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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/1
Lab Diagnosis of InfectiousDiseases and Proper Pre and
Post Testing Procedures
Donald Jungkind, Ph.D.Director, Clinical Microbiol ogy
Professor, Pathology and MicrobiologyThomas Jefferson University
Road Map for the Series• Lectures:
– Lab tests and specimen collection choices• What physi cians need to know.
– Antibiot ic susceptibil it y tes ting:• Wh it is more than ust “S” , I, and R
DJ
– Skin, soft ti ssue, and bloo dstream• Bacteria• Fungi/Parasites• Viruses
– Tie it together with ill ustrations!• Case studies
Diagnosis o f Infectious DiseasesNon-Specific Signs and Tests
• Physical signs – Fever – Pain
• General lab t ests – CBC
• Increased WBC count – Lymphocytes
DJ
– – Redness – Localized signs
• Headache – Many other signs
• Imaging
– eu r op es – Eosinophiles
– Non-specific tests:• Increased RBC sedimentation
rate• Elevated C-Reactive protein
– Localized changes• CSF: Increased protein• Abnorm al li ver func tio n
Test Accuracy vs. Precision
• Precision – How consistent
are the testvalues?
DJ
• Accuracy – How close are
the test valuesto the “true”value.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/2
Accuracy and Prec is ion: Graph
DJ
Sensitivity Vs. Specific ity
• Tests give overlappingbell curves. – Red = True osi tives
1
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– Blue = True negatives
• Test #3 is the best testbecause it would havefewest number of falsepositives andnegatives
2
3
Predictivevalue of apositive or
DJ
for a dis ease
Predictive Values Defini tion
• The predictive value of a test is a measure(%) of the times that the value (positive ornegative) is the true value.
DJ
– i.e. Percent of all positive tests t hat are true posit ivesis the Positi ve Predicti ve Value.
• __TP___ X 100 = Predict ive Value of a Posi tive Resu lt (%)• TP + FP
• __TN___ X 100 = Predi ct ive Value Negati ve Resul t (%)• FN + TN
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/3
Impact of Disease Incidenceon Test Sensitiv ity
• If a test is 98 % sensiti ve and specific it so undsgood. – If the population has a disease prevalence of 1% in the
general population, there will be 1 true positi ve and 2 false
DJ
p os v es p er r an o m es s .• That means 67% of the positives will be false positives.• The predictive value of a positive is only 33 %
– If by careful patient selection, you can i ncrease theprevalence in those tested to 10%, the test will performmuch better, and a positive Rx wi ll mean more.
Historical Perspective on SpecificTesting For Infectious Diseases
• First 100 years – Traditional cultures
DJ
– Slow answers. Manual labor intensive.• Last 30 years
– Traditional cultures plus:• Immunological detection of organisms.• Molecular tests for organisms.
Culture Takes Skill and Many SpecialReagents to Workup Signifi cant Colonies
DJ
Rapid Viral Culture SystemsRapid Viral Culture Systems
•• CytomegalovirusCytomegalovirus•• Herpes Simplex VirusHerpes Simplex Virus•• VaricellaVaricella--Zoster VirusZoster Virus
DJ
•• Respiratory VirusesRespiratory Viruses•• EnterovirusesEnteroviruses•• Others (Measles,Others (Measles,
Rubella)Rubella)
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/4
Traditional Methods Require Non-homogeneous Processes, Each WithBulky Equipment Such as Incubators
DJ
Traditional Methods Require Blood Agar,Extracts from Plants and Animals, andLive Animal Cells of Various Species.
• Sheep blood agar • Shell vial ti ssue culture
DJ
Blood CultureInstrument
DJ
Manual Identification of Colonies• Large test tub es with biochemicals vs.
biochemicals in small cups – Read as positi ve or negative and look in r eference
database for name.
DJ
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/5
Automated Al ternat ives
• MicroScan – Uses 96 well plasti c plates – Does identification – Susceptibilit y test
DJ
• Accurate – Rapid versions
• Slightly less accurate
Automated Al ternatives
• bioMérieux Vitek – Uses card size plastic plates – Does i dentification – Susceptibili ty test
-
DJ
-• Accuracy OK• Clinically useful
Automated Al ternat ives
• BD Phoenix System – Uses plastic test tube tray – Does identification – Susceptibilit y test
DJ
– -• Most accurate of rapid
systems – Upcoming system
Al ternatives to Culture:
• Immunoassays – Detection of specific microbes using
DJ
. – Detection of antibodies to microbes
using specific antibody capture.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/6
Diagnosis o f Infection UsingImmunoassays for Antibody
Timing of Antibody Response InNatural Infection
IgG i t e r
DJ
• IgM is marker of acute infection
• IgG can be a marker of recent or old infection.
