Labdiagnosisofbacterialinfections 130519094357-phpapp01
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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
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