Diagnostic Testing for Community-Acquired Pneumonia (CAP

1
Diagnostic Testing for Community-Acquired
Pneumonia (CAP) and Influenza

• Norman Moore, Ph.D.
• Director of Clinical Affairs
• norman.moore_at_invmed.com

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Objectives

• Discuss the etiological agents for pneumonia and
  which age groups are most prone to the infection.
• Describe what clinical samples should be taken
  and how they should be transported to the
  laboratory for analysis
• State the diagnostic testing methods recommended
  for community-acquired pneumonia and influenza
• Show how influenza can lead to pneumonia

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Infectious Disease in the US

• 1970 William Stewart, the Surgeon General of
  the United States declared the U.S. was ready to
  close the book on infectious disease as a major
  health threat modern antibiotics, vaccination,
  and sanitation methods had done the job.
• 1995 Infectious disease had again become the
  third leading cause of death, and its incidence
  is still growing!
• Pneumonia is the sixth leading cause of death in
  the US and
• the major cause of death from infectious disease
  in the US.

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Current Number of Pneumonia Cases (US)

• 37 million ambulatory care visits per year for
  acute respiratory infections (physician and ER
  visits combined)
• Community-Acquired Pneumonia (CAP)
• Each year 2 - 3 million cases of CAP result in
  10 million physician visits 500,000
  hospitalizations in the US
• Average mortality is 10-25 in hospitalized
  patients with CAP
• Nosocomial Pneumonia
• Standard definition onset of symptoms occurs
  approx 3 days after admission
• 250,000 - 350,000 cases of nosocomial pneumonia
  per year
• 25 - 50 mortality rate

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Etiological Agents

• Newborns (0 to 30 days)
• Group B Streptococcus, Lysteria monocytogenes, or
  Gram negative rods are common
• RSV in premature babies
• Infants and toddlers
• 90 of lower respiratory tract infections are
  viral with the most common being RSV, Influenza
  AB, and parainfluenza. Bacterial infections are
  rare, but could be S. pneumoniae, Hib, or S.
  aureus.

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Etiological Agents

• Outpatient
• S. pneumoniae, H. influenzae, M. pneumoniae, C.
  pneumoniae, and respiratory viruses
• Inpatient (non-ICU)
• With the above agents, add L. pneumophila
• Inpatient (ICU)
• S. pneumoniae, S. aureus, L. pneumophila,
  Gram-negative bacteria, and H. influenzae

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Streptococcus pneumoniae

• Types Over 90 serotypes exist, with 88 of
  disease covered in the 23-valent vaccine
• Incidence 100,000 to 135,000 cases of pneumonia
  requiring hospitalization up to the year 2000
• Around 80 of CAP
• Cases are dropping due to the S. pneumoniae
  vaccine
• Transmission Person to person
• Risk groups The young and elderly
• Most common identification Blood culture and
  sputum culture

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Haemophilus influenzae

• Types The original risk was H. influenzae Type
  B (Hib), but vaccine has dramatically reduced
  pneumonia due to Hib, but other types and
  non-typeable strains still cause disease
• Incidence Variable
• Transmission Person to person
• Risk groups The young and elderly
• Most common identification Blood culture and
  sputum culture

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Chlamydia pneumoniae

• Incidence Overall is unknown, but in the
  literature, it seems to go in cycles so high
  incidence in some years and low in others.
• Can be considered 3rd most common etiological
  agent in respiratory tract infections of young
  adults behind Mycoplasma pneumoniae and influenza
• Transmission Person to person
• Risk groups All age groups, but more common in
  school-age children.
• Most common identification Serology
• Personal contact with Barry Fields Chief of
  Respiratory infections from CDC rates of C.
  pneumoniae have been extremely low for years and
  he currently doesnt view this as a significant
  infection.

