Collection and Transport of Specimens for Microbiological Examinations

(Physician Assistants , Nurses & Public Health Students)

INNOCENT AFEKESCH. OF ALLIED HEALTH SCIENCES

UHAS

Philosophy:

The result of any laboratory examination is only as good as the sample received in the laboratory

Think about this

Upon completion of this topic, the student should be able to:

• Emphasize that obtaining sensitive and specific microbiology results begins with the

patient and not at the door of the microbiology laboratory

• Highlight the importance of proper collection and transport of specimens in both local

and referral environments

• Stress the importance of timely communication between the Microbiology laboratory

and those collecting specimens

• Describe common pitfalls in specimen collection and transport

• Discuss the 7 rules or principles one must be followed in order to collect microbiology

specimens which will accurately reflect the pathogenesis of the microbiological agent

Introduction• Specimens submitted for Microbi testing require proper handling from the time

of collection through all stages of transport, storage and processing

• Nurses and PAs are most often delegated to carry out these processes and hence needed to understand the requirements and consequences of such procedures

• There may be prior preparation of patient before collection of specimen

• Once the specimen is collected, it should be IMMEDIATELY dispatched to the Lab-delay in sending the specimen may cause delicate microbes to die from;Lack of nutrition, lowered or raised temperature, action of enzymes etc.

Influences of Sample Management

Good sample

management

Influences theaccuracy of laboratory diagnosis

Influences therapeutic

decisions

affects patient care

and outcome

Influences laboratory efficiency

Know the anatomical sites samples are collected for Microbiological investigations

Samples that are Collected for Microbiological Testing

BLOOD for:• Culture-preparation of site: critical-Multiple specimens: different sites-Bottle kept at RT or 35-37°C (Do not refrigerate)

• Serology-Serum/ Plasma: choice of bottle- Store frozen at -70°C

Field Work: Blood Collection & transport

• Collection of blood from large communities for research

-Dried Blood Spot for serology

- Blood film on slide for parasitology

- Unless necessary, take blood into sample tubes

CEREBROSPINAL FLUID (CSF) for:

• Cytology-first tapped specimen

• Biochemistry-Glucose & Protein-2nd tapped specimen

• Microbiology; culture-3rd tapped specimen-about 3mlNB CSF is emergency specimen and must be sent to lab immediately (No refrigeration except for viral cultures)

• Specimen collection is done by specialist

URINE for:• Routine examination (RE) for microorganisms e.g.-

Trichomonas vaginalis, Schistosoma ova, Candida etc.

-Collected into clean & dry universal bottle

• Culture and Sensitivity Testing (CS)-Patient preparation very critical-Clean mid-stream catch urine-Sterile universal bottle 2/3 full-Immediately refrigerate the specimen and submit to the lab within 24 hours of collection (maintain at 2-8 ºC when transporting)

• Immunology(ELISA) & Molecular testing (PCR)-First early morning voided specimen

SPECIAL CASES:• Paediatric urine collection kits are

also available

• Suprapubic Aspiration Specimen: needle aspiration through the abdominal wall into the bladder

• Catheter Collection Specimen can be used specially when patient cannot produce urine by him/her self

Field Work : Urine sample• For immunology test e.g. ELISA and for

Molecular test e.g. PCR ; for organisms like N. gonorrhoea, Chlamydia and viruses that are shared in urine-Take sample into conning tube and store on ice whilst on the field-Transport to the lab and store at -20oC till ready to work on them

• Urine samples for parasitology e.g. Schistosoma ova

- Preserve with 10% formalin and store at RT

• Conning Tubes

STOOL for:• RE for parasites e.g. amoeba, Tape worm,

Hookworm etc.-clean wide-mouth crew-capped container-work on immediately or store @ RT for months in fixative(buffered formalin)• C&S-Freshly passed stool samples

avoid specimens from a bed pan unless sterile one

-Use sterile or clean container do not clean with disinfectant

-Cary-Blair medium (transport medium)

-Storage &Transport: refrigerate at 4oC if testing within 48 hours

• FIELD WORK• Immunology & PCR-For Ag detection/PCR – no transport medium

-Transport on dry ice for Ag, PCR detection

- Store at -15oC for Ag detection and PCR; , -70oC if longer

Rectal swabs

Advantage: convenient, adapted to small children, debilitated patients and other situations where voided stool sample not feasible

Drawbacks: no macroscopic assessment possible & less material available

GENITOURINARY TRACT SPECIMENSOrganism/Syndrome Investigated Source of Specimen Collection

