Manual On Lab Referral For Outbreak Response-Draft 2

MANUAL ON LABORATORY REFERRAL FOR INFECTIOUS DISEASE OUTBREAK RESPONSE
This is a DRAFT manual. The final manual will be disseminated within second quarter of this year.
RESEARCH INSTITUTE FOR TROPICAL MEDICINE In cooperation with WORLD HEALTH ORGANIZATION OFFICE OF THE COUNTRY REPRESENTATIVE NATIONAL EPIDEMIOLOGY CENTER NATIONAL CENTER FOR HEALTH FACILITY DEVELOPMENT
Version 05 May 2011
MANUAL CONTENTS
I. LABORATORY REFERRAL SYSTEM a. Roles and Responsibilities of Different Laboratories b. Laboratory Referral Flow c. Requirements for Specimen Referral II. SPECIMEN COLLECTION AND TRANSPORT a. Specimen Collection Quick Guide b. General Guidelines for Specimen Collection c. International Guidelines for Transport of Specimen III. REPORTING OF RESULTS a. Guidelines for Reporting of Laboratory Result b. Guidelines for Interpretation of Result IV. ANNEXES a. Biosafety Guidelines b. Preparation of Transport Media c. Document Forms and Templates i. Template for Transmittal ii. Linelist of Referred Specimen iii. Laboratory Request Form iv. Template for Laboratory Results Cover Letter v. Official Laboratory Result Form
BACKGROUND AND RATIONALE
Laboratory support during outbreaks is essential for rapid and effective response. Information provided by the laboratory, aids not just in managing current outbreaks but also in enhancing control programs. Reference and sub-national laboratories must coordinate with each other to provide the National Epidemiology Center (NEC)-Department of Health, and other stakeholders with accurate, timely and quality assured results. The Research Institute for Tropical Medicine (RITM) is composed of different reference laboratories for emerging and re-emerging infectious diseases thus it is tasked to provide confirmatory tests, identify rare or dangerous pathogens, detect newly described or emerging pathogens, supply regional laboratories with uncommon transport media, and store outbreak isolates. Phenotypic and genotypic characterization may also be performed by RITM to investigate significant changes in the etiologic agent and to assess the epidemic strain. Sub-national laboratories, on the other hand, may perform specimen collection, initial processing, screening tests, simple diagnostic procedures, and specimen transport to RITM. Clinical samples may also be referred to nearby regional hospital capable of the required laboratory test provided that a percentage of the positive specimens are sent to RITM for confirmation. It is crucial that an efficient referral system between RITM and subnational laboratories be in place to aid stakeholders in the effective management of infectious disease outbreaks. RITM had been involved in the response to countless outbreaks in the country, such as Meningococcemia in 2004-5, Leptospirosis and Pandemic Influenza H1N1 in 2009, and more recently, suspected Anthrax in 2010. In many of the outbreaks, confusions regarding specimen collection, referral and information flow contributed to the underreporting of laboratory confirmed cases. Incidents of sending inappropriate specimens happened during the Pertussis outbreak of 2007, in which 50 serum samples were not tested because the referring institution failed to communicate with RITM regarding specimen collection guidelines, and the Leptospirosis outbreak of 2009. Misinterpretation of laboratory
results and non-compliance with proper specimen transport guidelines were also observed during the suspected Anthrax outbreak of 2010. It should also be noted that not all of the infectious disease outbreaks in the country had representative specimens sent to RITM. In order to assess the epidemic strain, it is critical that specimens from a sample of cases be sent to designated reference laboratory for investigation and repository. These occurrences illustrate the need to strengthen the specimen referral system in the country and to enhance the information link between different government health agencies.
PART I. LABORATORY REFERRAL SYSTEM Roles and Responsibilities of Key Institutions Qualified Laboratories Laboratory Referral Flow Requirements for Specimen Referral
ROLES AND RESPONSIBILITIES

NATIONAL REFERENCE LABORATORY - Performs routine laboratory diagnosis for outbreak specimens if there is no capable tertiary or subnational reference laboratory near the site of outbreak Sufficient aliquots of ALL collected clinical specimens should be sent to the corresponding NRL for testing. The number of specimen to be collected (all cases or cluster sampling) will be decided upon by NEC provided that they have information on the surge capacity of each NRLs. Three factors to consider for the control of influx of specimen: necessity of diagnosing all cases, collector, laboratory empowerment and surge capacity. - Conducts specialized diagnostic tests (e.g. Anthrax, Leptospirosis, viruses, emerging infections, newly identified pathogens) Sufficient aliquots of ALL collected clinical specimens should be sent to the corresponding NRL for testing. - Provides confirmatory tests to capable tertiary laboratories or sub-national reference laboratories BACTERIOLOGY: ALL positive outbreak isolates and 10% of negative clinical specimens - Provides peripheral/referring laboratories with transport media - Banks positive outbreak isolates nationwide (NRL as central repository of isolates nationwide for future investigative studies) BACTERIOLOGY: ALL positive outbreak isolates should be sent to corresponding NRL even if the laboratory referred to are already capable. Access by scientists from other institutions (request permission) - Assesses epidemic strains by characterizing isolates genetically or phenotypically (if applicable)
- Sets guidelines for laboratory testing; inform other laboratories of updates or alerts; provides technical assistance to other laboratories - Trains staff from other laboratories for specimen collection and testing. - Provides quality assessment to laboratories BACTERIOLOGY: If the tertiary laboratory is capable, ALL positive outbreak isolates should still be sent to RITM for isolate banking. For proficient SUB-NATIONAL LABORATORIES: - Reporting of data to BOTH RITM and NEC - Should they still send a percentage of the samples for subtyping or future characterization studies? - Informs NEC, NCHFD and other stakeholders of the capability of tertiary and sub-national reference laboratories to perform specific tests - Provides NEC with timely and quality-assured results. - Regularly reports summary of laboratory outbreak data to NEC. - Coordinates with international or local institutions for the characterization of isolates (if not available in RITM)
SUB-NATIONAL LABORATORY - Assists RITM in the timely detection and confirmation of Pandemic Influenza A/H1N1 using Real-time RT-PCR - Collects clinical specimen for testing - Performs routine testing for aerobic bacterial pathogens - Provides transport media to peripheral laboratories - Submits outbreak bacterial isolates to NRLs for banking
- Refers clinical specimen to NRL for confirmatory testing - Provides technical assistance to regional and peripheral laboratories (if applicable) - For emerging or newly-described pathogens: Specimens for initial cases should be sent to RITM for testing. If the NRL has exceeded its surge capacity, then sub-national laboratories will be tapped to perform the test. Sub-national laboratories should pass the QA tests of the NRL before they can perform the test. Initially, all positive [define number] and 10% of their negative specimens should be confirmed in the NRL - Participates in external quality assessment programs provided by NRLs - Provides NEC with timely and quality-assured results of tested specimens. - Regularly reports summary of data to BOTH RITM and NEC. [How regular?]
QUALIFIED REGIONAL LABORATORY - Collects clinical specimen for testing - Performs culture and sensitivity tests for aerobic, bacterial outbreak pathogens (if capable) - Performs preliminary tests for pathogens requiring specialized tests - Refers clinical specimens to NRLs for confirmatory testing - Submits positive bacterial isolates to NRLs for confirmatory testing, isolate banking and epidemic strain assessment - Provides transport media to tertiary and peripheral laboratories - Participates in external quality assessment programs provided by NRLs - Provides NEC with timely and quality-assured results of tested specimens - Regularly reports summary of data to NEC.
QUALIFIED TERTIARY LABORATORY - Collects clinical specimen for testing - Performs culture and sensitivity tests for aerobic, bacterial outbreak pathogens - Performs preliminary tests for pathogens requiring specialized tests - Refers clinical specimens to Regional laboratories for confirmatory testing - Submits positive bacterial isolates to NRLs for confirmatory testing, isolate banking and epidemic strain assessment - Participates in external quality assessment programs provided by NRLs - Provides NEC with timely and quality-assured results of tested specimens. - Provides assistance to peripheral laboratories
PERIPHERAL LABORATORY - Collects clinical specimen for testing - Performs preliminary testing on clinical specimens - Refers clinical specimen to the nearest capable tertiary, regional laboratories or sub-national reference laboratories
LIST OF QUALIFIED REGIONAL AND TERTIARY LABORATORIES FOR AEROBIC BACTERIAL CULTURE
The list is a record of the selected government tertiary hospitals that are qualified to perform aerobic bacterial culture and identification for outbreak specimens. This is based on the hospitals performance in both 2009 and 2010 NEQAS for Bacteriology results. This will be available in the RITM website (www.ritm.gov.ph) and will be updated annually. List of Qualified Tertiary Laboratories for Bacterial Culture (as of 04 May 2011). 1. NORTHERN MINDANAO MEDICAL CENTER 2. VETERANS REGIONAL HOSPITAL 3. DR JOSE FABELLA MEMORIAL HOSPITAL 4. NATIONAL CHILDRENS HOSPITAL 5. OSPITAL NG MAKATI MEDICAL CENTER 6. PASAY CITY GENERAL HOSPITAL 7. PHILIPPINE CHILDRENS MEDICAL CENTER 8. UP-PHILIPPINNE GENERAL HOSPITAL 9. JOSE R REYES MEMORIAL MEDICAL CENTER
References: 1. Research Institute for Tropical Medicine; Microbiology Department; National External Quality Assurance Scheme (NEQAS) Executive Report 2009 and 2010.
LIST OF QUALIFIED LABORATORIES FOR PANDEMIC INFLUENZA A/H1N1 TESTING

The list of qualified sub-national reference laboratories for Pandemic Influenza A/H1N1 Real-time PCR testing is based on the results of the external quality assessment scheme provided by RITM National Influenza Center. The sub-national reference laboratory will be considered qualified if they obtain a score of 100% in the proficiency test, which is offered biannually, and if they passed other quality assurance checks. This list will be available in the RITM MBL website and will be updated twice a year depending on the date of the release of proficiency test results by RITM NIC.
List of Qualified Sub-national Reference Laboratories for Pandemic Influenza A/H1N1 Real-time PCR testing (as of 04 May 2011).
INSTITUTION
San Lazaro Hospital Vicente Sotto Memorial Medical Center Southern Philippines Medical Center
LOCALE
NCR Visayas Mindanao
References: 1. Research Institute for Tropical Medicine; National Influenza Center; 2. DOH Department Personnel Order No. 2009-2441
LABORATORY REFERRAL FLOW

FOR OUTBREAKS a) Referring institution/CESU/PESU/MESU informs RESU of the outbreak and collects the specimen (based on the manual). b) RESU will inform NEC and also provide technical assistance to the referring institution. RESU will identify the testing laboratory to which the specimen will be referred to according to the list of qualified laboratories provided by the NRLs (available in RITM website). They will then contact the testing laboratory to inquire on the availability of the test and their office opening hours. c) If the testing laboratory agrees to the request for the referral, the RESU shall inform the referring institution/ESU where and how to refer the specimen. d) The referring institution will ship the specimen with the required documents to the selected testing laboratory according to the guidelines specified in the manual. e) The referring institution will notify RESU, NEC and the testing laboratory of the shipment, by phone AND fax, with the following details: a. Referring institution b. Requesting physician/health worker c. Outbreak name/details d. Outbreak site e. Test requested f. Purpose (e.g. routine testing for outbreak investigation, isolate banking, confirmatory testing) g. Specimen type and total number to be sent h. Expected date of arrival in testing laboratory i. Courier
j. Courier tracking number k. Shippers name, signature, position, institution and contact information Kindly refer to the template for transmittal document in the Annex section. f) The referring institution will ensure that the testing laboratory acknowledges the parcel is coming.
g) Upon receipt of shipment, the testing laboratory should check that the number and type of specimens confer with the transmittal document faxed to the testing laboratory. The specimen type and quality should also be in accordance to the guidelines specified in the manual. They should also ensure that all the received specimens have corresponding required documents. h) If there are any discrepancies or if a specimen should be rejected, the testing laboratory should immediately inform the referring institution or ESU for appropriate action. i) The testing laboratory shall contact the testing laboratory to ensure that they acknowledge the receipt of the parcel.
Outbreak
FLOWCHART
Referring Institution / CESU / MESU / PESU
Collects specimen Informs RESU of outbreak
RESU
Informs NEC of outbreak Provides technical assistance to referring institution or ESU Identifies testing laboratory based on the list of qualified laboratories provided by NRLs and inquires for the availability of the test and office opening hours Informs referring institution or ESU where and how to send the specimen
Referring Institution / CESU / MESU / PESU