Weeks Months Years10 2 4 2 3 4
IgM A n
t i b o
d y
Immuno diagnosis of AcuteInfections
• Draw an acute serum – Within the first week or as soon as possible before 14 days.
• Titers are usually zero or low at < 7 days.
• Draw a convalescent serum 3-6 weeks after the fi rstserum.
DJ
– Look for a 4 fold or greater titer increase due to IgG.• If initial titer is 1:4, the convalescent titer should be 1:16 or greater if
they are both tested the same way. – Usually titers go up to 1:64 or more after an acute infection.
• Most tests today use ELISA, so the serum “ti ter” i sgiven as an optical density.
– Each company will list the optic al density change that equals a>= 4 fold increase in titer.• The significant increase threshold should be printed on your lab report.
Antibody Titer Tests :Results Are HighestDilution of Serum StillGiving a Positive Rx:1:2, 1:4, 1:8 -----1:1024
DJ
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/7
ELISA Kit and InstrumentMicrowell Plate Result s: Optical Density
DJ
What to do i f you miss the acute titer ?
• Draw a convalescent titer at the peak. – 3-6 weeks after the start of the infection.
• A very high convalescent t it er, and the clin icals m toms a ree the infection can be
DJ
presumptively confirmed – A s ingle pos it ive ant ibody usually does n ot allow
a definitive confirmation of most acute infections. – Exceptions are the chronic infections:
• Lyme disease• HIV infection• Hepatitis infections
Diagnosis of Chronic Infections?The Acute Phase is Usually Missed
• For HIV and Lym e – Do a Western Blot ass ay to check for presence
of a variety of antibody types.
DJ
HIV Western Blot
DJ
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/8
Diagnosis of Chronic Infections?The Acute Phase is Usually Missed
• For hepatitis B – Check for several antibo dy markers.
• For HIV and Hepatit is B or C, you can also check
DJ
. – PCR for HIV, hepatiti s B, and h epatitis C – Ant igen immunoassay for hepati ti s B .
A Special Case:Congenital Infections
DJ
Natural Loss of Maternal Ab:No Congenital Infection in Baby
i t e r
Ant ibo dy i n baby du e to p lacen taltransfer from mom
DJ
Weeks Months Years10 2 4 2 3 4
IgG
No continued IgG orappearance of IgM witho utcongenital infection
A n
t i b o
d y
Ab t it er f rom mom drops by ½ each month aft er bir th .
Test for Congenital Infection• Check antibody t iter fo r agents suspected (CMV,
Toxoplasma etc.) – Test at birth (cord blood serum) – Test again after 3 or 4 mon ths.
DJ
– • IgM should not be present.• IgG from mother w ill di sappear at rate of 50% drop
every month. – In 3 or 4 months, the baby titers are low or negative.
• Initial titer 1:16 drops to 1:8, then 1:4, and 1:2, in 3 months.
– Baby positive for infection:• IgM will be positive and IgG titers will go up.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/9
Antibody Response In True CongenitalInfection
IgM i t e r
DJ
• Rising and continuous IgM is marker of acute infection
• IgG is usually a lifelong marker.
Weeks Months Years10 2 4 2 3 4
IgG
A n
t i b o
d y
Immunoassay Kits
• Generation 1 and 2 are the olderversions of agglutination, early
DJ
vers on an s m ar es sfrom the 50’s t ill the 1980’s – Accuracy was jus t “ OK” – Techniques complex and harder to
reproduce across people and labs.
Third-Generation Immunoassays• Very convenient to perform.
– Used in a point-of-care setting.
• Sensitivity and specificity still “ just OK” ,
DJ
typical of enzyme immunoassays. – Barely adequate or sometime inadequate!
• Single analyte tests.
– They easily detect one thi ng. – Solid phase or “ Membrane” based.
Binax NOW Influenza (Binax, Inc.)Binax NOW Influenza (Binax, Inc.)
• One of the easiest touse. – Only 1 step to the assay
• So easy to do that alaboratory license from
DJ
e governmen s norequired.
