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Clinically RelevantClinically Relevant
Microbiology Starts at theMicrobiology Starts at the
SourceSource
Mike Costello, PhD, MT(ASCP)Mike Costello, PhD, MT(ASCP)
ACL LaboratoriesACL Laboratories
847.349.7403847.349.7403
mike.ostello!a"#oatehealth.ommike.ostello!a"#oatehealth.om
Mar$ Dikema%, MT (ASCP)Mar$ Dikema%, MT (ASCP)
A&&i%it$ 'ealth S$stemA&&i%it$ 'ealth S$stem
90.738.3890.738.38
m"ikema%!a&&i%it$health.or*m"ikema%!a&&i%it$health.or*
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Program Objectives
Emphasize that obtaining sensitive and specificmicrobiology results begins with the patient and not at thedoor of the microbiology laboratory.
Accentuate the importance of proper collection andtransport of specimens in both local and referralenvironments
Stress the importance of timely communication betweenthe Microbiology laboratory and those collecting specimens
Describe common pitfalls in specimen collection and
transport Discuss What rules or principles must be followed in order
to collect microbiology specimens which will accuratelyreflect the pathogenesis of the microbiological agent. (hurchD. !he Seven "rinciples of Accurate Microbiology Specimen ollection. . algary#aboratory Services Microbiology $ewsletter. %olume &' ))*+
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Introduction!he practice of sensitive' specific and cost effective clinical
microbiology is intimately tied to the submission and properhandling of optimal specimens for analysis. ,nfortunately'these aspects of clinical microbiology are not as criticallycontrolled as our laboratory assays. -t is our responsibility
to educate and notify our healthcare colleagues whenspecimens arrive at the laboratory that will yield inferiorresults.
uality assurance of specimen collection and transport is anever ending battle and re/uires long term commitment ofyour time and resources' but the end results are betterpatient care and a more rewarding e0perience for those ofus who wor1 in the microbiology laboratory.
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collected "ith a minimum o contaminationcollected "ith a minimum o contamination
as close toas close to
site o inection as possiblesite o inection as possible
Specimen Source ofContamination Storage andTransport Solution/Monitor Education
Urine Culture All non surgicalsamples becomecontaminated withurogenital flora duringcollection.Contaminatingbacteria will replicate ifspecimen is notquickly transferred to apreservative tube orstored (4C.
!ransfer urine toa Urine"reservative tubewithin #$ minutesof collection (goodfor 4% hrs. atambient temp.&ess optimal'storetransporturines at 4 C forup to )4 hrs.
"atients must beinstructed to properlycleanse the peri*urethralgenital skin area prior tocollection of the mid*stream portion of theurine stream in order toget an accurate urineculture result. Use ofurine preservative tubes.
"romptfeedback toindividuals orsites whocollected urinefor culture.Urinepreservativetubes shouldbe used whenappropriate.
+lood Culture,bacterial,mycobacterial,fungal
-mproper cleaning ofskin or catheter prior todrawing specimen.!ransfer from "
tube to blood culturevial.Collection fromcatheter.
Ambient. /ust beincubated inautomatedsystem within #)
hours.
0ngoing educationprogram. /onitoringcontamination rates.&imit use " tubes.
1o not draw from catheterunless specificallyrequested (protocol2discard 3 cath. volume2then one culture set fromcatheter and one fromperipheral.
!imelyfeedback toindividualswho collected
specimen.