Packages specimen Ships specimen to testing laboratory Notifies testing laboratory, RESU and NEC of the shipment by phone AND fax (use transmittal template for the fax) Ensures that the testing laboratory acknowledges that the parcel is coming.
Testing Laboratory
Checks that type and number of specimens confer with transmittal document Ensures that all received specimens have corresponding required documents Verify that specimen type and quality are in accordance with the guidelines
Testing Laboratory
Are there any discrepancies, corrections, wrong specimen, missing documents? No
Yes
Referring Institution /ESU shall contact testing laboratory to ensure that they acknowledge the receipt of the parcel
Testing Laboratory will inform referring institution/ESU by phone or fax of any discrepancy/corrections for appropriate action
FOR SURVEILLANCE (From case reporting to specimen referral request)
The original flow from the PIDSR:
1. Identification of cases using the PIDSR Case Definitions
2. Classification of cases Immediately Notifiable (Category I Diseases) use Case Investigation Form (CIF) Weekly Notifiable (Category II Diseases) use Case Reporting Form (CRF)
3. Flow of Notification/Reporting depending on classification of cases is triggered (separate for Category I and Category II Diseases) [see below Figure 1 and 2]
4. Case based reporting using PIDSR Forms One (1) copy to be provided to the laboratory
5. Collection of appropriate sample
6. Proper packaging and transport
7. Delivery to Laboratory
Figure 1: Flow of Notification for Immediately Notifiable Diseases, Syndromes and Events
Cases from hospitals, clinics, ports, airports and laboratories
Cases from the community
Barangay Health Station (BHS)
Rural Health Unit/ City Health Office Feedback
Provincial Epidemiology and Surveillance Units (PESU)
Regional Epidemiology and Surveillance Units (RESU)
Legend: Weekly reporting Feedback Immediate notification (within 24 hrs)

National Epidemiology Center
World Health Organization
Taken from the PIDSR Manual
Figure 2: Flow of Weekly Reporting of Notifiable Diseases
Cases from the Community
Barangay Health Stations (BHS)
Barangay Health Stations (BHS)
Cases from local hospitals, clinics, ports, airports
Rural Health Units and City Health Offices in nonchartered cities
Cases from provincial and district hospitals, ports, airports
Provincial Epidemiology and Surveillance Units
City Epidemiology and Surveillance Units (CESU) in chartered cities
Cases from local hospitals, clinics, ports, airports
Regional Epidemiology and Surveillance Units (RESU)
Cases from level 3 and retained hospitals, ports, airports
Cases from referral hospitals, laboratories,
National Epidemiology Center
Taken from the PIDSR Manual
REQUIREMENTS FOR SPECIMEN REFERRAL

Proper specimen referral may be simple yet essential in obtaining accurate laboratory diagnosis of infectious diseases. There are three requirements that should be followed to ensure proper specimen referral. Specimen Documents Communication
1. SPECIMEN REQUIREMENTS a. Specimen Collection: The following requirements for specimen collection increase the likelihood of acquiring accurate laboratory diagnosis: i. Appropriateness of specimen type and date of collection: The specimen should be representative of the infectious process (e.g. sputum is the specimen for pneumonia and not saliva) and also suitable for the test method to be used. It is thus important to collect the appropriate type at the appropriate phase of disease. ii. Specimen quality Strict aseptic techniques should be practiced throughout the procedure. Hands must be washed before and after the collection. Specimen should be collected before the administration of drugs.
iii. Specimen quantity The specimen should have adequate quantity for the desired tests to be performed.
iv. Appropriateness of specimen container Liquids must be placed in a leak-proof, screw-capped container with a capacity less than 1 liter. Solids must be stored in a sift-proof container weighing less than four kilograms. Specimen should be placed aseptically in a sterile and/or appropriate container. The outside of the specimen container should always be clean and uncontaminated. v. Specimen Storage Storage temperature for the specimen depends on the type of test that will be performed. If the type of test requires a viable organism in the specimen, then the storage temperature should be ideal for the growth of the organism. Appropriate transport media should only be used if a viable organism in the specimen is required by the test procedure. Keep the specimen from direct exposure to the sun and extreme heat.
Detailed guidelines for appropriate specimen collection are included in the next section of this manual.
b. Specimen Transport: Detailed guidelines for specimen transport are available in succeeding sections of the manual. It should be noted that there are three essential practices that should be performed for specimen transport:
i. Label specimen properly by: completely and accurately writing the specimen id/patient name, specimen type, date of collection, age and sex using water proof stickers, label tape or markers writing in block letters
ii. Package specimen according to the guidelines detailed in the transport of specimen section. In emphasis: there should be three components for packaging of specimen: primary receptacle, secondary receptacle and rigid outer packaging; parcels should be properly labeled with: i. the sender and receivers addresses, ii. emergency contact (name and telephone, iii. Biological Substances, Category B iv. Orientation arrows placed on two opposite sides of package (one not shown) v. Net Weight or Volume of Sample if multiple packages being sent. iii. Arrange immediately for the transport of the specimen to the testing laboratory.
2. DOCUMENT REQUIREMENTS a. Required Accompanying Documents to Referred Specimens The accompanying documents to referred specimens should be inserted in a ziplock plastic separate from that of the specimen. i. For outbreaks: A copy of the transmittal document
Line list of specimens in the parcel with the following details: Specimen ID, patients name, age/sex, specimen type, and an optional information (additional unique identifiers) [Kindly refer to the template for the line list of referred specimen in the Annex section]
Testing Laboratorys Laboratory Request Form
ii. For surveillance: A copy of the transmittal document Line list of specimens in the parcel with the following details: Specimen ID, patients name, age/sex, specimen type, and an optional information (additional unique identifiers) [Kindly refer to the template for the line list of referred specimen in the Annex section] PIDSR Case Report Forms
b. Rules for completion of forms Use ball-point/fine black or blue pens Use block and legible letters Review data on the form before submission
3. COMMUNICATION REQUIREMENTS: It is the responsibility of the referring institution to: a. advise testing laboratory, RESU and NEC of intent to refer specimen and shipment details using the transmittal document (Kindly refer to the laboratory referral flow section and the annexes); b. ensure that testing laboratory acknowledges that parcel is coming through phone;
c. inform testing laboratory of patient and sample details through the required accompanying documents to referred specimens; d. ensure that testing laboratory acknowledges the receipt of the parcel through phone; and e. ensure that results are received through follow-ups upon expected date of release of results, which would be based on turn-around time for the requested test.
SPECIMEN REJECTION CRITERIA Missing or Incomplete request form Unlabelled or illegibly labeled specimen Inappropriate or leaking container Inappropriate specimen type Insufficient quantity Suspicion of contamination Inappropriate transport or storage Unknown time delay Hemolysed blood sample (for serology) Testing laboratory is not notified of the parcel shipment
References: 1.World health Organization; http://www.searo.who.int/en/Section10/Section17/Section53/Section482_1779.ht
PART II. SPECIMEN COLLECTION AND TRANSPORT

Specimen Collection Quick Guide General Guidelines for Specimen Collection International Transport Guidelines
SPECIMEN COLLECTION QUICK GUIDE

FOR MICROBIOLOGY
DISEASE
ETIOLOGIC AGENT/S
TESTS
APPROPRIAT E SPECIMEN
TIME OF
COLLECTION
QUANTITY
CONTAINER/ TRANSPORT
MEDIUM
STORAGE CONDITIONS PRIOR

TO TRANSPORT
TRANSPORT /TIME CONDITIONS

Within 1-2 hours at room temperature Within 3-6 hours in cold packs
IS THE TEST A CONFIRMATORY/ RAPID /PRESUMPTIVE TEST
TURNAROUND
TIME
TESTING CENTERS
E. coli 0157:07 (EHEC) ACUTE BLOODY DIARRHEA Shigella spp. Campylobacter spp. Salmonella spp.
Fresh Stool Culture
As soon as possible after onset of illness
2-5 mL/ peasized
Sterile, wide-mouthed, screw-capped, spillproof container
If placed in Cary Blair, store at room temperature or 4C for up to 48 hours Store at room temperature for up to 24 hours
Within 24 hours at room temperature Within 3-5 days at room temperature or 4C
CONFIRMATORY
2-3 days
RITM Any Qualified Tertiary Laboratory
Rectal Swab
1-2 swabs
Cary Blair Transport Medium in sterile, autoclavable screw capped, container
1-2 hours
Serogrouping/ Serotyping
Isolate
CONFIRMATORY
Except for Salmonella H-typing: minimum of 3 days
CHOLERA
Vibrio cholerae Other Vibrios
Culture
Fresh Stool
As soon as possible after onset
2-5 mL/peasized
Within 1-2 hours at room temperature
RITM CONFIRMATORY
2-3 days
Any Qualified
of illness If placed in Cary Blair, store at room temperature or 4C for up to 48 hours Cary Blair Transport Medium in sterile, autoclavable screw capped, container Store at room temperature for up to 24 hours
Within 3-6 hours in cold packs Within 24 hours at room temperature Within 3-5 days at room temperature or 4C CONFIRMATORY Within 1-2 hours at room temperature Within 3-6 hours in cold packs
Tertiary Laboratory
Rectal Swab Serogrouping/ Serotyping Isolate
1- 2 swabs
1-2 hours
Fresh Stool
TYPHOID SUSPECTS
Salmonella Typhi Culture Salmonella Paratyphi A Rectal Swab
2 to 3 week after onset of illness
nd
rd
2-5 mL/peasized
If stored with a holding medium, store at room temperature or 4C for up to 48 hours Store at room temperature for up to 24 hours
Within 3-5 days at room temperature or 4C Within a week at room temperature or 4C CONFIRMATORY
2-3 days
RITM Any Capable Tertiary Laboratory
2 swabs
Blood
1st week after onset of illness
Adult: 1:10 ratio with BCB Infant/ Child: 1:5 to 1:10 ratio with BCB Blood Culture Broth (BCB)
Store at room temperature until ready for transport
Within 3 days after collection at room temperature
7 days
Serogrouping/ Serotyping
Isolate 1. Four (4) tubes of EMJH or Fletchers medium (To be requested from referral laboratory if available). Label tubes from A to D.
CONFIRMATORY
Minimum of 3 days
Whole Blood (collected at bedside)
3 to 5 mL 2.
Inoculate: A: one (1) drop B: two (2) drops C & D: three (3) drops each
Store at room temperature in a dark area until ready for transport
Transport at room temperature within 24 hours
CONFIRMATORY
2- 6 weeks
LEPTOSPIROSIS
Leprospira sp.
Culture
Heparinized blood/ Defibrinated blood/ Triturated clotted blood
Within 10 days after onset of symptoms
3 to 5 mL
Sterile, leak-proof tubes
RITM CONFIRMATORY Any Capable Tertiary Laboratory
CSF (collected at bedside)
0.5 to 2 mL
Two (2) tubes of EMJH or Fletchers medium (To be requested from referral laboratory if available). 2. Inoculate 0.5 5 mL of the specimen * If culture medium is not available: Sterile, 2 mL screwcapped tube
1.
Store at room temperature in a dark area until ready for transport
CONFIRMATORY
6 weeks
Transport with ice packs as soon as possible
CONFIRMATORY
Urine
2 week up to 30 days after onset of symptoms
nd
15 L
Three (3) sterile leakproof container (preferably 50mL centrifuge tube)
Store in refrigerator until ready for transport
CONFIRMATORY
6 weeks Transport with ice packs as soon as possible