• Specially trainedpersonnel are notrequired to perform thetest.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/11
BD DirectigenBD DirectigenFLU A: 3FLU A: 3 stst GenGen FLU A + B: 4FLU A + B: 4 thth GenGen
DJ
Cultures and immunoassayswere either sensitive or fast,
but not both.
• We needed a breakthrough
DJ
. – Molecular amplif ication was that
breakthrough.• Discoverer of PCR won Nobel prize.
History of New Technology• Increasing sensitivi ty
EIA 100,000 - 10,000
RIA, FIA 5,000 - 1,000
DELFIA 100
NUCLEIC ACID AMPLIFICATION
Importance of Nucleic Acid Ampl if icat ion in Clinical Labs
• PCR:
PCR
– automation.
• PCR automation: – Key to lower cost – Could replace manual cultures.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/12
First Molecular Nucleic Acid Ampl ification Method:
• Target amplification – PCR
DJ
12 3 4
Target“fingerprint”
Detection Ampl if ic ation &Probe capture
First Automated PCR Amplification andDetection : COBAS Ampl icor , Early 1995.
DJ
COBAS AmpliPrep InstrumentRolled Out for Worldwide Distribution
at a Meeting in Prague in 2001
Evolutionary Change in Ampl if ied Target Detection
• Real-timequantitative
DJ
detection. – 1999 to 2009
and beyond.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/13
RealReal--Time PCRTime PCR•• Simple to performSimple to perform
– – Rx occurs in a single tubeRx occurs in a single tubethat does not have to bethat does not have to behandled or o pened afterhandled or o pened afterputting in the specimen.putting in the specimen.
••
DJ
– – Works with RNA and DNAWorks with RNA and DNAtargets.targets.
•• A probe with a A probe with afluorescent tag binds withfluorescent tag binds withthe RNA or DNA target.the RNA or DNA target. – – This binding results in buildThis binding results in build
up of fluorescent lightup of fluorescent lightcoming from the positivecoming from the positivesample tube.sample tube.
Roche LightCycler
DJ
DJ
Emerging DiagnosticTechnologies
• Microarrays – For microbial identification – For mi cr obi al t i n
DJ
– For detection of host responses to infection. – Unique to mi croarrays
• Abili ty to do 10 to 20,000 molecular tests on a
singl e specimen. One tiny dot on a glass slid eor one tiny bead in a liquid suspension isneeded to accomplish each of the many tests.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/14
Solid Chip MicroarrayCartridge
DJ
Types of Arrays
• Microbe oriented – Look for pathogen – Direct checks for multiple possible microbes.
• Host ori ented – Check for the patient’s response to infection.
DJ
• Look for pathogen indirectly by checking host’sRx profile to m atch pathogen Rx data bank.
– Check for host characteristics that predict:• Worse outcome with that microbe.• Problem wi th a particular antimicrobial drug.
– HIV infected patients with a unique genotype are 100Xmore likely to have a hypersensitivity Rx to abacavir.
Liquid Array Reactions inLiquid Format
DJ
MicroBeads Capture a Protein orNucleic Acid Target
DJ
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/15
Finally A Nucleic Acid Ampli fication (NAT) Test for
the Rest of Us
DJ DJPoint of Care NAT
Newest Technology Advance
• For about the effort and time that ittakes to do a Gram stain of abacterial colony, it is now possible
DJ
o en y e co ony. – Takes 15-30 minu tes and < $1.
• MALDI-TOF
– Matrix assisted laser desorptionionisation time of flight
MALDI-TOF
• MALDI – Based on the bombardment of sample
molecules with a laser light to bring aboutsample ionization.
DJ
• The time of flight of the charged particlesand their pattern is compared to adatabase to establi sh the genus andspecies. –Used in Europe and now making its way to USA
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/16
MALDI-TOF Principle
DJ
MALDI-TOF Instruments
DJ
Technology Summary• Cultures give pos . vs neg. answers
– An organism is present and grows or it is notpresent and does not grow .
• Chemistry style (molecular tests or
DJ
. – The endpoi nts are numerical values based on a
color producti on with varying degrees ofintensity.
– The same sample can be run multiple t imes wit h
different color intensity endpoints. – Positive vs. negative patients also give bellcurves showing a variety of color endpoints.
