Lab diagnosis of bacterial infections


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Lab diagnosis of bacterial infections

  1. 1.  Advantages in making a specific diagnosesbetter patient careappropriate antibioticsparing of expensespreventive measures can be initiated
  2. 2. Specimen collection &transport Most important Protect from contamination Selected media
  3. 3. Approch for identification ofinfectious agents5 different approaches detection of antibodies- identify NA using dna probes
  4. 4.  Gram stain: pneumococci, gonococci,staphylococci,meningococci Acid fast stain: mycobacteriumtuberculosis,leprae, nocordia, cryptosporidia
  5. 5.  Other new stainsAura mine rhodamine for mycobacteriaWright stains – malarial,& microfilarialIndian ink – capsular organismsSilver stains- fungi & pneumocystis
  6. 6.  Immune electron microscopy Direct detection of antigen Non cultivables like Rota virus, hepatitis A&NORWALK VIRUS
  7. 7.  In bacteremiaacute febrile illnesstyphoid,meningitis,pneumonia,osteomylitis,PUO 1% total blood volume At onset of chills
  8. 8.  Sputum cultureEarly morning samples(pooled overnight secretions likely tocontain pathogenic organisms)Heated or ultrasonic nebulised saline inducesputum production
  9. 9.  mid stream sample Reach lab by ½ hr Presence of more than 10000 colonies/mlsignifies true infection
  10. 10.  Gastric aspirates- TB Endoscopic biopsies-helicobacter pylori Stool samples
  11. 11.  BACTEC TB-460 : rapid,specific,&rapidculture method Both respiratory & non respiratory specimens 13-14 days 200 viable bacilli 14-17 days 20 viable bacilli
  12. 12.  Mycobacterial growth indicator tube(MGIT)960 Art fluorescent technology Rapidly 7-10 daysBased on O2 quenching of mycobacteria withfluorescent dye
  13. 13.  Uses specific Mycobacterial phages to detectviable bacteria within 48 hrs
  14. 14.  Serology helps in detecting either thespecific or non specific immune responsesof the host or the presence of the antigen inthe host
  15. 15. Specific Ig G &Ig M can be detected usingimmunologic techniques against virusesVarious types includeprecipitationAgglutination widal testComplement fixationimmunofluorescenceElisaWestern blot
  16. 16. Specific,sensitive,simple,inexpensible,& reproducibleUsed extensively to detect either Ag or AbAlso detects small quantities of AgUsed to diagnose TORCH,HIV,MEASLES,HEPATITIS(A,B,C,D,E)…….
  17. 17. Tag Ab with fluorescein isothiocynateAb-virus complex or viralantigensmicroscopically by UV illuminationDetects viruses which are uncultivable
  18. 18. Ω Assays are available for rapid detection ofbacterial Ags in various body fluidsΩ Useful when prior antibiotic therapy hasbeen initiated and cultures are negative after24 hrs of incubation
  19. 19. Detection of microbialantigens/products The following fluid/samples can be assayed CSF: Latex agglutination & counter-currentimmuno electrophoresis are used todemonstrate the soluble polysaccharide Agof cryptococcus &….
  20. 20. Detection of microbialantigens/products Serum- Pl.falciparum/vivax are detected atlevels of 100-200 parasite/űl Urine-strep.pneumonia legionella, Stool :helicobacter pylori,clostridiumdifficile,giardia
  21. 21. Detection of microbialantigens/products GLC : anaerobes HPLC : mycobacteria
  22. 22.  New developments Answer for organisms with growthcharacteristicsslow – mycobacteriadifficult – viruses,chlamydiafastidious- mycoplasma
  23. 23. Highly sensitive – detects low pathogen no’sas in meningitisUsed to monitor response to treatment(viral load assays for hepatitis B,C&HIV)
  24. 24. Nucleic acid probes :NA hybridization is powerful & widely usedtechniqueUsed to detect and locate specificDNA&RNA sequence in tissues orchromosomes by making use ofradioactive/fluorescent labelled DNA/RNAprobes complementary to the requiredsequence
  25. 25. Commercially available for the available foridentification of M.tuberculosiscomplex,avium,intracellulaire,kansasi,gordonae
  26. 26. Nucleic acid amplication3 typestarget amplificationsignal amplificationprobe amplification
  27. 27. Nucleic acid amplication- Target amplificationPCR : An in vitro method for amplifyingspecific DNA sequenceextremely minute amounts of NAgenerates billions of exact copiesmaking genetic analysis a simpleprocess
  28. 28. Target DNA acts as templatenucleotidesprimers&DNA polymerasegenerates copies by alternate heating &cooling for denturation, annealing, &extension
  29. 29. Nucleic acid amplication- Target amplificationReal time PCR : rapida fluorescent signal is used forreal time monitoring of amplificationTranscription mediated amplification Uses ribosomal rna as the target for reversetranscriptase
  30. 30. Nucleic acid amplication-signalamplification Increases the signal generated fromhybridized probe moleculeProbe amplificationEnd product is an amplified version of originalproduct includes LCR, cycling probetechnologyIn LCR phenotypic change in the organism suchas virulence or drug resistance
  31. 31. Disadvantages of molecularmethodsAmplification can amplify even minutequantities of contaminating DNA – false (+)Do not differentiate dead from livingorganismsFalse(-) results may due to low copy no’s ofmicroorganisims at site of infection