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$rine Culture Contamination Rates
+ri%e Cltre o%tami%atio% rates (-bateria at -00,000 C+) shol" be /0 A" 2"robe study (%alenstein " Meier 3. ,rine culture
contamination4 a ollege of American "athologists 2"robes study of contaminated urine cultures in 5)&
institutions. Arch "athol #ab Med. 655786469265+.. &9) participants collected information of 6**')9: urineculture specimens8 ).6; were considered contaminated(
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%lood Culture
!wo sets of blood cultures should be drawn.$umber of sets positive correlates with truesepsis (e0cept for coagulase negative Staph?+(lin Microbiol. =ev 654:7727)' ))&+
atheter drawn blood cultures atheter drawn blood cultures are e/ually li1ely to be
truly positive (associated with sepsis+' but more li1ely tobe colonized (@ lin Microbiol 9749959' ))6.+ ne drawn through catheter and other though vein ""% )f
5&; Both drawn from catheter ""%(positif predi1tif value+ )f
20 Both drawn through vein ""% of 57;
Study of positive coagulase negativeStaphylococcus cultures and sepsis(lin -nfect Dis.954999' ))C.+
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%lood Culture Contamination Rate%y Service &ra"ing Culture
+lood Cultures Collected +y -ndicated taff!ype (5
6o. of&abs
/ean +lood Culture Contamination7ate (5
Dedicated phlebotomy staff
$*)3 84 9.):);*:3 #): 9.$)
:;*#$$ #)$ 2.84Medical technologists or technicians
$ #;3 9.)3#*#$ ##9 ).83
##*#$$ ;$ 2.!"onlaboratory staff
$ 9; ).#:#*3$ )39 9.$$
3#*8$ 9; 9.4$8#*#$$ #: 4.2#
+erkeris & ?alsh, "6 @alenstein. !rends in +lood Culture Contamination.Arch "athol &ab /ed #)8'#)))*#)84, )$$3
What is an Acceptable Blood ulture ontamination =ate for Four #ab??
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Blood Culture Contamination in PediatricPatients
Young Children and Young Doctors
%ariable !rue "ositive 3alse "ositive "redicative %alue ofa "ositive =esult
E0perienced physician2older
child
8 74 0.23
E0perienced physician2younger
child
2 2 0.2
-ne0perienced physician2olderchild
9 28 0.37
-ne0perienced physician2youngchild
382 0.37
!otal 20 78
"ed -nfect Dis. ))&' *4&662&6C.
Foung hildren G 629* monthslder hildren G
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'hat is an (acceptable) blood culturecontamination rate*+
Contamination 7ate"opulation 6o. of &abs )3
th"ercentile 3$
th"ercentile (/edian :3
th"ercentile
Adults 9); ).)9 ).8) 9.%
6eonates )34 $.:3 ).$% 4.):
All "atients 93; ).#3 ).%8 9.;:
+lood culture is considered contaminated if # or more of the following organisms were identified
in only one of a series of blood culture specimens2 coagulase negative taphylococcus,
Propionibacterium acnes, /icrococcus spp., @iridans group treptococcus, Corynebacterium
spp., or +acillus spp. (not B. anthracis
CA" B*!racks (#888*)$$9 /edian contamination rate of ).8)5
?hat should your blood culture contamination rate be
#. tatic model. et a contamination rate (D95. 7ange ).)95*9.%5 Adults2 $.:35*4.):5
6eonates. 1efine an Eacceptable rateF and institute correct measures when rate drifts above
critical value
). Continuous Buality -mprovement /odel. et a rate that at which )9 can achieve, D).35.
0nce 835 of units achieve this rate lower it to ).$5. trive to be in the top #$ percentile
Ber1eris #H' @A !owore1' MI Walsh' "$ %alenstein. !rends in Blood ulture ontamination.Arch "athol #ab Med 6546265C' ))*
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Principle #1 !he specimen must be collected!he specimen must be collected
"ith a minimum o contamination as close to"ith a minimum o contamination as close to
site o inection as possible ,cont-.site o inection as possible ,cont-.
Specimen Source ofContamination Storage andTransport Solution/Monitor Education
7espiratoryCulture
-mproper mouthcare prior tocollection ofspecimen.&ack of deepcough to obtainlower respiratorymaterial.
Ambient for %hours.7efrigerated )4hours.omeorganisms, suchas Haemophilusinfluenzaeare
susceptible todrying or lowtemperature.
/onitor 5 reGected sputum.5 with oral contamination(epithelial cells2 multipletrep species, usually inclumps on gram stain andculture results.
putum culture @s. blood
culture results
All sputum samples arecontaminated to varyingdegrees with oropharyngealflora. 7insemouth with sterilesalinewater immediatelybefore eHpectoration reducesnumber of contaminatingbacteria. !imely feedback to
individuals who collectedspecimen. putum samplesof D) m& should not beprocessed unless obviouslypurulent.
?oundCulture
-mpropercleaning ofwound site prioror collection.
-n transportcontainer.Ambient for nolonger that )4hours.
/aHimiIetransport time.