Disease Prevalence in the Population Affec ts Pred ic tive Values
• Examples: – A test is 98 % sensit ive and specif ic so it has a
false ne ative and a false ositive rate of 2%.
DJ
• If incidence of di sease in pop ulation = 1%• If 100 people are randomly t ested, there will
be 1 true posi tive and 2 false posit ives.
– Of the 3 positives, 67% wil l be false positives. – The predicti ve value of a positi ve in that
situation is only 33%.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/17
Technology Summary• It important to make sure your patients
understand: – Tests are FDA approved wi th a certain % of
false pos. and neg. reactions as a given.•
DJ
tests OR better confi rmatory tests. – It is unus ual to have a single test that does it all – Always rely on your c linical j udgement and
don’t treat the test. Treat the patient – Be ready to recollect a sample and be ready to
repeat tests with different methods.
End of Lab CenteredTesting Phase
• On to the pre and pos ttes tin activi ties!
DJ
Working asa team
DJ
Activ it ies Related to Lab Tes ts1. Doctororders test 2. Someone
sees order and acts.
3. Order is enteredi nto c om uter o r o n
9. Results reported
10. Doctorprocesses resultsand acts.
DJ
paper request form.
4. Someone collectsspecimen at some point intime and from some
specific site5. Specimen is labeled and placedsomewhere for pickup by couri er.
6. Specimen istransferred to lab.
7. Lab initiates computer details to put specimen onwork lists for required tests
.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/18
Issues to Consider forOptimum Specimen Handling
• Appropriate specimen type.• Guidelines for collection.
DJ
– Quantity and ti ming – Device for collection and
transport – Transport tim e – Storage time
Responsibi lity of Lab whenRejecting Specimens
• Lab must screen specimens promptly. – Doctor / nurse must be notified of
DJ
specimen rejection immediately.• Name of persons involved should be
recorded.• Inadequate specimen should be saved
at 4 C for a short time.
Situations for Rejection of Specimensthat are Beyond Salvage
• No hope situations such as: – Misidentification of s pecimen – Culture s ecimens received in fixative
DJ
– Dried-out specimen on swab – Insufficient specimen quantity
• If specimen size is large, there may beplenty of material for multipl e types ofcultures and tests. If not, some cultureson the t est request list must be c anceled.
Criteria for Rejection of SeriouslyCompromised Specimens
• Improper sto rage temperature – Look out f or hot c ars in summer, freezing
conditions i n w inter, and unrefrigerated urine
DJ
• .• Improper transport time.
– Some microbes increase and some die.• Leaky specimen.
– Contamination of specimen – Biohazard to the technologist.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/20
“ Contaminated Specimens”• Two types:
– Expected contamination• Specimens which unavoidably contain
normal flora which needs to be distinguished
DJ
. – Unexpected contamination
• Sterile specimen containing introducedbacteria – Blood culture with inadequate cleaning of site – Cerebrospinal fluid with mouth flora from
“talkers” during collection.
Blood Cultures• Continuous bacteremia
– Volume dependent. – Collect 20 - 30 mL per set
• 2 or 3 sets / episode. –
DJ
– 1 anaerobic bott le per draw.
• Intermittent bacteremia – 2-3 sets before antibiotics.
• Collect each set from newvenipuncture site.
– Two sticks is minimum.
Things to Avoid In BC Collection
• Don’t disinfect collection site with alcohol only. – Use iodophor or chlorhexidine for 2 minutes.
• Don’t initially collect >= 5 blood cult ure sets todiagnose a single clini cal episode.
’
DJ
– so, on co ec on y oo cu ure se .• Don’t collect only 1 bottle per set.
– Collect one aerobic and one anaerobic bottle• Don’t collect all sets from a 1 needle stick.
– Don’t collect blood cultures from femoral veins orvenous catheters.
Blood Culture Issues• 3 % increase in yield /mL.
– Collect 20 ml / set and 2-3 sets.• Central venous catheters
– Semiquantitative culture of CVC tip is
DJ
culture cultur e from a peripheral vein is taken.• > 15 colonies from CVC tip i s signi ficant.• Matching positi ve blood culture is us eful .
• Postmortem blood rarely useful. – Organisms leave normal flora sites and spread
to “ sterile” sites within an hour.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/22
Using Sputum to DiagnoseBacterial Pneumonia
• Goal of sputum culture is to identify
DJ
– Cough sputum has lower respiratory
tract organism plu s saliva,nasopharyngeal secretions, food, andoropharyngeal bacteria.
