乙型鏈球菌篩檢的檢驗標準作業流程ƒ安靜.pdf ·...

乙型鏈球菌篩檢的檢驗標準作業流程

林口長庚醫院醫檢教研部

郭安靜

2012/9/15

2

大綱

The 2010 CDC Early Onset GBS Disease Prevention GuidelinesSpecimen collection

Enriched brothChromogenic agar

Identification for Group B streptococcusAntimicrobial Susceptibility Testing Conclusion

2

The 2010 CDC Early Onset GBS Disease Prevention Guidelines:

Prenatal Specimen Collection & Processing Recommendations

4

GBS Maternal ColonizationGBS is a common colonizer of genital, GI tracts in women

10-30% of all women colonizedRates higher in African-American women

Colonization difficult to detect clinicallyAsymptomaticDynamic conditionCannot determine from history or physical

GBS maternal colonization is a strong risk factor for early onset GBS disease in infants

5

Mother to Infant Transmission of GBS

GBS colonized mother

Non-colonized newborn

50%

Colonized newborn

50%

Asymptomatic98%

Early-onset sepsis, pneumonia, meningitis

2%

20-30%

2~3.5‰

6

Early-onset GBS Disease in the U.S.

MMWR,2010, Nov.19

:

2010

7

(Van Dyke 2009; NEJM; 360:2626-36)

2003-2004

10 U.S. states, 819,528live births

8

Labs Play a Critical Role in Success of Universal Screening

Correct laboratory processing of specimens critical to the success of universal screening Most cases of early onset GBS disease now occur in infants born to women who screened negative (Van Dyke 2009; NEJM; 360:2626-36)

Due in part to false negative prenatal screening results

To prevent as many cases as possible, it is important to optimize specimen collection and processing procedures

9

Group B Streptococcus DiseaseCauses invasive disease in young infants, pregnant women and older adultsIn 1970s, emerged as most common cause of sepsis and meningitis in infants

10

1999~2010年林口長庚醫院GBS血流感染分佈

76

8

11

54

3 3 3 34

2

4

76

45

32

5

7

2

6

4

0

2

4

6

8

10

12

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 年

個

案

數

7day內 7d-3m

Specimen collection

12MMWR,2010, Nov.19

13

Prenatal Specimen CollectionLab feedback to clinicians can help optimize specimen collectionTiming: 35 to 37 weeks gestationVaginal-rectal swabs

14

N=826Yancey et al., OB GYN 1996;88:811-5.

When to Culture? Colonization can be transientNegative predict value 95-98%

35 to 37 weeks gestation

15

•Vaginal-rectal swabsSwab lower third of vaginalInsert through anal sphincterSingle swab or two swabs can be usedDo NOT collect by speculumSelf collection an option

16

Prenatal Specimen TransportInoculate swabs into nonnutritive transport medium

Appropriate transport systems are commercially available (Amies’ or Stuarts’)Specimens in transport media may remain viable at room temperature for up to 4 days, however:

Results most sensitive when processed within 24 hoursResults most sensitive when refrigerated prior to processing

Specimens should be labeled clearly GBS specimen, penicillin allergy status

17

EnrichmentRemove swabs from transport mediumInoculate into enrichment broth

Non-pigmented brothLim broth (Todd-Hewitt/nalidixic acid/colistin), TransVag broth (Todd-Hewitt/nalidixic acid/gentamicin),TransVag broth should be supplemented with 5% defibrinated sheep blood

Pigmented broth- β-hemolysis StrepB carrot broth, Granada biphasic broth

Incubate inoculated broth for 18-24 hr at 35-37°C ambient air or 5% CO2

18

Why Enrich?Remember: for prenatal samples, accurate results much more important than rapid resultsEnrichment in appropriate broth media for 18-24 hours critical step for recovery GBS cultures~50% of women will have a false negative result if sample is not incubated for 18-24 hours in enrichment brothDirect plating can be done, but only as an additional step to broth enrichmentIf the direct plate is negative for GBS then the enrichment broth should be processed

20

加入LIM broth,

陽性率可提高 4.3-5.5%,

陽性件數增加20-26%

21

Testing—Non-Pigmented BrothOptions for further testing after incubation in broth:

Direct test of broth – DNA probe, latex agglutination or NAAT testingSubculture to appropriate agar plate (e.g., sheep blood agar plate, CNA, commercial chromogenic agar) and incubate for 18-24 hours**If subculture negative, and GBS is not identified after 18-24h on BAP, re-incubate and inspect at 48 hrs; if still negative, report as GBS negative5% CO2 at 35-37 °C

22

Pigmented Broth — Positive ResultPositive color change

Photo courtesy of Dr. Lesley McGee, CDC

StrepB Carrot Broth™

J CLIN MICROBIOL,2008, 46: 2780–2782

a unique characteristic of hemolytic GBS due to reaction with substrates such as starch, peptone, serum, and folate pathway inhibitors.