Neisseria gonorrhoeae Endocervical-womenUrethral-men

Direct cultureAmies Transport Swab

Yeast, Bacterial Vaginosis, T. vaginalis Post Vaginal Vault Direct examination

Syphilis/Treponema pallidum Aspirates/Scrapings from ulcerative lesions

Transport immediately for dark-field microscopy

Prenatal Screening for Group B Streptococcus at 35-37 weeks gestation

Combined Vaginal/Rectal Amies Transport Swab

• Sampling-FEMALE • Sampling-MALE

RESPIRATORY SPECIMENSa) Lower Respiratory: Sputum for TB Culture• Do not rinse mouth with water as saprophytic mycobacteria in tap water may

produce false positive results• Collect an early morning specimen from a deep, productive cough on three

consecutive days into the empty 90 mL sterile or clean container (?Ghana)

• Carefully and tightly replace the cap• Wash your hands after collecting the specimen• Do not pool specimens• Store refrigerated at 2-8°C • Submit to the PHL laboratory within 24 hours

of collection with a completed PHL requisition

FIELD WORK: TB

• Use Mantoux test for screening

• Measurement of reaction spot

b) Upper Respiratory:Throat swab(posterior pharyngeal swab)

-Hold tongue away with tongue depressor

-Locate areas of inflammation and exudate in posterior pharynx, tonsillar region of throat behind uvula

-Avoid swabbing soft palate; do not touch tongue

-Rub area back and forth with cotton or Dacron swab WHO/CDS/EPR/ARO/2006.1

c) Upper Respiratory:

Nasopharyngeal swab

-Tilt head backwards

-Insert flexible fine-shafted polyester swab into nostril and back to nasopharynx

-Leave in place a few seconds

-Withdraw slowly; rotating motion

-This procedure is done by a trained physician

d) Upper Respiratory:

Naso-pharyngeal aspirate (Lavage)-Tilt head slightly backward

-Instill 1-1.5 ml of sterile normal saline into one nostril

-Use aspiration trap

-Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril

-Procedure normal done by a trained Physician

Collection of Wound Swabs• Gentle cleansing of a skin wound prior to sample

collection is recommended to reduce commensal flora contamination

• Purulent exudates must be expressed onto swabs

• Place the swab into the transport media

• Label swab with patient’s full name, source of specimen and date and time of collection.

• Maintain swabs at room temperature and submit to the lab within 24 hours of collection

• Deep wound specimens (aspirates) are optimal as the predictive value of

superficial swabs is low

• Investigation of deep wounds for anaerobes requires a special anaerobic collection kit

• Anaerobic GasPak System

Anaerobic bags/ containers

Tissue for Culture

• Tissue for routine C&S should be collected in a sterile container

• For small samples add several drops of sterile saline to the container to

maintain moisture

• Maintain at room temperature and submit to the laboratory within 24 hours of

collection

Surgical Specimens (Other Shared Specimens)

TISSUETISSUE FLUIDFLUID

Specimen size of pea or largerSpecimen size of pea or larger

Divide Divide

Anaerobic transport tubeAnaerobic transport tubeHoldHold upright, upright, uncap,uncap,insert specimen and insert specimen and recaprecap

AnaerobicAnaerobicCultureCulture

Keep moist by Keep moist by adding 1adding 1--2 mL 2 mL sterilesterilesalinesaline

Aerobic cultureAerobic cultureand Gram stainand Gram stain

Aerobic cultureAerobic culture

Fungus cultureFungus cultureand stainand stain

AFB CultureAFB Cultureand stainand stain

Virus CultureVirus Culture

Specimen smaller than a peaSpecimen smaller than a pea(i.e. Needle biopsy)(i.e. Needle biopsy)

Put entire specimen intoPut entire specimen intoAnaerobic transport tubeAnaerobic transport tubeOn piece of On piece of telfatelfa backed gauzebacked gauze

Aerobic cultureAerobic cultureand Gram stainand Gram stain

Aerobic cultureAerobic culture

Fungus cultureFungus cultureand stainand stainAFB CultureAFB CultureAnd stainAnd stain

Viral CultureViral Culture

Pleural PeritonealPleural PeritonealAbscess CSF, etc.Abscess CSF, etc.

4 drops 4 drops –– 2 mL2 mLAnaerobicAnaerobicTransport Transport TubeTube

11--20 mL20 mLSterile Specimen tubeSterile Specimen tubeor cup Containeror cup Container

Aerobic cultureAerobic culture

Fungus cultureFungus cultureand stainand stain

AFB CultureAFB Cultureand stainand stain

Virus CultureVirus CultureIn Viral TransportIn Viral TransportMediumMedium

Anaerobic Anaerobic CultureCulture

Indicate sourceIndicate sourceOn requisition (Cath., On requisition (Cath., cysto, bladder,cysto, bladder,R ureter, L. ureter)R ureter, L. ureter)MaximumMaximum2 hr transport2 hr transportto labto lab

URINE

COLLECTION AND HANDLING OF OPERATING ROOM SPECIMENS FOR MICROBIOCOLLECTION AND HANDLING OF OPERATING ROOM SPECIMENS FOR MICROBIOLOGYLOGY

Know the 7 rules or principles must be followed in order to collect microbiology specimens which will accurately reflect the pathogenesis of the microbiological agent

