COLLECTION, STORAGE AND TRANSPORTATION OF MICROBIOLOGICAL SPECIMENS

Dr. Nabilah Ismail Jabatan Mikrobiologi dan Parasitologi Perubatan PPSP

Outlines
Introduction Methods for detection Specimen collection Specimen transportation

Introduction
Importance of microbial diagnosis Established etiologic agents targeted therapy Monitoring of response to treatment Establishing carrier state
Establishing epidemiology of disease and its

transmission Infection control -Prevent transmission of infection and outbreak

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 of

non pathogen

Methods used for detection of microorganism

Infectious diseases Direct detection Culture & isolation Antigen/ antibody detection (immunodiagnostic)
EIA/ELISA DFA/IFA Latex agglutination ICT

Molecular diagnostic assay

PCR NASBA PFGE DNA finger-printing Pyrosequencing

Cell mediated immunity

Blood Sputum Urine CSF

Light microscopy

Fluorescent microscopy

Darkfield microscopy

Electron microscopy

Clinician who request the specimen should know

1. WHAT lab examination to request 2. WHEN to take the specimen 3. HOW to take the specimen 4. HOW TO TRANSPORT the specimen 5. HOW to interpret the results.

Interpretive problems
1. 2. 3. 4. 5. 6.

Organism isolated from non-sterile body site contamination, colonization or infection Organism isolated from normally sterile body site significant and cause infection Poor specimen may give misleading results -CONS, sputum samples with lots of epithelial cells. Urine depends on colony count mixed with normal flora. Influenced by specimen and transportation. Wound swab easily contaminated by adjacent skin/structures, mixed growth ETT secretions colonization if no evidence of infection

Collection of good quality specimen for microbiological examination is crucial

Good microbiology results depend on:

1. GOOD QUALITY of the specimen Appropriate specimen collection Appropriate specimen transportation

2. TECHNICAL PROFICIENCY of the laboratory personnel 3. APPROPRIATE METHOD of detection or test Direct, culture, detection of ag/ab, detection of nucleic acid

The importance of specimen collection

The proper practice of microbiological specimen

collection begins in the ward/clinic The quality of the result that we obtain is limited by the quality of the specimen collected by staff in clinic/ward

COLLECTION OF GOODQUALITY SPECIMENS

The proper timing of specimen collection The correct types of specimen Proper technique avoid contamination Adequate quantities and an appropriate number of

specimens Clearly labelled and safe specimens

The proper timing of specimen collectionin order to recover the pathogen(s) of interest

Collect all microbiology specimens before the start of

antibiotics Blood culture & BFMP- just as temperature starts to rise Infective endocarditis- 3 sets of blood culture collected separately 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 sera 4-fold or greater rising antibody titre in convalescent sera

Specimen

Optimal Time

Comments Or not have urinated in several hours Sputum not saliva

Urine for culture First morning midstream specimen preferred. Sputum for AFB Three consecutive specimens & culture collected 8-24 hours apart, with at least one being an early morning specimen Urine for GC/Chlamydia, First voided urine of day. Early stream of urine

Not midstream urine

The correct types of specimen

TEST GBS screening Sinusitis Decubitus Anaerobic culture APPROPRIATE Low vaginal/rectal Sinus Tissue Tissue, deep wound, Fluid NOT APPROPRIATE Cervical /hvs Nose Swab Voided urine, stool, sputum, swab, superficial site

Appropriate specimen?
1. Wound swab 2. Skin ulcer 3. Catheter tips/blood from catheter 4. ETT secretion Produce mixed culture of colonizing/indigenous flora, misinterpreted and leads to inappropriate therapy

Proper technique- avoid contamination

from the normal flora of the patient or the person collecting the

specimens, collected as close to site of infection as possible

Specimen Urine Culture Source of Contamination Samples become contaminated with urogenital flora during collection. Contaminating bacteria will replicate if specimen is not quickly transported within 2 hours

Solution/Monitor Patients must be instructed to properly cleanse the periurethral 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 to Ongoing education program. drawing specimen. Monitoring contamination rates. Collection from catheter. Do not draw from catheter unless specifically requested.

Aspirate from abscess/pus swab from wound

Aspirate if possible; an aspirate is always superior to a

swab specimen - using a sterile needle and syringe transfer into sterile container . If abscess is open, ensure all pus and cellular debris is removed, then swab deep into the lesion and firmly sample the lesions advancing edge. Swab samples are suboptimal for bacterial culture (aerobic or anaerobic) because of low specimen collection volume, risk of contamination and exposure to oxygen (anaerobes).

Anaerobic culture
The greatest chance for recovery of anaerobic bacteria

is by protecting the specimen from any contact with atmospheric oxygen before inoculation in the laboratory. For wound swab specimens, it must be submitted in an appropriate anaerobic transport medium.

