Laboratory diagnosis of PUO


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Pyrexia of Unknown origin!

Laboratory diagnosis of PUO

  2. 2. APPROACH TO THE DIAGNOSIS OF PUO 1. Duration and pattern of fever 2. Age of patient 3. Sexual history 4. Contact with other ill people 5. Vaccination history 6. Travel history 7. Animal / insect exposure 8. Previous treatment including blood products
  3. 3. PHYSICAL EXAMINATION  Clinical features: 1. Sinus tenderness 2. Mouth ulceration 3. Chorioretinitis 4. Chest- pneumonitis 5. Abdomen- hepatic tenderness, splenomegaly 6. Lymphadenopathy 7. Arthritis 8. Rash
  5. 5. BACTERIAL INFECTIONS Specimens:  Blood: for blood cultures, peripheral blood smear, haematology, serology and other tests  Urine analysis: for Urinary Tract Infections  Sputum: in cases of lung infections  Pus: in localised abscesses
  6. 6. SPECIMENS Specimens to be collected for ZN staining:  pulmonary secretions: sputum, bronchioscopic aspirations  A series of early morning sputum specimens are to be collected over a 3 day period.  ideal amount for mycobacterium= 5-10 mL of sputum
  7. 7. OTHER SPECIMENS  fecal specimens  tissue and body fluids: pleural, pericardial and peritoneal fluids)  CSF  bone marrow aspirates  *Note: Blood and stool specimens are usually cultured from AIDS patient
  8. 8. BACTERIAL INFECTIONS  Collection  All the specimens should be collected preferably prior to antibiotic therapy.  These specimens are to be collected in a sterile containers under aseptic conditions  Blood is collected in blood culture bottles for culture and in a sterile vial for serology.  Mid stream urine specimen should be collected in a sterile universal container.
  9. 9. BACTERIAL INFECTIONS Sterile Vial containers (30 ml): Used for collecting Blood
  10. 10. BACTERIAL INFECTIONS Sterile Universal Container: Used for collecting Urine
  12. 12. BLOOD CULTURE: Procedure:  Take 5 ml of blood in each bottle of 50 ml of glucose broth + 50 ml taurocholate broth  Incubate these broths at 37ºC for 24 hours  Subcultures are made on Blood agar from (glucose broth) and MacConkey agar (from taurocholate broth)
  13. 13. Blood Agar MacConkey Agar Blood agar and MacConkey agar are incubated at 37ºC for 24 hours
  14. 14. BLOOD CULTURE  Febrile agglutinins  refers to serologic studies for salmonellosis, brucellosis, and rickettsial diseases.  These studies are useful, having low sensitivity and variable specificity.  Multiple blood samples (no fewer than three and rarely more than six, including samples for anaerobic culture) should be cultured in the laboratory for at least 2 weeks to ensure that any HACEK group organisms that may be present have time to grow
  15. 15. BLOOD CULTURE  Lysis-centrifugation blood culture techniques should be employed in cases where prior antimicrobial therapy or fungal or atypical mycobacterial infection is suspected.  Blood culture media should be supplemented with L-cysteine or pyridoxal to assist in the isolation of nutritionally variant streptococci.  It should be noted that sequential cultures positive for multiple organisms may reflect self-injection of contaminated substances.
  16. 16. URINE CULTURE  A calibrated volume of midstream urine specimen is inoculated on:  blood agar  MacConkey agar •Incubated at 37ºC for 24 hours *In renal tuberculosis, culture should be performed in Lowenstein Jensen Urine cultures, including cultures for mycobacteria, fungi, and CMV, are indicated.
  17. 17. SPUTUM CULTURE •Sputum is inoculated on blood agar and MacConkey agar plate •Incubated at 37 C for 24 hours •In case of TB, specimen is cultured in LJ medium
  18. 18. CSF COLLECTION The patient lies on his or her side, with knees pulled up toward the chest, and chin tucked downward. After the back is cleaned, local anesthetic will be injected into the lower spine. A spinal needle is inserted, usually into the lower back area at the level of L3 and L4 Once the needle is properly positioned, CSF pressure is measured and a sample is collected. The needle is removed, the area is cleaned, and a bandage is placed over the needle site. The person is often asked to lie down for a short time after the test.
  19. 19. CSF COLLECTION AND DISTRIBUTION Tube 1 Cell count Tube 2 Stat gram stain and culture Tube 3 Glucose and protein Tube 4 Cell count Tube 5 (optional) Virology, mycology and cytology cerebrospinal fluid can be tested for: •Herpes virus, with use of the polymerase chain reaction (PCR) to amplify and detect viral nucleic acid •recurrent fevers with lymphocytic meningitis (Mollaret's meningitis)
  20. 20. BACTERIAL AND VIRAL MENINGITIS viral infections that can lead to meningitis include mumps, herpesvirus (such as Epstein-Barr virus, herpes simplex viruses, and varicella-zoster virus—the cause of chickenpox and shingles), measles, and influenza.