Transport at room temperature as soon as possible Transport chilled or with ice packs as soon as possible Transport chilled or with ice packs as soon as possible
*PostMortem Samples
As soon as possible after death
Sterile, screw-capped container
Store in refrigerator until ready for transport
CONFIRMATORY
Whole Blood
3 to 5 mL
EDTA Tube (Purple top)
CSF qPCR
Within 10 days after onset of symptoms
0.5 to 2 mL
Sterile, 2 mL screwcapped tube
CONFIRMATORY
2-3 days
a. Serum 0.5mL b.
Sterile, screwcapped tube OR Properly capped red top tube
Urine
2 week up to 30 days after onset of symptoms
nd
15 mL
Sterile, leak-proof container (preferably 50mL centrifuge tube)
Transport chilled or with ice packs as soon as possible

CONFIRMATORY
*PostMortem Samples
As soon as possible after death
2-3 days
MAT/ELISA
Serum
5-10 days or later after onset of symptoms (acute), 5-10 days after collection of acute serum (convalescen t) * Paired sera collection
a. 0.5mL b.
CONFIRMATORY
14 days after onset of symptoms * Single serum collection 0.5mL
a.
b.
CONFIRMATORY
Vesicular Fluid/ Eschar Material
Sterile Swab
Refrigerate
3-5 days
Blood As soon as possible after onset of illness
Adult: 1:10 ratio with BCB Infant/ Child: 1:5 to 1:10 ratio with BCB
Blood Culture Broth (BCB)
Within 3 days after collection at room temperature Within 1-2 hours at room temperature
3-7 days
ANTHRAX
Bacillus Anthracis
Culture
CONFIRMATOR Y
RITM
Stool
2-5 mL/peasized
Within 3-6 hours at 4C
3-5 days
If placed in Cary Blair, store at room temperature or 4C for up to 48 hours Store at room temperature for up to 24 hours
Within 24 hours at room temperature Within a week at room temperature
Rectal Swab
2 swabs
3-5 days
or 4C
Sputum
> 1 ml
Sterile Container
Refrigerate
Transport with ice packs as soon as possible Transport with ice packs as soon as possible
Within 3 days after collection at room temperature Within 1-2 hours at room temperature Within 3-6 hours at 4C
3-5 days
Vesicular Fluid/ Eschar Material
Sterile Swab
Refrigerate
Blood As soon as possible after onset of illness Stool
PCR
2-5 mL/peasized
Sterile, wide-mouthed, screw-capped, spillproof container If placed in Cary Blair, store at room temperature or 4C for up to 48 hours Cary Blair Transport Medium in sterile, autoclavable screw capped, container Store at room temperature for up to 24 hours
3 working days
Within 24 hours at room temperature Within a week at room temperature or 4C
Rectal Swab
2 swabs
Sputum
> 1 ml
Sterile, leak-proof Container
Refrigerate

Transport at room temperature as soon as possible Transport at room temperature as soon as possible
Blood Onset of illness
Culture
CONFIRMATORY
7 days
RITM or any qualified Tertiary Laboratory
CSF
0.5-1ml
T-I using a sterile syringe and needle
vent the T-I using a sterilized venting needle and incubate at 37C with 5% CO2
CONFIRMATORY
3 days minimum
Invasive Meningococcal DIseae
Neisseria meningitidis
Whole Blood
3 to 5 mL Onset of illness
EDTA Tube (Purple top)
PCR Serum
a. 0.5mL
Sterile, screwcapped tube OR b. Properly capped red top tube
Freeze
CONFIRMATORY
3 working days
RITM
CSF
Serology (Latex
0.5-1ml Onset of Illness 0.5-1ml
Sterile Container Sterile Container Refrigerated (at 2 8 up to C)

Transport chilled or
CSF
PRESUMPTIVE
1 working day
RITM or any
Agglutination Test)
Whole Blood
3 to 5 mL a.
EDTA Tube (Purple top) Sterile, screwcapped tube OR b. Properly capped red top tube
several hours Or Freeze at -20 C for longer periods
with ice packs as soon as possible
qualified Tertiary Laboratory
Serum
0.5mL
Serogrouping
Isolate Throat swab & Nasal Swab
Plates Incubated at 37C
Transport at room temperature
CONFIRMATORY
1-2 hours
RITM
RITM or any capable Tertiary Laboratory
Culture DIphtheria
Corynebacterium diphtheria
(If present, collect pseudomembrane PCR Throat Swab & Nasal Swab Nasopharyngeal Swab Culture Nasopharyngeal Aspirate
Onset of Illness
2 swabs (1 throat and 1 nasal)
Amies without charcoal
Refrigerate
CONFIRMATORY
2 days minimum
PRESUMPTIVE 2 Dacron swabs (left and right nostrils) 0.5mL 2 Dacron swabs (left and right nostrils) 0.5mL Transport chilled or with ice packs as soon as possible Transport chilled or with ice packs as soon as possible
3 working days
RITM
< 2 weeks post-cough onset
Regan Lowe Transport Media Refrigerate Sterile Container
CONFIRMATORY
5 days minimum
RITM
Pertussis
Bordetella pertussis Nasopharyngeal Swab PCR Nasopharyngeal Aspirate < 4 weeks post-cough onset
Regan Lowe Transport Media or Sterile Container
Refrigerate
CONFIRMATORY
3 working days
RITM
Bacterial Meningitis
S. pneumoniae H. influenzae N.meningitidis Culture
Blood
Onset of illness
Transport at room temperature as soon as possible Transport at room temperature as soon as possible
CONFIRMATORY
7 days
CSF
0.5-1ml
T-I using a sterile syringe and needle EDTA Tube (Purple top) Sterile, screwcapped tube OR b. Properly capped red top tube Sterile Container Sterile Container EDTA Tube (Purple top) Sterile, screwcapped tube OR b. Properly capped red top tube
vent the T-I using a sterilized venting needle and incubate at 37C with 5% CO2
CONFIRMATORY
3 days minimum
Whole Blood Onset of illness PCR Serum
3 to 5 mL a.
0.5mL
Freeze
CONFIRMATORY
3 working days
RITM
CSF CSF Whole Blood

Serology (Latex Agglutination Test)
0.5-1ml 0.5-1ml 3 to 5 mL Onset of Illness a.
Serum
0.5mL
Refrigerated (at 2 8 C) up to several hours or Freeze at 20 for C longer periods
PRESUMPTIVE
1 day working day
Serogrouping
Isolate
Plates Incubated at 37C
Transport at room temperature
CONFIRMATORY
1-2 hours
RITM
FOR VIROLOGY CATEGORY I (IMMEDIATELY NOTIFIABLE DISEASES)

IS THE TEST A CONFIRMATORY / RAPID / PRESUMPTIVE TEST
DISEASE/SYNDROME
UNDER SURVEILLANCE
ETIOLOGIC AGENT/S
TESTS
SPECIMEN TYPE
VOLUME/ QUANTITY
TIMING OF COLLECTION
CONTAINER
STORAGE CONDITIONS
PRIOR TO TRANSPORT
TRANSPORT CONDITIONS
TURNAROUND TIME
TESTING CENTERS
Acute Flaccid Paralysis
Polio Virus and other entero viruses
Virus Isolation
Stool
2 samples thumb size (8-10 grams); 5-10 mL if diarrheic
Within 14 days from onset of paralysis collected 24 hours apart
Separately in clean, leak-proof, screw cap-type containers
Keep refrigerated prior to transport
Transport in ice within 72 hours
Confirmatory Test
2 weeks
RITM Department of Virology National Polio Reference Laboratory
Serum
At least 1-2 mL
Red top (plain) tube
Allow to clot in refrigerator; Keep refrigerated until transport

Allow blood spots to dry thoroughly (at least 60 minutes); Do not expose to heat. Place DBS card in a re-sealable bag with desiccant individually; Keep refrigerated prior to transport.
Transport in ice within 24-48 hours
Serology Measles Measles Virus Dried Blood Spot (DBS) 3 dried blood spots in 1 DBS card per suspect case
Obtain a single sample at the first contact with the health care system at any time within 28 days after onset of rash
Place individually packed DBS in envelope big enough to accommodate the number of DBS. Ship the DBS samples via ordinary postage.
Confirmatory Test
5-7 days
Individually in resealable bag with dessicant
RITM Department of Virology National Reference Laboratory for Measles and other Exanthems
Virus Isolation
NPS/ OPS
1 Nasopharyngeal
Within the first 3 days after onset
VTM
Keep refrigerated prior to transport
Transport in ice within 72 hours
Confirmatory Test
2-3 weeks
Swab and/or 1 Oropharyngeal Swab in the same VTM container 1 Nasopharyngeal Swab and/or Virus Isolation Antigen Detectio n PCR
Adenovirus
of rash
Influenza-like Illness/ Human Avian Influenza/ Severe Acute Respiratory Syndrome
RSV Influenza A/B

Parainfluenza 1/2/3
NPS/ OPS
1 Oropharyngeal Swab in the same VTM container 1.5-2 mL
VTM Within 5 days from onset of illness Mucus trap/ extractor VTM Keep refrigerated prior to transport Transport in ice within 72 hours Confirmatory Test RITM National Influenza Center
2-3 weeks
Rhinovirus
NP
aspirate/
wash Lung aspirate
1 mL
CATEGORY II (WEEKLY NOTIFIABLE DISEASES)

DISEASE/SYNDROME
UNDER SURVEILLANCE
ETIOLOGIC AGENT/S
TESTS
SPECIMEN TYPE
VOLUME/ QUANTITY
TIMING OF COLLECTION Within 5 days from onset of illness
CONTAINER
STORAGE CONDITIONS
PRIOR TO TRANSPORT
IS THE TEST A CONFIRMATORY/ RAPID / PRESUMPTIVE TEST
TURNAROUND TIME
TESTING CENTERS
Acute Encephalitis Syndrome
Japanese
Encephalitis Virus
Serology
Serum
At least 1-2 mL
Allow to clot in refrigerator; Keep refrigerated until transport
Confirmatory Test
RITM 5-7 days National JE Lab
CSF Within 5 days from onset of illness
Dengue
Dengue Virus
Virus Isolation
Serum
1-2 mL
Allow to clot in refrigerator; Keep refrigerated
Confirmatory Test
2 weeks
RITM National Reference Laboratory for
Serology
Whole Blood
5-10 mL
Within Days 5-14 from onset of illness Within 5 days from onset of illness
until transport
Dengue
5-7 days PCR Serum 1-2 mL
FOR PARASITOLOGY FOR SPECIAL PATHOGENS LABORATORY STORAGE CONDITIONS

