Download Current Status of Pneumonia and Influenza Diagnostics

Diagnostic Testing for
Community-Acquired
Pneumonia (CAP) and
Influenza
Norman Moore, Ph.D.
Director of Clinical Affairs
[email protected]
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
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.
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
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
A&B, and parainfluenza. Bacterial infections are
rare, but could be S. pneumoniae, Hib, or S.
aureus.
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, Gramnegative bacteria, and H. influenzae
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
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
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 doesn’t
view this as a significant infection.
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
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
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
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
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
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
Specimen Collection
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
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 aerosolinduced specimen
Blood culture
• Usually done with fever spike
• Standard is to take two sets of blood cultures one hour
apart
Urine
• Urine can be used for Legionella and Streptococcus
pneumoniae
– Antigen test
– Noninvasive sample
– Does not need to be qualified like a sputum
sample
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 @ 48°C
– If the sample cannot be evaluated in this time period, the
sample should be frozen @ -70°C.
Diagnostic Methods Available
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.”
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*
Common Diagnostic Tests
•
•
•
•
•
•
•
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Gram stain
Sputum culture
Blood culture
Latex agglutination assays
DFA/IFA
PCR
Serology
Urinary antigen
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
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 >1 week
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
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
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
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 >1 week
Many bacterial infections don’t
progress to bacteremia
Latex Agglutination
•
•
Detecting antigen associated w/certain serogroups
Polystyrene latex particles coated with antibodies
Pros:
Cons:
Relatively simple
Rapid (~15 minutes)
Does not detect all serogroups of S. pneumoniae
Procedure associated with urine is cumbersome
Interpretation of results can be subjective
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)
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)
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 1:128 in single serum specimen
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
Test Procedure for Urinary Antigen
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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
Diagnostic Methods for Influenza
•
•
•
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Culture
DFA
PCR
Rapid Tests
Viral Culture
• Pro
– Highly sensitive as long as sample is properly
handled
• Con
– Can’t give same day result to help monitor
therapy
– High level of difficult/equipment
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
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.
Rapid Tests
• Pro
– Tests take minimal time
– Some tests are so simple that they can be CLIAwaived
– Can be used to triage patients
– Positive results can be used to rule out other
issues like pneumonia so don’t give unnecessary
chest x-ray, antibiotics, etc.
• Con
– Performance is not as good as culture, PCR, and
DFA
The Connection Between
Influenza and S. pneumoniae
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.
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.
S. pneumoniae and Penicillin
Penicillin Breakpoint
Minimum Inhibitory Concentration (MIC) (mcg/mL)
Susceptible (S)
Intermediate (I)
Resistant (R)
Updated
≤2
4
≥8
Previous
≤0.06
0.12-1.0
≥2
• IV Penicillin
– Less expensive than broad spectrum antibiotics
– Reduce broad spectrum antibiotic resistance
– Less liver/kidney resistance
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; 44:S27-72.
• 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 &
Scott’s Diagnostic Microbiology 12th Edition.