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Mycoplasma pneumoniae

• Incidence Estimated 2 million cases and 100,000
  pneumonia related hospitalizations in US
• Transmission Person to person by respiratory
  secretions, usually close contact
• Outbreaks in crowded conditions like military and
  college which can last several months
• Risk groups All age groups, but more common in
  school-age children and young adults.
• Most common etiological agent for adults younger
  than 30
• Most common identification - Serology

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Legionella pneumophila

• Incidence Estimates vary greatly from 15,000
  per year to 100,000 per year in US
• Transmission Contaminated water
• Outbreaks in hospitals, ships, hotels, etc.
• Risk groups Usually elderly, smokers
• Most common identification Urinary antigen

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Viral pneumonia

• Adults may get viral pneumonia by influenza,
  adenovirus, cytomegalovirus, parainfluenza,
  varicella, rubeola, or respiratory syncytial
  virus, particularly during epidemics
• Viral pneumonia, especially influenza, may cause
  a secondary bacterial disease, such as
  pneumococcal pneumonia

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Influenza AB

• Impact of influenza in the US
• Hospitalizations up from 114,000 to 226,000
• 36,000 deaths annually
• Influenza target population 188MM in US
• 5-20 of US population affected by influenza each
  year
• Most deaths affect elderly and young children
• Also affects otherwise healthy individuals

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Influenza Treatment

• Antiviral drugs are available
• Must be administered within 48 hr of onset of
  symptoms
• Generally reduce duration of symptoms by one day
• First generation drugs (amantidine, rimantidine)
  are cheaper but only treat influenza A
• Second generation drugs (Tamiflu, Relenza) are
  more expensive but treat both influenza A and B
• Reason to differentiate between influenza A and B

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Respiratory Syncytial Virus

• Almost all children with have RVS by their second
  birthday
• 25 to 40 will have signs or symptoms of
  bronchiolitis or pneumonia
• 0.5 to 2 will require hospitalization
• Recovery is in 1 to 2 weeks, but they can spread
  virus for 1 to 3 weeks
• The elderly can get a usually mild RSV infection
  due to a weakened immune system
• Rapid tests are not recommended on this population

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Specimen Collection
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Swab collection

• Swab should remain moist and cultured within 4
  hours
• If longer than 4 hours to get to culturing,
  should use transport medium
• Refrigeration, not frozen

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Sputum Collection

• Quality of specimen
• Care should be taken in collection since a lower
  respiratory tract sample can be contaminated with
  upper unless collected by an invasive technique
• Collection
• Patient is instructed to give a deep coughed
  specimen
• Put into sterile container, trying to minimize
  saliva
• Transport to lab immediately
• Patient unable to give specimen can be given an
  aerosol-induced specimen

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Blood culture

• Usually done with fever spike
• Standard is to take two sets of blood cultures
  one hour apart

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Urine

• Urine can be used for Legionella and
  Streptococcus pneumoniae
• Antigen test
• Noninvasive sample
• Does not need to be qualified like a sputum sample

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Influenza Sample Collection

• Appropriate specimens
• Nasal wash/aspirate, nasopharyngeal swab, or
  nasal swab
• Throat swabs have dramatically reduced
  sensitivity
• Samples should be collected within first 24 to
  48 hours of symptoms since that is when viral
  titers are highest and antiviral therapy is
  effective
• Testing can be done immediately with rapids or
  sample placed in transport media
• Infectivity is maintained up to 5 days when
  stored _at_ 4-8C
• If the sample cannot be evaluated in this time
  period, the sample should be frozen _at_ -70C.

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Diagnostic Methods Available
23
Infectious Disease Society of America/American
Thoracic Society Consensus Guidelines on the
Management of Community-Acquired Pneumonia in
Adults (2007)

• Diagnostic Testing
• Suggestive clinical features combined with a
  chest radiograph or other imaging technique is
  required for the diagnosis of pneumonia
• It is recommended that patients with CAP should
  be investigated for specific pathogens that would
  significantly alter standard (empirical)
  management decisions, when the presence of such
  pathogens is suspected on the basis of clinical
  and epidemiologic clues.

