LAB DIAGNOSIS OF BACTERIAL

INFECTIONS

Advantages in making a specific diagnoses

better patient care

appropriate antibiotic

sparing of expenses

preventive measures can be initiated

Specimen collection &transport

Most important Protect from contamination Selected media

Approch for identification of infectious agents

5 different approaches Microscopy: Culture Serology detection of antibodies Detection of microbial antigens & products Molecular technology

- identify NA using dna probes

Microscopydirect

Gram stain: pneumococci, gonococci, staphylococci,meningococci

Acid fast stain: mycobacterium tuberculosis,leprae, nocordia, cryptosporidia

Microscopydirect

Other new stains

Aura mine rhodamine for mycobacteria

Wright stains – malarial,& microfilarial

Indian ink – capsular organisms

Silver stains- fungi & pneumocystis

Microscopydirect

Immune electron microscopy Direct detection of antigen Non cultivables like Rota virus, hepatitis A

&NORWALK VIRUS

CULTURE BLOOD CULTURE In bacteremia

acute febrile illness typhoid,meningitis,pneumonia,osteomylitis,PUO

1% total blood volume At onset of chills

CULTURE

Sputum culture

Early morning samples

(pooled overnight secretions likely to contain pathogenic organisms)

Heated or ultrasonic nebulised saline induce sputum production

Urine culture

mid stream sample Reach lab by ½ hr Presence of more than 10000 colonies/ml

signifies true infection

Gastro –intestinal culture

Gastric aspirates- TB Endoscopic biopsies-helicobacter pylori Stool samples

Automated liquid culture methods

BACTEC TB-460 : rapid,specific,&rapid culture method

Both respiratory & non respiratory specimens 13-14 days 200 viable bacilli 14-17 days 20 viable bacilli

Automated liquid culture methods

Mycobacterial growth indicator tube (MGIT)960

Art fluorescent technology Rapidly 7-10 days

Based on O2 quenching of mycobacteria with fluorescent dye

TB-PHAGE ESSAY Uses specific Mycobacterial phages to detect

viable bacteria within 48 hrs

SEROLOGY

Serology helps in detecting either the specific or non specific immune responses of the host or the presence of the antigen in the host

SEROLOGY

Specific Ig G &Ig M can be detected using immunologic techniques against viruses

Various types include

precipitation

Agglutination widal test

Complement fixation

immunofluorescence

Elisa

Western blot

Specific,sensitive,simple,inexpensible,& reproducible

Used extensively to detect either Ag or Ab

Also detects small quantities of Ag

Used to diagnose TORCH, HIV,MEASLES,HEPATITIS(A,B,C,D,E)…….

Tag Ab with fluorescein isothiocynate

Ab-virus complex or viral antigens

microscopically by UV illumination

Detects viruses which are uncultivable

Detection of microbial antigens/products

Ω Assays are available for rapid detection of bacterial Ags in various body fluids

Ω Useful when prior antibiotic therapy has been initiated and cultures are negative after 24 hrs of incubation

Detection of microbial antigens/products

The following fluid/samples can be assayed

CSF: Latex agglutination & counter-current immuno electrophoresis are used to demonstrate the soluble polysaccharide Ag of cryptococcus &….

Detection of microbial antigens/products

Serum- Pl.falciparum/vivax are detected at levels of 100-200 parasite/űl

Urine-strep.pneumonia legionella, Stool :helicobacter pylori,clostridium

difficile,giardia

Detection of microbial antigens/products

GLC : anaerobes HPLC : mycobacteria

Molecular methods

New developments Answer for organisms with growth

characteristics

slow – mycobacteria

difficult – viruses,chlamydia

fastidious- mycoplasma

Molecular methods

Highly sensitive – detects low pathogen no’s as in meningitis

Used to monitor response to treatment

(viral load assays for hepatitis B,C&HIV)

Molecular methods

Nucleic acid probes :

NA hybridization is powerful & widely used technique

Used to detect and locate specific DNA&RNA sequence in tissues or chromosomes by making use of radioactive/fluorescent labelled DNA/RNA probes complementary to the required sequence

Commercially available for the available for identification of M.tuberculosis complex,avium,intracellulaire,kansasi,

gordonae

Nucleic acid amplication3 types

target amplification

signal amplification

probe amplification

Nucleic acid amplication - Target amplification

PCR : An in vitro method for amplifying specific DNA sequence

extremely minute amounts of NA

generates billions of exact copies

making genetic analysis a simple process

Target DNA acts as template

nucleotides

primers&DNA polymerase

generates copies by alternate heating & cooling for denturation, annealing, &extension

Nucleic acid amplication - Target amplification

Real time PCR : rapid

a fluorescent signal is used for real time monitoring of amplification

Transcription mediated amplification Uses ribosomal rna as the target for reverse

transcriptase

Nucleic acid amplication -signal amplification

Increases the signal generated from hybridized probe molecule

Probe amplificationEnd product is an amplified version of original

product includes LCR, cycling probe technology

In LCR phenotypic change in the organism such as virulence or drug resistance

Disadvantages of molecular methods

Amplification can amplify even minute quantities of contaminating DNA – false (+)

Do not differentiate dead from living organisms

False(-) results may due to low copy no’s of microorganisims at site of infection