Presentation on theme: "Director of Clinical Affairs"— Presentation transcript:
1Director of Clinical Affairs Diagnostic Testing for Community-Acquired Pneumonia (CAP) and InfluenzaNorman Moore, Ph.D.Director of Clinical Affairs
2ObjectivesDiscuss 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 analysisState the diagnostic testing methods recommended for community-acquired pneumonia and influenzaShow how influenza can lead to pneumonia
3Infectious 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 andthe 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
4Current 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 USAverage mortality is 10-25% in hospitalized patients with CAPNosocomial PneumoniaStandard definition: onset of symptoms occurs approx 3 days after admission250, ,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
5RSV in premature babies Etiological AgentsNewborns (0 to 30 days)Group B Streptococcus, Lysteria monocytogenes, or Gram negative rods are commonRSV in premature babiesInfants and toddlers90% 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.
6With the above agents, add L. pneumophila Etiological AgentsOutpatientS. pneumoniae, H. influenzae, M. pneumoniae, C. pneumoniae, and respiratory virusesInpatient (non-ICU)With the above agents, add L. pneumophilaInpatient (ICU)S. pneumoniae, S. aureus, L. pneumophila, Gram-negative bacteria, and H. influenzae
7Streptococcus pneumoniae Types – Over 90 serotypes exist, with 88% of disease covered in the 23-valent vaccineIncidence – 100,000 to 135,000 cases of pneumonia requiring hospitalization up to the year 2000Around 80% of CAPCases are dropping due to the S. pneumoniae vaccineTransmission – Person to personRisk groups – The young and elderlyMost common identification – Blood culture and sputum culture
8Haemophilus 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 diseaseIncidence – VariableTransmission – Person to personRisk groups – The young and elderlyMost common identification – Blood culture and sputum cultureManual of Clinical Microbiology 8th Edition. Page
9Chlamydia pneumoniaeIncidence – 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 influenzaTransmission – Person to personRisk groups – All age groups, but more common in school-age children.Most common identification – SerologyPersonal 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.
10Mycoplasma pneumoniae Incidence – Estimated 2 million cases and 100,000 pneumonia related hospitalizations in USTransmission – Person to person by respiratory secretions, usually close contactOutbreaks in crowded conditions like military and college which can last several monthsRisk groups – All age groups, but more common in school-age children and young adults.Most common etiological agent for adults younger than 30Most common identification - SerologyBailey & Scott’s Diagnostic Microbiology 12th Edition. Forbes, B.A., D.F. Sahm, and A.S. Weissfeld editors. Page 804.
11Legionella pneumophila Incidence – Estimates vary greatly from 15,000 per year to 100,000 per year in USTransmission – Contaminated waterOutbreaks in hospitals, ships, hotels, etc.Risk groups – Usually elderly, smokersMost common identification – Urinary antigenHorwitz, 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.
12Viral pneumoniaAdults 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 pneumoniaBailey & Scott’s Diagnostic Microbiology 9th Edition. Page 227.
13Influenza A&B Hospitalizations up from 114,000 to 226,000 Impact of influenza in the USHospitalizations up from 114,000 to 226,00036,000 deaths annuallyInfluenza target population: 188MM in US5-20% of US population affected by influenza each yearMost deaths affect elderly and young childrenAlso affects otherwise healthy individuals
14Influenza TreatmentAntiviral drugs are availableMust be administered within 48 hr of onset of symptomsGenerally reduce duration of symptoms by one dayFirst generation drugs (amantidine, rimantidine) are cheaper but only treat influenza ASecond generation drugs (Tamiflu®, Relenza®) are more expensive but treat both influenza A and BReason to differentiate between influenza A and B
15Respiratory Syncytial Virus Almost all children with have RVS by their second birthday25% to 40% will have signs or symptoms of bronchiolitis or pneumonia0.5% to 2% will require hospitalizationRecovery is in 1 to 2 weeks, but they can spread virus for 1 to 3 weeksThe elderly can get a usually mild RSV infection due to a weakened immune systemRapid tests are not recommended on this population
17Swab should remain moist and cultured within 4 hours Swab collectionSwab should remain moist and cultured within 4 hoursIf longer than 4 hours to get to culturing, should use transport mediumRefrigeration, not frozenBailey & Scott’s Diagnostic Microbiology 9th Edition.Page 224.
18Sputum 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 techniqueCollectionPatient is instructed to give a deep coughed specimenPut into sterile container, trying to minimize salivaTransport to lab immediatelyPatient unable to give specimen can be given an aerosol-induced specimenBailey & Scott’s Diagnostic Microbiology 9th Edition.Page 228.
19Blood culture Usually done with fever spike Standard is to take two sets of blood cultures one hour apartBailey & Scott’s Diagnostic Microbiology 12th Edition.Page 786.
20Does not need to be qualified like a sputum sample UrineUrine can be used for Legionella and Streptococcus pneumoniaeAntigen testNoninvasive sampleDoes not need to be qualified like a sputum sample
21Influenza Sample Collection Appropriate specimensNasal wash/aspirate, nasopharyngeal swab, or nasal swabThroat swabs have dramatically reduced sensitivitySamples should be collected within first 24 to 48 hours of symptoms since that is when viral titers are highest and antiviral therapy is effectiveTesting can be done immediately with rapids or sample placed in transport mediaInfectivity is maintained up to 5 days when 4-8°CIf the sample cannot be evaluated in this time period, the sample should be -70°C.
