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8/12/2019 Pneumonia and Influenza
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Diagnostic Testing for
Community-Acquired
Pneumonia (CAP) and
Influenza
Norman Moore, Ph.D.
Director of Clinical Affairs
norman.moore@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 pneumoniaand 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 Statesdeclared the U.S. was ready to close the book on infectiousdisease as a major health threat; modern antibiotics, vaccination,and sanitation methods had done the job.
1995: Infectious disease had again become the third leading causeof 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 respiratoryinfections (physician and ER visits combined)
Community-Acquired Pneumonia (CAP)
Each year 2- 3 million cases of CAP result in ~ 10 million physicianvisits & 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 afteradmission
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 commonRSV in premature babies
Infants and toddlers
90% of lower respiratory tract infections are viral
with the most common being RSV, Influenza
A&B, 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, addL. pneumophila
Inpatient (ICU)
S. pneumoniae, S. aureus,L. pneumophila, Gram-
negative bacteria, andH. influenzae
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Streptococcus pneumoniae
TypesOver 90 serotypes exist, with 88% of disease
covered in the 23-valent vaccine
Incidence100,000 to 135,000 cases of pneumonia
requiring hospitalization up to the year 2000
Around 80% of CAP
Cases are dropping due to the S. pneumoniaevaccine
TransmissionPerson to person
Risk groupsThe young and elderly
Most common identificationBlood culture and sputum
culture
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Haemophilus influenzae
TypesThe original risk was H. influenzaeType B
(Hib), but vaccine has dramatically reduced pneumonia
due to Hib, but other types and non-typeable strains still
cause disease
IncidenceVariable
TransmissionPerson to person
Risk groupsThe young and elderly
Most common identificationBlood culture and sputum
culture
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Chlamydia pneumoniae
IncidenceOverall is unknown, but in the literature, itseems to go in cycles so high incidence in some yearsand low in others.
Can be considered 3rd
most common etiological agent in respiratorytract infections of young adults behindMycoplasma pneumoniaeandinfluenza
TransmissionPerson to person
Risk groupsAll age groups, but more common in
school-age children. Most common identificationSerology
Personal contact with Barry FieldsChief of Respiratoryinfections from CDCrates of C. pneumoniaehave
been extremely low for years and he currently doesntview this as a significant infection.
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Mycoplasma pneumoniae
IncidenceEstimated 2 million cases and 100,000
pneumonia related hospitalizations in US
TransmissionPerson to person by respiratory
secretions, usually close contact
Outbreaks in crowded conditions like military and college which can
last several months
Risk groupsAll 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
IncidenceEstimates vary greatly from 15,000 per year
to 100,000 per year in US
TransmissionContaminated water
Outbreaks in hospitals, ships, hotels, etc.
Risk groupsUsually elderly, smokers
Most common identificationUrinary 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 A&B
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, shoulduse 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 Legionellaand 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 @ 4-
8C
If the sample cannot be evaluated in this time period, the
sample should be frozen @ -70C.
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Diagnostic Methods Available
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Infectious Disease Society of America/American Thoracic
Society Consensus Guidelines on the Management ofCommunity-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 inpatientswith productive cough*
All adult patients with severe CAP, should have
blood culture, sputum culture,Legionellaurinary
antigen and S. pneumoniaeurinary 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 CultureBacterial Culture
Pros: Inexpensive
Standard media for mostSheep blood agar, MacConkeyagar, and chocolate agar, BCYE for Legionella
Allows for antibiotic susceptibility testing
Cons: Requires live bacteriaantibiotics can affect resultsDifficult to get good sample
Requires dedicated tech time / experienced personnel
Results take 24 hours to >1 week
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LegionellaCulture
Legionel la
Legionellaneeds specific growth conditions
Buffered charcoal yeast extract (BCYE) plate
Clinical sample may need to be acid treated to reduce generalmicroflora
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 bacteriaantibiotics can affect resultsRequires dedicated tech time / experienced personnel
Results take 24 hours to >1 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 Observefor 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/3rdshifts
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 sensitivecan 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 isconsidered suggestive of recent infection
Mycoplasma pneumoniae
A fourfold rise from acute to convalescent serum or complement
fixation titer of 1:128 in single serum specimen
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Urinary antigen
Tests are available for S. pneumoniaeand L.
pneumophila serogroup1
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 Legionellatest
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 unnecessarychest x-ray, antibiotics, etc.
Con
Performance is not as good as culture, PCR, and
DFA
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The Connection BetweenInfluenza 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.
pneumoniaeinvasion.
S. pneumoniaehas 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 50years were made.
1957 and 1997 pandemic strains that were related
to bacterial pneumonia had highest levels of
neuraminidase activity.
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S. pneumoniaeand Penicillin
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Penicillin Breakpoint
IV Penicillin
Less expensive than broad spectrum antibiotics
Reduce broad spectrum antibiotic resistance
Less liver/kidney resistance
Minimum Inhibitory Concentration (MIC) (mcg/mL)
Susceptible (S) Intermediate (I) Resistant (R)
Updated 2 4 8
Previous 0.06 0.12-1.0 2
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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, andC.G. Whitney. Infectious Diseases Society of
America/American Thoracic Society ConsensusGuidelines on the Management of Community-AcquiredPneumonia in Adults. Clinical Infectious Diseases.2007; 44:S27-72.
Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaller,and R.H. Yolken. Manual of Clinical Microbiology 8thEdition.
Forbes, B.A., D.F. Sahm, and A.S. Weissfeld. Bailey &Scotts Diagnostic Microbiology 12thEdition.
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