6umber of squamousepithelial cells @s. "/6sseen on
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Respiratory Cultures
ommunity Ac/uired "neumonia J Sputum re>ection rate andculture correlation with gram stain *C; of all samples were >udged to be of good /uality. "resence of a (predominant morphology+ "M on Hram stain was
predictive of whether the sputum culture could demonstrate apathologic organism. -n the presence of a positive "M' 7&; of culturesyielded a pathologic organism' while a positive culture was obtained in
65.*; of Hram stains without a predominant organism. S.pneumoniaewas the most common infection' growing in **.:; ofpositive sputum cultures.
!he sensitivity and specificity of finding Hram2positive diplococci for apositive culture of S. pneumoniaewere &); and 5:.&;' respectively(Arch Intern Med. ))C86&C46:*26::' 67):26766+
%entilator associated pneumonia (%A"+ J appropriate specimen
Blood cultures highly specific but not sensitive (positive in K6); of%A"+ uantitative cultures of lower respiratory tract specimens show a
closer clinical correlation than sputum subcultures (linical Microbiol.=ev. 654&9:2&*:' ))&.+
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/iral Respiratory Cultures Collect Sample 0rom Site o Inection
Comparison of nasal swab, nasopharyngeal swab, and nasopharyngeal wash specimens with an
expanded gold standard in the Quidel QuickVue influenza test
No. of positive samplesno. of samples !"#
$pecimen %ype $ensitivity $pecificity &ositive &redictive Value Negative &redicative Value
Nasal $wab '()* !+# '(' !*(# (-+' !# -/+- !#
Nasopharyngeal Swab 50/59 (85%) 50/51 (98 62/71 (87) 112/122 (92)
Nasopharyngeal 0ash '-)* !(*# '-' !*# (/ !+# -/1- !'#
2 Clin 3icrobiol. //(4 ''5(16('-
Low do you 1now that an ade/uateSpecimen was submitted for rapidE-A assays???
!hroat swabs are even worse
S l i i
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Samples or &iagnosis o/iral Respiratory Inections
$ung biopsy
%ronchial al&eolar
la&age/'ash/brush
"asopharygeal secretion
"asopharygeal 'ash
(nduced sputum
"asopharygeal s'ab
"asal 'ash
Throat s'ab
)adeno&irus only*
Sali&a
%lood+
SputumSputum
1LAM-A 0-A Culture
LRTC*
present
LRTC cells
absent ,eagentCost1LA NCulture NNM-A NNNNN
0-A NNNN7!*"C7 NNNNN
"C7Costeffort ofcollection
ource ofinfection
,emote
sampling
-iral Titer
(
-iral Titer
$01
Sample2tility
as$
'ar"
#=! G lower respiratory tract cells(columnar epithelial cells' alveolar macrophages+
Site of
infection
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Submit Specimens 3"ot S'abs
"ancy Cornish MD
'''.cap.org
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Sin and Sot !issue ,'ound.Cultures
ollect with steel (needle aspirate or scalpel+ Discourage the use of swabs -f infection $! suspected' D$N! culture Het infected tissue or body fluid O discourage swabs P
2use something sharp ( syringe' scalpel' etc + 2close doesnNt count DonNt culture the surface Q get deep infected sample =emove needles Q send capped syringe with aspirate Share specimen4 Microbiology2Surgical "ath2ytology
#abel specimen and site accurately Hive appropriate history(Mat1os1i . Sharp SE' Iis1a D#. Evaluation of the Score and 9C Systems for cost2effective and
clinically relevant interpretation of wound cultures. @ lin Microbiol ))&8CC467&5267:+
d t
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order torecover the pathogen,s. o
interest
Specimen 0ptimal Time Comments
Urine Lirst morning specimen preferred. 0r not have urinated in severalhours
+lood Culture Collect prior to administration of antibiotics.Collect )*9 sets of blood cultures from differentsites. -f suspect bacterial endocarditis and
initial cultures are negative at 4% hours thencollect )*9 additional cultures from differentsites.uspected bacteremia or fungemia withpersistently negative blood cultures
-nterpretation of one positiveculture problematic, especially ifisolate is coagulase negative
taphylococcus.
Consider laternative blood culturemethods dsigned to enhancerecovery of mycobacteria, fungi,and other rare and fastidiousmicroorganisms
AL+ Culture !hree consecutive specimens collected %*)4hours apart, with at least one being an earlyA/ specimen
putum not saliva
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collected at the optimal time,s. in orderto recover
the pathogen,s. o interest ,cont.