Cough Sputum Cultures
• Avoid saliva. – Small quantities of clear thin material is
usually saliva from the mouth.
DJ
• bacteria from saliva may be misleading. – Saliva can be detected because it has
squamous epithelial cells and few whit eblood cells (pus cells).
Advantages of Using GramStain to Evaluate Sputum
• Can reject poor specimens – Ratio of pus cells to epithelial cells.
DJ
– aves me an e or .
• Eventually trains staff to col lect betterspecimens. – Avoids consideration of wrong “ pathogen”
for treatment.
Murray and Washington System
• Number of cells / low power fieldGroup Epithelial Cells Pus Cells
1 >25 10
DJ
2 >25 10-253 >25 <254 Ref 1 10-25 >25
5 Ref 2 < 10 25
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/25
Diarrhea Arising in HospitalizedPatients Is Not the Same as
Outpatient Reasons for Diarrhea
• Community acquired diarrhea: – Occurs before 3 da s in hos ital
DJ
• May be due to:
– Parasites – Enteric bacterial pathogens
• Salmonella, Shigella, Campylobacter . – Viral causes
Hospitalized Patients withDiarrhea
• C. difficil e toxin
DJ
– have been in hospi tal >3 days• Patients with recent antibioti cs
and/or chemotherapy
Laboratory Stool Exams
• Cultures routinely detect: – Salmonella spp., Shigella spp.,
Campylobacter spp.
DJ
• By special request only: – E. coli 0157:H7, Vibrio, Yersinia .
• If you suspect these, tell the lab!
• C. diffi cile test is a toxin assay on stoo l.
Neisseria gonorrhoeae• Place genital swab in
transport medium. – Do not refrigerate. – -
DJ
.• Plate rectal swabs on
selective medium. ie:Thayer Martin.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/26
Urethral Specimens in Males• Use special flexible metal shaft sw ab with
small dacron tip.• Insert approximately 2 cm. and rotate 10 X.
DJ
– .
• Chlamydia PCR on u rine specimen makesurethral swabs unnecessary.
Endocervical Specimens• Cervical os cleaning swab
is good for GC.• Chlamydia cultures
DJ
. – Sample endocervical canal
• Vigorously rotate > 10 X.• Cytobrush gets cells.
“Wound” Specimens• Tell lab the anatomical site.• Surface woun ds have skin
flora as pathogens andcontamination.
DJ
– Don’t sample superficial pus. – Do sample advancing edge of
deep lesion o r abscess• Deep wound sample OK.
– Requires invasive procedureto collect deep specimen.
Do’s and Don’ts For LikelyContaminated Surface Wounds
• Don’t swab superfic ial layers – Don’t plunge swab thr ough contaminated
zone tr in to o dee er.
DJ
• Don’t use surgical technique to take asuperficial specimen with surfacecontamination. – Do use surgical procedure or biopsy to take
deep specimen w hile bypassingcontamination zone.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/27
Ideally , send the specimen ,not a swab of the specimen.
– Karin McGowan, Ph.D.,’
DJ
ren s osp a ,Philadelphia, PA
During an invasive procedure like surgery,remove a bit of tissue, or scrape the infectedsurface of a prosthetic device like a joint.
Anaerobic Bacterial Cultures• Anaerobes live 1- 3 hours at 20
C in large specimens.• Anaerobic transpor t devices
extend survival to 8-24 hours
DJ
a .• Avoid swabs.
– Tissue or several mL of flui d isbetter for stains & gr owth
Rejections Related to Anaerobes
• Don’t culture these for anaerobes: – Fecal contaminated specimens. – Midstream and cath urine.
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– . – Throat, nose, oropharyngeal swabs. – Gastric contents. – Vaginal and cervical swabs
– Superficial material from skin:• Wounds, ulcers, eschar, sinus tracts.
Time to move on to SusceptibilityTesting
DJ
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/28
Correlation of In Vitro withIn Vivo Result s
• Doctor must kno w expected blood level. – Dose and excretion dependent.
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• You must know the antibiotic penetrationlevel into i nfected area. – Other site specific issues can change levels.
• pH, blood supply• Know previous cl inical trial results.
Reference Antib iotic Susceptibili ty Tests(Gold Standards)
• Minimum Inhibitory Concentration. – MIC
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• Least antibiotic concentration that inhibits growth. – Ant ib ioti c c onc entr ation m easur ed in ug/ml .
