Lower respiratory tract specimens are submitted to the laboratory for the diagnosis of airway infections includingbut not limited to community and hospital acquired pneumonia including ventilator associated pneumonia, bronchitis,chronic airway infection in cystic fibrosis patients, lung abscess, and tuberculosis.
Specimen types submitted for the detection of etiology of these infections include:
Sputum, induced sputum, tracheal aspirates, non-bronchoscopic bronchoalveolar lavage (BAL) from intubated patients, and bronchoscopically obtained specimens including bronchial washes and BAL, and pleural fluid.
Other specimens discussed in the literature but essentially never done at UNC Hospitals include transbronchial biopsy for detection of PCP, fungi, mycobacterium, and viruses; protected bronchial brush for anaerobic bacteria, fungi, viruses, transthoracic needle aspiration and open lung biopsy.
Importantly only 60% of patients with CAP can produce sputum.
Chest tube drainage is sometimes sent but we don’t know what to do with it.
Pre-analytical stage:
Specimen quality determined by gram stain-
Sputum gram stain screening-ratio of WBC to epithelial cells as seen on low power field must be greater than 1.
Screening is not done in cystic fibrosis patients (sputum producers almost always have purulent sputa and we are looking for a limited number of organisms);
Patients suspected of either M. tuberculosis or Legionella pneumophilia.
Try to tell morphology and what it is consistent with.
For example, Streptococcus pneumoniae has a distinctive morphology, lancet shaped diplococcus; we should report: gram positive, lancet shaped diplococci consistent with Streptococcus pneumoniae.
Recent study of pneumococcal pneumonia showed 16% had inadequate specimens, gram stain was positive in 63% and culture in 86%.
Other organisms with distinctive gram stains include:
Gram positive cocci in clusters-Staphylococcus aureus
Thin gram negative rods- Pseudomonas aeruginosa
Pointed gram negative rods-Fusobacterium
Barnching, beaded gram positive rods- Nocardia
Mixed gram positive cocci and thin gram negative rods-mixed anaerobic infections
Pleiomorphic gram negative coccobacilli-Haemophilus
Gram negative diplococci-Moraxella
Tracheal aspirate screening-as per sputum with the additional proviso that the specimen must have organisms seen on oil immersion.
Over 90% of specimens that have “no organisms seen” on gram stain will not grow a pathogen, so they are only cultured by request.
Likewise, if a specimen shows only yeast, it too is not cultured because it is likely to represent endotracheal tube colonization which is common.
Candida pneumonia is so rare that it needs to be diagnosed by other means as tracheal aspirate culture will have poor positive predictive value
What specimen when trying to detect specific organism:
CAP organisms- sputum and tracheal aspirates culture of good quality specimen will be useful for detection of most bacterial agents
Legionella-induced sputum may help if high index of suspicion such as positive urinary antigen test otherwise sputum, even of poor quality is OK-BCYE added to all BAL specimens but must ask for it on tracheal aspirates and sputum
Pneumocystis- induced sputum OK in HIV positive patients; otherwise use BALs: not a fan of tracheal aspirates, rarely positive
Nocardia- should grow from sputum specimen; will grow on most laboratory isolation method but holding BCYE works best
Anaerobes- gram stain that showed mixed gram positive and negative organisms and grows oropharyngeal flora only is a tip off
Can look for them on BAL but must be requested as is not routinely done.
Protected bronchial brush probably the optimal specimen-rarely done but if anaerobes sought this is the specimen of choice.
Will also culture pleural fluids but rarely positive
Anthrax-organism will be detected in blood cultures not respiratory specimens
Culture set-up of different specimens
Sputum/tracheal aspirates-HBA, CNA, MacConkey
Pleural fluid- as with sputum + anaerobic medium; very low yield in part because specimens are typically obtained when patients have been on antimicrobial therapy.May be a good specimen for bacterial detection by direct sequencing
BAL- cultured quantitatively; work up organisms >104 cfu/ml if pathogens add BCYE (will grow pertussis, Nocarida, Francisella) and Inhibitory Mould agar to plates used in sputum culture
Protected bronchial brush- quantitative on aerobic and anaerobic plates otherwise like BAL; use 103 cfu/ml as our cut-off
Bronchial wash-cultured like a sputum
CF specimens-as above with BCSA (for Burkholderia cepacia complex organism) and Mannitol salts (for Staphylococcus aureus
Analytical stage:
Key organisms :
Streptococcus pneumoniae- can be seen in patients with bronchitis and both hospital and community pneumonia; infrequent in CF patients.
Characteristic gram stain can be of value.
Other important facts- pneumococcal penicillin breakpoints have changed from 1 to 2 ug/ml.