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Infectious Disease Society of America/American
Thoracic Society CAP Guidelines 2007

• When to apply diagnostic tests
• Optional for outpatients with CAP
• Blood culture and sputum culture for inpatients
  with productive cough
• All adult patients with severe CAP, should have
  blood culture, sputum culture, Legionella urinary
  antigen and S. pneumoniae urinary antigen tests

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Common Diagnostic Tests

• Gram stain
• Sputum culture
• Blood culture
• Latex agglutination assays
• DFA/IFA
• PCR
• Serology
• Urinary antigen

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Gram stain

• Apply sample to microscope slide
• Apply stains view using standard microscope
• Pros Inexpensive
• Rapid (15 minutes)
• Cons Difficult to get good sample (50 are
  inadequate)
• Should have less than 10 squamous
  epithelial cells per low power field (100x)
• Requires trained personnel to read

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Sputum Culture Bacterial Culture

• Pros Inexpensive
• Standard media for most Sheep blood agar,
  MacConkey agar, and chocolate agar, BCYE for
  Legionella
• Allows for antibiotic susceptibility testing
• Cons Requires live bacteria antibiotics can
  affect results
• Difficult to get good sample
• Requires dedicated tech time / experienced
  personnel
• Results take 24 hours to gt1 week

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Legionella Culture

• Legionella
• Legionella needs specific growth conditions
• Buffered charcoal yeast extract (BCYE) plate
• Clinical sample may need to be acid treated to
  reduce general microflora
• May take 3 to 10 days to get result

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Cell culture for Chlamydia pneumoniae

• Chlamydia cultures should be transported in
  2-sucrose phosphate or other transport medium
• Use HeLa cell line rather than McCoy that is used
  for C. trachomitis
• May take 3 to 10 days and is labor-intensive

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Culture for Mycoplasma pneumoniae

• Specialty media
• May take over 3 weeks for result
• Vial is inspected daily and is prone to
  contamination (usually indicated by color shift
  in first 5 days)
• Needs subculturing to agar
• Highly labor intensive

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Blood Culture

• Pros Inexpensive
• Allows for antibiotic susceptibility testing
• High specificity
• Cons Requires live bacteria antibiotics can
  affect results
• Requires dedicated tech time / experienced
  personnel
• Results take 24 hours to gt1 week
• Many bacterial infections dont
• progress to bacteremia

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Latex Agglutination

• Detecting antigen associated w/certain serogroups
• Polystyrene latex particles coated with
  antibodies
• Pros Relatively simple
• Rapid (15 minutes)
• Cons Does not detect all serogroups of S.
  pneumoniae
• Procedure associated with urine is cumbersome
• Interpretation of results can be subjective

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Fluorescent Antibody (DFA/IFA)

• Performed directly from sample on microscope
  slide
• Sputum, pleural fluid, aspirated material, or
  tissue
• Add fluorescent-tagged antibody specific for
  specific bacteria Observe for fluorescence using
  a special microscope
• Pros Relatively quick turn around time (1
  hour)
• Cons More labor intensive than rapid tests
• Requires trained technologist and special
  microscope
• Few labs equipped to perform DFA on
• 2nd/3rd shifts
• Sensitivity can be poor (25 to 75
• on Legionella)

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Polymerase Chain Reaction (PCR)

• Molecular technique using a clinical sample
• Extract and amplify nucleic acid (DNA or RNA) of
  specific pathogen
• Pros Extremely sensitive can detect one
  microorganism
• Detects both live and dead pathogens
• Cons Requires highly trained technologist,
  expensive equipment
• More labor intensive than rapid tests
• Prone to cross-contamination (false positives)

