Slides+for+Posting+Lectures+21+ +22+Diagnoses+of+Infectious+Diseases+I+ +II

8/13/2019 Slides+for+Posting+Lectures+21+ +22+Diagnoses+of+Infectious+Diseases+I+ +II

http://slidepdf.com/reader/full/slidesforpostinglectures21-22diagnosesofinfectiousdiseasesi-ii 1/36

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.




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

DJ

– 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




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)




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




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.




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




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




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


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.




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.


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.






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.




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.




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.




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




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




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%.




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

.




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.






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.






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








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.




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.




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.

DJ

– . – 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




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.

DJ

• 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

DJ

• 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

DJ

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.




MICR570/DJ/F12 21-22/29

Disk Diffusion Test

• Developed by Drs. Bauer, Kirby.• Mueller Hinton agar plate.

– Seeded with organism f rom patient.

DJ

• 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

DJ

BK Plate and Ruler for Measurements

DJ

Bauer Kirby TestInterpretation

SS

DJ

RR

II




MICR570/DJ/F12 21-22/30

Problems with Bauer KirbyDisk Tests

• Overnight incubation required.•

DJ

– 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.

DJ

• . – 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

DJ

Automated Bac ter ial ID andSusceptibility Testing

• BD Phoenix System – Uses plastic test tube tray – Does identification – Susceptibility test

DJ

– Rapid 4-12 hours• Perhaps most

accurate of rapidsystems

– Upcoming

system




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

DJ

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

DJ

Cefotaxime plus abeta-lactamase

inhibitor

Confirmation of Extended Spectrum Beta -Lactamase in Gram Negative Rods

A A+

DJ

B B+




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

DJ

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:

DJ

– • 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.




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

DJ

Klebsiella Pneumoni ae Carbapenemase

• KPC is a -lactamase – Confers resistance to all -lactams incl uding extended-

spectrum cephalosporins and carbapenems

DJ

• 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






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. –

DJ

• 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.

DJ

– 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.




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%

DJ

(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:

DJ

– 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.

Documents

Slides+for+Posting+Lectures+21+ +22+Diagnoses+of+Infectious+Diseases+I+ +II