CD W1 02. Microbiological Specimen Collection & Handling

8/13/2019 CD W1 02. Microbiological Specimen Collection & Handling

1/43

Dr. Nabilah IsmailJabatan Mikrobiologi dan Parasitologi Perubatan

PPSP

COLLECTION,STORAGE AND

TRANSPORTATION OF

MICROBIOLOGICALSPECIMENS


2/43

Outlines Introduction

Methods for detection

Specimen collection Specimen transportation


3/43

IntroductionImportance of microbial diagnosis

Established etiologic agents targeted therapy

Monitoring of response to treatment Establishing carrier state

Establishing epidemiology of disease and itstransmission

Infection control -Prevent transmission of infectionand outbreak


4/43

Problems

Not all microorganisms are cultivable Hepatitis B virus, Hep C virus

Cultivable organisms might not be cultured due to Death Very scanty

Fastidious

Cultivable organism might not be the aetiologic agent

Colonization Contamination

Normal flora

Pathogens are cultured but masked by overgrowth ofnon pathogen


5/43

Methods used for detection of

microorganismInfectious diseases

Directdetection

Culture &isolation

Antigen/ antibodydetection(immunodiagnostic)

Moleculardiagnosticassay

Light

microscopy

Fluorescent

microscopy

Darkfield

microscopy

Electron

microscopy

Cellmediatedimmunity

EIA/ELISA

DFA/IFALatex agglutinationICT

PCR

NASBAPFGEDNA finger-printingPyrosequencing

Blood

SputumUrineCSF


6/43

Clinician who request the specimen

should know

1. WHAT lab examination to request

2.WHEN to take the specimen

3. HOW to take the specimen4. HOW TO TRANSPORT the specimen

5. HOW to interpret the results.


7/43


8/43

Collection of good quality specimen for

microbiological examination is crucial


9/43

1. GOOD QUALITY of the specimen

Appropriate specimen collection

Appropriate specimen transportation

2. TECHNICAL PROFICIENCY of the laboratorypersonnel

3.APPROPRIATE METHOD of detection or test

Direct, culture, detection of ag/ab, detection ofnucleic acid

Good microbiology results depend on:


10/43

The proper practice of microbiological specimencollection begins in the ward/clinic

The quality of the result that we obtain is limited bythe quality of the specimen collected by staff inclinic/ward

The importance of specimen

collection


11/43

COLLECTION OF GOOD-

QUALITY SPECIMENS The proper timing of specimen collection

The correct types of specimen

Proper technique avoid contaminationAdequate quantities and an appropriate number of

specimens

Clearly labelled and safe specimens


12/43

The proper timing of specimen collection-in order to recover the pathogen(s) of interest

Collect all microbiology specimensbefore the start ofantibiotics

Blood culture & BFMP-just as temperature starts torise

Infective endocarditis- 3 sets of blood culture collectedseparately at no less than hourly intervals within 24h

irrespective of temperature Specimen for detection or isolation of viruses- during

acute stage of the disease

Serology- need paired sera4-fold or greater rising

antibody titre in convalescent sera


13/43

Specimen

Optimal Time Comments

Urine for culture First morning midstream specimen

preferred.

Or not have urinated in

several hours

Sputum for AFB

& culture

Three consecutive specimens

collected 8-24 hours apart, with at

least one being an early morning

specimen

Sputum not saliva

Urine for

GC/Chlamydia,

First voided urine of day. Early stream

of urine

Not midstream urine


14/43


15/43


16/43

TEST APPROPRIATE NOT APPROPRIATE

GBS screening Low vaginal/rectal Cervical /hvs

Sinusitis Sinus Nose

Decubitus Tissue SwabAnaerobic culture Tissue, deep wound,

FluidVoided urine, stool,sputum, swab,superficial site

The correct types of specimen


17/43

1. Wound swab

2. Skin ulcer3. Catheter

tips/blood from

catheter

4. ETT secretion

Produce mixed culture of

colonizing/indigenousflora, misinterpreted and

leads to inappropriate

therapy

Appropriate specimen?


18/43

Proper technique- avoid contamination from the normal f lora of the patient or the person collecting the

specimens, collected as close to site of infection as possible

Specimen Source of Contamination Solution/MonitorUrine Culture Samples become contaminated

with urogenital flora during

collection. Contaminating

bacteria will replicate if specimen

is not quickly transported within

2 hours

Patients must be instructed to

properly cleanse the peri-

urethral genital skin area prior to

collection of the mid-stream

urine and the urine sample

should be transported to the lab

within 2 hours.

