Conventional and rapid methods in the laboratory diagnosis of pyrexia of unknown origin

MICROBIOLOGY / By /

Introduction to Pyrexia of Unknown Origin (PUO):

Pyrexia of unknown origin (PUO) is a temperature above 38.3 degrees Celsius, lasting more than 3 weeks, and there is failure to reach a diagnosis despite a week of inpatient investigation.  PUO could be classic PUO (temperature >38.3 degrees Celsius for more than 3 weeks and evaluation of at least 3 outpatient visits and 3 days in hospital  is made) or Nosocomial ( temperature >38.3 degrees Celsius where there was hospitalization for more than 24 hours and evaluation is made for at least 3 days)

PUO is mostly seen in cases of Endocarditis, Occult bacteremia, Tuberculosis, Typhoid, and Scarlet fever. Mostly, blood is taken as a specimen for the detection of PUO. Other specimens include urine, sputum, throat swab, and stool.

Selection of specimens for PUO:

Specimens like blood, sputum, urine, throat swab, and stool are collected for the diagnosis of pyrexia of unknown origin.

Specimen Selection Criteria for PUO Diagnosis:

  • All the specimens selected should be contained in an appropriate sterile container to be suitable for diagnosis
  • All specimens are best selected when they are collected without the administration of antimicrobial therapy
  • Sputum should not be contaminated with saliva
  • Sputum should not have >10 epithelial cells and <25 Neutrophils per low-power field
  • Blood collected after proper asepsis (2% tincture of iodine) should be selected for diagnosis
  • Stool contaminated with urine should not be selected.
  • The throat swab collected without the use of any antibacterial mouthwash, 8 hours before swabbing, should be collected.
  • The throat swab mixed with saliva should not be selected.
  • The patient information on the label should be matched with the information on the requisition form for selection.

Collection of specimens for PUO:

All specimens should be collected in appropriate sterile containers.

Collection of blood

About 10-20 ml of blood in adults, 2-4 ml in infants, and 1-2ml blood in neonates should be collected by venipuncture by proper skin disinfection with 2% tincture of iodine and mixed with an anticoagulant like SPS to prevent blood clotting.

Blood is best collected without the administration of antimicrobial therapy.

For pyrexia of unknown origin, 2 or 3 sets of blood should be collected at about 1 hour apart in 24 24-hour period. In case of culture negative in the first 24 hours, 2-3 more sets of blood are withdrawn the next day.

Collection of sputum

The early morning sputum (no less than 2 ml) should be collected without the use of mouthwash wash making sure there is no contamination with saliva.

The patient is instructed to cough deeply.

If the patient is not able to produce sputum, aerosol (saline) induction is done, as in cases of children.

For pulmonary tuberculosis detection, up to three specimens are suggested.

In case of failure to produce sputum, bronchoscopy can be done to aspirate bronchoalveolar lavage.

Collection of throat swab

For a collection of a throat swab, the patient is seated in a sitting posture and under good light, a tongue depressor is used to depress the tongue and look for inflammation.

The affected area is swabbed using a sterile cotton swab, while avoiding contact with saliva, and then placed into transport media.

Collection of stool sample

The patient is given a sheet of newspaper along with a pair of wooden sticks.

About 5 grams of stool should be passed onto the newspaper, then carefully transferred into the provided container using the sticks.

It is important to ensure the stool sample is free from any urine contamination.

Collection of urine

Clean catch Mid-stream urine (15ml) should be collected in a clean, sterile wide container.

Before the urine collection, the hands should be washed with soap and water, and the urethral opening thoroughly water-washed to avoid contamination.

Transportation and Storage of Specimens:

  • It is desired for all the bacterial specimens to be processed within 1-2 hours of collection.
  • In case of delay, transport media are used.
  • For stool specimens, Cary-Blair transport medium is employed in case of delay.
  • In case of urine, boric acid preservation (10g /lit) is used to maintain the viability of the specimen, but no growth.
  • Sputum and throat swab sample is placed into Amies transport media after collection in case of delay.

Storage

  • Blood should not be refrigerated
  • Specimens like urine, sputum, and stool can be refrigerated for storage.

Conventional diagnosis of PUO includes:

Macroscopy Examination

The appearance of sputum is seen and examined by the eyes, whether it is purulent, mucopurulent, mucosalivary, or bloody.

The colour, consistency, and presence of blood and mucus are observed in stool.

For the urine sample, the colour of the urine and the cloudiness of the urine, if any, are observed. Cloudy urine indicates a bacterial infection.