PRIOR TO TRANSPORT
DISEASE
ETIOLOGIC AGENT/S
TESTS
APPROPRIATE SPECIMEN Routine: Brain Tissue
DATE OF COLLECTION
CONTAINER
IS THE TEST A CONFIRMATORY/ RAPID/ PRESUMPTIVE TEST
RAPID TEST
TESTING CENTERS RITM San Lazaro Hospital Bureau of Animal Industry Philippine Animal Health Center Department of Agriculture Regional Animal Disease Diagnosis
Rabies
Lyssavirus
Antigen Detection Test
Direct Fluorescent Antibody Test (dFAT)
Research PurposesOnly: 1. Salivary Glands 2. Nuchal Biopsy 3. Corneal Imprints
Antemortem/ Postmortem
Sterile specimen container No preservatives
-20C or lower freezer temperature
On ice 2-8C refrigerator temperature 2hours
CONFIRMATORY
N/A
Laboratory Research Purposes Only: 1. Brain Tissue 2. Salivary Glands 3. Nuchal Biopsy 4. Corneal Imprints At least 7-10 days after the last dose of rabies vaccine Antemortem At least 7-10 days after the last dose of rabies vaccine Antemortem Time: 4-6 hours interval Serial: 3-5 samples Single Collection 2 to 8C refrigerator temperature for 2 hours On ice 2-8C refrigerator temperature 2hours Serum separator tube/red top tubes without additives Sterile specimen container Serum separator tube/red top tubes without additives RITM US Centers for Disease Control and Prevention National Institute of Infectious Diseases RITM US Centers for Disease Control and Prevention
Direct Rapid Immunohistochemistry Test (DRIT)
Antibody Detection test
Rapid Fluorescent Focus Inhibition Test (RFFIT)
Routine: Serum
Research Purposes Only: CSF
2 to 8C refrigerator temperature for 2 hours
CONFIRMATORY
Platelia Rabies II Kit
ELISA (Enzyme Linked Immunosorbent Assay)
Routine: Serum
N/A
RITM
RT PCR and Nested PCR
Research Purposes Only: 1. Liquid matrices (e.g. saliva, VTM for oral swabs, urine) 2. Others (e.g. CSF)
Sterile specimen container
N/A
RITM
3. Tissues (e.g. brain tissue, salivary glands, etc) Research Purposes Only: 1. Tissues (e.g. liver, kidney) 2. Liquid matrices (e.g. serum, whole blood in EDTA or heparin tube/whole blood on strips For Research and Routine Testing: 1. Serum 2. Whole Blood on strips
Postmortem
Postmortem
Sterile container
-20C or lower freezer temperature on ice 2-8C referigerator temperature 2hours N/A
Antigen Detection Test
Antemortem
With or without signs/ symptoms When exposure is suspected
Antigen ELISA
Tube without anticoagulant EDTA/Heparin anticoagulated Tube Filter strips placed inside Conical tube Tube without anticoagulant 2 to 8C refrigerator temperature for 2 hours
RITM US Centers for Disease Control and Prevention
Ebola
Ebola Reston
IgG ELISA Antibody Detection Test
Antemortem
With or without signs/ symptoms When exposure is suspected
IgM ELISA
Nobuto strips placed inside Conical tube
on ice 2-8C referigerator temperature 2hours
FOR IMMUNOLOGY ETIOLOGIC AGENT/S Hepatitis A virus STORAGE CONDITIONS PRIOR TO