Blood culture for bacterial and

fungal Improper cleaning of skin prior todrawing specimen.Collection from catheter.

Ongoing education program.

Monitoring contamination rates.Do not draw from catheter

unless specifically requested.


19/43


20/43

Aspirate from abscess/pus swab from

woundAspirate if possible; an aspirate is always superior to a

swab specimen - using a sterile needle and syringetransfer into sterile container .

If abscess is open, ensure all pus and cellular debris isremoved, then swab deep into the lesion and firmlysample the lesions advancing edge.

Swab samples are suboptimal for bacterial culture(aerobic or anaerobic) because of low specimencollection volume, risk of contamination and exposureto oxygen (anaerobes).


21/43

Anaerobic culture The greatest chance for recovery of anaerobic bacteria

is by protecting the specimen from any contact withatmospheric oxygen before inoculation in the

laboratory.

For wound swab specimens, it must be submitted in anappropriate anaerobic transport medium.

ff d b f


22/43

Sufficient quantitiy and number of

specimens

Sufficient quantity of specimenCLSI indicated that bloodvolumeis the single, most importantfactor in the sensitivityof blood culture

Type of blood culture

medium

Use Optimal blood

volume

Bactec Plus +

Aerobic/F

For adults, aerobic

culture

8-10ml

Bactec Plus +

Anaerobic/F

For adults, anaerobic

culture

8-10ml

Bactec Peds Plus/F For neonates &

paediatric, aerobic

cultureAnaerobic culture is

usually not necessary

in this population.

1-3mlMinimum volume

for neonates is

0.5ml (0.1 ml may

be acceptable in

certain

circumstances)

Bactec Myco/F-Lytic For fungal culture (on

request)3-5ml


23/43

Sufficient number of specimen CLSI recommends 2-3 blood culture sets for initial

evaluation (adult sepsis patient)

NEVER*draw only 1 blood culture set during the initial

evaluation of a septic patient.

In patients with suspected bacterial endocarditis, 3 blood

culture setsshould be collected separately at no less

than hourly intervals within 24 hours- sufficient to isolate

the etiologic agent.

Number of positive blood culture sets correlates with

true sepsis (Clin Microbiol. Rev 19:788-802, 2006)

Note: A setis defined by the number of independent

venapunctures.


24/43

The cumulative yield of pathogens from three blood cultures, with a blood

volume of 20 mL each (excluding patients with infective endocarditis),

was 65% from the first culture, 80% from two cultures, and 96% with

three cultures (Cockerill, 2004).


25/43

Reasons for negative blood culture in sepsispatients

Poor timing of collection

A potential issue with intermittent bacteremias

Too low blood volume collected Patient on antibiotics

Infection is contained locally

Host defenses are containing the infection at the 1site


26/43

Each specimen shall have a label firmly attachedto the specimen container and bearing thefollowing information:

Patients name

Patients registration number (R/N)

Ward or clinic

Type of specimen (including specific anatomicsite)

Date of collection

Time of collection

Test requested

Clearly labelled & safe

specimens


27/43

Completing microbiology request form

Patients name, age and sex Registration number and identification card/passport

number

Name of ward or clinic

*Relevant clinical summary and provisional diagnosis

Doctors name, signature and contact number

Type of specimen

Date and hour of specimen collection

Test requested (to state clearly the specific test required)

Antibiotics, if any, that the patient is receiving

Consult lab staff/microbiologist if in doubt

Note: Provide a relevant patient history. The information is

important to accurately interpret results and relate the

results to patient care.


28/43

Safe specimen- Placed in leaked-proof containers and enclosed in a

plastic bag in a separate pocket from the request form

- Microbiological hazards to staff handling leakingcontainers, e.g:

Enteric infection- feaces

TB- sputum from an open case of pulmonary TB

Virus- leaking blood Biohazard risk label clearly attached and request form

should also similarly labelled


29/43

Transport immediately to lab (ideal within 30 min to 2h) Rapid transport:

a. Ensures the survival and isolation of fastidious organismsand prevents overgrowth by more hardy bacteria.

b. Shortens the duration of specimen contact with any localanesthetics used in collection that might have antibacterialactivity.

c. Provides a more accurate assessment of the number of

organisms present in the infectious disease process. Use correct transport media

If delay anticipated, keep in refrigerator but not CSF forC&S (N. meningitidis, H. influenzae sensitive to cold)

Transportation


30/43

Specimen preservation - viral culture, fastidious organism(egNeisseria gonorrhoea)- put in transport media orbedside inoculation

Delay: misleading result1. Decreased recovery of causative agent

2. Overgrowth of contamination or colonizing flora

Transportation


31/43

Appropriate container Appropriate collection devices and specimen containers

must be used to ensure recovery of all organisms

Example, blood culture- BACTEC bottle. Blood culture

from heparin or EDTA bottle- not acceptable. Heparin istoxic to bacteria.

