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 automation Dr.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. tissue Dr.Sarookhani

NA CHIP TECHNOLOGY Dr.Sarookhani

MICRO ARRAY TECHNOLOGY Dr.Sarookhani

Application of microarray for pathogen detection Dr.Sarookhani

DNA SEQUENCING Dr.Sarookhani

(NAAT) ‏‏‏‏‏ ‏‏Nucleic Acid Amplification ‏ ‏‏ ‏Technologies (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

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

MOLECULAR TECHNIQUES APPLICATIONS OF MOLECULAR TECHNIQUES 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) Dr.Sarookhani

MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA (2) Dr.Sarookhani

MOLECULAR METHODS FOR IDENTIFICATION OF BACTERIA (3) 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 identityDetection of identity of strains genotypesdetection of genotypes unusualdetection of emergence & spread of strains of an organism with unusual resistance patterns or pathogenicity efficiencydetermining the efficiency of infection control procedures sourceidentification of source in outbreaks Dr.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-PCR Dr.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 for Influenza Test Method Time to Results Comments Serology >2 wks Retrospective, requires paired sera Culture 1-10 days Still gold standard(?), requires expertise, provides virus for studies Molecular(RT-PCR) 2-4 hrs Becoming gold standard(?), requires expertise & expensive equipment Antigen Detection (IF) 2-4 hrs Requires reading expertise & IF microscope Antigen Detection (Rapid EIA-like) min Widely available, requires little expertise Dr.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

MOLECULAR DETECTION OF PARASITES &PROTOZOA Dr.Sarookhani

Nested PCR for Leishmania diagnosis Dr.Sarookhani

PCR-BASED DETECTION OF TOXOPLASMA GONDII Dr.Sarookhani

MOLECULAR DETECTION OF FUNGI T.verrocosum :HSP 70 :PCR Candida sp. :PCR Cryptococcus neoformans:nested PCR(CSF) Histoplasma capsulatum: probe Coccidioides immitis :probe Blastomyces dermatidis :probe Acanthamoeba :PCR Dr.Sarookhani

Detection of Cryptococcus neoformans by nested PCR Dr.Sarookhani

MOLECULAR METHODS FOR CO-IDENTIFICATION OF MULTIPLE AGENTS Dr.Sarookhani

Clinical manifestation(s) and/or specimen Pathogens targeted Infectious agent Genital ulcer disease HSV, H. ducreyi, and T. pallidum Combination (All agents) Genital swabs HPVs, HSV, and C. trachomatis Keratoconjunctivitis Adenovirus, HSV, and C. trachomatis Acute respiratory tract infection EV, influenza viruses A and B, RSV, PIV types 1 and 3, adenovirus, M. pneumoniae, and C. pneumoniae Dr.Sarookhani

Application of multiplex PCR for diagnosis of viral infections (viruses in CNS) Viruses and/or other agent(s) targeted Specimen(s) Clinical manifestation(s) HSV-1, HSV-2, and CMV HSV and VZV; EBV and HHV-6 HSV-1, HSV-2, VZV, CMV, HHV-6, and EBV CSF Meningitis, encephalitis, and/or meningoencephalitis HSV-1, HSV-2, VZV, CMV, HHV-6, EBV, and Ent.V six herpesviruses that may infect the CNS that may infect the CNS CMV, EBV, HHV-6, HHV-7, and 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 specimens Dr.Sarookhani