Blood cultures positive in approximately 30% of patients with pneumococcal pneumonia- antigen test for pneumococcal urinary antigen has a sensitivity of 50 to 80%;
false positives can occur in nasopharyngeal carriers
Haemophilus influenzae/Moraxella catarrhalis- important agent of bronchitis especially in those folks with COPD; 90%+ of Moraxella are beta-lactamase positive-
H. influenzae- 20% are ampicillin resistance with most of the resistance beta-lactamase mediated; non-typable strains predominant; H. infuenzae uncommon cause of CAP- ranges of 2 to 12% have been reported
Legionella pneumophilia- will grow in 3 to 5 days on BCYE agar; isolated infrequently- urinary antigen has a sensitivity of 80% at our institution and very good specificity but numbers or low so data should be considered carefully;
test can remain positive for months nosocomial infections are rare at our institution; only know of 1 documented nosocomial case since I have been here
Staphylococcus aureus- important CF pathogen, CAP secondary to influenza, nosocomial infection; CA-MRSA found in 3% of CF patients in a 2 year prospective study-negative gram stain/cultures reported to be adequate to rule out MRSA
Pseudomonas aeruginosa- key organism in chronic airway infections in CF patients- highly adaptive, can survive some disinfectants; makes a unique morphotype called “mucoid.” Mucoid variant grow in a biofilm; organisms growing in biofilm are highly adaptive to CF lung environment;organisms are refractory to antimicrobial therapy and immune clearance; organism may be seen in older women with COPD; also the most common agent of ventilator associated pneumonia; strains may become resistant to all known antimicrobials with the exception of colistin- colistin resistance is rare.
Burkholderia cepacia complex- important agent in cystic fibrosis and chronic granulomatous disease patients; environmental bacteria; highly adaptable and resistant to many antimicrobials and disinfectants requires special medium for isolation (BCSA is the one we use here);
organism is resistant to wide array of antimicrobials and some strains develop resistance to all available antimicrobials;
Limited antimicrobials for which susceptibility guidelines are available
Stenotrophomonas/Achromobacter-glucose non-fermenters of low virulence; environmental bacteria; highly adaptable and resistant to many antimicrobials and disinfectants.
Can colonize CF patients and are likely colonizers rather than pathogens in patients on ventilators.
Outside the CF population, found most common in tracheal aspirate cultures. As with B. cepacia complex, few antimicrobials have susceptibility guidelines for Stenotrophomonas
Acinetobacter- becoming an important agent of nosocomial pneumonia- glucose non-fermenting gram negative rod; can appear as a diplococcus on gram stain. Strains being found in BICU and SICU that are only susceptible to colistin.
Mycoplasma/Chlamydophila pneumoniae- important agents of CAP; no satisfying diagnostic test so is primarily a diagnosis of exclusion
Post analytical stage:
Call the floor when specimen of poor quality is collected so re-collection can be attempted
Try to limit the organisms that we report so that the report will make sense
Specimen types submitted for the detection of etiology of these infections include:
Sputum, induced sputum, tracheal aspirates, non-bronchoscopic bronchoalveolar lavage (BAL) from intubated patients, and bronchoscopically obtained specimens including bronchial washes and BAL, and pleural fluid.
Other specimens discussed in the literature but essentially never done at UNC Hospitals include transbronchial biopsy for detection of PCP, fungi, mycobacterium, and viruses; protected bronchial brush for anaerobic bacteria, fungi, viruses, transthoracic needle aspiration and open lung biopsy.
Importantly only 60% of patients with CAP can produce sputum.
Chest tube drainage is sometimes sent but we don’t know what to do with it.
Pre-analytical stage:
Specimen quality determined by gram stain-
Sputum gram stain screening-ratio of WBC to epithelial cells as seen on low power field must be greater than 1.
Screening is not done in cystic fibrosis patients (sputum producers almost always have purulent sputa and we are looking for a limited number of organisms);
Patients suspected of either M. tuberculosis or Legionella pneumophilia.
Try to tell morphology and what it is consistent with.
For example, Streptococcus pneumoniae has a distinctive morphology, lancet shaped diplococcus; we should report: gram positive, lancet shaped diplococci consistent with Streptococcus pneumoniae.
Recent study of pneumococcal pneumonia showed 16% had inadequate specimens, gram stain was positive in 63% and culture in 86%.
Other organisms with distinctive gram stains include:
Gram positive cocci in clusters-Staphylococcus aureus
Thin gram negative rods- Pseudomonas aeruginosa
Pointed gram negative rods-Fusobacterium
Barnching, beaded gram positive rods- Nocardia
Mixed gram positive cocci and thin gram negative rods-mixed anaerobic infections
Pleiomorphic gram negative coccobacilli-Haemophilus
Gram negative diplococci-Moraxella
Tracheal aspirate screening-as per sputum with the additional proviso that the specimen must have organisms seen on oil immersion.
Over 90% of specimens that have “no organisms seen” on gram stain will not grow a pathogen, so they are only cultured by request.
Likewise, if a specimen shows only yeast, it too is not cultured because it is likely to represent endotracheal tube colonization which is common.