Whole blood in EDTA bottle- molecular testing. (heparin inhibitor).

Fluids- sterile container. NO preservatives. Swabs- Transport medium. Avoid dry.


32/43

Suggested Transport Media General CommentsMedium Utility Comments

Amies Medium (for

swab only)

Most aerobic and facultative

anaerobes

Good general purpose media. Yield

for facultative anaerobes may behigher than from Stuarts.

Amies with Charcoal

(for swab only)

GC Best media for GC

Cary-Blair (for stool

specimens)

Good medium for transport of stool

pathogens (Salmonella, Shigella,

Vibro, Campylobacter, Yersinia,(C. difficile toxin A/B some

assays).

All stool specimens that can not be

setup within 1 hour should be placed

in Cary-Blair media Cary-Blair mediaespecially useful for Campylobacter.

Anaerobic Transport

Media (for anaerobic

specimens)

Many Types. Recommend media with oxygen

indicator. General transport media

are not good for strict anaerobes. Do

not refrigerate.

Ova and Parasite

media (PVA, SAF,

10% formalin,

Alcohol

Protozoa quickly disintegrate in

unpreserved stool

Media that do not contain mercury or

formalin are more environmentally

friendly.

Viral Transport

Media (for swab

only)

Viral culture Most contain antibiotics which

renders then unusable for bacterial

culture.


33/43


34/43

Anaerobic cultureCommercially available container

BBL Port-A-Cul1. Tubesare for swab specimens. Swab

specimens are inserted into areduced solidified holding medium.

2. Transport Jarsare for tissue andbiopsy specimens. The wide-mouthjar allows for easier insertion of thespecimen into the reduced solidifiedholding medium.

3. Vialsare for fluid specimens. Fluidspecimens are injected directly ontothe solid agar surface. The Port-A-CulFluid Collection Setprovides asterile-packed syringe and needle.


35/43

STAT SPECIMEN (URGENT PRIORITY

SPECIMEN)

Blood for culture

Blood for detection of malariaparasites (BFMP)

Cerebrospinal fluid fromlumbar puncture procedure ordirect sampling in operating

theatre Surgical specimens - from

operating theater

Eye specimens - in cases ofendophthalmitis

Joint fluids - if septic arthritisis diagnosed

Pericardial fluid

Transtracheal aspirate - fromtranstracheal procedure

Amniotic fluid - fromamniocentesis procedure

Serum specimen from victimof

needle-prick injury for anti-HBs Serum specimen from sourceof

needle-prick injury for anti-HIV

Serum specimen from sourceofneedle-prick injury for HBsAg

Specimen from patient with potentially life-threatening illnesses

requiring immediate attention


36/43

SPECIMEN REJECTIONS

Wrong registration number (R/N) Improper temperature of transportation Improper transport medium Prolonged transport time Unlabeled, mislabel or improperly labeled specimen

Leaking specimen Cracked or broken container Obvious contamination with foreign materials Dried specimens Inappropriate specimen for a given test Specimen received in fixative Inadequate specimen volume Oropharyngeal-contaminated sputum, i.e. salivary specimens Duplicate specimens within a 24 hour period Specimen unsuitable for culture, i.e., routine swab for anaerobe culture or

Foleys catheter tip.


37/43

Bartletts score Sputum appearance: mucoid/mucopurulent/bl. stained +1

Squamus epi. cell >25 -2

10-25 -1

25 +2

10-25 +1


38/43


39/43


40/43


41/43

Acinetobacter baumanii

MDR strain


42/43

Take home messages1. Contamination, Colonization Vs infection2. Organism isolated from normally sterile body site

significantand cause infection

3. Poor specimenmay give misleading results4. Urine contaminationwith normal flora, influenced

by specimen and transportation.5. Wound swab easily contaminated by adjacent

skin/structures, mixed growth6. ETT secretions colonizationif no evidence of

infection7. Take at least 2 sets of blood culture from pt with sepsis


43/43

Documents

CD W1 02. Microbiological Specimen Collection & Handling