Microscopy Examination

Bacteria like Mycobacterium tuberculosis, Brucella, Salmonella Typhi, Coxiella burnetii, Streptococcus pneumoniae, and Streptococcus pyogenes are the common bacterial etiologies of PUO.

Sputum

Gram staining is performed by using the purulent part of the sputum. Then neutrophils and predominant bacteria are looked for (S pneumoniae– gram-positive diplococci, Hemophilus influenzae– gram-negative diplococci)

Acid-fast staining is performed to detect the presence of M tuberculosis.

Urine

In case of sterile pyuria, when there are neutrophils but not culture positive on regular media, AFB staining is done to detect M. tuberculosis by urine centrifugation.

Feces

The feces are subjected to Gram stain in PUO organisms like Salmonella Typhi, Clostridium difficile (gram-negative rods) are mostly seen.

Blood

Gram staining of the buffy coat can be performed to observe bacteria that have entered the blood (bacteremia, either transiently, intermittently, or continuously). In endocarditis, bacteria are continuously released from the endothelial vegetation.

Culture

Culture is the mainstay for bacterial diagnosis.

Blood

The drawn blood sample is introduced into a blood culture broth at a dilution ratio of 1 part blood to 10 parts broths. Columbia biphasic broth is mostly used for aerobic blood pathogens. The organisms are incubated for 2 weeks in the case of Endocarditis. In case of Brucellosis, the Tryptic Soy broth is incubated for 4 weeks at 37 degrees Celsius and is examined twice daily.

Blind subculture is done at 1st, 3rd, and if no growth is seen till the 5th day, the culture is negative. In case of endocarditis, 2 more blood samples can be drawn as prior antibiotic therapy could have suppressed bacteria in the vegetation, but it is not able to kill them completely.

Sputum

  • When M tuberculosis is suspected in sputum by AFB staining, it is cultivated on Lowenstein-Jensen medium for 6 weeks in 5% carbon dioxide. If it is positive for Mycobacterium tuberculosis, it gives rough, buff, and touch colonies.
  • Streptococcus pneumoniae, Haemophilus influenzae can be cultured on Blood Agar with 5% carbon dioxide. The Optochin disc is placed on the secondary. On BA with H. influenza suspicion, Staphylococcus aureus is primarily streaked to provide X and V factors.
  • S. pneumoniae are alpha-hemolytic and are optochin sensitive.
  • H influenzae shows nonhemolytic satellite colonies.

Urine

In case of sterile pyuria, when there are neutrophils but not culture positive on regular media, urine is cultured on LJ medium for M. tuberculosis detection.

Stool

After pre-enrichment on Selenite F broth, the sample is cultivated on SS media. Salmonella Typhi shows pink colonies with a black centre.

Throat swab

S. pyogenes on BA show beta hemolysis and are sensitive to the bacitracin disc.

Biochemical Characterization

  • Streptococci tests negative for catalase and oxidase
  • The bile solubility test is done to differentiate S. pneumoniae from other alpha-hemolytic viridans.
  • Salmonella Typhi is catalase positive, oxidase negative, IMVic -+– Alk/A gas – H2S +
  • Brucella abortus is Catalase and oxidase positive, IMViC – – – +

Antibiotic susceptibility testing

  • Antibiotic usage before diagnosis is discouraged
  • Empirical therapy is suggested in cases of CNS or military TB and infective endocarditis.

Rapid Diagnostic Methods for PUO:

  • It could employ samples like serum, sputum for rapid diagnosis of PUO.
  • AFB staining employed before culture of Mycobacterium tuberculosis is a rapid preliminary method in the identification of M tuberculosis.
  • The sputum or urine sample in case of pulmonary and extrapulmonary Tuberculosis is stained with carbol fuchsin, decolorized with 3% acid alcohol, and counterstained with Methylene blue. Because M tuberculosis is fast, it resists decolorization and is seen as a bright rod on AFB staining.
  • Similarly, Auramine phenol technique can also be used to identify M tuberculosis.
  • Bipolar staining can be used to identify Yersinia pestis after staining with Wayson’s stain as safety pin-shaped bacteria with pink ends.
  • Modified tube agglutination test that only detects IgG (1:80) is used for Rapid diagnosis of Brucella melitensis.
  • The ASO test for semiquantitative determination of S. pyogenes is a rapid test to identify S pyogenes.
  • PUO Screen test uses standardized, killed, stained, smooth antigen of Salmonella Typhi, Brucella, and Rickettsia spp antigen for rapid antibody detection.

References:

Leave a Comment

Your email address will not be published. Required fields are marked *