TRANSPORT
DISEASE
TESTS
SPECIMEN TYPE
CONTAINER serological tubes
TRANSPORT CONDITIONS in ice
IS THE TEST A CONFIRMATORY/RAPID /PRESUMPTIVE TEST Rapid
TURN-AROUND TIME
TESTING CENTERS Dept of Immunology, RITM
Hepatitis
HAV IGM
serum
4C
24 hours
GENERAL GUIDELINES FOR SPECIMEN COLLECTION
OVERVIEW 1. Reference laboratories should usually receive isolates, rather than clinical specimens. However, in the event that the local laboratories cannot perform culture, clinical samples maybe accepted. Proper specimen referral may be simple yet essential in obtaining accurate laboratory diagnosis of infectious disease. Emphasis in two important aspects in specimen collection should be followed. 2. 1. Specimen should be collected before administration of antimicrobial agents 2. Aseptic technique should be followed to prevent contamination of externally present microorganism or normal flora of the body. 3. 4. General rules for collection and transport of samples: 5. 1. Apply strict aseptic techniques throughout the procedure. 2. Wash hands before and after the collection. 3. Collect the specimen at the appropriate phase of disease. 4. Make certain that the specimen is representative of the infectious process (e.g. sputum is the specimen for pneumonia and not saliva) and is adequate in quantity for the desired tests to be performed. 5. Collect or place the specimen aseptically in a sterile and/or appropriate container. 6. Ensure that the outside of the specimen container is clean and uncontaminated. 7. Close the container tightly so that its contents do not leak during transportation. 8. Label and date the container appropriately and complete the requisition form. 9. Arrange for immediate transportation of the specimen to the laboratory.
BLOOD During outbreaks, blood and serum are the most common collected specimen. Venous blood can be used for isolation and identification of the pathogen in culture and by inoculation, or separated into serum for the detection of genetic material (e.g. by Polymerase Chain Reaction), specific antibodies (by serology), antigens or toxins (e.g. by immunofluorescence). For most specimens used for diagnosis of viral pathogens, serum is preferable to whole blood otherwise directed. When specific antibodies are being assayed, it is often helpful to collect paired sera, i.e. an acute sample at the onset of illness and a convalescent sample one to four weeks later. Blood can also be collected by finger prick for the preparation of slides for microscopy or for absorption onto special filter paper discs for analysis. Whenever possible, blood specimens for culture should be taken before antibiotics are administered to the patient.
Materials Needed for Collection: 1. 70% alcohol (isopropyl alcohol, ethanol) or 10% povidone iodine, swabs, gauze pads, medical plaster strips. (for skin disinfection) 2. Disposable latex or vinyl gloves 3. Tourniquet, Vacutainer or similar vacuum blood collection devices, or disposable syringes and needles. 4. Vacutainer or sterile screw cap tubes (or cryotubes if indicated), blood culture bottles (50ml for adults, 25ml for children) with appropriate media. 5. Labels and indelible marker pen. Method of Collection: 1. Place a tourniquet above the venipuncture site. 2. Disinfect the rubber stopper of blood culture bottles.
3. Palpate and locate the vein. It is critical to disinfect the venipuncture site meticulously with 10% povidone iodine or 70% isopropyl alcohol by swabbing the skin concentrically from the center of the venipuncture site outwards. Let the disinfectant evaporate. Do not re-palpate the vein again. 4. Perform venipuncture. 5. If withdrawing using conventional disposable syringes, the volume recommended is: Adults: 5-10 ml of whole blood Children: 2-5ml of whole blood Infants: 0.5-2ml of whole blood. 6. Using aseptic technique, transfer the specimen to appropriate transport tubes and culture bottles. Secure caps tightly. 7. If withdrawing using vacuum systems, withdraw the desired amount of blood directly into each transport tube and culture bottle. 8. Remove the tourniquet. Apply pressure to site until bleeding stops, and apply medical plaster strips. 9. Label the tube, including the unique patient identification number, using indelible marker pen. 10. Do not recap used sharps. Discard directly into the sharps disposal container. 11. Complete the case investigation and the laboratory request forms using the same identification number.
HANDLING AND TRANSPORT
Blood specimen bottles and tubes should be transported upright and secured in a screw cap container or in a rack in a transport box. They should have enough absorbent paper around them to soak up all the liquid in case of spill.
If the specimen can be transported within 24 hours, transport at room temperature. However, if specimen will be transported for >24 hours, keep at 4-8C unless it is a cold-sensitive pathogen.
SERUM
ADDITIONAL MATERIALS REQUIRED 1. Sterile Pasteur pipettes and rubber bulb, or disposable transfer pipettes. 2. Sterile screw-cap tubes (2 per sample).
METHOD OF SEPARATION 1. Draw 10 ml of venous blood using the materials and methods indicated previously and transfer to a screw cap tube without anti-coagulant. 2. Alternatively, blood may be collected directly into a proprietary collection and transport tube (e.g., Vacutainer, Monovette, etc.). 3. Let the blood specimen clot for 30 minutes at room temperature, then place in a cool box to retract at 4 to 8C for a minimum of 1 to 2 hours (it may be stored at this temperature for 48-72 hours). 4. Centrifuge the specimen at low speed (1000g for 10 minutes) to remove residual blood cells. When performing serum separation in a field laboratory proper safety precautions should be taken. 5. Always make sure that the centrifuge is in good condition and the tubes are properly closed and balanced to prevent breakage and spilling. If a viral haemorrhagic fever is strongly suspected, samples should only be processed in properly equipped, specialized laboratories. It is recommended to discuss with the laboratory whether a separation gel blood tube (see Note) would be acceptable in this case. 6. Separate the serum aseptically from the clot using a sterile Pasteur pipette and bulb or soft, disposable transfer pipette. Transfer equally to 2 plastic screw cap tubes. Secure the caps tightly.
7. If a centrifuge is not available and there will be a delay before samples can be transported to a laboratory, serum may still be separated carefully from the retracted clot using a disposable transfer pipette. Allow 4-6 hours to pass after taking the blood sample to ensure adequate clot retraction. 8. Using a transfer pipette, remove the clear yellow serum while taking care to keep the tip as far as possible from the clot, and avoid agitating the blood tube during the removal process. Transfer to plastic screw cap tubes and secure caps tightly. 9. Label the tubes clearly with the same patient details that appear on the blood sample tube.
NOTE: In some cases it may be acceptable to use a special blood tube containing a separation gel, which encourages separation of serum from clot. In this case, the centrifugation step is eliminated. This has advantages for ease and safety of specimen processing under field conditions, but it is important to check with the laboratory in advance to ensure that these devices are appropriate for your particular investigation. HANDLING AND TRANSPORT
If serum will be required for testing, separation from blood should take place as soon as possible, within 24 hours at room temperature.
If the specimen will not reach a laboratory for processing within 24 hours, serum should be separated from blood prior to transportation. Sera may be stored at 4-8C for up to 10 days.
Serum samples may be frozen if testing is delayed for a long period of time. URINE
Materials Needed for Collection:
1. Sterile plastic cup with lid (50 ml or more) 2. Clean, screw capped specimen transport containers 3. Gauze pads 4. Soap and clean water (or normal saline) if possible. 5. Labels and indelible marker pen
Method of Collection: 1. Instruct the patient clearly to pass urine for a few seconds and then hold the cup in the urine stream for a few seconds to catch a mid-stream urine sample. This method decreases the risk of contamination from organisms living in the urethra. 2. Instruct the patient to avoid touching the inside or rim of the specimen cup to decrease the risk of contamination from normal skin flora. Tighten the cap firmly when finished. 3. For hospitalized or debilitated patients, it may be necessary to wash the external genitalia with soapy water to reduce the risk of contamination. If soap and clean water are not available, the area may be rinsed with normal saline. Dry the area thoroughly with gauze pads before collecting the urine. 4. Urine collection bags may be necessary for infants. If used, transfer urine from the urine bag using a disposable transfer pipette to specimen containers as soon as possible to prevent contamination with skin bacteria. 5. Label the specimen containers.
Handling and Transport: Urine must be transported to the laboratory within 30 minutes of collection.
If this is not possible, refrigerate the specimen at 4-8C for no longer than 24 hours. Do not freeze. This will decrease the risk of overgrowth of contaminating organisms.
Ensure that transport containers are leak-proof and tightly sealed. Transport on wet ice or refrigerant gel packs.
STOOL
Stool specimens are most useful for microbiological diagnosis if collected soon after onset of diarrhoea (for viruses < 48 hours and for bacteria < 4 days), and preferably before the administration of antimicrobial agents. If required, two or three specimens may be collected on separate days. Stool is the preferred specimen for culture of bacterial, viral, and parasitic diarrheal pathogens. Rectal swabs showing feces may also be used. In general, rectal swabs are not recommended for the diagnosis of viruses.
MATERIALS FOR COLLECTION 1. Clean, dry, leak-proof screw cap container and tape 2. Appropriate bacterial transport media for (Cary-Blair) 3. Parasitology transport pack: 10% formalin in water, polyvinyl isopropyl alcohol (PVA).
METHOD OF COLLECTING A STOOL SPECIMEN 1. Using a sterile, leak-proof container, collect freshly passed stool - - - 5 ml liquid or 5 g solid (peasize) 2. Label the container properly.
METHOD OF COLLECTING A RECTAL SWAB FROM INFANTS 1. Moisten a swab in sterile saline. 2. Insert the swab tip just past the anal sphincter and rotate gently. 3. Withdraw the swab and examine to ensure that the cotton tip is stained with feces. 4. Place the swab in sterile tube/container containing the appropriate bacterial or viral transport medium. 5. Break off the top part of the stick without touching the tube and tighten the screw cap firmly.
6. Label the specimen tube.
HANDLING AND TRANSPORT Stool specimens should be transported at 4-8C. If specimens are cannot be processed within 12 days, bacterial yields may fall significantly. Shigella sp. is particularly sensitive to elevated temperatures. If specimens will be examined within 48 hours, refrigerate at 4C, otherwise store at -70C and ship on dry ice.
ABSCESS/SKIN LESIONS
For most dermatological conditions, diagnosis may be established on the basis of physical examination and clinical history without the collection of diagnostic specimens. However, it is necessary to collect specimens from rashes and/or skin lesions in cases of indeterminate diagnoses, unusual presentations, and some rare conditions. For cases of vesicular rashes, specimens for microscopy and culture are taken directly from vesicles. In other exanthemata (macular and/or papular), the diagnosis may be more readily established from alternative specimens (e.g. blood cultures, serology). In suspected cutaneous anthrax or bubonic plague, specimens from the skin lesions (eschars and buboes, respectively) and blood cultures may be taken.
MATERIALS FOR COLLECTION 1. Sterile saline 2. Sterile swabs and appropriate transport media 3. Sterile screw-cap vials 4. Sterile lancets or needles (for piercing of vesicles) 5. Syringe with wide-bore needle (for aspiration of abscesses/buboes) 6. Wide-mouth screw-cap containers (for biopsy specimens) 7. Glass slides and slide boxes.
METHOD OF COLLECTION A. Vesicular or vesiculo-pustular rash (for diagnosis of viral infections) 1. Using a sterile lancet, pierce the roof of fluid-containing vesicle. 2. Swab fluid with sterile swab. Try to get a good amount of fluid onto the swab. 3. Take a clean labeled microscope slide and make a smear with the swab in the central area of the slide. Make 2 slides if possible. The slides should be left to dry in air.
4. Place swab directly into virus transport medium. 5. Label the bottles or tubes containing swabs in transport media. 6. When glass slides have dried, place carefully into a plastic slide box. Do not refrigerate or freeze the slides during storage or transport. Keep in the closed container at room temperature.
B. CRUSTING STAGE 1. Gently remove the crust with a lancet or scalpel and a pair of disposable forceps. 2. Take 5-10 crusts; place them in a plastic screw-cap vial. Make sure the lid is tightly closed. 3. Label the specimen containers. 4. Discard forceps, lancets, and scalpels into sharps disposal container. Do not re-use forceps on specimens from another patient.
Note: If cutaneous anthrax is suspected, the vesicular fluid under the eschar is a better diagnostic specimen than a piece of the eschar.
C. ASPIRATION OF ABSCESSES Note: Aspiration of abscesses should only be performed by experienced personnel.
1. Disinfect the skin overlying the abscess/bubo with 70% isopropyl alcohol. 2. Aspirate the fluid from the abscess with a sterile needle and syringe. Collect enough fluid to perform the diagnostic tests. 3. Transfer the aspirate aseptically into a sterile tube with transport medium. HANDLING AND TRANSPORT
For bacteriological analysis, specimens should be transported in Stuarts or Amies medium. For suspected viral pathogens, swabs should be transported in virus transport medium. If processing takes longer than 2 hours, bacteriology specimens can be stored at room temperature for 24 hours. On the other hand, specimens for virus isolation may be refrigerated at 4-8C, and transported to the laboratory as rapidly as possible.
RESPIRATORY TRACT SPECIMENS
Collection of specimen from the respiratory tract depend primarily on the site of infection. Upper respiratory tract pathogens, both viral and bacterial, are isolated from the throat and the nasopharyngeal areas. Lower respiratory tract pathogens, on the other hand, are detected in sputum samples. In the case of a suspected outbreak of Legionella, where culture is timely and difficult, urine is instead collected for detection of the bacterial antigen.
Materials needed for collection: 1. Transport media for bacteria and virus 2. Sterile dacron or rayon swabs with plastic shafts or if available, flocked swabs (calcium alginate swabs or swabs with wooden sticks are not recommended as they may contain substances that inactivate some viruses and inhibit some molecular assays) 3. Tongue depressor 4. Suction apparatus or 20-50 ml syringe 5. Sterile screw-cap tubes, and dry, wide-mouthed clean sterile jars (minimum volume 25ml)
Upper respiratory tract specimens Method of collection for throat swab: 1. Hold the tongue down with the depressor. Use a strong light source to locate areas of inflammation and exudate in the posterior pharynx and the tonsillar region of the throat behind the uvula. 2. Rub the area back and forth with a swab. Withdraw the swab without touching cheeks, teeth or gums. 3. Immediately place the swabs in the container with transport medium. Break off the top part of the applicator sticks without touching the tube and tighten the screw cap firmly.
4. Label the specimen containers. 5. Complete the laboratory request form.
Method of collection for nasopharyngeal swab 1. Seat the patient comfortably and tilt the head back. 2. Insert a flexible swab through the nares parallel to the palate (not upwards) until resistance is encountered or the distance is equivalent to that from the ear to the nostril of the patient indicating contact with the nasopharnyx. Gently, rub and roll the swab. Leave the swab in place for several seconds to absorb secretions. 3. Carefully remove the swab and insert it into the tube containing the transport medium, without antibiotics. Break off the top part of the applicator sticks without touching the tube and tighten the screw cap firmly. 4. Label the specimen tube, indicating left or right side. 5. Complete the laboratory request form. 6. Repeat on the other side.
Lower respiratory tract specimens
Method of collection for sputum 1. Instruct patient to first rinse the mouth with water, then take a deep breath and cough up sputum directly into a wide-mouth sterile container. Avoid saliva or postnasal discharge. Minimum volume should be about 1 ml. 2. Label the specimen container. 3. Complete the laboratory request form.
Handling and transport Specimens must be accurately and properly labeled. Transport as quickly as possible to the laboratory. If specimens will be examined within 48 hours after collection, keep specimen at 4C and ship on wet ice or refrigerant gel-packs, otherwise store frozen at -70C and ship on dry ice. Avoid freezing and thawing of specimens
TISSUE
Materials needed for collection: 1. Container with the appropriate size 2. Fixative (10% buffered formalin) 3. Saline solution 4. Transport media for virus
Method of collection for non-fixed tissues (preferred for culture) 1. Aseptically place the tissue after removal in a container filled with sterile saline and/or viral transport media. Use a separate sterile instrument for each collection site and put each specimen in separate sterile containers. Close the lid tightly to prevent leakage during transport. 2. Label the specimen container. 3. Complete the laboratory request form.
Handling and Transport Specimens must be accurately and properly labeled. Transport as quickly as possible to the laboratory. If specimens will be examined within 48 hours after collection, keep specimen at 4C and ship on wet ice or refrigerant gel-packs, otherwise store frozen at -70C and ship on dry ice.
Method of collection for fixed tissues 1. Place the tissue immediately after removal in a container filled with 10% buffered formalin. The ratio of the fixative and tissue must be 20:1. Close the lid tightly to prevent leakage during transport. A specimen in formalin can be kept 1-2 days before processing. No refrigeration is needed. 2. Label the specimen container. 3. Complete the laboratory request form.
Handling and Transport Specimens must be accurately and properly labeled. If the fixed specimen has been stored in formalin for more than two weeks, the paraffinembedded tissue is preferred for analysis because long fixation may interfere with molecular assays and immunohistochemical testing. Fixed tissues from different organs can be placed in one container. Fixed tissues must be stored and shipped at room temperature. An exemption can be made though in shipping paraffin blocks during a hot weather. Cold packs can be included to prevent melting of the paraffin. References World Health Organization. Guidelines for the collection of clinical specimens during field investigation of
outbreak. Geneva: WHO; 2000.
Fixed tissues must never be frozen.
INTERNATIONAL GUIDELINES FOR TRANSPORT OF INFECTIOUS SUBSTANCES