• Usually takes more labor and expense to producethese answers.
Example of MIC1ug 2 ug 4ug 8ug 16ug 32 ug1 64 ug 128 ug
Cloudy TubesCloudy Tubes Clear TubesClear Tubes
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First tube with no growth (clear)
MIC = The lowest concentration of antibiotic thatstill inhi bits growth of the organism.What is the MIC in thi s case? ____ ug/ml.
Minimum bacteriocidal concentration MBC :
Problems with MIC Tests
• Can be expensive.• Each d rug t akes multiple tubes.
DJ
• Needed: – Quick, inexpensive way to approximate
MIC interpretations.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/29
Disk Diffusion Test
• Developed by Drs. Bauer, Kirby.• Mueller Hinton agar plate.
– Seeded with organism f rom patient.
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• Swab soaked with organism.• Position paper disks
– Impregnated with antibiotic.• Measure zone sizes in mm.
– Interpret zone sizes as: S, I, R.
BK Dispenser and Plate Incubator
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BK Plate and Ruler for Measurements
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Bauer Kirby TestInterpretation
SS
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RR
II
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/30
Problems with Bauer KirbyDisk Tests
• Overnight incubation required.•
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– Misses 1 out of 12 results.
• Increasing numbers of Bug/Drugspecific contraindications limit itsability to be a universal test.
Other Ways to Produce an MIC
• E-test – Antibiot ic is impregnated onto a rectangular
plastic strip.
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• . – Test is done li ke a BK test except that an E-test
strip is placed on the agar rather than paperdisks.• MIC can be estimated by reading the test.
E-Test MIC vs. Bauer Ki rby Disk Test
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Automated Bac ter ial ID andSusceptibility Testing
• BD Phoenix System – Uses plastic test tube tray – Does identification – Susceptibility test
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– Rapid 4-12 hours• Perhaps most
accurate of rapidsystems
– Upcoming
system
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/31
Extended SpectrumBeta- Lactamase (ESBL):
• ESBL's are plasmid-mediated B-lactamases that confer resistance topenicill ins, cephalosporin s, and aztreonam
DJ
– Mainly in Klebsiella pneumon iae, Klebsiellaoxytocia, Escherichia coli
– In a few other genera of the familyEnterobacteriaceae that are usuallysusceptible to these agents.
ESBL Enterobacteriaceae• Appear suscept ib le in-vitro to second
generation cephalosporins (cefoxitin) andresistant to fir st (cephalothin) and many ofthe thi rd enerat ion cefotaxime
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cephalosporins. – For test interpretation for all ESBL-producing
strains, they should be regarded as clinicallyresistant for all penicillins, cephalosporins, andaztreonam regardless of the in-vitro result.
Confirmation of ESBL’sPositive Pattern
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Cefotaxime plus abeta-lactamase
inhibitor
Confirmation of Extended Spectrum Beta -Lactamase in Gram Negative Rods
A A+
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B B+
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/32
How to Report ESBL’s
• Presumptive identification of ESBL. – Lab computer automatically adds a c omment
to the re ort al er tin ou to re ard an
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sensitive in-vitro B -lactam antibiotic resultsas possibly clinically resistant.
• Confirmed identification of ESBL. – Lab automatically reports all Beta-lactams as
resistant to avoid confusion.
Inducible Beta- Lactamase:
• An enzyme conferring res is tance topenicillins and cephalosporins in certaingram negative rods.
DJ
– This resistance is insi dious in that it is delayedresistance that develops slow ly and is oftenmissed in susceptibility testing.• There is NO economical way to detect this
resistance by rou tine susceptibility testingprocedures.
Inducible Beta- Lactamase
• Sometimes occurs w ith certain speciessuch as:
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– • Klebsiella,, Enterobacter, some E. coli
– Other organisms – less common:• Citrobacter, Aeromonas, Pseudomonas ,
and a few other genera .
How to Report Possibility ofInducible Beta -Lactamase?
• Include a message in the lab reportalerting the physician. – Routine laboratory tests do not reliably detect
DJ
e pr esence o n uc e - ac am ases nthese species.• Physicians must be aware of the possi bility
of emergence of resistance in-vivo if patientis being tr eated with B-lactam antibiot ics. – Carefully monitor the patient.
