1. Director of Clinical Affairs
Diagnostic Testing for Community-Acquired Pneumonia (CAP) and Influenza
Norman Moore, Ph.D.
Director of Clinical Affairs

2. 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

3. 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.
(1) New Eng Jr of Medicine, Bartlett & Mundy – Community-Acquired Pneumonia; 1995, vol. 333, no. 24, pp

4. 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 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 cases of nosocomial pneumonia per year
% mortality rate
(1) Annals of Internal Medicine, 2001; 134: Ralph Gonzales, et.al. “Principles of Appropriate Antibiotic Use for Treatment of Nonspecific Upper Respiratory Tract Infections in Adults: Background”
(2) Bartlett et. al. IDSA Guidelines for CAP in Adults; Clin Inf Dis 2000; 31:
(3) New Eng Jr of Medicine, Bartlett & Mundy – Community-Acquired Pneumonia; 1995, vol. 333, no. 24, pp
(4) Sherwood L. Gorbach, et.al. Guidelines for Infectious Diseases in Primary Care, 1999

5. RSV in premature babies
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.

6. With the above agents, add L. pneumophila
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

7. 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

8. 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
Manual of Clinical Microbiology 8th Edition. Page

9. 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.
Bailey & Scott’s Diagnostic Microbiology 12th Edition. Forbes, B.A., D.F. Sahm, and A.S. Weissfeld editors. Page 804.

10. 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
Bailey & Scott’s Diagnostic Microbiology 12th Edition. Forbes, B.A., D.F. Sahm, and A.S. Weissfeld editors. Page 804.

11. 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
Horwitz, M.A., et.al. Prospects for vaccine development. Presented at the 4th International Symposium on Legionella, Legionella  - Current Status and Emerging Perspectives – ASM; editors – J. Barbaree, et.al.

12. 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
Bailey & Scott’s Diagnostic Microbiology 9th Edition. Page 227.

13. Influenza A&B Hospitalizations up from 114,000 to 226,000
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

14. 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

15. 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

16. Specimen Collection

17. Swab should remain moist and cultured within 4 hours
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
Bailey & Scott’s Diagnostic Microbiology 9th Edition.
Page 224.

18. Sputum Collection Quality of specimen Collection
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
Bailey & Scott’s Diagnostic Microbiology 9th Edition.
Page 228.

19. Blood culture Usually done with fever spike
Standard is to take two sets of blood cultures one hour apart
Bailey & Scott’s Diagnostic Microbiology 12th Edition.
Page 786.

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

21. 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 4-8°C
If the sample cannot be evaluated in this time period, the sample should be -70°C.

22. 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.”

24. Optional for outpatients with CAP
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*

25. Common Diagnostic Tests
Gram stain
Sputum culture
Blood culture
Latex agglutination assays
DFA/IFA
PCR
Serology
Urinary antigen

26. 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

27. 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

28. 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

29. 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
Bailey & Scott’s Diagnostic Microbiology 9th Edition.
Page 232.
Page 556.

30. 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
Bailey & Scott’s Diagnostic Microbiology 9th Edition.
Page 558.

31. 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

32. 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

33. 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)

34. 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)

35. Serology Chlamydia pneumoniae Mycoplasma 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
Bailey & Scott’s Diagnostic Microbiology 9th Edition.
Pages

36. 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
Kohler, R.B., W.C. Winn, Jr., and L.J. Wheat. Onset and duration of urinary antigen excretion in Legionnaires’ disease. J. Clin. Microbiol : 20:

37. 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

38. Diagnostic Methods for Influenza
Culture
DFA
PCR
Rapid Tests

39. Highly sensitive as long as sample is properly handled
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

40. Usually considered to have high level of sensitivity
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

41. For respiratory specimens, high performance Same day results
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.

42. Tests take minimal time
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 don’t give unnecessary chest x-ray, antibiotics, etc.
Con
Performance is not as good as culture, PCR, and DFA

43. The Connection Between Influenza and S. pneumoniae

44. 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.

45. 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.

46. S. pneumoniae and Penicillin

47. Penicillin Breakpoint
Minimum Inhibitory Concentration (MIC) (mcg/mL)
Susceptible (S)
Intermediate (I)
Resistant (R)
Updated ≤2 4 ≥8
Previous
≤0.06 ≥2
IV Penicillin
Less expensive than broad spectrum antibiotics
Reduce broad spectrum antibiotic resistance
Less liver/kidney resistance

48. 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 ; 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.