The guidelines written hereof are based on WHO 2009 2010 Guidance on Regulations for The Transport of Infectious Substances. These guidelines facilitate compliance with current international regulations for the transport of infectious substances and patient specimens by all modes of transport, both nationally and internationally. It has included the changes that apply from 1 January 2009. Specimens need to be transported safely, timely, efficiently and legally from the place where they are collected to the place where they will be analyzed. Human and animal specimens should be packaged and transported in such a way as to protect those engaged in transportation from the risk of infection. Shippers, carrier and consignee must work together, understand their specific obligations and be familiar with the current regulatory requirements. All personnel involved in transport of infectious substances are required to undergo appropriate training and education.
CLASSIFICATION
1. Infectious substance, Category A
Infectious substance, Category A is defined as an infectious substance which is transported in a form that, when exposure to it occurs (e.g. substance is released outside of the protective packaging, resulting in physical contact with humans or animals), is capable of causing permanent disability, lifethreatening or fatal disease in otherwise healthy humans or animals. Indicative examples of substances that meet these criteria are given in the table in Annex 2. Infectious substances, including new or emerging pathogens, which do not appear in the table but which meet the same criteria shall be assigned to Category A. In addition, if there is doubt as to whether or not a substance meets the criteria it shall be included in Category A.
Infectious substances meeting the criteria of infectious substance, category A classification are assigned with UN number and Proper shipping names according to their hazard classification and their composition. Proper shipping names are used to clearly identify the dangerous article or substance.
Infectious substance, Category A which: causes disease in humans or both in humans and animals
UN number
Proper Shipping Name
UN 2814
INFECTIOUS SUBSTANCE, AFFECTING HUMANS
cause disease only in animals
UN 2900
INFECTIOUS SUBSTANCE, AFFECTING ANIMALS only
Assignment to UN 2814 or UN 2900 shall be based on the known medical history and symptoms of the source human or animal, endemic local conditions, or professional judgment concerning individual circumstances of the source human or animal.
2. Infectious substance, Category B
Is defined as an infectious substance which does not meet the criteria for inclusion in Category A. Infectious substances in Category B shall be assigned to UN 3373 with proper shipping name of BIOLOGICAL SUBSTANCE, CATEGORY B.
Exceptions
The following substances of biological origin are exempted from dangerous goods requirements and regulations due to relatively low hazard: Substances that do not contain infectious substances or will not cause disease in humans or animals substances containing microorganisms that is not pathogenic to humans or animals Substances in a form in which any pathogens present have been neutralized or inactivated such that they no longer pose a health risk Environmental samples (including food and water samples) that are not considered to pose a significant risk of infection Blood and/or blood components collected and shipped for the purposes of transfusion and/or transplantation Dried blood spots and faecal occult blood screening tests Decontaminated medical or clinical wastes. Exempt Human/Animal Specimens Human or animal specimens with minimal likelihood that pathogens are present are not subject to strict regulation. It is only requires that specimens be transported in a packaging system which will prevent any leakage. Outer packaging must be marked with the words Exempt human specimen or Exempt animal specimen, as appropriate.
GENERAL PREPARATION OF SHIPMENTS FOR TRANSPORT Packaging, labeling and documentation requirements vary for each category. The current packaging requirements are described and outlined below. Category A Packaging PI 602 Instruction UN 2814 UN Number UN 2900 UN 3373 ---PI 602 PI 650 ---Category A Category B Exempt
Infectious Proper Substance Shipping Affecting Humans Name
Infectious Substance Affecting Animal Only Biological Substance, Category B
Exempt Human Specimen / Exempt Animal Specimen Required if
Training
Required
Required
Required
shipping with dry ice
Leakproof
Leakproof Leakproof
Leakproof
Capable of Primary withstanding the Container temperature required
Capable of Capable of withstanding withstanding the the temperature temperature required required
Capable of withstanding the temperature required
Include Absorbent material in sufficient quantity to absorb the entire content between
primary and secondary container (except for solid infectious substance). Multiple and or fragile primary receptacles must be individually wrapped or separated to prevent contact. Primary container must be kept on upright position and secured inside the secondary container with cushioning material.
Leakproof
Leakproof
Leakproof
Leakproof
Secondary Itemized list of contents must be enclosed between the secondary and outer packaging. Container Secondary container must be kept on upright position and secured inside the secondary container with cushioning material. Primary or Shall be capable of withstanding without leak an internal pressure Secondary producing a differential of not less than 95kPa and temperature Container range -40 C to + 55C (-40F to +130 F) --------
Must be Rigid
Must be rigid
Either secondary or Not less than Outer Container Drop tested at 9meters Puncture Tested Drop tested at 9meters Puncture Tested Drop tested at 1.2 100mm (4inches) Not less than outer container is rigid 100mm (4inches)
Must be of adequate strength
--------meters -----------------
Other dangerous goods shall not be packed in the same packaging as Division 6.2 infectious substances unless they are necessary for maintaining the viability, stabilizing or preventing degradation or neutralizing the hazards of the infectious substances. A quantity of 30 ml or less of dangerous goods included in Classes 3 (flammable liquids), 8 (corrosives) or 9 (miscellaneous dangerous substances and articles) may be packed in each primary receptacle containing infectious substances.
Inner packaging containing infectious substances shall not be consolidated with inner packaging containing unrelated types of goods. Shippers name and address Markings Receivers name and address Name and telephone of responsible person (who is available 24 hours a day until shipment arrives) Proper Shipping Name and UN Number Proper Shipping Name UN Specification Marking ---------------------------------
Infectious substance label Package orientation label (only used Label when primary container exceeds 50ml) Shippers Declaration of Dangerous --------Goods -------------------------
Air transport
Airway bill maximum 50ml or 50g per package for maximum 4kg or 4l per package -maximum 1l per primary container for passenger or cargo aircraft Permit (import/export) --------
Limitation of transport by air
passenger aircraft maximum 4kg or 4l per package for cargo aircraft
Other Documents
Letters (authorization) Certifications Others required by the receiving party / area of destination and portal of exits Subject to specific
Hand carriage
strictly prohibited by international air carriers regulation of the transport mode
Regulatory requirements presented in the table may change in time. The shipper, couriers and consignee of any infectious substances must be updated with the current guidelines prior transport of any infectious substances. Local transport of infectious substances by land requires the basic packaging requirements set by the international regulation except for the documentation requirement.
Source : WHO 2009 2010 Guidance on regulations for The Transport of Infectious Substances
PART III. REPORTING OF LABORATORY RESULT

Guidelines for Reporting of Laboratory Result Guidelines for Interpretation of Result
GUIDELINES FOR REPORTING OF LABORATORY RESULT

a) The testing laboratory should prepare the following documents after finalizing the laboratory results: Official Laboratory Results (kindly refer to the Annex section for the template) Official results should include the following information: A. Patient info: name, age, sex, date of onset of symptoms, B.Specimen info: type, date of specimen collection, C. Referring institutions info: name, requesting physician/health worker D. Test performed: type (e.g. culture), kit (if commercial kit is used) E. Test Results F. Date Reported G. Signatories: Technologist, Lab Supervisor, Pathologist/MD Cover letter addressed to the CHD Regional Director through the RESU Head, and copied to the referring institution/ESU, NEC, and testing laboratorys division chief and institutional director (kindly refer to the Annex section for the template) b) The testing laboratory should fax those two documents to the following institutions: referring institution or ESU, RESU, and NEC. - The testing laboratory should provide a copy of the cover letter to their division chief and institutional director. RITM NRLs should provide copies of results to the RITM LRD Chief and RITM Director. c) Official results may only be sent to authorized fax numbers or emails. As per RITMs SOP, results may not be released over the phone or through SMS text messages.
d) NEC will provide Infectious Disease Office, and other stakeholders, with a summary of the outbreak reports for appropriate action.
LABORATORY RESULTS
Testing Laboratory should prepare the following documents:

Official Laboratory Result Form Cover letter - Addressed to: CHD Regional Director - Through: RESU Head - Copied to: i. Referring Institution/RESU ii. NEC iii. Division Chief of testing laboratory iv. Institutional Director of the testing laboratory
Fax
Give a copy
RESU
Referring Institution / ESU
NEC
Testing Laboratorys Division Chief
Testing Laboratorys Institutional Director
NEC will prepare a summary of Outbreak Report
Infectious Disease Office
GUIDELINES OF INTERPRETATION OF RESULTS

FOR BACTERIOLOGY
Clinical impression Acute bloody diarrhea
Bacterial Etiologic agent Shigella spp.*
Positive test result Isolation of the organism from the clinical specimen by culture and confirmation by serology
Interpretation Positive
Negative test result Non-isolation of organism
Interpretation Negative
Remarks If Shigella dysenteriae 1 is not isolated during a suspected outbreak, the laboratory should test for E. coli O157:H7
Enterohemorrhagic E. coli 0157:H7*
Isolation of the organism from the clinical specimen by culture and confirmation by serology
Positive
Non-isolation of organism
Negative
Enteroinvasive E. coli
Isolation of the organism from the clinical specimen by culture and confirmation by serology
Positive
Negative
Campylobacter jejuni
Isolation of the organism from the clinical specimen by culture
Positive
Negative
Cholera
Vibrio cholera 01* and 0139*
Isolation of the organism from the clinical specimen by culture and confirmation by serology Isolation of the organism from the clinical specimen by culture and confirmation by serology Isolation of the organism from the clinical specimen by culture and confirmation by serology Isolation of the organism from the clinical specimen by culture
Positive
Negative
During an outbreak or epidemic, it is necessary to document the biotype and serotype of the isolate
Vibrio non-01 and Vibrio non-0139
Positive
Negative
Vibrio parahemolyticus
Positive
Negative
Yersinia enterocolitica
Positive
Negative
Typhoid and paratyphoid fever
Salmonella typhi
Isolation of the organism from the clinical specimen by culture and confirmation by serology Isolation of the organism from the clinical specimen by culture and confirmation by serology
Positive
Negative
Proper and timely collection of specimen is very important
Salmonella nontyphi
Positive
Negative
Anthrax
Bacillus anthracis
Isolation of the organism from the clinical specimen by culture( or detection of DNA/RNA by PCR when culture is not available)
Positive
Negative
Importance of Biosafety practice as this organism has spores to easily infect
Meningococcal disease
Neisseria meningitides
Isolation of the organism from the clinical specimen by culture (or detection of DNA/RNA by PCR when culture is not available)
Positive
Negative
Diptheria
Corynebacterium diphtheriae
Isolation of the organism from the clinical specimen by culture (or detection of DNA/RNA by PCR when culture is not available) Isolation of the organism from the clinical specimen by culture (or detection of DNA/RNA by PCR when culture is not available)
Positive
Negative
Pertussis
Bordetella pertussis
Positive
Negative
Choice of the diagnosis depends on the age and the immune status of the suspected patients. Infants: culture is priority or PCR where culture is not available but serology is unreliable Children: Culture or PCR for non-vaccinated children (first days of coughing) or serology (if vaccination was not performed during the last 3 years) Adults: Serology (if vaccination was not performed during the last three years)
Bacterial meningitis
Haemophilus influenzae
Streptococcus pneumoniae
Listeria monocytogenes
Isolation of the organism from the clinical specimen by culture (or detection of DNA/RNA by PCR when culture is not available) Isolation of the organism from the clinical specimen by culture (or detection of DNA/RNA by PCR when culture is not available) Isolation of the organism from the clinical specimen by culture
Positive
Negative
Positive
Negative
Positive
Negative
* most common cause of the outbreak/disease Before concluding whether a suspected outbreak case is positive or negative for a certain bacterial etiologic agent, other significant data must be considered. These include proper specimen collection, epidemiological reports, other laboratory tests and clinical assays.
References Centers for Disease Control and Prevention and World Health Organization. Manual for Laboratory identification and antimicrobial susceptibility testing of bacterial pathogens of public health importance in the developing world Haemophilus influenzae, Neisseria meningitides, Streptococcus pneumoniae, Neisseria gonorrhea, Salmonella serotype typhi, Shigella, Vibrio cholerae. Georgia: CDC; 2003. Centers for Disease Control and Prevention. Basic laboratory protocols for the presumptive identification of Bacillus anthracis. Georgia: CDC; 2001. Centers for Disease Control and Prevention. Guide to Confirming a diagnosis in Foodborne disease. CDC Surveillance Summaries. Georgia: CDC; 2000. MMWR 2000;49 (No. SS-1) Efstration A, Maple PAC. Manual for the laboratory diagnosis of diphtheria. Copenhagen: Expanded Programme on Immunization in the European Region of WHO, 1994. (ICP/EPI038 (0)). Global Task Force on Cholera control. Guidelines for cholera control. Geneva: World Health Organization, 1992. Publication no. WHO/CDD/SER/80.4 Rev 4. World Health Organization and ILS (International Leptospirosis Society). Human leptospirosis: Guidance for diagnosis, surveillance and control. Geneva: WHO; 2003. World Health Organization. Epidemic diarrheal disease preparedness and response: training and practice. Participants manual. Geneva: WHO; 1997. Publication no. WHO/EMC/DIS/97.3 World Health Organization. Guidelines for the control of epidemics due to Shigella dysenteriae 1. Geneva: WHO; 1995. Publication no. WHO/CDR/95.4 World Health Organization. Laboratory manual for the diagnosis of whooping cough caused by Bordetella pertussis/B. parapertussis. Geneva: WHO;2004. Publication no. WHO/IVB/04.14 World Health Organization. Prevention and control of enterohemorrhagic Escherichia coli (EHEC)
infections. Report of a WHO Consultation. Geneva, Switzerland, 28 April-1 May 1997. WHO/FSF/FOS/97.6
FOR MOLECULAR BIOLOGY

