Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections
By:Mohammad Rahbar (PhD)
Department Of Microbiology Reference Laboratory of Iran
Anatomy of Respiratory Tract
“ The culture of lower respiratory specimens may result in more unnecessary microbiologic effort than any other type of specimen.”
Raymond C Bartlett
Lower Respiratory Tract Infections
Epidemiology
* Pneumonia is the sixth leading cause of death in US
* Increasing numbers of patients at risk
o Aging population
o Increase in patients with immunocompromising conditions
* Overtreatment has lead to resistance
o Multidrug resistant Streptococcus pneumoniae
o Resistance among hospital acquired pathogens such as Acinetobacter, Pseudomonas aeruginosa E.coli K.pneumonia (ESBLs) MRSA and others
* Major sections
o Clinical aspects of diseases of LRT
o Specimen collection
o Specimen processing
o Interpretation of bacterial cultures
o Most common pathogens
o Methods for implementing change
o Guidelines for frequency of testing
o Public health issues
o Reimbursement codes
Categories of Lower Respiratory Tract Infections
* Acute bronchitis
* Community acquired pneumonia
* Hospital acquired pneumonia
* Pneumonia in the immunocompromised host
Community Acquired Pneumonia Etiologic Agents
Community Acquired Pneumonia Diagnosis
Available Test Methodologies
* Sputum Gram stain and culture
* Blood cultures
* Serologic studies
* Antigen detection tests
* Nucleic acid amplification tests
Sputum Gram Stain and Culture
Proponents
* Demonstration of predominant morphotype on Gram stain guides therapy
* Accuracy is good when strict criteria are used
* Cheap, so why not?
Antagonists
* Poor specimen collection
* Intralaboratory variability (Gram stain interpretation)
* Low sensitivity and specificity
* Empiric treatment guidelines
* Not cost effective
Sputum Collection
* Proper patient instruction
o Food should not have been ingested for 1-2 h prior to expectoration
o The mouth should be rinsed with saline or water
o Patient should breathe and cough deeply
o Patient should expectorate into a sterile container
* Transport container immediately to lab
* Perform Gram stain and plant specimen as soon as possible
Sputum collection
* Sputum of less than 2ml should not be processed unless obviously purulent
* Only 1 sputum per 24hr .submitted
* Some scoring system should be used to reject specimen that re oral contamination.
* Transportation in <2 hr is recommended with refrigeration if delays anticipated.
* Handle all samples using universal precautions.
* Perform Gram stain and plant specimen as soon as possible
Induced sputum
Patients who are unable to produce sputum may be assisted by respiratory therapy technician. Aerosol induced specimen are collected by allowing the patient to breath aerosolized droplets of a solution of 15% sodium chloride and 10% glycerin for approximately 10 minute . obtaining such specimen may avoid the need for a more invasive procedures ,such as bronchoscopy or needle aspiration, in many cases.
Gastric aspiration
* The gastric aspiration is used exclusively for isolation of acid-fast bacilli and may be collected from patients who are unable to produce sputum, particularly young children. The relative resistance of mycobacteria allows them to remain viable for a short period. Gastric lavage must be delivered to the lab immediately so that the acidity can be neutralized. Specimen can be first neutralized and then transported if immediate delivery is not possible.
Sputum Gram Stain Unacceptable
Sputum Gram Stain Good Quality
Good quality specimens
* Quantify number and types of inflammatory cells
* Note presence of bronchial epithelial cells
* Concentrate on areas with WBCs when looking for organisms
* Determine if there is a predominant organism (> 10 per oil immersion field)
o Semiquantitate and report organism with descriptive
o If no predominant organism is present, report “mixed gram positive and gram negative flora”
Utility of the Gram Stain in Diagnosis of Pneumonia
Roson, B, et. al. 2000. Clin Infect Dis 31:869-74.
* Prospective study
* Non immunocompromised patients hospitalized with CAP
* 1,000 bed hospital in Spain
* ER physicians instructed on sputum collection for Gram stain and culture
* Sputum collected under supervision of nurse or resident
* Sputum collected under supervision of nurse or resident
o Samples were processed immediately
o Screened for epithelial cells
o Screened for predominant morphotype (> 75% of the organisms seen)
o Sputum planted to blood agar, chocolate agar and MacConkey agar
* Strictly defined clinical and diagnostic parameters
Utility of the Gram Stain in Diagnosis of Pneumonia
Roson, B, et. al. 2000. Clin Infect Dis 31:869-74
Results
* 190/533 (35.6%) patients had no sputum sample submitted (these patients were included in the calculations)
* 133/533 (25%) patients had a poor quality specimen
* 210/533 (39.4%) patients had a good quality specimen
* Overall sensitivity and specificity for pneumococcal pneumonia: 57% and 97%
* Overall sensitivity and specificity for H. influenzae pneumonia: 82 % and 99%
* Gram stain gave presumptive diagnosis in 80% of patients who had a good specimen submitted
* > 95% of patients in whom a predominant morphotype was seen on Gram stain received monotherapy
Gram Stain Reports
* Be as descriptive as possible
o Moderate neutrophils
o Moderate Gram positive diplococci suggestive of Streptococcus pneumoniae
o Few bacteria suggestive of oral flora
* Keep report short—avoid line listing of all morphotypes present
Sputum and Endotracheal Suction Culture Evaluation
* Identify and perform susceptibility testing on 2-3 potential pathogens seen as predominant on Gram stain
* Alpha strep—rule out S. pneumoniae
* Yeast—rule out Cryptococcus neoformans only
* S. aureus, Gram negative bacilli
o < normal flora, quantify and limit ID; no susceptibility
o Add comment that organism not predominant on stain
* ID mould, Mycobacteria or Nocardia spp.