Specimen 0ptimal Time Comments
0va and
"arasites
?ait #$ days if barium or oil present.
Lor multiple samples, collect every other day.
"lace stool in preservative (#$5
formalin, "@A, AL, McofiH within
one hour of collection.
-nstruct patient.
tool Cultures 7ecommend ) samples on consecutive days.
"rior to 9 days post admission.
"lace in enteric preservative (Cary*
+lair immediately.tool specimens that are obtained 9
days after admission are not usually
helpful for the diagnosis of hospital
acquired diarrhea
+lood "arasites Collect during a febrile episode or every ; hours
for a )4 hour period.
ubmit finger stick !hick P !hin
slides or peripheral blood in an
M1!A tube within )4 hours. tore at
ambient temperature.@iral Culture Collect as soon after onset of symptoms as
possible.
!he first 9 days is best.
i i l i i i h
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Principle #4 3 suicient 5uantity o thespecimen must be obtained to perormthe re5uested tests
Culture !n!"u"
#e$u!re"ents
Co""ent
7lood Culture -/ ml of aerobic5 -/
ml for anaerobic
bottle
$ensitivity of a blood culture is directly related to the
volume of blood submitted. %wo blood culture sets !-/
m8 in both aerobic and anerobic bottles# before
administration of antibiotics is *" sensitive !2. Clin.
3icrobiol. -** 1(4 ()+6((-#.
9ne swab for
multiple
cultures
: separate swab!s#
for each culture
;nough material must be submitted for gram stain, if
re
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%lood Cultures
6olme o& bloo" "ra5% is the si%*le mostim1orta%t &ator i%&le%i%* se%siti#it$. Asingle set for an adult blood culture consists ofone aerobic and one anaerobic bottle. ptimally0 mL o& bloo" shol" be i%olate" i%to
eah bottle. %olume of blood for a pediatricculture can be related to the infants weight
Solitary blood cultures should be less than *;(Arch "athol #ab Med. ))6 6*465)265C+
-f only enough blood can be drawn for one bottle'
inoculate the aerobic bottle. &CC positive blood cultures' *5.7; from both bottles'5.7; from aerobic bottle only and 6).C; fromanaerobic bottle only (@ -nfect hemother 54:' ))9+.
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Recommended Pediatric Blood Culture Volumes By Patient WeightWeight(KG) ofPatient
Weight(LB) ofPatient
MinimumVolume(mL)
OnePediatricBottle
Two AdultBottles(aerobic
andanaerobic)
25.0 Kg >55 Lb. 20.0 Ml No Yes (10 mLin each)
Pediatric %lood Cultures 6/olume
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Specimens.
T(SS2ET(SS2E 4$2(D4$2(D
pecimen siIe of pea or largerpecimen siIe of pea or larger
Di&ideDi&ide
Anaerobic transport tubeAnaerobic transport tubeKoldKold upright5upright5uncap,uncap,insert specimen andinsert specimen andrecaprecap
AnaerobicAnaerobicCultureCulture
>eep moist by>eep moist byadding #adding #**) m&) m&sterilesterilesalinesaline
Aerobic cultureAerobic cultureand
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3cceptable Specimens 0or3naerobic Culture
Site 7cceptable Specimens 2nacceptableSpecimens
Abdomen Peritoneal fluid obtained by needle and syringeAbscess aspirate obtained by needle and syringeBileBiopsy material surgically obtainedAnaerobic swab surgically obtained
Aerobic swabs
Body Fluids Ascitic fluid, bile, blood, bone marrow, CSF, pericardial,
pleural, seminal, synovial fluid, throacentesis, transudatesBone and joint Aspirate obtained by needle and syringeBiopsy material surgically obtainedAnaerobic swap surgically obtained
Superficial materialcollected with swabs
Central nervoussystem
Abscess aspirate obtained by needle and syringeBiopsy material surgically obtainedAnaerobic swab surgically obtained
Aerobic swabs
Female genital
tract
Culdoscopy specimens
Endometrial aspirate obtained by suction or protectedcollectorAbscess aspirate obtained by needle and syringeBiopsy material surgically obtainedAnaerobic swabs surgically obtainedIUD forActinomyces species
Vaginal or cervical swabs
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3cceptable Specimens 0or3naerobic Culture
Site 7cceptable Specimens nacceptable Specimens
Head andneck
Abscess aspirate obtained by needle and syringeafter surface decontaminationBiopsy material surgically obtainedAnaerobic swab surgically obtained whenaspiration is not feasible
