1. Hospital Acquired Pneumonia Mohammadali Boroumand M.D. Associate Professor of Pathology Tehran Heart Center Tehran University of Medical Sciences

2. Laboratory Diagnostic Criteria For VAE and VAP/PNEU Leukocytosis Leukocytosis Gram stain of direct smears from lower respiratory tract specimens Gram stain of direct smears from lower respiratory tract specimens Culture of lower respiratory tract specimens Culture of lower respiratory tract specimens Blood or pleural fluid cultures Blood or pleural fluid cultures Molecular or immunoassay methods Molecular or immunoassay methods

3. SPECIMEN COLLECTION, TRANSPORT, AND HANDLING  Collection of lower lung samples by ET tube aspirates(ETA)  Bronchoscopically obtained specimens Bronchoalveolar lavage (BAL) Bronchoalveolar lavage (BAL) Protected specimen brushing (PSB) Protected specimen brushing (PSB) Transbronchial biopsy specimens Transbronchial biopsy specimens

4. ET Tube Aspirates 1.Connect suction catheter to rubber tubing side on aspirating trap and connect the other side of aspirating trap to the tubing from wall suction 1.Connect suction catheter to rubber tubing side on aspirating trap and connect the other side of aspirating trap to the tubing from wall suction 2. Draw up the appropriate amount of preservative free sterile saline for irrigation into a syringe.( 2mL. )Pour the remaining normal saline for irrigation into graduated cup. 2. Draw up the appropriate amount of preservative free sterile saline for irrigation into a syringe.( 2mL. )Pour the remaining normal saline for irrigation into graduated cup. 3.Suction the patient by putting on sterile gloves, instilling saline from syringe (without needle), suction patient while keeping the aspirating trap in a vertical position. 3.Suction the patient by putting on sterile gloves, instilling saline from syringe (without needle), suction patient while keeping the aspirating trap in a vertical position. 4.If the specimen is in the catheter, suction a small amount of normal saline for irrigation from graduated cup to move secretions into aspirating trap. Disconnect aspirating trap from suction tubing and catheter

5. Collection of lower lung samples by bronchoscopy  Precautions Obtain BAL specimens before brushing or biopsy specimens Obtain BAL specimens before brushing or biopsy specimens Avoid suctioning through the working channel before retrieval of specimens Avoid suctioning through the working channel before retrieval of specimens Avoid the injection of topical anesthetic agents as much as possible, Aerosol Avoid the injection of topical anesthetic agents as much as possible, Aerosol application of anesthetic agents is preferred application of anesthetic agents is preferred

6. Notes The laboratory will not be able to distinguish a BAL from a bronchial wash based on the appearance of the container or the fluid in the container The laboratory will not be able to distinguish a BAL from a bronchial wash based on the appearance of the container or the fluid in the container In BAL Discard the initial fluid as contaminated and submit the rest for culture and staining In BAL Discard the initial fluid as contaminated and submit the rest for culture and staining Place the PBS in a sterile vial containing 1 ml of nonbacteriostatic saline Place the PBS in a sterile vial containing 1 ml of nonbacteriostatic saline

7. Transport of Specimen BAL specimens should be immediately transported and handled by the laboratory as soon as possible after collection, since saline may be toxic to the recovery of some primary pathogens BAL specimens should be immediately transported and handled by the laboratory as soon as possible after collection, since saline may be toxic to the recovery of some primary pathogens Store specimens at 2 to 8C until samples can be transported or processed by the laboratory Store specimens at 2 to 8C until samples can be transported or processed by the laboratory (A delay in processing of more than 1 to 2 h may result in a decreased ability to recover fastidious pathogens) (A delay in processing of more than 1 to 2 h may result in a decreased ability to recover fastidious pathogens)

8. Rejection Criteria for Endotracheal Aspirate Specimens  Reject duplicate specimens received on the same day  Gram Stain Criteria Examine 20 to 40 fields from endotracheal Smears and average the number of cells in representative fields that contain cells and Examine 20 to 40 fields from endotracheal Smears and average the number of cells in representative fields that contain cells and Purulent respiratory secretions defined as secretions from the lungs, bronchi, or trachea that contain >25 neutrophils and 25 neutrophils and <10 squamous epithelial cells per low power field [lpf, x100]

10. Rejection Criteria for PSB & BAL Specimens PSB and BAL specimens should never be rejected by the laboratory PSB and BAL specimens should never be rejected by the laboratory For specimens delayed in transit more than 2 hours without refrigeration, indicate on the report that the delay in transit may compromise the culture results For specimens delayed in transit more than 2 hours without refrigeration, indicate on the report that the delay in transit may compromise the culture results

11. ETA,BAL & PSB samples should be cultured quantitatively for bacterial pathogens ocorresponding semi- quantitative result should be reported ETA,BAL & PSB samples should be cultured quantitatively for bacterial pathogens or corresponding semi- quantitative result should be reported

12. Processing and Culture Examination of ET Aspirate Specimens Perform a Gram stain of the specimen as soon after receipt as possible to microscopically assess cellular and bacterial components Perform a Gram stain of the specimen as soon after receipt as possible to microscopically assess cellular and bacterial components Identify the organisms that grow in significant amounts, defined as bacterial morphotypes that are potential primary pathogens that may or not be part of the normal respiratory microbiota Identify the organisms that grow in significant amounts, defined as bacterial morphotypes that are potential primary pathogens that may or not be part of the normal respiratory microbiota

13. BAL & PSB Use a cytocentrifuge to prepare BAL specimens for Gram stain Use a cytocentrifuge to prepare BAL specimens for Gram stain Perform quantitative cultures on BAL samples Perform quantitative cultures on BAL samples The original Gram stain result should be used to guide further analysis of BAL and PBS cultures The original Gram stain result should be used to guide further analysis of BAL and PBS cultures