23Infectious Disease Society of America/American Thoracic Society Consensus Guidelines on the Management of Community-Acquired Pneumonia in Adults (2007)Diagnostic TestingSuggestive clinical features combined with a chest radiograph or other imaging technique is required for the diagnosis of pneumoniaIt 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.”
24Optional for outpatients with CAP Infectious Disease Society of America/American Thoracic Society CAP Guidelines 2007When to apply diagnostic testsOptional for outpatients with CAPBlood 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*
26Gram stain Apply sample to microscope slide Apply stains & view using standard microscopePros: InexpensiveRapid (~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
27Sputum Culture – Bacterial Culture Pros: InexpensiveStandard media for most – Sheep blood agar, MacConkey agar, and chocolate agar, BCYE for LegionellaAllows for antibiotic susceptibility testingCons: Requires live bacteria – antibiotics can affect resultsDifficult to get good sampleRequires dedicated tech time / experienced personnelResults take 24 hours to >1 week
28Legionella Culture Legionella Legionella needs specific growth conditionsBuffered charcoal yeast extract (BCYE) plateClinical sample may need to be acid treated to reduce general microfloraMay take 3 to 10 days to get result
29Cell culture for Chlamydia pneumoniae Chlamydia cultures should be transported in 2-sucrose phosphate or other transport mediumUse HeLa cell line rather than McCoy that is used for C. trachomitisMay take 3 to 10 days and is labor-intensiveBailey & Scott’s Diagnostic Microbiology 9th Edition.Page 232.Page 556.
30Culture for Mycoplasma pneumoniae Specialty mediaMay take over 3 weeks for resultVial is inspected daily and is prone to contamination (usually indicated by color shift in first 5 days)Needs subculturing to agarHighly labor intensiveBailey & Scott’s Diagnostic Microbiology 9th Edition.Page 558.
31Blood Culture Pros: Inexpensive Allows for antibiotic susceptibility testingHigh specificityCons: Requires live bacteria – antibiotics can affect resultsRequires dedicated tech time / experienced personnelResults take 24 hours to >1 weekMany bacterial infections don’tprogress to bacteremia
32Latex Agglutination Detecting antigen associated w/certain serogroups Polystyrene latex particles coated with antibodiesPros: Relatively simpleRapid (~15 minutes)Cons: Does not detect all serogroups of S. pneumoniaeProcedure associated with urine is cumbersomeInterpretation of results can be subjective
33Fluorescent Antibody (DFA/IFA) Performed directly from sample on microscope slideSputum, pleural fluid, aspirated material, or tissueAdd fluorescent-tagged antibody specific for specific bacteria Observe for fluorescence using a special microscopePros: Relatively quick turn around time (~1 hour)Cons: More labor intensive than rapid testsRequires trained technologist and special microscopeFew labs equipped to perform DFA on2nd/3rd shiftsSensitivity can be poor (25% to 75%on Legionella)
34Polymerase Chain Reaction (PCR) Molecular technique using a clinical sampleExtract and amplify nucleic acid (DNA or RNA) of specific pathogenPros: Extremely sensitive – can detect one microorganismDetects both live and dead pathogensCons: Requires highly trained technologist, expensive equipmentMore labor intensive than rapid testsProne to cross-contamination (false positives)
35Serology Chlamydia pneumoniae Mycoplasma pneumoniae Measurement usually of acute and convalescent serumA four-fold rise in titer is considered diagnosticA single IgM titer of 16 or greater or IgG of 512 or greater is considered suggestive of recent infectionMycoplasma pneumoniaeA fourfold rise from acute to convalescent serum or complement fixation titer of 1:128 in single serum specimenBailey & Scott’s Diagnostic Microbiology 9th Edition.Pages
36Urinary antigenTests are available for S. pneumoniae and L. pneumophila serogroup 1With Legionella, antigen appears in the urine 1 to 3 days after infectionNoninvasive sampleEasy-to-useKohler, R.B., W.C. Winn, Jr., and L.J. Wheat. Onset and duration of urinary antigen excretion in Legionnaires’ disease. J. Clin. Microbiol : 20:
37Test Procedure for Urinary Antigen Collect urine sample (no pre-treatment i.e. concentrating, boiling, filtering, etc.)Open device pouch and lay flatDip provided sampling swab into urinePlace swab in lower hole of swab well and push upAdd required number of drops of Reagent A (2 drops for Legionella test and 3 for S. pneumoniae)Close deviceWait 15 minutesInterpret results
38Diagnostic Methods for Influenza CultureDFAPCRRapid Tests
39Highly sensitive as long as sample is properly handled Viral CultureProHighly sensitive as long as sample is properly handledConCan’t give same day result to help monitor therapyHigh level of difficult/equipment
40Usually considered to have high level of sensitivity DFAProUsually considered to have high level of sensitivityCan usually test for other respiratory pathogens at the same timeResults can be achieved in same dayConLabor intensive needed experienced usersTurn-around time from lab usually takes many hours
41For respiratory specimens, high performance Same day results PCRProFor respiratory specimens, high performanceSame day resultsConTurn around time from lab is extensive, especially if batching specimensExpensiveRequires experienced technicians, labs, dedicated equipment, etc.
42Tests take minimal time Rapid TestsProTests take minimal timeSome tests are so simple that they can be CLIA-waivedCan be used to triage patientsPositive results can be used to rule out other issues like pneumonia so don’t give unnecessary chest x-ray, antibiotics, etc.ConPerformance is not as good as culture, PCR, and DFA
43The Connection Between Influenza and S. pneumoniae
44Pandemic outbreaksIn 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.
45Synergy 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.