Principle #1: The specimen must be collected with a minimum of contamination as close to site of infection as possible

• BLOOD CULTURES:-Improper cleaning of skin or catheter prior to drawing blood-Acceptable blood culture contamination <3%

• URINE CULTURES:-Males have a lower contamination rate than females (11.2% Vs 22.8%)???NB. patient preparation/education very important

• Sputum for AFBs-Sputum not saliva

Principle #2: A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest oUrine Bact. Culture: 1st morning Mid-Stream Clean Catch

oUrine Gono/chlymedia PCR/RE: 1st voided , first stream urine of day

oBlood Bact. Culture: - Collect prior to admin. of antibiotics - During Chills and To spikes - Collect 2-3 bottles from different sites (times)?? Transient bacteraemia endocarditis/ pathogenic role of saprophytes e.g. S. epidermidis

oBlood Parasites: Collect during febrile episode or every 6hrs for 24hrs

oSputum for AFBs Microscopy & Culture: -3 consecutive specimen collected 8-24hrs apart-At least one must be early morning specimen-In Ghana, one early morning + One spot specimen at clinic/Lab.

oViral cultures: Collect as soon after onset of symptoms as possible

oStool specimena) For parasites ;vegetative form e.g. Entamoeba histolytica: freshly stool

specimenb) For ova, cysts and Oocyst: preserve stool within an hour of collection

Principle #3: A sufficient quantity of the specimen must be obtained to perform the requested tests • BLOOD-Adult : 10ml for aerobic, 10ml for anaerobic bottle-Child: 2-5ml-Infant: 0.5-2ml why??

• CSF-At least 0.5ml in 1st bottle, 2ml each into bottles 2 &3

• URINE- At least 12ml (2/3 of the container, do not fill it)

Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of specific organisms

• Anaerobic Culture-Best collected with metal; needle aspiration or with a scalpel

• Viral culture sample must not be transported in bacterial transport media• Skin Parasites: place skin scraping in a clean dry container, cap tightly and

transport to lab within 24hrs at RT

• Blood culture from Heparin or EDTA tube-Heparin is toxic to many organisms-Transferring from these tubes increases risk of contamination

Principle #5: Collect all microbiology test samples prior to the institution of antimicrobial agents• Hair, Skin and Nails for fungal culture: -Collect sample before antifungal therapy -if already started, discontinue at least 5 days before taken sample• Urine Culture:-Antibiotics may cause a transient decrease in bacterial concentration resulting in a false negative report• Blood Culture:- Take blood sample before antibiotic therapy- State on the lab form if you have started therapy before blood taken

Principle #6: The specimen container must be properly labeled and sealed prior to transport

Situation Comments Any unlabeled or improperly labeled specimen sent to the lab

May decide to have the individual who collected the specimen to label specimen. Label only on bag not allowed.

Any leaking container Reject. Each sample must have at least two unique identifiers

Unique identifies may be name, medical record number, age, patient ID number, etc. Upholds patient safety initiatives.

Slides transferred– recommend two identifiers

Name and specimen number.

Identify what is in the container Swab from where? Body fluid or urine?

Principle #7: Minimize transport time or maximize transport media. There is always some loss of viability during transport

Specimen Maximum Transport Time not in Transport Media

Maximum Transport time in Transport media

All Specimens Process within one hour Place in transport media. Store and transport as recommended

Stool Culture 2 hours Cary-Blair 48 hours GC Cultures Immediately place swab in Amies with

charcoal or other GC approved transport medium.

Not more than 24 hours in Amies with charcoal. Store/transport at ambient temperature.

Respiratory Viral Cultures

Nasopharyngeal secretions or aspirates, BAL; 24 hours at 4°C

Not more than 48 hours if specimen transferred to Viral Transport Media

Clostridium difficile Toxin Assay

2 hours at ambient temperature 72 hours at 4°C; 1 week, frozen

Cary-Blair one week (check with manufacturer)

Urine for CMV 24 hours. Store at 4°C Not recommended Minimize transport time and maximize use of transport media as much as possible

Organism Most Likely Specimen Comment Shigella spp. Stool Immediate processing recommended N. gonorrhoeae Genital Sensitive to cold. Need 5-10% CO2.

Immediate processing recommended N. meningitidis CSF Sensitive to cold. Immediate processing

recommended H. influenzae CSF, eye, ear, throat Sensitive to cold. Immediate processing

recommended

Criteria For Rejection of Microbiological Specimens

• Unlabeled or improperly labeled specimen• Prolonged storage or transport• Improper or damaged container• Specimen received in fixative• Oropharyngeal contaminated sputum• Duplicate specimens stools, sputum) within a 24 hour period. Exceptions

cleared by the laboratory• Specimens unsuitable for culture request (anaerobic culture from not

acceptable source, urine from Foley catheter)• Dry Swab• 24-hr collection of urine or sputum for AFB or fungal culture