Throat or nasopharyngeal swabsGingival swabsSuperficial material collected with swabs
&ungs Transtracheal aspirateMaterial from percutaneous lung puncture
Biopsy material surgically obtainedBronchoscopic specimen obtained by protectedbrushThoracatomy specimenAnaerobic swab surgically obtained
Expectorated sputumInduced sputum
Endotracheal aspirateBronchoscopic specimens not speciallycollected
Soft tissue Aspirate obtained by needle and syringeBiopsy material surgically obtainedAspirate from sinus tract obtained by needle and
small plastic catheterDeep aspirate of open-wound obtained throughdecontaminated skinDeep aspirate of surface ulcer obtained throughdecontaminated skin
Superficial material collected from skinsurfaces or edges of wound
Urinarytract
Suprapubic aspirate Voided urineCatheterized urine
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devices and specimen containers mustbe used to
ensure recovery o all organisms
Culture/S!tuat!on Co""ents
:naerobic Culture :naerobic cultures are best collected with metal !needle aspiration or
with a scalpel#. :spirates of pus or fluids could be left in syringe if
not a long distance transport. : large piece of tissue )6-/ mm will
protect anaerobes in center. $pecimen received in aerobic transport
media. $tuart>s and :mies media will allow for isolation of facultative
anaerobes !:mies giving slightly better yields#. ?se of true anaerobictransport media will result in the best yields of all anaerobes. Consider
re@ection of swabs not in anaerobic transport.
Chlamydia or AC Culture $pecimen received in N:% transport tube can not be cultured.
Collect Chlamydia in 36', ?%3.
Collect AC culture in :mies B charcoal and or transport immediately.
to lab at ambient temperature for immediate plating.
%issue sent in preservative 7acterial culture ordered on tissue placed in formalin. Culture is notan option. e
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Recovery o 3naerobic %acteria Placedin in 3erobic83naerobic !ransport
Media
%" G opan %i2"a1 Amies Agar Hel collection and transport swabsSSS G Starple0 StarSwab --'"A G BB# "ort2A2ult
Low Does !ransport !ime Affect Fields?
R 3 bi % t i Pl d
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Recovery o 3naerobic %acteria Placedin in 3erobic83naerobic !ransport
Media ,Cont.
Low Does !ransport !ime Affect Fields?
R 3 bi % t i Pl dff
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Recovery o 3naerobic %acteria Placedin in 3erobic83naerobic !ransport
Media ,Cont.
@ lin Microbiol. ))6495 9::297)
Low Does !ransport !ime Affect Fields?
devices and specimen containers must
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devices and specimen containers mustbe used to
ensure recovery o all organisms
Culture/S!tuat!on Co""ents
Viral culture sent in
bacterial transport media
Viral culture sent in 7acterial transport media. e
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devices and specimen containers must beused to
ensure recovery o all organisms ,Cont.
Culture/S!tuat!on Co""ents
&neumocystis @aroveci!carinii#
7ronchoscopy or induced sputum preferred. &lace sample in a sterile,tightly capped container and storetransport refrigerated within '
hours
$kin parasites &lace skin scraping in a clean dry container, cap tightly and transport
to lab within ' hours at ambient temperature
3ycoplasma pneumoniae
Culture
espiratory sample or C$= in sterile cup. %ransfer specimen into 36'
or ?%3. $toretransport at 'FC. %ransport time should not exceed 'hours.
Consider amplified nucleic acid assay
7lood culture from
Heparin or ;D%: tubes
Heparin is toxic to many organisms.
Increased risk of contamination during transfer.
Suggested !ransport Media 9eneral
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Suggested !ransport Media 9eneralComments
Medium 2tility Comments
tuartOs /edium /ost aerobic and some facultativeanaerobes.
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Principle #: Collect all microbiologytest samples prior to the institution o
antibiotics
Specimen Comments
+lood Culture Collect two sets at same time from different sets. 10 60! collect both sets from
the same site (assessment for contamination
Kair, skin and nailsLungal Culture
Collect before antifungal therapy or discontinue treatment for at least 3 days.
