Applications of PCR in Diagnostic Medicine

Done by Mohamed Khalid 09B212 Rajathilagam 09B213 Rajeeva Lochan 09B214 Sasi Devi 09B215 Sridhar 09B219

Outline

Introduction Need for new diagnostic methods Terms frequently used in diagnosis Molecular Diagnosis (PCR-based) Advantages and pitfalls of PCR as a diagnostic tool

Need for new diagnostic methods

Microscopy gives false positive results - T.vaginalis, N.gonorrhoeae Intracellular pathogens viruses, M.genitalium Low sensitivity Chlamydia sp.,Neisseria sp. Seropositivity is common Chlamydia sp. Subtyping is mandatory HSV, HPV, HCV Microbial growth is slow M. Tuberculosis False negative results in patients receiving antimicrobials Bacterial meningitis

Terms frequently used in diagnosis

Diagnostic sensitivity is defined by the percentage of persons who have the disorder of interest who have positive results on the assay. Diagnostic specificity is defined by the percentage of persons who do not have the condition of interest who have negative results on the assay. Note:- Do not confuse diagnostic & analytical parameters

The analytical sensitivity of an assay represents the smallest amount of a substance that can be accurately measured in a biological sample; analytical specificity is the assay's ability to measure a particular organism or substance, rather than another, in a sample. These characteristics are distinct from diagnostic sensitivity and specificity. In the clinical setting, diagnostic sensitivity is defined by the percentage of persons who have the disorder of interest who have positive results on the assay. Although one might expect that an analytically sensitive assay should more readily identify those persons, the ability to measure a very small quantity of a substance does not always translate into high diagnostic sensitivity. This apparent contradiction results from the shortcomings of sampling a very small volume, variations in the clinical spectrum of disease, and possible difficulties with specimen preparation and technical performance of the assay. Diagnostic specificity is defined by the percentage of persons who do not have the condition of interest who have negative results on the assay. False-positive reactions diminish the diagnostic specificity; these reactions may be particularly likely to occur in molecular assays as a result of contamination with amplified material from other reactions (carryover).

Detection Of Pathogens

Molecular Diagnosis
Polymerase chain reaction (PCR) is the best-known and most successfully implemented diagnostic molecular technology to date. It can detect slowgrowing, difficult-to-cultivate, or uncultivatable microorganisms and can be used in situations in which clinical microbiology diagnostic procedures are inadequate, time-consuming, difficult, expensive, or hazardous to laboratory staff.

 PCR diagnosis can be used to detect a specific pathogen or for identification of classes of pathogens. To detect a specific pathogen, say Chlamydia trachomatis, primers specific for C.trachomatis are used and checked for amplification To identify a broad range of pathogens, say all bacteria, primers specific for 16S rRNA gene (evolutionarily conserved in all bacterial species) are used and checked for amplifiaction

Viability assesment using PCR

Nucleic acid detection assays is not a reliable indicator for assesing pathogen viability. If pathogen viability is to be assessed , PCR analysis at the RNA level provides information about transcription activity of the organism, which is a marker of viability. Limitation:- can't be used for RNA viruses

Biomarkers for diseases

Biomarkers or gene signatures are genes which are specifically induced during disease and can identify a disease process with high confidence. For example , cytokines or chemokines and their transcription factors and receptors are selectively induced and can be used to detect an infectious disease process without the presence of pathogen itself. Additionally , pathogen specific genes encoding for antimicrobial resistance or toxins can be used as diagnostic biomarkers

Single pathogen detection vs. Panel strategy

Selecting a single PCR assay greatly relies on the ability of the clinicians to indicate the suspected organism , is a hit-or-miss approach and carries a risk of misdiagnosing the patient Thus instead of selecting a single suspect pathogen the clinician selects a syndrome-specific panel of PCR assays that broadly covers all the potential pathogens , greatly increasing it's diagnostic power (multiplex pcr)

Examples of PCR assays for diagnosis of infectious diseases

Microbial pathogen
Anaplasma phagocytophila

Tissue submission
Whole blood

Target gene
16S rRNA Phospholipase D exotoxin Glycoprotein B 18S rRNA Envelope gene 16S rRNA 18S rRNA Haemagglutinin

Corynebacterium tuberculosisAspirate from abcess Equine herpes virus 1 Sarcosystis neurona West nile virus Lawsonia intracellularis Neospora hughesi Equine influenza virus NPS , whole blood , TTW , BAL , CSF CSF Whole blood , CSF Feces CSF NPS , TTW , BAL

Limitations of PCR assay diagnosis

FALSE POSITIVES :Arises due to background contamination due to exogenous sources of DNA , mostly carry over DNA. Contamination is more pronounced in those assays which use universal primers those targetting 16S rRNA gene. None of the methods(UV treatment , enzyme digestion ,chemical treatment) has been shown to be entirely effective without significant diminution of assay sensitivity Self contained micro-chip platforms hold promise for best means of decontamination and overall assay efficiency.

False negatives :May be due to two reasons Relatively small sample volume permissible for PCR reactions Problems associated with PCR processing The problems can be encountered by optimising the concentration of target DNA for PCR reaction and by monitoring the presence of any PCR inhibitors and inducing various chaotropic , enzymatic and thermal methods of cell lysis to effectively liberate microbial DNA content

Limited detection space for characterising detected pathogen

While PCR product detection and analysis have typically been achieved using gel-electrophoresis and sequencing techniques, these approaches are laborious and timeconsuming, which detracts from clinical applicability. Even real-time PCR (unfortunately!) has a limited ability to spectrally differentiate multiple fluorescent signals.

Other novel approaches to analyse the amplified products

MICROARRAY TECHNOLOGY :-DNA microarrays are constructed by spatially isolating specific genome sequences to prearranged areas on a microchip. Flourescently labelled amplification products are then allowed to anneal to complementary sequences on the chip, and the resultant pattern is spectrally analysed. The main advantage of using microarrays for pathogen detection is the potentially large number of target sequences the system can discriminate simultaneously. The use of microarray technology for pathogen detection is still in the development phase however.

 MASS SPECTROMETRY :In MALDI-TOF- MS, the organic moleculefor example an amplified productis ionised and subsequently identified based on its mass-to-charge ratio .The advantages of MALDI-TOF-MS lie in the inherent accuracy and the high-speed (one second) of signal acquisition, making this technology an attractive candidate for high-throughput DNA analysis.

References
Samuel Yang and Richard E Rothman(2004)PCR
based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings.THE LANCET, Infectious Diseases,Vol-4,June 2004 Pusterla N, Magigan JE, Leutenegger CM(2006)Real time polymerase chain reaction:Novel molecular diagnostic tool for equine infectious diseases.J Vet Intern Med 2006;20:3-12.

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