Ultra Lecture Tissue and Body Fluids.pptx

Tissue and Body Fluid Collection

1. Lecture 3 Tissue & Body Fluids “Be the super man & look for hidden foci & Investigate for the source culprit”

2. Under IAP President’s Action Plan 2023 D Dr Upendra Kinjawadekar – President IAP 2023 Dr Vineet Saxena – Hon Secretary General Dr Bhaskar Shenoy – National Convenor Dr Maninder S Dhaliwal – Joint National Convenor Dr Vasant M Khalatkar – National Co- ordinator

3. Preanalytical 60-70% Postanalytical 20-25% Analytical <10%

4. The quality of clinical specimen can impact the value of the tests & results C hoice of the specimen depends on the site of infection & likely pathogens. Good Collection of the specimen MUST Prior to initiation of antimicrobial therapy Use sterile Technique to avoid contamination with colonizer Take specimen deep & as close to the site of infection. Provide an adequate volume of material Use separate & appropriate containers Label and seal Correctly, carefully with relevant clinical information T ransport time-best minimum, within reco . deadline, use of transport media

5. Selection Decision- Second/Third line Collection, Storage, Transport- Specific needs Handling, Processing, TAT, Cost, Equipment, Technology Diagnostic Value - Principles, Accuracy, limitations Interpretation- decisive PRECIOUS Invasive & One time Expensive

6. Specimen Body Fluids- Pleural Peritoneal Pericardial Articular Tissue- Aspirate/Biopsy LN Bone Marrow Pleura/peritoneum pericardium/synovium Liver, bone, Bowel, Kidney, skin, Lung, brain/nerve Tests Tests: Gross Exam Cell study Biochemistry Microbiology- Stains- Gram,ZN , Fungal,special Cultures-Ae/an, myco , fungal Serology Molecular Histopathology Histochemistry Immunohistochemistry

7. Rapid, inexpensive, simple & Direct microbiological test for visualization, with basic identification of bacteria & some yeasts. Used to differentiate between different types of bacteria based on the biochemical properties of their cell walls, stain colors & morphology . Useful for guiding empiric clinical management for bacterial infections pending definitive culture data and/or molecular data. Need further tests for speciation & culture for definition.

8. Gram Positive bacteria Cell wall contains more & thick peptidoglycans Shrink on dehydration & seal pores to block stain movement across Resist decolorization to Retain primary stain CV+I complex inside Appear purple/blue due to primary stain Gram Negative Bacteria Cell wall contains less & small peptidoglycans but more lipids. Pore size increases to allow free stain movement across Decolorization removes primary stain CV+I complex from inside Appear pink/magenta-due to secondary stain- safranin

9. Gram- positive bacteria retain crystal violet and appear purple Gram- negative bacteria do not retain crystal violet but take up safranin counterstain and thus appear pink/magenta. Gram variable bacteria- may stain either negative or positive Non visualized bacteria - as either lack of cell wall (Mycoplasma sp.) or cell wall does not retain Gm stain reagents (Chlamydia, Mycobacterium Sp.) Some nonbacterial species stain purple with Gram stain (Candida sp.)

10. Shape - spherical "cocci,“ rod-shaped "bacilli," "coccobacilli,“ “curved”, “branched “, “ filamentous”, “pleomorphic” Size/Length - Micro/Macro, short, medium, Long, plum, thin Number - semiquantitative - -, +, ++, +++ or absent, occasional, small, moderate, large Arrangement Pattern- "chains" or "clusters“

11. Sterile Site Specimen - CSF, pleural, pericardial, synovial, peritoneal fluid Any bacteria in sterile site specimen is considered significant A negative Gram stain does not exclude infection Prior cytospin centrifugation may increase sensitivity ( esp.CSF ) Non sterile site specimen- sputum, throat, wound, and genital swabs High-quality- high no. of white cells, few epithelial cells; Low-quality- high no. of epithelial cells, few white cells

12. 2 yrs girl, chickenpox : 8 days back, One lesion on thigh worsened in last 2days, Febrile, Toxic , Swelling- 7cms, tender, red-dark brown, hot with crusty discharge at edge.. Gram stain pic. Shown Next Action? A- Send CBC,CRP start oral Co- Amoxy clav (HD) B- Send pus culture & start ceftriaxone + klox C- Send pus & blood culture, Start Linezolid + Mero + Metro, consider IVIG D- Send blood + pus culture, start IV penicillin

13. A- Send CBC,CRP start oral Co- Amoxy clav (HD) needs culture & sensitivity, parenteral Ab, no use of BL-BLI in GAS B- Send pus culture & start ceftriaxone + klox needs blood culture also, is it staph – no – therefore why clox C- Send pus & blood culture, Start Linezolid + Mero + Metro, consider IVIG – not in ICU/ inotropes No need of upfront Mero D- Send blood + pus culture, start IV penicillin Most GAS strains are universally susceptible to Penicillin