Urine Culture Antibiotics may cause a transient decrease in bacterial concentration resulting in a
false negative report
Principle #; !he specimen container
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Principle #; !he specimen containermust be properly labeled and sealed
prior to transport
S!tuat!on Co""ents
:ny unlabeled or improperlylabeled specimen sent to the lab
3ay decide to have the individual who collected thespecimen to label specimen. 8abel only on bag not allowed.
:ny leaking container e@ect.;ach sample must have at least
two uni
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or ma>imi=e transport media- !here isal"ays some
loss o viability during transport
Specimen Ma6imum Transport Time not inTransport Media
Ma6imum Transport time in Transportmedia
All pecimens "rocess within one hour "lace in transport media. tore andtransport as recommended
tool Culture ) hours Cary*+lair 4% hours
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?nvironmentally 0ragileOrganisms
rgan!s" ost &!'ely Spe!"en Co""ent
Shigella spp. $tool Immediate processing recommended
N. gonorrhoeae Aenital $ensitive to cold. Need )6-/" C9.
Immediate processing recommended
N. meningitidis C$= $ensitive to cold. Immediate processing
recommended
H. influenzae C$=, eye, ear, throat $ensitive to cold. Immediate processing
recommended
A monitor??
Principle #@ Special
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Principle #@ Specialhandling8Collection instruction must be
ollo"ed
Specimen Special (nstructions
+lood Culture +eware of decentraliIed phlebotomy
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Principle #@ Specialhandling8Collection instruction must be
ollo"ed ,Cont-.
3irst' communicate with those that are doingcollections.
ollection instructions are written and available.
Het involved with nursing orientationQeducationdays and as1 to have the instructions given out8poster board learning8 /uiz or competencies.
!al1 to providers when there are problems with
specimen collection8 they sometimes do not 1nowthey could do it better.
or Ordered !est
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or Ordered !est
Specimen "ot acceptable Specimen 7cceptableSpecimen
Comments
Lungal +loodCulture
7outine blood cultures fordetection of Kistoplasmacapsulatum, Blastomycesdermatitidis, Coccidioides immitis,or Malassezia furfur
Lungal +loodCulture
7outine blood cultureswill detect maGority ofpatients withcandidemia.
Lungal7espiratorypecimens
putum swabs for AL+ or fungus AL+' putumLungus' putum,Cryptococcus only.!hrush
6eed tissue to make adiagnosis of fungalpneumonia.1iagnosis of thrushusually only requiresgram stain.
AnaerobicCulture
Autopsy material, respiratory,decubitus, environmental, stool,urine (not aspirate, vaginalsecretions, superficial wounds
ee Anaerobicspecimen table
"olymicrobial
lide for gram
stain
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Criteria 0or Rejection o MicrobiologicalSpecimens
riteria for re>ection must be readily available andlaboratory specific
,nlabeled or improperly labeled specimen "rolonged storage or transport
-mproper or damaged container Specimen received in fi0ative ropharyngeal contaminated sputum Duplicate specimens stools' sputum+ within a C hour
period. E0ceptions cleared by the laboratory Specimens unsuitable for culture re/uest (anaerobic culture
from not acceptable source' urine from 3oley catheter+ Dry Swab C2hr collection of urine or sputum for A3B or fungal
culture ther criteria specific to your laboratory
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Cultures !hat Should Include a 9ramStain
S3 or sterile body fluid (cytospin+
Eye
"urulent discharge
Sputum or transtracheal aspirate All surgical specimens
!issue
,rethral e0udates (male only' intracellular
gonococcus++ %aginal specimens
Wounds
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Summary
"ublish specific rules for specimencollection !here will be e0ceptions
Ma1e physician or healthcare provider aware ofimplications of culturing suboptimal specimens
ommunicate' communicate'communicate
=eal time feedbac1 ontact the health care wor1er who collected
the suboptimal specimen
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References linical Microbiology "rocedures Landboo1. nd Edition. .
LD -senberg ed. ASM. umitechs. ASM "ress. Wash. D.
Manual of linical Microbiology' 5th Edition. ASM "ress.Wash. D. )):.Miller M@.
A Huide !o Specimen Management in linical Microbiology.ASM "ress. Wash. D. 6555.