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Serology

• Chlamydia pneumoniae
• Measurement usually of acute and convalescent
  serum
• A four-fold rise in titer is considered
  diagnostic
• A single IgM titer of 16 or greater or IgG of 512
  or greater is considered suggestive of recent
  infection
• Mycoplasma pneumoniae
• A fourfold rise from acute to convalescent serum
  or complement fixation titer of 1128 in single
  serum specimen

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Urinary antigen

• Tests are available for S. pneumoniae and L.
  pneumophila serogroup 1
• With Legionella, antigen appears in the urine 1
  to 3 days after infection
• Noninvasive sample
• Easy-to-use

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Test Procedure for Urinary Antigen

• Collect urine sample (no pre-treatment i.e.
  concentrating, boiling, filtering, etc.)
• Open device pouch and lay flat
• Dip provided sampling swab into urine
• Place swab in lower hole of swab well and push up
• Add required number of drops of Reagent A (2
  drops for Legionella test and 3 for S.
  pneumoniae)
• Close device
• Wait 15 minutes
• Interpret results

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Diagnostic Methods for Influenza

• Culture
• DFA
• PCR
• Rapid Tests

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Viral Culture

• Pro
• Highly sensitive as long as sample is properly
  handled
• Con
• Cant give same day result to help monitor
  therapy
• High level of difficult/equipment

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DFA

• Pro
• Usually considered to have high level of
  sensitivity
• Can usually test for other respiratory pathogens
  at the same time
• Results can be achieved in same day
• Con
• Labor intensive needed experienced users
• Turn-around time from lab usually takes many hours

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PCR

• Pro
• For respiratory specimens, high performance
• Same day results
• Con
• Turn around time from lab is extensive,
  especially if batching specimens
• Expensive
• Requires experienced technicians, labs, dedicated
  equipment, etc.

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Rapid Tests

• Pro
• Tests take minimal time
• Some tests are so simple that they can be
  CLIA-waived
• Can be used to triage patients
• Positive results can be used to rule out other
  issues like pneumonia so dont give unnecessary
  chest x-ray, antibiotics, etc.
• Con
• Performance is not as good as culture, PCR, and
  DFA

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The Connection Between Influenza and S. pneumoniae
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Pandemic outbreaks

• In 1957 and 1968 influenza pandemic outbreaks, it
  was shown that a bacterial agent was present in
  approximately 70 of the serious
  (life-threatening or death) cases.
• In contrast, in non-pandemic years, only 25 of
  serious cases had a secondary bacterial infection.

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Synergy Between Influenza and S. pneumoniae

• Influenza neuraminidase found to prime lung for
  S. pneumoniae invasion.
• S. pneumoniae has its own neuraminidase that it
  uses to promote binding to cells.
• In a mouse model, if neuraminidase inhibitors
  were added, then mortality went down.
• Recombinant versions of influenza strains of past
  50 years were made.
• 1957 and 1997 pandemic strains that were related
  to bacterial pneumonia had highest levels of
  neuraminidase activity.

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S. pneumoniae and Penicillin
47
Penicillin Breakpoint

• IV Penicillin
• Less expensive than broad spectrum antibiotics
• Reduce broad spectrum antibiotic resistance
• Less liver/kidney resistance

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Reference

• Mandell, L.A., R.G. Wunderink, A. Anzueto, J.G.
  Bartlett, G.D. Campbell, N.C. Dean, S.F. Dowell,
  T.M. File, D.M. Musher, M.S. Niederman, A.
  Torres, and C.G. Whitney. Infectious Diseases
  Society of America/American Thoracic Society
  Consensus Guidelines on the Management of
  Community-Acquired Pneumonia in Adults.
  Clinical Infectious Diseases. 2007 44S27-72.
• Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A.
  Pfaller, and R.H. Yolken. Manual of Clinical
  Microbiology 8th Edition.
• Forbes, B.A., D.F. Sahm, and A.S. Weissfeld.
  Bailey Scotts Diagnostic Microbiology 12th
  Edition.