14. Direct Smear of BAL If laboratory processes respiratory specimens such as bronchoalveolar lavage fluid using a centrifugation procedure, a report indicating the presence of white blood cells, without quantitation, is sufficient to meet the purulent secretions criterion If laboratory processes respiratory specimens such as bronchoalveolar lavage fluid using a centrifugation procedure, a report indicating the presence of white blood cells, without quantitation, is sufficient to meet the purulent secretions criterion ≥5% BAL-obtained cells contain intracellular bacteria on direct microscopic exam (e.g., Gram’s stain) ≥5% BAL-obtained cells contain intracellular bacteria on direct microscopic exam (e.g., Gram’s stain)

15. Significant growth is defined as follows

16. ETA specimen just use for VAE and PVAP surveillance system and not acceptable for VAP/PNEU diagnosis ETA specimen just use for VAE and PVAP surveillance system and not acceptable for VAP/PNEU diagnosis

17. These values have significance only when the samples have been obtained >72 hours before the initiation or a change of antibiotic therapy These values have significance only when the samples have been obtained >72 hours before the initiation or a change of antibiotic therapy

18. Loop Method  PBS (1) Vortex for 30 to 60 s. (2) Place 100µ (2 drops) of specimen onto each plate marked “×10.” Each colony from this plate = 10 CFU/ml. (3) Using a 1:100 loop, place 10 µ on a plate labeled “× 100.” Each colony from this plate= 100 CFU/ml.  ETA & BAL fluid (1) Vortex for 30 to 60 s. (2) Using a 1:100 loop, place 10µ on a plate labeled “× 100.” Each colony from this plate = 100 CFU/ml. (3) Using a 1:1,000 loop, place 1µ on a plate labeled “× 1,000.” Each colony from this plate = 1,000 CFU/ml

19. Positive culture of virus, Legionella or Chlamydia from respiratory secretions  Positive non culture diagnostic laboratory test of respiratory secretions or tissue for virus, Bordetella, Chlamydia, Mycoplasma, Legionella (e.g., EIA, FAMA, shell vial assay, PCR, micro-IF) Fourfold rise in paired sera (IgG) for pathogen (e.g., influenza viruses, Chlamydia) Fourfold rise in Legionella pneumophila serogroup 1 antibody titer to ≥1:128 in paired acute and convalescent sera by indirect IFA Detection of L. pneumophila serogroup 1 antigens in urine by RIA or EIA

20. Interpretation Normal respiratory tract microbiota is defined as viridans group streptococci, commensal Neisseria spp., N. meningitidis, diphtheroids, coagulase-negative staphylococci, Rothia, group F streptococci, anaerobes, Haemophilus spp. (not H. influenzae), enterococci, Candida spp., Eikenella, Actinobacillus, Capnocytophaga, and Moraxella Normal respiratory tract microbiota is defined as viridans group streptococci, commensal Neisseria spp., N. meningitidis, diphtheroids, coagulase-negative staphylococci, Rothia, group F streptococci, anaerobes, Haemophilus spp. (not H. influenzae), enterococci, Candida spp., Eikenella, Actinobacillus, Capnocytophaga, and Moraxella

21. Coagulase-negative Staphylococcus species, Enterococcus species, Candida species or yeast not otherwise specified can only be used to meet PNEU definitions when isolated from pleural fluid obtained during thoracentesis or initial placement of chest tube (not from an indwelling chest tube) or lung tissue Coagulase-negative Staphylococcus species, Enterococcus species, Candida species or yeast not otherwise specified can only be used to meet PNEU definitions when isolated from pleural fluid obtained during thoracentesis or initial placement of chest tube (not from an indwelling chest tube) or lung tissue

22. Pleural Fluid Culture Bedside inoculation into blood culture bottles has become an established practice Bedside inoculation into blood culture bottles has become an established practice This is acceptable providing that the manufacturer’s guidelines are followed with respect to the volume inoculated This is acceptable providing that the manufacturer’s guidelines are followed with respect to the volume inoculated If blood culture bottles are used, an additional sample should be sent to the microbiology laboratory for Gram stain and culture of nonbacterial pathogens when indicated If blood culture bottles are used, an additional sample should be sent to the microbiology laboratory for Gram stain and culture of nonbacterial pathogens when indicated

23. Procalcitonin Tends to rise quickly as bacterial infections (but not viral infections) develop Tends to rise quickly as bacterial infections (but not viral infections) develop Increase with the severity of infection Increase with the severity of infection Decline as bacterial infections resolve Decline as bacterial infections resolve Should only support clinical judgment, not replace it Should only support clinical judgment, not replace it Increasingly appears to be a useful tool to help physicians prescribe antibiotics to the right patients, and to stop antibiotics when they're no longer helpful Increasingly appears to be a useful tool to help physicians prescribe antibiotics to the right patients, and to stop antibiotics when they're no longer helpful

24. BRAHMS PCT Serum collected using standard sampling tubes or tubes containing separating gel Serum collected using standard sampling tubes or tubes containing separating gel Li ‑ heparin, K2 ‑ EDTA and K3 ‑ EDTA plasma Li ‑ heparin, K2 ‑ EDTA and K3 ‑ EDTA plasma Stable for 24 hours at 2 ‑ 8 °C and 3 months at -20 °C Stable for 24 hours at 2 ‑ 8 °C and 3 months at -20 °C Due to possible evaporation effects, samples should be analyzed/measured within 2 hours Due to possible evaporation effects, samples should be analyzed/measured within 2 hours

25. THANK YOU FOR YOUR ATTENTION ANY QUESTION?