  21. 21. PUS CULTURE  Pus is inoculated in glucose broth, blood agar and MacConkey agar.  Incubate at 37°C for 24 hours  For M. tuberculosis, pus should be cultured on LJ medium.  If suspecting anaerobic organism, culture of pus should be performed under anaerobic conditions
  22. 22. IDENTIFICATION  On the basis of:  Colony morphology  Gram Staining  Ziehl-Neelsen Staining- For M. tuberculosis  Biochemical reactions  Agglutination
  23. 23. ZIEHL-NEELSEN STAINING  The ZN stain is mostly used to identify acid-fast mycobacteria, the most important of which is Mycobacterium Tuberculosis, the organism responsible for tuberculosis (TB).  the tubercle bacilli having a lipid-rich cell wall that takes up phenol-dye solutions (eg. carbol fuchsin, the main dye used in the ZN stain) and after subsequent differentiation, retains the phenol-dye.
  24. 24. ZIEHL-NEELSEN STAINING Solutions Concentrated carbol fuchsin 20% Sulphuric acid 2% methylene blue
  25. 25. ZIEHL-NEELSEN STAINING- PROCEDURE 1. Flood the slide with concentrated carbol fuchsin and heat slide from below intermittently until steam arises (Strains should not boil or evaporate). Heating  better penetration of strain into the cell. 2. Decolorize the smear with 20% Sulphuric acid and wash with water. Repeat this step till the red/pink colour stops coming out 3. The smear is counterstained with 2% methylene blue for 1-2 minutes 4. Wash with water and air dry.
  26. 26. ZIEHL- NEELSEN STAINING Identification of Mycobacteria spp. by qualified clinical laboratories entails several of the following:  Confirmation that the isolate recovered in broth or on solid media is an acid-fast organism.  Categorize (presumptively) the isolate by phenotypic characteristics, such as colony morphology, photoreactivity, growth rate, and optimum growth temperature.  Identification through tests based on enzyme systems of the organism, metabolic by-products, and inhibition of growth by exposure to selected biochemicals.  Chromatographic detection of mycolic acid.  Identification by DNA hybridization (e.g., Gen-Probe-San Diego, Calif.)  Identification by PCR (polymerse chain reaction) tests.
  27. 27. BIOCHEMICAL TESTS The biochemical tests most often utilized are:  niacin accumulation  nitrate reduction  TCH (inhibition of growth when exposed to thiophene-2-carboxylic acid hydrazide)  growth in 5% NaCl, tellurite reduction  growth on MacConkey agar  Catalase  hydrolysis of Tween 80  iron uptake  tests for the enzymes aryl-sulfatase, urease, and pyrazinamidase.
  28. 28. SEROLOGY  Useful in:  Infectious mononucleosis- Paul-Bunnell test  Enteric fever  Hepatitis A, B infections  CMV infections  Amoebiasis
  29. 29. PARASITIC INFECTION Stained peripheral blood smears (thick and thin) will help in diagnosis of:  Malaria  Leishmaniasis  Filariasis  Toxoplasmosis  Tripanoma  Entamoeba histolytica Wet blood film may show microfilaria in filarsis
  30. 30. VIRAL INFECTION Paul- Bunnell test is useful in infectious mononucleosis CMV Epstein barr virus Hep A and B viruses Arboviruses Enteroviruses Adenovirsues Myoxviruses Human immunodeficiency virus Haemorrhagic fever viruses
  31. 31. FUNGAL CAUSES  Candida albicans  Streptococcus neoformans  Histoplasm  Aspergillous spp  Coccidioides innipil neocystis carinii
  32. 32. FUNGAL INFECTION Specimens maybe cultured on Sabouraud’s Dextrose Agar or Brain- Heart infusion agar. Fungal growth on Sabouraud’s agar Brain heart infusion agar
  33. 33. OTHER TESTS 1. Skin tests 2. Haematology 3. Immunologic tests 4. Biopsy
  34. 34. Mantoux Skin Test using a needle and syringe to inject 0.1 ml of 5 tuberculin units of liquid tuberculin between the layers of the skin (intradermally), usually on the forearm
  35. 35. MANTOUX TEST The induration (raised area) is what is measured. NOT the erythema (red area).
  36. 36. MANTOUX TEST
  37. 37. MANTOUX TEST  An induration of 5 to 15 millimeters is considered as a positive reaction for the following people:  People with HIV infection  Close contacts of people with infectious TB  People with chest x-ray findings suggestive of previous TB disease  People who inject illicit drugs and whose HIV status is unknown  If more than 15 millimeters, the patients with positive reaction with no risk factor for TB.
  40. 40. IMMUNOLOGIC TESTS LE cell phenomenon Antinuclear antibody test SLE
  41. 41. BIOPSY Biopsy of Lymph node or other tissues
  42. 42. TREATMENT  vital-sign instability or neutropenia is an indication for empirical therapy with a fluoroquinolone plus piperacillin  If the PPD skin test is positive or if granulomatous hepatitis or other granulomatous disease is present, then a therapeutic trial with isoniazid and rifampin, with treatment usually continued for up to 6 weeks.
  43. 43. BIBLIOGRAPHY  Dr. Fauci and Dr. Longo. “Harrison’s Internal Medicine”. America: The McGraw-Hill Companies, Inc 2008.  Skin Pathonline. “Ziehl Neelsen Stain for AFB”. 9 May. 2011 < ehl%E2%80%93neelsen-stain-for-acid-fast- organisms-method-and-tips/>.  fast-culture  Kesson%20PUO.pdf