Molecular diagnostic assays have greatly enhanced the sensitivity of pathogen detection in clinical samples. However, interpretation of results must be within the clinical context and performance of the assay. An understanding of the limitations of the assay, as well as the microbiology and pathogenesis of the target organism, is critical for the proper interpretation of the results.1 Molecular assays are based mainly on the polymerase chain reaction (PCR) technology, which detects the presence of the microorganism by amplification of minute amounts of its nucleic acid. Hence, a positive result in a molecular diagnostic test means that the target nucleic acid (deoxyribonucleic acid, DNA, or ribonucleic acid, RNA) has been detected, and conversely, a negative result means that the target nucleic acid was not detected in the clinical sample. Typical results would contain the following information: TEST RESULTS _(Pathogen)_ DNA/RNA detected OR Positive POSITIVE for _(Pathogen)_DNA/RNA _(Pathogen)_ DNA/RNA NOT detected OR Negative NEGATIVE for _(Pathogen)_DNA/RNA INTERPRETATION
These results would have to be interpreted in the context of available epidemiological and clinical data. A positive result from a sample without epidemiological association does not prove that the person or item was a source or vehicle of infection, while a positive result with corresponding epidemiological or clinical data strongly suggests that the person or item was a source or vehicle of infection. In the same manner, a negative result also does not necessarily mean there is no association, but only means that the pathogen was not detected in the clinical sample tested.2 Apart
from the absence of the organism in the sample, this might be caused by any one of the following reasons: 1) inappropriate volume or type of specimen 2) presence of PCR inhibitors 3) inadequate sample collection, transport or processing or 4) inappropriate timing of sample collection. Aside from positive or negative results, there are some cases when the assay internal control would fail, invalidating the test. In this case, the result would be reported as: TEST RESULTS Invalid result due to sample quality INTERPRETATION Invalid result
When this result is released, another sample collection is requested in order to perform a repeat of the assay. In interpreting molecular test results, it is important to know the target gene/s of the assay. Assays for identification of bacterial and parasite DNA can have both genus and species specific gene targets. The molecular detection of Bacillus anthracis, the causative agent of anthrax, for instance, may employ assays for the detection of the genus Bacillus and subsequently, B. anthracis-specific gene targets. If it is positive only for the genus and not the B. anthracis species, the clinical specimen may harbor only the non-pathogenic Bacillus spp. Example:
TEST RESULTS Positive for Bacillus sp. DNA; Negative for Bacillus anthracis DNA Positive for Bacillus sp. DNA; Positive for Bacillus anthracis DNA. Negative for Bacillus sp. DNA; Negative for Bacillus anthracis DNA.
INTERPRETATION Sample is NEGATIVE for B. anthracis but is positive for OTHER Bacillus species Sample is POSITIVE for B. anthracis Sample is NEGATIVE for Bacillus species (including B. anthracis)
For viruses, the detection of specific subtypes or serotypes of certain viruses have significant public health implications. Influenza subtype detection is important during outbreak investigations since novel or highly pathogenic subtypes may cause pandemics. REFERENCES
1
Forbes BA. Introducing a molecular test into the clinical microbiology laboratory. Arch Pathol
Lab Med 2003 Sep; 127:1106-1111.

2
Institute of Environmental Science and Research Limited. Disease outbreak manual. Porirua:
2002. 168p. ABBREVIATIONS / ACRONYMS PCR-polymerase chain reaction DNA-deoxyribonucleic acid RNA-ribonucleic acid
ANNEXES
Biosafety Guidelines Preparation of Transport Media Document Forms and Templates Template for Transmittal Linelist of Referred Specimen Laboratory Request Form Template for Laboratory Results Cover Letter Official Laboratory Result Form
GENERAL BIOSAFETY GUIDELINES
Operational biosafety practices and techniques must be used to minimize the occurrence of infection as a result of improper handling of infectious microorganisms and its by-products. The term "containment" is used in describing safe methods for managing infectious materials in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents. Primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by both good microbiological technique and the use of appropriate safety equipment. The use of vaccines may provide an increased level of personal protection. Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials, is provided by a combination of facility design and operational practices. Therefore, the three elements of containment include laboratory practice and technique, safety equipment, and facility design. The risk assessment of the work to be done with a specific agent will determine the appropriate combination of these elements.
Laboratory Practice and Technique. The most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with infectious agents or potentially infected materials must be aware of potential hazards, and must be trained and proficient in the practices and techniques required handling such material safely.
Standard Microbiological Practices Use of pipettes and pipetting aids 1. A pipetting aid must always be used. Pipetting by mouth must be prohibited. 2. All pipettes should have cotton plugs to reduce contamination of pipetting devices. 3. Air should never be blown through a liquid containing infectious agents. 4. Infectious materials should not be mixed by alternate suction and expulsion through a pipette. 5. Liquids should not be forcibly expelled from pipettes. 6. Mark-to-mark pipettes are preferable to other types as they do not require expulsion of the last drop. 7. Contaminated pipettes should be completely submerged in a suitable disinfectant contained in an unbreakable container. They should be left in the disinfectant for the appropriate length of time before disposal. 8. A discard container for pipettes should be placed within the biological safety cabinet, not outside it. 9. Syringes fitted with hypodermic needles must not be used for pipetting. 10. Devices for opening septum-capped bottles that allow pipettes to be used and avoid the use of hypodermic needles and syringes should be used. 11. To avoid dispersion of infectious material dropped from a pipette, an absorbent material should be placed on the working surface; this should be disposed of as infectious waste after use.
Avoiding the dispersal of infectious materials 1. In order to avoid the premature shedding of their loads, microbiological transfer loops should have a diameter of 23 mm and be completely closed. The shanks should be not more than 6 cm in length to minimize vibration.
2. The risk of spatter of infectious material in an open Bunsen burner flame should be avoided by using an enclosed electric microincinerator to sterilize transfer loops. Disposable transfer loops, which do not need to be resterilized, are preferable. 3. Care should be taken when drying sputum samples, to avoid creating aerosols. 4. Discarded specimens and cultures for autoclaving and/or disposal should be placed in leakproof containers, e.g. laboratory discard bags. Tops should be secured (e.g. with autoclave tape) prior to disposal into waste containers. 5. Working areas must be decontaminated with a suitable disinfectant at the end of each work period.
Avoiding ingestion of infectious materials and contact with skin and eyes 1. Large particles and droplets (> 5 m in diameter) released during microbiological manipulations settle rapidly on bench surfaces and on the hands of the operator. Disposable gloves should be worn. Laboratory workers should avoid touching their mouth, eyes and face. 2. Food and drink must not be consumed or stored in the laboratory. 3. No articles should be placed in the mouth pens, pencils, chewing gum in the laboratory. 4. Cosmetics should not be applied in the laboratory. 5. The face, eyes and mouth should be shielded or otherwise protected during any operation that may result in the splashing of potentially infectious materials.
Avoiding injection of infectious materials 1. Accidental inoculation resulting from injury with broken or chipped glassware can be avoided through careful practices and procedures. Glassware should be replaced with plastic ware whenever possible. 2. Accidental injection may result from sharps injuries e.g. with hypodermic needles (needle-sticks), glass Pasteur pipettes, or broken glass.
3. Needle-stick injuries can be reduced by: (a) minimizing the use of syringes and needles (e.g. simple devices are available for opening septum-stoppered bottles so that pipettes can be used instead of syringes and needles; or (b) using engineered sharp safety devices when syringes and needles are necessary. 4. Needles should never be recapped. Disposable articles should be discarded into punctureproof/puncture-resistant containers fitted with covers. 5. Plastic Pasteur pipettes should replace those made of glass.
Separation of serum 1. Only properly trained staff should be employed for this work. 2. Gloves and eye and mucous membrane protection should be worn. 3. Splashes and aerosols can only be avoided or minimized by good laboratory technique. Blood and serum should be pipetted carefully, not poured. Pipetting by mouth must be forbidden. 4. After use, pipettes should be completely submerged in suitable disinfectant. They should remain in the disinfectant for the appropriate time before disposal or washing and sterilization for reuse. 5. Discarded specimen tubes containing blood clots, etc. (with caps replaced) should be placed in suitable leakproof containers for autoclaving and/or incineration. 6. Suitable disinfectants should be available for clean-up of splashes and spillages
Opening of ampoules containing lyophilized infectious materials Care should be taken when ampoules of freeze-dried materials are opened, as the contents may be under reduced pressure and the sudden inrush of air may disperse some of the materials into the atmosphere. Ampoules should always be opened in a biological safety cabinet. The following procedures are recommended for opening ampoules. 1. First decontaminate the outer surface of the ampoule.
2. Make a file mark on the tube near to the middle of the cotton or cellulose plug, if present. 3. Hold the ampoule in alcohol-soaked cotton to protect hands before breaking it at a file scratch. 4. Remove the top gently and treat as contaminated material. 5. If the plug is still above the contents of the ampoule, remove it with sterile forceps. 6. Add liquid for resuspension slowly to the ampoule to avoid frothing.
Storage of ampoules containing infectious materials Ampoules containing infectious materials should never be immersed in liquid nitrogen because cracked or imperfectly sealed ampoules may break or explode on removal. If very low temperatures are required, ampoules should be stored only in the gaseous phase above the liquid nitrogen. Otherwise, infectious materials should be stored in mechanical deep-freeze cabinets or on dry ice. Laboratory workers should wear eye and hand protection when removing ampoules from cold storage. The outer surfaces of ampoules stored in these ways should be disinfected when the ampoules are removed from storage.
Standard precautions with blood and other body fluids, tissues and excreta Standard precautions (which include universal precautions) are designed to reduce the risk of transmission of microorganisms from both recognized and unrecognized sources of infection.
Collection, labelling and transport of specimens 1. Standard precautions should always be followed; gloves should be worn for all procedures. 2. Blood should be collected from patients and animals by trained staff. 3. For phlebotomies, conventional needle and syringe systems should be replaced by single-use safety vacuum devices that allow the collection of blood directly into stoppered transport and/or culture tubes, automatically disabling the needle after use.
4. The tubes should be placed in adequate containers for transport to the laboratory and within the laboratory facility. Request forms should be placed in separate waterproof bags or envelopes. 5. Reception staff should not open these bags.
Opening specimen tubes and sampling contents 1. Specimen tubes should be opened in a biological safety cabinet. 2. Gloves must be worn. Eye and mucous membrane protection is also recommended (goggles or face shields). 3. Protective clothing should be supplemented with a plastic apron. 4. The stopper should be grasped through a piece of paper or gauze to prevent splashing. Glass and sharps 1. Plastics should replace glass wherever possible. Only laboratory grade (borosilicate) glass should be used, and any article that is chipped or cracked should be discarded. 2. Hypodermic needles must not be used as pipettes. Films and smears for microscopy Fixing and staining of blood, sputum and faecal samples for microscopy do not necessarily kill all organisms or viruses on the smears. These items should be handled with forceps, stored appropriately, and decontaminated and/or autoclaved before disposal. References World Health Organization Geneva. Laboratory Biosafety Manual, Third edition. 2004 U.S. Department of Health and Human Services Public Health Service, Centers for Disease Control and Prevention and National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories, Fourth Edition. 1999
PREPARATION OF TRANSPORT MEDIA
Amies transport medium A semi-solid media used for the collection, transport and preservation of microbiological samples, preferably those from the throat, vagina and wound. Amies incorporated charcoal in the formula upon discovering that Neisseria gonorrhea increased its survival rate when charcoal is used. Components: Sodium thioglycolate Sodium chloride Potassium chloride Calcium chloride Magnesium chloride Potassium phosphate, monobasic Sodium phosphate, dibasic Agar with charcoal Demineralized/distilled water Final ph 7.4 + 0.2 at 25C Procedure : Suspend the ingredients in the water and dissolve by boiling. Dispense in tubes and autoclave for 15 minutes at 121C. Prior to solidification, the tubes should be inverted to distribute the charcoal evenly. Store in refrigeration. 1.00 g 3.00 g 0.20 g 0.10 g 0.10 g 0.20 g 1.15 g 4.00 g 10.00 g 1L
Cary Blair transport medium A semi-solid media almost exclusively used for the transport of fecal samples and rectal swabs for the isolation of enteric pathogens. It is a modification of the Stuart formulation. Components: Sodium thioglycolate Disodium phosphate Sodium chloride Agar Demineralized/distilled water Procedure : Prepare in a clean glassware rinsed with Sorensen 0.067 M buffer (pH 8.1). Heat with agitation until the solution just becomes clear. Cool to 50C, add 9 ml of freshly prepared aqueous 1% CaCl2, and adjust the pH to about 8.4. Dispense 7 ml into previously rinsed and sterilized 9-ml screw cap vials. Steam vials for 15 minutes, cool, and tighten the caps. Calcium chloride is already incorporated in commercially prepared powder form Carry Blair medium. 1.5 g 1.1 g 5.0 g 5.0 g 991.0 ml
Virus Transport Media (VTM)

