; no. 352, HPV 16, 26, and MM8; no. 353, HPV 16; no. 354, HPV 16, 51, and 66; no. 355, HPV negative; no. 357, HPV 39; no. 359, HPV MM7; no. 361, HPV 16 and 52; and no. 373, HPV 18, 56, and 58.

1=HPV 162=HPV 183=HPV 264=HPV 315=HPV 336=HPV 357=HPV 398=HPV 459=HPV 5110=HPV 5211=HPV 5512=HPV 5613=HPV 5814=HPV 5915=HPV 6816=MM417=MM718=MM919=High B Globin20=Low B Globin21=HPV 622=HPV 1123=HPV 4024=HPV 4225=HPV 5326=HPV 5427=HPV 5728=HPV 6629=MM8

HPV 16HPV 18HPV 26HPV 31HPV 33HPV 35HPV 45HPV 51HPV 52HPV 55HPV 56HPV 58HPV 59HPV 68MM4MM7MM9HPV 6HPV 11HPV 40HPV 42HPV 53HPV 54HPV 57HPV 66MM8K562No DNA

1=H

PV

16

2=H

PV

18

3=H

PV

26

4=H

PV

31

5=H

PV

33

6=H

PV

35

7=H

PV

39

8=H

PV

45

9=H

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10=H

PV

52

11=H

PV

55

12=H

PV

56

13=H

PV

58

14=H

PV

59

15=H

PV

68

16=M

M4

17=M

M7

18=M

M9

19=H

igh

B G

lobi

n20

=Low

B G

lobi

n

Hig

h B

Glo

bin

Low

B G

lobi

nH

PV

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– DNA Sequencing• Traditional DNA sequencing

– Use traditional Sanger sequencing or sequencing by termination– Radioactive reporter molecules fluorescently labeled reporter

molecules, automated– Clinical Applications

» HCV, HIV genotyping, to determine the presence of acquired mutations in the genome of HIV resistance to antiretroviral agent, resistance associated mutation in CMV

» Identify bacteria : Mycobacteria, Nocardia and fungi rDNA genes as the genetic targets

• Sequencing by synthesis (Pyrosequencing)– Sequencing by synthesis sequencing based on nucleotide

incorporation into the newly synthetized strand of DNA– The pyrophospate is converted into light by the enzymatic reaction

the light generated is recorded by the instrument and the sequence is determined

– The limitations are its ability to generate only relatively short sequence (~ 30bp)

POST AMPLIFICATION ANALYSIS

– DNA Sequencing• Sequencing by synthesis (Pyrosequencing)

– Clinical Applications» The identification and typing of bacteria : H. Pylori, Listeria

monocytogenes» To identify mycobacteria, Nocardia and fungi the 16S rDNA

gene» To detect resistance associated genetic mutations

• Microarray analysis– A wide variety of microarrays gene chips hybridization sites

signal generated may be fluorescent or electrical– Data generated require computers and advanced software packages

for data analysis– The advantage of microarrays is its ability to examine thousands of

hybridization reactions simultaneously.– To detect a multitude of signals simultaneously and genetic (DNA

differences or differences in expression (mRNA)– Products of broad range PCR or RT-PCR are applied to microarrays

POST AMPLIFICATION ANALYSIS

– DNA Sequencing• Microarray analysis

– Clinical Applications» Microarrays are very useful for research and discovery have

not yet been introduced into routine use in the clinical microbiology laboratory for diagnosis or assessment of patient with infections

» To identify bacteria, mycobacteria, fungi and viruses» To detect the genetic determinants of resistance» To study host response to infection» To discover new drugs that may be useful to cure infections» To study HIV viral gene expression and the changes into the

host cell expression profile after infection

POST AMPLIFICATION ANALYSIS

DNA SEQUENCING

Automasi Susunan DNA

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

DNA SEQUENCING

Pyro Sequencing

Adapted from Bryant et al., 2004, Lancet infectious disease

cDNA Microarray analysis

Clinical Cancer Research 11:2625-2636, 2005

REAL TIME NUCLEIC ACID AMPLIFICATION

• Evidence of amplification occurs during the PCR reaction in “real time”

• More rapid than traditional methods of amplicon detection, significant reduction of in the chance of amplicon contamination

• Methods of Detecting the Products of Real Amplification :• SYBR Green, a dye that binds to the minor groove of

double stranded DNA generate fluoresecence when bound to DNA, nonspecific detection

REAL TIME NUCLEIC ACID AMPLIFICATION

• Hybridization Probes– Taqman or Hydrolysis Probes, hydrolisis oligonucleotide

probe is labelled with both a fluorophore and a quencher molecule.

– Fluorescence Resonance Enargy Transfer (FRET) probes– Molecules Beacon, like Taqman

• Clinical Application• To detect and differentiate microorganisms ~

traditional PCR• To rapidly detect newly recognized and emerging

viral pathogens (SARS)• The quantitative assays to predict disease and

monitor the viral load in response to therapy

Real-time PCR Principles

* based on the detection and quantitation of a fluorescent reporter

* the first significant increase in the amount of PCR product (CT - threshold cycle) correlates to the initial amount of target template

(www)

STRAIN TYPING– Non amplification Based Typing

• Pulsed Field Gel Electrophoresis (PFGE)– The restriction endonucleases cut the chromosomal DNA into a

variety of pieces, depending on the number of restriction sites present in that particular strain

– The fragments of chromosomal DNA are separated using specialized type of electrophoresis profile

– Amplification Based Typing• PCR Restriction Fragment Length Polymorphism (PCR-

RFLP) product PCR enzymes restriction profile• Repetitive PCR (repPCR) repetitive DNA elements in

prokaryotic genomes form the basis for repPCR the hybridization sites for the primers used in the repPCR

– Clinical Applications• To investigate outbreaks in hospital, in cities and across the

nation

Pulsed Field Gel Electrophpresis

PFGE Results

Restriction Fragment Length Polymorphism (RFLP)

RFLP

RFLP Analysis

RFLP results

RFLP results

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