14. 12yr old boy , fever for 4 days, wet cough 2days, scanty blood stained sputum today , Febrile, RR 28/min, Reduced BS Right Inframammary area: fine crepts , BB+ Fair oral intake, stable vitals, Sputum gram stain shown Next Action ? A- No invest, PO Amoxycillin+ Para + ORS, close FU B- CBC, CRP, CXR, PO Azi + Cefpodoxime + Neb C- Admit, CBC,CRP, TT, CXR, Sputum Culture, AFB IV Ceftriaxone + Amikacin + Fluvir D- Investigate, PO Linezolid + Azithromycin + Fluvir

15. A- No Investigation, Oral Amoxycillin + Para + ORS, close FU DOC for Non Severe CAP B- Investigate, Oral Azithromycin + Cefpodoxime + Neb Inappropriate Ab & neb C- Admit, Investigate, IV Ceftriaxone + Amikacin + Fluvir OPD manageable, No role of Amikacin D- Investigate, Oral Linezolid + Azithromycin + Fluvir No upfront empiric Linezolid/azithromycin

16. A 4yrs boy of SDNS has recurrent pustular skin lesions last treated with cefpodoxime + mupirocin 2 weeks ago. Now has a 5.5cms diameter, minimally discharging abscess on abdomen without fever. Gram Stain pic. Shown Next Action? A- Oral Cefadroxil + serratiopeptidase + Fusidic acid B- Admit, Investigate, IV Ceftriaxone + klox + source control C- Admit investigate, IV Vancomycin + Klox + source control D- Admit, Investigate, IV Ceftriaxone + clindamycin

17. A- Oral Cefadroxil + serratiopeptidase + Fusidic acid No Oral Ab, No MRSA cover with First GC B- Admit, Investigate, IV Ceftriaxone + klox No MRSA cover with 3GC/ or klox C- Admit investigate, IV Vancomycin + Klox DOC upfront Monotherapy for MRSA SSTI But we don’t have culture, so klox will be added now and after culture – will narrow antibiotics D- Admit, Investigate, IV Ceftriaxone + clindamycin No MRSA cover with 3GC, Clindamycin resistance high, only after D test

18. A 1.5 yrs old girl with perforated appendicitis with shock underwent emergency Laparotomy. Started on ceftriaxone + Amikacin + Metronidazole. Peritoneal fluid gram stain shown in pic. Next action? A - Piptaz + ciprofloxacin B- Colistin +Amikacin C- Ceftazidime + Avibactam + Aztreonam D - Meropenem + Vanco & consider Xpert Carba R (if indicated)

19. Simple, Rapid & inexpensive diagnostic test for tuberculosis Also used to monitor the treatment of Tuberculosis in Smear + ve cases. Special stain for MTB detection in specimens - sputum, body fluids & tissue. Not useful in choice of ATT as no information of drug resistance. Acid-fastness is related to lipid Mycolic acid in bacterial outer capsule. Take up the dye fuchsin, not decolorized by acid alcohol-acid-fastness. Also detects NTM, M.Leprae , Nocardia, cryptosporidia

20. Mycobacterial Load/Conc. In the test specimen determines the sensitivity of ZN smear results LOD- 500-1000 bacilli/ml Sensitivity 5-60% Low in children & EPTB High specificity >95%

21. A 12 yr girl has PUO since 6weeks, No H/O TB contact Small pleural effusion Rt. side & Rt. Paratracheal globular shadow on Xray chest. Pleural fluid- straw color, 80 lymphocytes , P 2.5g (normal <2g/ dL ), S 80, ADA 30 AFB smear negative, GeneXpert Negative. What next ? A- Start 2HRZE & prednisolone B- Give levofloxacin for 10days & review C- Do Gastric Lavage/Induced Sputum 2 days for AFB, CBNAAT, MGIT, & consider needle pleural biopsy D- CECT chest & Pulmonology consult for Bronchoscopy BAL

22. A- Start 2HRZE & prednisolone No MTB/ DRTB evidence B- give levofloxacin for 10days & review No quinolones empirically when MTB is a possibility C- Gastric Lavage/Induced Sputum 2 days for AFB, CBNAAT, MGIT ~30% cases have Sputum MTB detected by all 3 Pleural fluid AFB yield is poor, pleural biopsy has high diagnostic yield D- CECT chest & Pulmonology consult for Bronchoscopy BAL No additional benefit over TB suggestive CXR, BAL/TBNA- Next resort

23. Two Induced Sputum specimen negative AFB & CBNAAT CECT - C aseating Rt. Paratracheal & subcarinal LN+, Rt.Pleural thickening Bronchoscopy BAL & EBUS TBNA: done BAL scanty AFB +, TBNA – nonspecific , GenXpert Ultra- ve What Next? A- Start HRZE B- Wait for MGIT reports

