This document outlines the diagnostic approach and laboratory tests for evaluating a patient presenting with pyrexia of unknown origin (PUO). It describes collecting relevant clinical history and performing a physical exam. Specimens including blood, urine, sputum, CSF and tissues may be obtained for bacterial, viral, parasitic and fungal cultures and stains. Tests like blood cultures, urine cultures, sputum smears and cultures, and CSF analysis can help identify potential infectious causes. Serology, skin tests, hematology, immunology and biopsy may also provide diagnostic clues. Empiric antibiotic therapy is guided by risk factors and test results.

1. LABORATORY DIAGNOSIS OF
PYREXIA OF UNKNOWN

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. 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. BACTERIAL INFECTIONS

6. 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

7. 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

8. 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

9. 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.

10. BACTERIAL INFECTIONS
Sterile Vial containers (30 ml): Used for collecting Blood

11. BACTERIAL INFECTIONS
Sterile Universal Container: Used for collecting Urine

12. CULTURING
BACTERIAL INFECTIONS

13. 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)

14. Blood Agar MacConkey Agar
Blood agar and MacConkey agar are
incubated at 37ºC for 24 hours

15. 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

16. 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.

17. 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.

18. 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

19. 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.

20. 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)

21. 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.

22. 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

23. IDENTIFICATION
 On the basis of:
 Colony morphology
 Gram Staining
 Ziehl-Neelsen Staining- For M.
tuberculosis
 Biochemical reactions
 Agglutination

24. 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.

25. ZIEHL-NEELSEN STAINING
Solutions
Concentrated carbol fuchsin
20% Sulphuric acid
2% methylene blue

26. 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.

27. 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.

28. 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.

29. SEROLOGY
 Useful in:
 Infectious mononucleosis- Paul-Bunnell
test
 Enteric fever
 Hepatitis A, B infections
 CMV infections
 Amoebiasis

30. 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

31. 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

32. FUNGAL CAUSES
 Candida albicans
 Streptococcus neoformans
 Histoplasm
 Aspergillous spp
 Coccidioides innipil neocystis carinii

33. 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

34. OTHER TESTS
1. Skin tests
2. Haematology
3. Immunologic tests
4. Biopsy

35. 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

36. MANTOUX TEST
The induration (raised area) is what is measured.
NOT the erythema (red area).

37. MANTOUX TEST

38. 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.

39. SKIN TESTS FOR HISTOPLASMOSIS

40. HAEMATOLOGY
TLC AND DLC

41. IMMUNOLOGIC TESTS
LE cell phenomenon
Antinuclear antibody test
SLE

42. BIOPSY
Biopsy of Lymph node or other tissues

43. 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.

44. 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
<http://skinpathonline.wordpress.com/2011/05/09/zi
ehl%E2%80%93neelsen-stain-for-acid-fast-
organisms-method-and-tips/>.
 http://www.enotes.com/nursing-encyclopedia/acid-
fast-culture
 http://www.virusesinmay.com/docs/2006/VIM06%20
Kesson%20PUO.pdf