Legionella Pneumophilia (Monofluo IFA) Test

The MONOFLUO legionella Immunoflourescent Antibody Test Kit is used to detect and identify L. pneumophila directly in patient specimens or in culture isolates, confirming a diagnosis of Legionnaires disease.

Principle:
The MONOFLUO Anti – Legionella pneumophilia staining reagent contains a single monoclonal antibody that is labeled with fluorescein isothiocyanate. This antibody reacts with a protein present in the outer membrane of all known serogroups of L. pneumophilia.

Smears are prepared on microscope slides as either directly from patient specimen or from organisms isolated in culture. The smears are stained with MONOFLUO Anti-Legionella Staining Reagent. The labeled monoclonal antibody in this reagent binds immunologically to any L. pneumophilia in the smear. A subsequent rinse step removes unbound antibody from specimen. When the samples are viewed with a fluorescein MICROSCOPE, l. Pneumophilia appear as brightly fluoresceing apple-green coccobacili. Other organisms will appear dark or dull gold due to counter.

Procedure:
Smear Preparation for Direct Test on Patient Specimens

• Clean and label a two-well MONOFLUO Fluorescein Microscopy Slide. 
• Prepare two smears per patient specimen, as described below. 
• Tissue (fresh or fresh-frozen). 
• Choose an area of dense gray or reddish consolidation. Use one of both of the following techniques to prepare two smears:  

1. Grind the tissue with sterile deionized water to create an approximate 10% tissue homogenate. 
2. Using a sterile wooden applicator stick, make thin smears covering the wells on the slides.  
3. Using a sterile forceps and scalpel, cut a fresh face of tissue (If too wet to give a thin smear, blot on                                              sterile gauze.) with a forceps. 
4. Press and squeeze the tissue against the slide to fill the wells. If culture is to be performed, do so                                 before making smears. 

• Lung Exudates (Fresh or fresh-frozen sputum, transtracheal, aspirates, bronchial washings, etc.) 

1. Select a viscous portion of the specimen that is milky or bloody in appearance. 
2. With a sterile wooden applicator stick, prepare two thin smears. 

• Pleural Fluid and Cerebrospinal Fluid. 

1. Centrifuge the specimen in a safety cup at about 4,000 rpm for 30 minutes. 
2. Discard all but 0.5 ml of supernatant, re-suspend and prepare two thin smears. 
3. Air dry the smears, ten heat-fix the samples by rapidly passing slide through a flame. 
4. Place the slide in a moisture chamber and cover each smear with 10% formalin in saline. 
5. Fix for 10 minutes at room temperature. 
6. Drain following the formalin and briefly dips the slide in de-ionized water. 
7. Soak the slide in fresh de-ionized water for two minutes to remove the remainder. 
8. Air dry then proceed with Fluorescence Staining Procedure. 

Smear Preparation for Culture Confirmation

• Clean and label a MONOFLUO Fluorescence Microscopy Slide. 
• Remove a distinct colony of suspect L. pneumophilia from the culture plate with a bacteriological loop.
• Suspend the bacteria in a test tube containing very small amount of 1% formalin in saline to the turbidity of a Mac Farland No. 1 standard. 
• Using a Pasteur pipette, apply 2-3 drops of the suspension to a well on the slide, and then remove with the same Pasteur pipette remaining a thin film of organisms on the well. 
• Repeat steps 1-3 to make a separate smear of suspect colony, using individual slides. 
• Air dry the smears, then heat-fix by rapidly passing the slide through a flame. 
• After drying, proceed with the Fluorescence Staining Procedure. 

Smear Preparation for Positive Control

• Clean and label a MONOFLUO Fluorescence Microscopy Slide NOTE: The positive Control smear should be processed separately from patient test smears. 
• Shake the bottle containing the Positive Control Antigen Suspension to re-suspend cells. 
• Add 1-2 drops of the suspension to a well on the slide then using a fresh Pasteur pipette, remove the liquid from the well and discard. 
• Air dry, and then heat the sample by rapidly passing the slide through a flame and then 
• Proceed with Fluorescence Staining Procedure. 

Fluorescence Staining Procedure

• Apply a sufficient amount of the MONOFLUO Anti-Legionella pneumophilia staining reagent to cover the specimen (1-2 drops depending on the type of specimen, keeping within the boundary of the well).
• Place the slide in a moisture chamber and incubate for 30 minutes at 35-37ºC. 
• Incubate the Positive Control Slide and specimen slides in separate moisture chambers .
• Remove excess Staining Reagent by a method such as aspiration and briefly dip the slide in de-ionized water .
• Clean hand or change gloves between handling the positive control slide and specimen slides. 
• Air dry slides .
• Add 1-2 drops of Mounting Medium to the slide and apply a cover slip 

Interpretation:

• Stained L. pneumophilia will appear as intra or extracellular fluorescing, apple-green rods or coccobacilli. Samples are Negative when organisms appear dark red or dull gold. L. pneumophilia are pleomorphic organisms that can range from very short coccobacillary forms to long, filamentous rods. Antibiotic therapy may result in organisms with uncharacteristic morphology. 
• The staining characteristics and morphology of positive organisms resemble the L. pneumophilia positive control. In a culture confirmation test, overcrowding of cells in the smear may significantly reduce the staining intensity. If necessary, examine the outer edges of the well or portions of the smear containing fewer cells. Culture conditions, especially age of culture, may result in cell-to-cell variation in the fluorescence intensity. Some cells may nit stain uniformly over their entire surface. In all cases, however, positively stained cells will fluoresce in a pattern that reveals definite cellular morphology. Cultures are confirmed for L. pneumophilia based on the distinctive appearance of the colonies, the morphology of the fluorescent organisms, and the result of the gram stain.