Department of Virology - RITM Marianne R. Morales, RMT; Ma.Anne-Lesley Valencia, RMT; Analisa N. Bautista, RMT; Hazel O. Galang, RMT; Amado O. Tandoc III, MD, DPSP
Virus Transport Medium is used for the transport of clinical specimens collected for detection and isolation of suspected viral agents including: Herpes Simplex Type I, Herpes Simplex Type II, Cytomegalovirus (CMV), Influenzae A, Influenzae B, Parainfluenzae, Respiratory Syncytial Virus (RSV), Rhinovirus, Enterovirus, Adenovirus, et cetera. Appropriately prepared and used, such media maintain suitable conditions for virus isolation and contain antimicrobials to prevent growth of other organisms. Specimens that require VTM are those that are collected using swabs and/or those that easily dry up/dessicate. It is important to avoid repeated freezing and thawing as such may cause deterioration and changes in pH which may reduce recovery of viable organisms. Shelf life of the medium is 3 months from the date of preparation.
MATERIALS Triple Distilled Water Modified Hanks Balance Salt Solution (without CaCl2, MgSO4, & NaHCO3) -formulated to contain 9.5g of powder/liter of medium (SIGMA, Cat. #: H2387) Gelatin powder from porcine skin Type A, SIGMA, Cat. #: G1890 Sodium Bicarbonate (NaHCO3), SIGMA, Cat. #: S5761 Mycostatin/Fungizone (Generic: Nystatin), -concentration: 100,000 units/mL, ready-mix, oral-suspension -can be purchased over-the-counter in drugstores Penicillin-Streptomycin, liquid, 100ml/bottle -concentration: 100,000 units of Penicillin (base) and 10,000 g of Streptomycin (base)/ml, GIBCO Cat#: 15140-122 or
Benzylpenicillin sodium, 1,000,000 units and streptomycin sulfate, 1g powder, lyophilized Sterile Media Bottles: 1000ml, Pyrex Cat #: CZ-34514-25; 250 ml, Pyrex Cat #: CZ-34514-23 Weighing Scale Graduated Cylinder, 1L Disposable Serological Pipettes, 5 mL or 10 mL Membrane Filter, 0.2 m - Vacucap w/ tubing attached, PALL, Cat #: TA4632; or - Sterivex (Sterile Filter Unit w/ Filling Bell), Millipore, Catalog #: SVGVB1010 Vacuum pump (used together w/ Vacucap Filter); or Sterile Disposable Syringe (used together w/ Sterivex) Water Bath; Autoclave; Micropipettor 100-1000 L volume Sterile Screw Capped Tubes, 13x100 mm Glass: Pyrex, 7.5 mL, Catalog No. CZ-34579-21 with Screw Caps, Pyrex, Catalog #: CZ-34581-21 or Polystyrene: Falcon, 8 mL, Catalog No. F2027, disposable Tube (optional: for aliquoting VTM for pH measurement) 2 tubes of 5ml. Thioglycollate Medium -for sterility test 1N HCl 1N NaOH (sterile)
DEFINITION OF TERMS Modified Hanks Balanced Salt Solution (HBSS) - use to maintain the pH and osmotic balance in the medium and to provide the cells with water and essential inorganic ions. Sodium Bicarbonate (NaHCO3) - buffer used to maintain the pH
Gelatin powder from porcine skin Type A - nutrient agar Penicillin/Streptomycin Solution - antibacterial Mycostatin/Fungizone (Generic: Nystatin) antifungal
RESPONSIBILITY The procedure of VTM preparation shall be carried out by staff assigned for cell culture and maintenance. The supervisor will oversee the procedures, verify completion of records, and troubleshoot problems.
PROCEDURE FOR PREPARATION: STEP NO. 1 Aliquot 800 mL of triple distilled water to a 1 Liter sterile media triple distilled water into another sterile media bottle. bottle and 150 mL of TASK/ACTIVITIES
2 3 4
Weigh 2.5 grams of gelatin powder and 0.35 grams of Sodium Bicarbonate. Add 2.5 grams of gelatin powder to the aliquoted 150 mL of triple distilled water. Autoclave the bottle containing gelatin powder at 115C for 10 mins. Or you may dissolve in the waterbath at 100C, check periodically the gelatin solution if its completely dissolved, swirl the container to mix. To the 800 mL triple distilled water, add 9.5 grams HBSS (Hanks Balanced Salt Solution) and 0.35 grams Sodium Bicarbonate. Rinse original Hanks container with a small amount of triple distilled water to remove all traces of powder. Stir until dissolved. Add lukewarm gelatin solution to the solution in step 5. Stir until dissolved. Do not heat.
Sterilize immediately by filtration using a 0.22 m filter membrane into another sterile bottle. You may use a vacuum pump or a syringe for sterilization.
8 9
Add 50 mL Penicillin/Streptomycin Solution and 250 L Mycostatin. Aliquot 3 mL of VTM to a tube for pH measurement. The use of 1N HCl or 1N NaOH is recommended to adjust the pH to 7.4 Note: Color of the VTM is Salmon pink, with a pH of 7.4 0.3 (pH 7.1- 7.7).
10
Aseptically dispense 2.5 mL into a sterile screw capped tubes inside the Biosafety Cabinet II or Laminar-Flow Hood. Be sure that there will be a remaining 2ml volume of VTM for sterility test. Store VTM at -20C. For sterility testing of the VTM:
11
Dispense 1 mL of VTM into each tube of thioglycollate medium. Incubate at 37C incubator and room temperature, respectively. Observe both tubes for bacterial or fungal growth for 7 days. If the thioglycollate medium is clear, the VTM is ready to use. If there is turbidity or any signs of bacterial growth, discard the VTM.
Preparation of specimens and culture for Leptospira
Specimen source: Urine a. Prepare three (3) test tubes of semi-solid EMJH or Fletchers medium. b. Inoculate 1-2 drops to each tube: Tube #1 : undiluted urine Tube #2 : 1:10 dilution with PBS Tube #3 : 1:100 dilution CSF a. Prepare two (2) tubes if semi-solid of EMJH or Fletchers medium. b. Inoculate 0.5 5ml of CSF specimen. Blood Maybe collected : 1. Whole blood at bedside 2. Heparinized blood 3. Defibrinated blood 4. Triturated clotted blood a. Prepare four (4) tubes of EMJH or Fletchers medium. b. Inoculate : Tube #1 : one (1) drop Tube #2 : two (2) drops Tube #3 and #4 : three (3) drops each
Tissues (Liver or Kidney) a. Prepare three (3) tubes of semi-solid EMJH or Fletchers medium. b. Macerate tissue by a glass grinder or mortar and pestle. c. Inoculate 1-2 drops each : Tube #1 : direct inoculation Tube #2 : 1:10 dilution with NSS Tube #3 : 1:100 dilution with NSS
Do not shake or disturb the medium. Incubate the tubes at 28C - 30C (room temperature) in the dark.

Manual On Lab Referral For Outbreak Response-Draft 2

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MANUAL ON LABORATORY REFERRAL FOR INFECTIOUS DISEASE OUTBREAK RESPONSE

Version 05 May 2011

BACKGROUND AND RATIONALE

ROLES AND RESPONSIBILITIES

LIST OF QUALIFIED LABORATORIES FOR PANDEMIC INFLUENZA A/H1N1 TESTING

LABORATORY REFERRAL FLOW

Referring Institution / CESU / MESU / PESU

Are there any discrepancies, corrections, wrong specimen, missing documents? No

FOR SURVEILLANCE (From case reporting to specimen referral request)

The original flow from the PIDSR:

1. Identification of cases using the PIDSR Case Definitions

5. Collection of appropriate sample

6. Proper packaging and transport

Cases from hospitals, clinics, ports, airports and laboratories

Cases from the community

Barangay Health Station (BHS)

Rural Health Unit/ City Health Office Feedback

Provincial Epidemiology and Surveillance Units (PESU)

Regional Epidemiology and Surveillance Units (RESU)

Legend: Weekly reporting Feedback Immediate notification (within 24 hrs)

World Health Organization

Taken from the PIDSR Manual

Figure 2: Flow of Weekly Reporting of Notifiable Diseases

Cases from the Community

Barangay Health Stations (BHS)

Barangay Health Stations (BHS)

Cases from local hospitals, clinics, ports, airports

Rural Health Units and City Health Offices in nonchartered cities

Cases from provincial and district hospitals, ports, airports

Provincial Epidemiology and Surveillance Units

City Epidemiology and Surveillance Units (CESU) in chartered cities

Cases from local hospitals, clinics, ports, airports

Regional Epidemiology and Surveillance Units (RESU)

Cases from level 3 and retained hospitals, ports, airports

Cases from referral hospitals, laboratories,

National Epidemiology Center

Taken from the PIDSR Manual

REQUIREMENTS FOR SPECIMEN REFERRAL

Testing Laboratorys Laboratory Request Form

References: 1.World health Organization; http://www.searo.who.int/en/Section10/Section17/Section53/Section482_1779.ht

PART II. SPECIMEN COLLECTION AND TRANSPORT

SPECIMEN COLLECTION QUICK GUIDE

STORAGE CONDITIONS PRIOR

TRANSPORT /TIME CONDITIONS

IS THE TEST A CONFIRMATORY/ RAPID /PRESUMPTIVE TEST

Fresh Stool Culture

As soon as possible after onset of illness

2-5 mL/ peasized

Sterile, wide-mouthed, screw-capped, spillproof container

Within 24 hours at room temperature Within 3-5 days at room temperature or 4C

RITM Any Qualified Tertiary Laboratory

Cary Blair Transport Medium in sterile, autoclavable screw capped, container

Except for Salmonella H-typing: minimum of 3 days

Vibrio cholerae Other Vibrios

As soon as possible after onset

Sterile, wide-mouthed, screw-capped, spillproof container

Within 1-2 hours at room temperature

Rectal Swab Serogrouping/ Serotyping Isolate

Salmonella Typhi Culture Salmonella Paratyphi A Rectal Swab

2 to 3 week after onset of illness

Sterile, wide-mouthed, screw-capped, spillproof container

Cary Blair Transport Medium in sterile, autoclavable screw capped, container