Candida pneumonia is so rare that it needs to be diagnosed by other means as tracheal aspirate culture will have poor positive predictive value
What specimen when trying to detect specific organism:
CAP organisms- sputum and tracheal aspirates culture of good quality specimen will be useful for detection of most bacterial agents
Legionella-induced sputum may help if high index of suspicion such as positive urinary antigen test otherwise sputum, even of poor quality is OK-BCYE added to all BAL specimens but must ask for it on tracheal aspirates and sputum
Pneumocystis- induced sputum OK in HIV positive patients; otherwise use BALs: not a fan of tracheal aspirates, rarely positive
Nocardia- should grow from sputum specimen; will grow on most laboratory isolation method but holding BCYE works best
Anaerobes- gram stain that showed mixed gram positive and negative organisms and grows oropharyngeal flora only is a tip off
Can look for them on BAL but must be requested as is not routinely done.
Protected bronchial brush probably the optimal specimen-rarely done but if anaerobes sought this is the specimen of choice.
Will also culture pleural fluids but rarely positive
Anthrax-organism will be detected in blood cultures not respiratory specimens
Culture set-up of different specimens
Sputum/tracheal aspirates-HBA, CNA, MacConkey
Pleural fluid- as with sputum + anaerobic medium; very low yield in part because specimens are typically obtained when patients have been on antimicrobial therapy.May be a good specimen for bacterial detection by direct sequencing
BAL- cultured quantitatively; work up organisms >104 cfu/ml if pathogens add BCYE (will grow pertussis, Nocarida, Francisella) and Inhibitory Mould agar to plates used in sputum culture
Protected bronchial brush- quantitative on aerobic and anaerobic plates otherwise like BAL; use 103 cfu/ml as our cut-off
Bronchial wash-cultured like a sputum
CF specimens-as above with BCSA (for Burkholderia cepacia complex organism) and Mannitol salts (for Staphylococcus aureus
Analytical stage:
Key organisms :
Streptococcus pneumoniae- can be seen in patients with bronchitis and both hospital and community pneumonia; infrequent in CF patients.
Characteristic gram stain can be of value.
Other important facts- pneumococcal penicillin breakpoints have changed from 1 to 2 ug/ml.
Blood cultures positive in approximately 30% of patients with pneumococcal pneumonia- antigen test for pneumococcal urinary antigen has a sensitivity of 50 to 80%;
false positives can occur in nasopharyngeal carriers
Haemophilus influenzae/Moraxella catarrhalis- important agent of bronchitis especially in those folks with COPD; 90%+ of Moraxella are beta-lactamase positive-
H. influenzae- 20% are ampicillin resistance with most of the resistance beta-lactamase mediated; non-typable strains predominant; H. infuenzae uncommon cause of CAP- ranges of 2 to 12% have been reported
Legionella pneumophilia- will grow in 3 to 5 days on BCYE agar; isolated infrequently- urinary antigen has a sensitivity of 80% at our institution and very good specificity but numbers or low so data should be considered carefully;
test can remain positive for months nosocomial infections are rare at our institution; only know of 1 documented nosocomial case since I have been here
Staphylococcus aureus- important CF pathogen, CAP secondary to influenza, nosocomial infection; CA-MRSA found in 3% of CF patients in a 2 year prospective study-negative gram stain/cultures reported to be adequate to rule out MRSA
Pseudomonas aeruginosa- key organism in chronic airway infections in CF patients- highly adaptive, can survive some disinfectants; makes a unique morphotype called “mucoid.” Mucoid variant grow in a biofilm; organisms growing in biofilm are highly adaptive to CF lung environment;organisms are refractory to antimicrobial therapy and immune clearance; organism may be seen in older women with COPD; also the most common agent of ventilator associated pneumonia; strains may become resistant to all known antimicrobials with the exception of colistin- colistin resistance is rare.
Burkholderia cepacia complex- important agent in cystic fibrosis and chronic granulomatous disease patients; environmental bacteria; highly adaptable and resistant to many antimicrobials and disinfectants requires special medium for isolation (BCSA is the one we use here);
organism is resistant to wide array of antimicrobials and some strains develop resistance to all available antimicrobials;
Limited antimicrobials for which susceptibility guidelines are available
Stenotrophomonas/Achromobacter-glucose non-fermenters of low virulence; environmental bacteria; highly adaptable and resistant to many antimicrobials and disinfectants.
Can colonize CF patients and are likely colonizers rather than pathogens in patients on ventilators.
Outside the CF population, found most common in tracheal aspirate cultures. As with B. cepacia complex, few antimicrobials have susceptibility guidelines for Stenotrophomonas
Acinetobacter- becoming an important agent of nosocomial pneumonia- glucose non-fermenting gram negative rod; can appear as a diplococcus on gram stain. Strains being found in BICU and SICU that are only susceptible to colistin.
Mycoplasma/Chlamydophila pneumoniae- important agents of CAP; no satisfying diagnostic test so is primarily a diagnosis of exclusion
Post analytical stage:
Call the floor when specimen of poor quality is collected so re-collection can be attempted
Try to limit the organisms that we report so that the report will make sense