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/33
One Of the Several Induc ible Beta-LactamasesJust Became Ready for Limited Routine
Testing: Amp C
• Inducible Amp C beta-lactamase is on aplasmid in some Enterobacteriaceae : – Exam le if Klebsiella has these susce tibilit test
DJ
characteristics consider testing for Amp C:
• Cefoxitin intermediate or resistant.• Ceftazidime and cefoxitin intermediate or resistant
and ESBL confi rmatory t est negative.• Reduced susceptibility to cefotetan.
Another Form of Resis tance:Inactivation of Carbapenems:
Imipenem, Meropenem, Ertapenem
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Klebsiella Pneumoni ae Carbapenemase
• KPC is a -lactamase – Confers resistance to all -lactams incl uding extended-
spectrum cephalosporins and carbapenems
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• Occurs in Enterobacteriaceae – Most commonly in Klebsiella pneumoniae – Also repo rted in: K. oxytoca , Citrobacter freundii ,
Enterobacter spp., Escherichia coli , Salmonella spp.,
Serratia spp.,• Rarely in Pseudomonas aeruginosa
Susceptibility Profile of KPC-Pos. K. pneumoniae
Ant imi cro bia l Int erp ret atio n Ant im icr obi al Int erp ret atio n Amik acin I Chlor amphen ico l R Amox/ clav R Cipro flo xacin R Ampi cil lin R Ertapen em R Aztreo nam R Gentam ici n RCefazolin R Imipenem R
DJ
Cefpodoxime R Meropenem RCefotaxime R Pipercillin/Tazo RCetotetan R Tobramycin RCefoxitin R Trimeth/Sulfa RCeftazidime R Polymyxin B MIC >4 g/ml
Ceftriaxone R Colistin MIC >4 g/mlCefepime R Tigecycline S
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Diagnoses of Infectious Diseases I & II
MICR570/DJ/F12 21-22/35
When to Suspect a KPC-Producer
• Enterobacteriaceae – especially Klebsiellapneumoniae that are resistant to extended-spectrum cephalosporins: –
DJ
• Mixture of susceptible, intermediate, or
resistant answers. – Ertapenem is most often the firs t to be “ R”
– Resistant or intermediate to:• Cefoxitin and Cefepime.
Confirmation of Carbapenemase Activity
• PCR for KPC gene – Gold Standard accuracy. –
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• In-vitro methods exist but are not routinely
and widely available to physicians.
Antibiograms: How To In terpretThem
DJ
Empiric Antimicrobial Therapy is BasedUpon the Following:
• Historical susceptibility patterns. – Antibiograms from the reg ion.
• Bod site and acuit of disease.
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– Tempo of disease and antibiotic penetration i ntoaffected organ.
• Likely organism(s) involved.
• Clinical trial data. – Published cure rates for different antibiotics insimilar types of infection.
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Diagnoses of Infectious Diseases I & II
Susceptibili ty Report on anIndividual Patient
E. coli Antibiotic MIC Interpretation
Ampici ll in 128 ug/ml Resistant
DJ
Imipenem 1 ug/ml Susceptible
Piperacillin 64 ug/ml Resistant
Annual Antibiogram for Hospital
Organism Number Amp. Imip. Pip.
E. coli 2835 59% 100% 66%
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(63) (100) (62)Klebsiellapneumonia
1014 2%(1%)
100%(100)
63%(73)
Pseudo.aeruginosa
1677 00
83%(76)
89%(80)
Putting It All Together - 1
• Clinical signs and/or history point to infection possibi lity – May be supported by non-specific tests.
• Clinician orders specific microbiology tests to detect theorganism:
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– Stains, cultures, PCR, immunoassays for organism.• Susceptibility testing, is performed on micr obes where the
susceptibility/resistance pattern is not highly predictable. – Many bacteria are tested. – Only a few viruses and f ungi are tested.
• Tests for antibody are not ordered for most infections if moredirect tests work well.
Putting It All Together - 2• Clinician decides whether to w ait for test r esults
or to tr eat. Almost 100% of time they init iatetreatment. – For acute infection, the decision often is made to treat
before the specific lab test results are available.
DJ
• Treatment choi ces are made on empiri crecommendations and review of the literature.
• When results of identification and susceptibilitytesting co me back, the treatment can be modif ied.
• The data and experience of that infecti on goesinto helping direct future diagnosis and treatment – Personal experience.