Lower Respiratory Tract Infection, LRTI - Lab diagnosis, Specimen Collection, Processing, Exclusion criteria, Culture

Laboratory diagnosis of LRTI

The laboratory diagnosis of Lower Respiratory Tract Infection (LRTI) begins with the collection of samples.

Exclusion criteria

Etiologic agent’s discovery (especially in pneumonia) is important but in 50% of cases, it is not identified despite extensive diagnostic testing. No single test is capable of identifying all LRT pathogens.

Samples that contain predominantly upper respiratory tract materials should be rejected. Previously, only expectorated sputum was allowed to be rejected on the basis of microscopy screening. However, endotracheal aspirates (ETAs) criteria for rejection include greater than 10 squamous epithelial cells per low power field of no organisms seen under oil immersion (1000x).

However, in Legionella pneumonia, the sputum may be scant and watery with few or no host cells. Such specimens give the result by direct fluorescent antibody stain and culture and they should not be subject to screening procedures.

The number of WBC is not relevant because the patient may be severely neutropenic. On the other hand, the presence of 25 or more polymorphonuclear leukocytes per 100x field, together with a few squamous epithelial cells implies an excellent specimen.

Specimen collection

• expectorated sputum

• sputum obtained by postural drainage of thoracic percussion (induced)

• tracheostomy aspirates/suction

• transbronchial biopsy

• transtracheal aspirates

• The specimen can be lower BAL, BB, BW

  BAL = bronchoalveolar lavage

  BW = bronchial washing

  BB = bronchial brushing

Transport

Transport of Lower Respiratory Tract Infection (LRTI) samples should be done by:

• sterile screw-top container used for the collection

• should be transported to the lab in less than 2 hours if decay is foreseen. Can be stored at 4°C for up to 24 hours

• BA, CA, Mac, CA can be used for plating.

Specimen processing

Direct visual examination

Fungal elements can be visualized under phase microscopy with 10% potassium hydroxide (KOH), with UV light with calcofluor white, or using periodic acid-Schiff-stained smears.

For other evaluations, the Lower Respiratory Tract Infection (LRTI) specimens must be fixed and stained. Bacteria and yeast can be recognized on Gram stain. To determine the quality of expectorated sputum received, gram stain can be used as a screening procedure.

Respiratory secretion may need to be concentrated before staining. Specimens are centrifuged and the sediment is used for visual examination and cultures. A Cytocentrifuge instrument is an alternative to conventional centrifugation.

For screening, the presence of ciliated columnar bronchial epithelial cells, goblet cells, and pulmonary macrophages in specimens obtained by BAL or bronchoscopy indicate Lower Respiratory Tract (LRT)specimen.

The respiratory specimen may also be stained for acid-fast bacilli with either classic Ziehl-Nelson or the Kenyon carbol fuchsin stain. Auramine or Auramine-rhodamine may also be used to detect acid-fast organisms. Since they are fluorescent, they are more sensitive and are preferred for rapid screening.

Direct fluorescent Ab (DFA) has been used to detect Legionella spp. in LRT specimens.

Culture

Following the screening of the Lower Respiratory Tract Infection (LRTI) samples, culture is done on blood agar, chocolate agar, and MacConkey agar at 37°C for 24-48 hours.

The isolates are then subjected to identification on the basis of colony morphology, gram staining, grown in selective media, biochemical tests, etc.