IDSA Practice Guidelines
Diagnostic Tests for CAP
* Outpatients
o Empiric therapy with a macrolide, doxycycline, or a fluoroquinolone
* Hospitalized patients with CAP
o Gram stain and culture of sputum
o 2 pretreatment blood cultures
o Studies for Mtb, Legionella in select patients
Bartlett JG. 2000. Clin Infect Dis 31:347-82.
* Rationale
o To improve patient care
o Advance knowledge of epidemiologically important organisms
o Prevent antibiotic abuse
o Reduce antibiotic expense
Bartlett JG. 2000. Clin Infect Dis 31:347-82.
ATS Guidelines Diagnostic Tests for CAP
* Empiric therapy for outpatients
o Macrolide or tetracycline
* Hospitalized patients with CAP
o 2 sets of pre-treatment blood cultures
o Pleural fluid Gram stain/culture when appropriate
o Studies for Legionella, Mtb, fungi in select patients
o Sputum Gram stain/culture only if resistant or unusual pathogen is suspected
o Avoid extensive testing
ATS. 2001. Am J Respir Crit Care Med 163: 1730-1754.
Hospital Acquired Pneumonia
* Most frequent nosocomial infection (30-33% of cases) among combined medical surgical intensive care units
* 83% are ventilator associated
* Etiologic agents Frequency (%)
o Gram positive cocci
+ S. aureus 17
+ S. pneumoniae 2-20
AGENTS OF HAP
* Aerobic gram-neg bacilli 60
o Pseudomonas aeruginosa
o Enterobacter sp.
o Klebsiella pneumoniae
o Acinetobacter
o Legionella
o Anaerobes 10-20
o Fungi 0-10
Modified from: Carroll KC. 2002. J Clin Microbiol 40: 3115-3120.
Hospital Acquired Pneumonia Diagnosis
* American College of Chest Physicians: Clinical findings are not sufficient for definitive diagnosis
* Qualitative culture or endotracheal sputum has poor predictive value
* Bronchoscopy is recommended by many pulmonologists
o Bronchial brushings
o Bronchial washes
o Protected specimen brushing
o Bronchoalveolar lavage specimens (BAL)
o Transbronchial biopsy
Respiratory Specimens
* Protected Brush Specimen
o To procure uncontaminated lower airway secretions
o Brush within 2 catheters
* Bronchoalveolar Lavage (BAL)
o Samples large area of the lung
o Performed using a bronchoscope
o 100 to 250 ml of saline injected
o Injected saline along with secretions is collected by aspiration
* Transthoracic Aspiration
o Involves percutaneous introduction of a needle directly into the infiltrate
Bronchoalveolar Lavage (BAL) Specimen Acceptability
* Microscopic examination of Gram-stained smear
o Acceptable
+ <1% of cells present are squamous epithelial cells
o Unacceptable
+ >1% of cells present are squamous epithelial cells
Thorpe JE et. al. 1987. Bronchoalveolar lavage for diagnosing acute bacterial
pneumonia. J. Infect. Dis. 155:855-861
Processing Bronchoscopy Specimens
* Bronchoscopy brush protected
o Aerobic bacterial culture and Gram stain
o Anaerobic bacterial culture
o Limited volume
* Bronchoscopy brush, unprotected
o No anaerobic culture
o Limited volume
* Bronchial washings
o Useful only for pneumonia caused by strict pathogens
o Reasonable requests: Mtb, Fungi, Legionella, Pneumocystis
* Bronchoalveolar lavage
o No anaerobe culture
o Amenable to extensive testing for all opportunistic pathogens
Interpretation of Quantitative PSB/BAL
* Dilution Method
o Quantify each morphotype present and express as CFU/ml
* Calibrated Loop Method
o Quantify each morphotype present and express as log10 colony count ranges
* Thresholds for significance
o PSB > 103 CFU/ml
o BAL > 104 CFU/ml
Bronchoscopy Samples Quantitative Methods
Routine culture
* Most of the commonly sought etiologic agents of lower respiratory tract infection will isolated on routinely used media : 5% sheep blood agar ,MacConkey agar for isolation and differentiation of gram-negative bacilli ,and chocolate agar for Neisseria spp and Haemophilus
* Because of contaminating oral flora ,sputum specimens ,specimens obtained by bronchial washing, and lavage trachestomy, or endotracheal tube aspirates are not inoculated to enriched broth or incubated anaerobically. Only specimens obtained by percutaneous aspiration (including transtracheal aspiration )and by protected bronchial brush are suitable for anaerobic culture: he latter must be done quantitatively for proper interpretation.
* Transtracheal and percutaneous lung aspiration material may be inoculated to enriched thioglycollate ,as well as to solid media. For suspected cases of legionnaires disease buffered charcoal –yeast extract (BCYE) agar and selective BCYE are inoculated.
* Sputum specimens from patients known to have cystic fibrosis should be inoculated to selective agar ,such as manitol salt agar for recovery of S .aureus and selective horse blood-bacitracin ,incubated anaerobically and aerobically ,for recovery of H,influenzae that may be obscured by the mucoid P,aeroginosa on routine media. The use of selective medium for B.cepacia ,such as PC or OFPBL agar ,is also recommended
Immunocompromised Patients
Suggested BAL Protocol
* Aerobic Gram stain quantitative bacterial culture
* Fungal stain and culture
* Mycobacterial stain and culture
* Viral culture/Respiratory DFA
* Pneumocystis DFA
* Legionella culture
Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections.ppt
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