LABORATORY DIAGNOSIS OF

PYREXIA OF UNKNOWN

Presented By: Dr. Trisha

APPROACH TO THE DIAGNOSIS OF PUO

1. Duration and pattern of fever2. Age of patient3. Sexual history4. Contact with other ill people5. Vaccination history6. Travel history7. Animal / insect exposure8. Previous treatment including blood

products

PHYSICAL EXAMINATION Clinical features:

1. Sinus tenderness2. Mouth ulceration3. Chorioretinitis4. Chest- pneumonitis5. Abdomen- hepatic tenderness,

splenomegaly6. Lymphadenopathy7. Arthritis8. Rash

BACTERIAL INFECTIONS

BACTERIAL INFECTIONSSpecimens:

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

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

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

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.

BACTERIAL INFECTIONS

Sterile Vial containers (30 ml): Used for collecting Blood

BACTERIAL INFECTIONS

Sterile Universal Container: Used for collecting Urine

CULTURING BACTERIAL INFECTIONS

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)

Blood Agar MacConkey Agar

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

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

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.

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 medium*

Urine cultures, including cultures for mycobacteria, fungi, and CMV, are indicated.

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

CSF COLLECTIONThe 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 L4Once 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.

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)

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.

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

IDENTIFICATION

On the basis of: Colony morphology Gram Staining Ziehl-Neelsen Staining- For M.

tuberculosis Biochemical reactions Agglutination

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.

ZIEHL-NEELSEN STAINING

SolutionsConcentrated carbol fuchsin20% Sulphuric acid2% methylene blue

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.

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.

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.

SEROLOGY Useful in:

Infectious mononucleosis- Paul-Bunnell test

Enteric feverHepatitis A, B infectionsCMV infectionsAmoebiasis

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

VIRAL INFECTION

Paul- Bunnell test is useful in infectious mononucleosis

CMVEpstein barr virusHep A and B virusesArbovirusesEnterovirusesAdenovirsuesMyoxvirusesHuman immunodeficiency virusHaemorrhagic fever viruses

FUNGAL CAUSES

Candida albicans Streptococcus neoformans Histoplasm Aspergillous spp Coccidioides innipil neocystis carinii

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

OTHER TESTS

1. Skin tests2. Haematology3. Immunologic tests4. Biopsy

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

MANTOUX TEST

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

MANTOUX TEST

MANTOUX TEST

An induration of 5 to 15 millimeters is considered as a positive reaction for the following people:

People with HIV infectionClose contacts of people with infectious TBPeople with chest x-ray findings suggestive

of previous TB diseasePeople 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.

SKIN TESTS FOR HISTOPLASMOSIS

HAEMATOLOGYTLC AND DLC

IMMUNOLOGIC TESTS

LE cell phenomenon

Antinuclear antibody test

SLE

BIOPSY

Biopsy of Lymph node or other tissues

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.

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/ziehl%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%20Kesson%20PUO.pdf