23

Testing — Pigmented BrothIf color change present (orange), report as GBS positiveIf color change NOT present:

Important to realize that chromogenic methods may only identify beta-hemolytic GBS ~96%Must try to identify non-beta hemolytic GBS by either:

Subculturing broth onto appropriate agar plate, incubating 18-24 hours, and identifying organisms suggestive of GBS*Direct testing of broth using appropriate methods

24

GBS認證實驗室 5-6月培養陽性率

Identification for GBS

26MMWR,2010, Nov.19

27

Tests to identify GBS from subcultureInspect subculture plates for β-hemolysis

Generally β-hemolytic on blood agar but non-hemolytic GBS are also possible

Additional tests to perform on selected likely colonies

Gram stain yielding gram positive cocci Catalase negativePresumptive identification: CAMP positive (also hippurate positive)Confirmatory identification: streptococcal grouping latex agglutination or other antigen detection tests (e.g., GBS Accuprobe); commercial kit

28

Streptococcus agalactiae(Group B streptococcus)

StreptococcusComplete genome ~2,000kb, ~2,100 proteins Gram-positive cocciSpherical or ovoid cells --

29


Facultatively anaerobicColony : gray to whitish and are usually glistering in appearanceLarge colonies (>0.5 mm in diameter after 24h of incubation)5% CO2β or γ-haemolysis

Gr.B streptococcus

30


α-Streptococcus β-Streptococcus γ- Streptococcus

S. pneumoniaeviridans streptococcusGr.D streptococcusEnterococcusLactococcusAerococcusGemellaLeuconstocPediococcus

Gr A streptococcusGr B streptococcusGr.C streptococcusGr.G streptococcusGr.D streptococcusGr.F streptococcusEnterococcus

EnterococcusGr.B streptococcusGr.D streptococcus

31

Comparison of β- haemolysis

GAS GBS

S. aureus β-strepto. non ABD

32

Gram Stain – Gram (+) cocci in chain

Photo courtesy of ASM MicrobeLibrary

33

Catalase – (-)

Streptpcpccus– catalase (-)

Staphylococcus – catalase (+)

Photos courtesy of Dr. Lesley McGee, CDC

3% H2O2

34

CAMP Test (+)

Negative reaction for GAS

Positive zone of enhanced hemolytic activity (GBS)

S.aureus ATCC25923 –β-haemolysin


35

Rapid Hippurate Test (+)

Purple color is positive for hippurate hydrolysis


36


Streptococci can also be classified by the type of carbohydrate contained in the cell wall, a system called the Lancefieldclassification. Group A – S. pyrogenesGroup B – S. agalactiaeGroup CGroup D

37

Commercial Agglutination Tests

negative positiveLancefield grouping of β-hemolytic isolated---A,B,C,D,F,G…

38

Commercial kitPhoenix

Vitex II

API/ATB

39

MALDI-TOF MSMALDI-Biotyper system- Bruker Daltonics (Leipzig, Germany) (who have recently partnered with Becton-Dickinson, Franklin Lakes, NJ)Vitex-MS - Shimadzu Corp. (Kyoto, Japan) and bioMerieux SA (Marcyl’Etoile, France)

39

40

Chromogenic agarA: Granada agarC: commercial chromogenic agarE: CNAsheep blood agar plate,

41Tazi et al. 2008. J Microbiol Methods 73:263-5

COH-BAPStrepto B ID®- chromogenic agar: 5 false(-),2 false(+)StreptoB agar: 2 false(-)

Antimicrobial Susceptibility Testing

43

Antimicrobial Susceptibility TestingCLSI recommends using either:

Disk diffusionBroth microdilution

FDA-cleared/approved commercial system may also be used

Testing for inducible clindamycin resistance D-zone or other validated test

44

Antimicrobial Susceptibility: Etest & Disk DiffusionMcFarland No. 0.5; MH-BAPCO2 incubation at 36℃ for 28~24h