Sufficient quantitiy and number of specimens

Sufficient quantity of specimen
CLSI indicated that blood volume is the single, most important factor in the sensitivity of blood culture
Type of blood culture medium Bactec Plus + Aerobic/F Bactec Plus + Anaerobic/F Bactec Peds Plus/F Use For adults, aerobic culture For adults, anaerobic culture For neonates & paediatric, aerobic culture
Anaerobic culture is usually not necessary in this population.

Optimal blood volume 8-10ml 8-10ml 1-3ml

Minimum 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

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 sets should 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.

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).

Reasons for negative blood culture in sepsis patients

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

Clearly labelled & safe specimens

Each specimen shall have a label firmly attached to the specimen container and bearing the following information: Patients name Patients registration number (R/N) Ward or clinic Type of specimen (including specific anatomic site) Date of collection Time of collection Test requested

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.

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 leaking containers, 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

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

a. Ensures the survival and isolation of fastidious organisms and prevents overgrowth by more hardy bacteria. b. Shortens the duration of specimen contact with any local anesthetics used in collection that might have antibacterial activity. 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 for C&S (N. meningitidis, H. influenzae sensitive to cold)

Transportation
Specimen preservation - viral culture, fastidious organism

(eg Neisseria gonorrhoea)- put in transport media or bedside inoculation Delay: misleading result 1. Decreased recovery of causative agent 2. Overgrowth of contamination or colonizing flora

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 is toxic to bacteria. Whole blood in EDTA bottle- molecular testing. (heparin inhibitor). Fluids- sterile container. NO preservatives. Swabs- Transport medium. Avoid dry.

Suggested Transport Media General Comments

Medium Amies Medium (for swab only) Amies with Charcoal (for swab only) Cary-Blair (for stool specimens) Utility Most aerobic and facultative anaerobes GC Good medium for transport of stool pathogens (Salmonella, Shigella, Vibro, Campylobacter, Yersinia, (C. difficile toxin A/B some assays). Many Types. Comments Good general purpose media. Yield for facultative anaerobes may be higher than from Stuarts. Best media for GC All stool specimens that can not be setup within 1 hour should be placed in Cary-Blair media Cary-Blair media especially useful for Campylobacter. Recommend media with oxygen indicator. General transport media are not good for strict anaerobes. Do not refrigerate. Media that do not contain mercury or formalin are more environmentally friendly. Most contain antibiotics which renders then unusable for bacterial culture.

Anaerobic Transport Media (for anaerobic specimens) Ova and Parasite media (PVA, SAF, 10% formalin, Alcohol Viral Transport Media (for swab only)

Protozoa quickly disintegrate in unpreserved stool

Viral culture

Anaerobic culture Commercially available container BBL Port-A-Cul 1. Tubes are for swab specimens. Swab specimens are inserted into a reduced solidified holding medium. 2. Transport Jars are for tissue and biopsy specimens. The wide-mouth jar allows for easier insertion of the specimen into the reduced solidified holding medium. 3. Vials are for fluid specimens. Fluid specimens are injected directly onto the solid agar surface. The Port-ACul Fluid Collection Set provides a sterile-packed syringe and needle.

STAT SPECIMEN (URGENT PRIORITY SPECIMEN)

Specimen from patient with potentially life-threatening illnesses requiring immediate attention

Blood for culture Blood for detection of malaria parasites (BFMP) Cerebrospinal fluid from lumbar puncture procedure or direct sampling in operating theatre Surgical specimens - from operating theater Eye specimens - in cases of endophthalmitis Joint fluids - if septic arthritis is diagnosed

Pericardial fluid Transtracheal aspirate - from transtracheal procedure Amniotic fluid - from amniocentesis procedure Serum specimen from victim of needle-prick injury for anti-HBs Serum specimen from source of needle-prick injury for anti-HIV Serum specimen from source of needle-prick injury for HBsAg

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.

Bartletts score
Sputum appearance: mucoid/mucopurulent/bl. stained Squamus epi. cell

+1

>25

-2

10-25 <10
Pus cell

-1 0
+2

>25

10-25 <10
Reject if score < 1

+1 0

Exception: 1. Specimens from neutropenic patients 2. Request for culture of Mycobacterium tuberculosis

Acinetobacter baumanii MDR strain

Take home messages

1. Contamination, Colonization Vs infection 2. Organism isolated from normally sterile body site 3. 4. 5. 6. 7.

significant and cause infection Poor specimen may give misleading results Urine contamination with normal flora, influenced by specimen and transportation. Wound swab easily contaminated by adjacent skin/structures, mixed growth ETT secretions colonization if no evidence of infection Take at least 2 sets of blood culture from pt with sepsis