24. A- Start HRZE No conclusive evidence of MTB, non caseating LN, TBNA- no MTB on ZN & CBNAAT AFB scanty + ve can be NTM contaminating bronchoscope No information on DSTB/DRTB B- Wait for MGIT reports Prudent option with close clinical monitoring for conclusive MTB/NTM & Susceptibility /AMR evidence

25. The rapid, simple, inexpensive method to identify fungi & yeasts from skin/mucosal scrapings, hair roots, nail, urine, body fluids, tissues. KOH is a strong alkali, dissolves epidermal keratinocytes & cement substance to separates the fungal elements from intact cells to enable visualization & identification of the hyphae and conidia (spores) of fungi. Indicated to identify dermatophyte (tinea pedis, manus, corporis, cruris, capitis, onychomycosis) & yeast infections (pityriasis versicolor, Candida). May yield false-negative results in 12 to 24% of cases , highly corrosive chemical, handled with great care

26. Clean the skin of any lotions/creams with an alcohol preparation Obtain a specimen- 1 S kin - Number 15 blade/side of a glass slide to scrape material loose 2 P ustules/vesicles- scrape the roof onto a glass slide. 3 O ral candidiasis- a tongue blade to scrape white plaques onto a slide. 4 N ai l - remove maximum nail with clippers to expose the most proximal area, scrape subungual debris on slide with 1/2 mm curette/no.15 blade. 5 H air - pluck 5-10 hairs from active scaling area with a hemostat / rub 2x2 gauze vigorously on alopecia/scaling, place broken-off hairs on a slide .

27. A 12 yrs IDDM boy had Rt. maxillary pain , tender swelling, foul smelling discharge since 6 weeks, not responding to antibiotics. PNS xray showed opacification & erosion of Rt. maxillary sinus. Discharge smear had pus cells & wet KOH ppn . shows filamentous fungi without septae & wide angle branching. Next Step? A- Blood Galactomannan, Oral Fluconazole B- BDG, CT scan PNS & Oral Voriconazole C- CT scan PNS, RFT, IV amphotericin B

28. A- Galactomannan assay, Oral Fluconazole Galactomannan only for aspergillus, Fluconazole not effective B- BDG, CT scan PNS & Oral Voriconazole BDG- Cost, nonspecific, Voriconazole not effective for mucor C- FESS, culture, CT scan PNS, RFT, IV amphotericin B Source control, extension evaluation, DOC for invasive mucor

29. Specimen quality & quantity is paramount for microbiology culture. The accurate identification of the infectious microorganisms from surgical specimens is vital to ensure the correct antimicrobial therapy. Proper collection, prompt delivery to the laboratory are critical factors Tissue & aspirates are preferred over swab specimen Sensitivity can be improved by inoculating the fluid into the liquid media of a blood culture bottle and adding appropriate supplementation.

30. SWABS Tissue & Aspirate fluids Location of infection Superficial lesions, tissue or fluid is preferred Deep or invasive infections Care setting Discouraged from surgeries; acceptable in clinic if aspirate /tissue difficult Specimen of choice, esp. from invasive procedures, surgeries Risk of contamination High, contamination with N flora, colonizers Lower risk of contamination Volume 50–100 micro L released is inadequate for uniform inoculation of all media; fibers trap microorganisms & contents are difficult to release 3–4 mm3 of tissue- sufficient to adequately inoculate all required multiple culture media Sensitivity 70% 93% Specificity 89% 98% PPV 68% 93% NPV 90% 98%

31. Must be interpreted in context of patient’s clinical condition & accuracy of the test. Additional interpretation & tests are often required R elatively few microorganisms are unequivocal pathogens Factors to consider are: • Whether the microorganism is isolated in abundance? • Isolated in pure culture? • Isolated from deep/superficial site , normally sterile/unsterile? • Isolated on more than one occasion ? • Is it a microorganism that is a normal commensal /colonizer? • Is there clinical or laboratory evidence of local inflammation? • Does the presence of the microorganism fits with the clinical picture ?

32. Case

33. Two questions……. Is sending a throat swab culture & starting antibiotics justified ? Comment on antibiotic sensitivity report of the isolate.

34. Gold standard (rule out test) for diagnosing strep pharyngitis Negative throat culture result: very high NPV; false-negative rates < 10%. Positive result does not distinguish acute Strep. pharyngitis from asymptomatic carriage. Send only for clinically suspected strep pharyngitis. 18-48 hours TAT : not really a drawback As delay in antibiotic therapy for up to 9 days, does not decrease the ability to prevent rheumatic fever. Throat culture – use and interpretation

35. Case

36. Wound swab – Culture sensitivity report What kind of MRSA is this? What are your antibiotic(s) choices?