2.  Advantages in making a specific diagnoses
better patient care
appropriate antibiotic
sparing of expenses
preventive measures can be initiated

3. Specimen collection &transport
 Most important
 Protect from contamination
 Selected media

4. Approch for identification of
infectious agents
5 different approaches


 detection of antibodies


- identify NA using dna probes

5.  Gram stain: pneumococci, gonococci,
staphylococci,meningococci
 Acid fast stain: mycobacterium
tuberculosis,leprae, nocordia, cryptosporidia

6.  Other new stains
Aura mine rhodamine for mycobacteria
Wright stains – malarial,& microfilarial
Indian ink – capsular organisms
Silver stains- fungi & pneumocystis

7.  Immune electron microscopy
 Direct detection of antigen
 Non cultivables like Rota virus, hepatitis A
&NORWALK VIRUS

8. 
 In bacteremia
acute febrile illness
typhoid,meningitis,pneumonia,osteomylitis,P
UO
 1% total blood volume
 At onset of chills

9.  Sputum culture
Early morning samples
(pooled overnight secretions likely to
contain pathogenic organisms)
Heated or ultrasonic nebulised saline induce
sputum production

10.  mid stream sample
 Reach lab by ½ hr
 Presence of more than 10000 colonies/ml
signifies true infection

11.  Gastric aspirates- TB
 Endoscopic biopsies-helicobacter pylori
 Stool samples

12.  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

13.  Mycobacterial growth indicator tube
(MGIT)960
 Art fluorescent technology
 Rapidly 7-10 days
Based on O2 quenching of mycobacteria with
fluorescent dye

14.  Uses specific Mycobacterial phages to detect
viable bacteria within 48 hrs

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

16. 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

17. 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)…….

18. Tag Ab with fluorescein isothiocynate
Ab-virus complex or viral
antigens
microscopically by UV illumination
Detects viruses which are uncultivable

19. 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

20. 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 &….

21. 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

22. Detection of microbial
antigens/products
 GLC : anaerobes
 HPLC : mycobacteria

23.  New developments
 Answer for organisms with growth
characteristics
slow – mycobacteria
difficult – viruses,chlamydia
fastidious- mycoplasma

24. Highly sensitive – detects low pathogen no’s
as in meningitis
Used to monitor response to treatment
(viral load assays for hepatitis B,C&HIV)

25. 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

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

27. Nucleic acid amplication
3 types
target amplification
signal amplification
probe amplification

28. 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

29. Target DNA acts as template
nucleotides
primers&DNA polymerase
generates copies by alternate heating &
cooling for denturation, annealing, &extension

30. 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

31. Nucleic acid amplication
-signal
amplification
 Increases the signal generated from
hybridized probe molecule
Probe amplification
End 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

32. 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