Etest

Erythromycin MIC = 0.19µg/ml

Disk Diffusion

Zone of inhibition of growth for clindamycin is ≥19 mm (susceptible)

Zone of inhibition of growth for erythromycin is ≥21 mm (susceptible)


45

Measuring zonesReflected light

Hold the plate a few inches above a black nonreflecting surfaceEnterobacteriaceae, other gram-nagative bacilli, staphylococci, enterococci (except for oxacillin and vancomycin), streptococci (remove the lid)

46

Interpreting resultsThe zone of growth, not hemolysis

47

Antimicrobial Susceptibility Test Broth Microdilution Dilution

Low High concentration

Clindamycin MIC >32 µg/ml

Penicillin MIC 0.06 µg/ml

Erythromycin MIC 8 µg/ml

Growth Control

Sterile control

(MIC) Minimum Inhibitory Concentration


48

β-hemolysis streptococci

DiskS I R

MICS I R

Penicillin ≥ 24 - - ≤0.12 - -

Ampicillin ≥ 24 - - ≤0.25 - -

Clindamycin ≥ 19 16-18 ≤15 ≤0.25 0.5 ≥ 1

Erythromycin

≥ 21 16-20 ≤15 ≤0.25 0.5 ≥ 1

Vancomycin ≥ 17 - - ≤1 - -

CLSI M100-S22,2012

49

Streptococcus spp.

Organism or group

Category INot report or only rarely reported to

dateβ-streptococcus

Ampicillin or penicillin – NSExtended-spectrum cephalosporine-NSDaptomycin – NSErtapenem or meropenem -NSLinezolid – NSVancomycin - NSCLSI M100-S22,2012

50

Mechanism Determinant Ery Clin

Efflux msrA, mefA R S

Ribosome modification erm C R S**

Ribosome modification erm B R Rconstitutive

* * Groups A, B, C, GGroups A, B, C, G**requires induction to show resistance**requires induction to show resistance

Staphylococcus, β-hemolytic Streptococci* Erythromycin / Clindamycin

51

Procedure for D-zone Testing to Detect Inducible Clindamycin Resistance

Erytromycin(R),Clindamycin (S)Inducible clindamycin resistance(erm-mediated)

Routine disk diffusion test:Place 2 μg clindamycin disk 12 mm (for β-streptococcus) from edge of 15 μg erythromycin disk.

12 12 mmmm

No inductionNo induction

InductionInduction

E

E CC

CC

52

D-zone Test Result for GBS

Blunting of the inhibition zone indicating inducible clindamycin resistance


53

Clindamycin/Erythromycin Susceptibility Testing for GBS

Testing only required for women with PCN allergy who are at high risk for anaphylaxisRoom for improved implementation

In a review of U.S. births in 2003-4, 84% of mothers at high risk for penicillin anaphylaxis received clindamycin Only 18% had documented susceptibility testing (Van Dyke 2009; NEJM; 360:2626-36)

54

Clindamycin & Erythromycin Resistance amongGBS isolates, ABCs sites, 2000-2008

*Isolates are from CO, GA, MD, MN, NY, and OR. 2007 data excluded

55

2002~2011(1-6)年林口長庚醫院P/CC/E對GBS的抗藥性

0102030405060

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

(1~6)年

抗

藥

%

Penicillin

Clindamycin

Erythromycin

57

Key Methods for Laboratories Specimen transport options clarified

GBS colonization status should be determined by collecting both vaginal and rectal specimens at 35–37 weeks’ gestation. A single combined vaginal-rectal specimen can be collected

Enrichment step critical ALL prenatal samples must be enriched in broth media for 18-24 hoursEnrichment greatly enhances sensitivity of culture

Accurate results are much more important than rapid turnaround time for antenatal screening.

58

Key Methods for Laboratories48 total hours of incubation for subculture plate important for negative samples

Higher likelihood of false negatives without 48 hours incubation

Pigmented media to detect GBSColor change in presence of beta-hemolytic GBS

Clindamycin susceptibility testing for penicillin-allergic women at high risk of anaphylaxis

Increasing rates of clindamycin resistance make testing extremely important for disease prevention

59

Key GBS ResourcesCDC's GBS Internet page

http://www.cdc.gov/groupbstrepMMWR: http://www.cdc.gov/mmwr/GBS Association home page

http://www.groupbstrep.orgAmerican Society for Microbiology

http://www.asm.orgFDA website (includes GBS molecular diagnostic tests)

http://www.amp.org/FDATable/FDATable.doc

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