guidelines on collection and transport of specimens for suspected ...

Research Institute for Tropical Medicine 

LABORATORY RESEARCH DIVISION 

MICROBIOLOGY DEPARTMENT 

Document: 

Introduction: 

Guidelines for Collection and Transport of Specimen for Laboratory 

Diagnosis of Pathogenic Leptospira spp. 

Leptospirosis is an acute bacterial infection caused by organism belonging to 

the genus Leptospira. They are zoonotic organisms often affecting animals 

but can be transmitted directly or indirectly from animals to humans. 

Leptospirosis is a biphasic disease. The first phase, acute phase or phase of 

leptospiremia occurs from the start of the disease and may last for 4-7 days. 

During this phase leptospires multiply in the blood and spread to different 

organs. Recovery rate of leptospires from blood or other tissues or body 

fluids is usually high during this stage. It is also during the acute phase 

wherein seroconversion starts but may only be detectable after 4-7 days 

after onset of illness. 

The second phase, convalescent phase, immune phase or leptospirurea 

phase occurs 3-5 days after the acute phase and may last up to 30 days after 

the onset of illness. During this phase leptospires are being excreted in the 

urine and may only persist in the blood at very low concentration. During 

the convalescent phase seroconversion reaches its peak and antibodies are 

at their highest detectable level after which they slowly decline and may last 

for years at very low levels. 

Available Tests: 

Real-Time PCR (qPCR) 

- Molecular detection of pathogenic Leptospira spp. DNA from blood, CSF 

and urine. 

Microscopic Agglutination Test (MAT) 

- Serological test for the detection of leptospire specific antibodies present 

in the serum. 

Culture 

- Identification of live leptospires from blood, CSF and urine. 

Rates: Samples with PIDSR form Free of charge 

Samples without PIDSR form 

Real-Time PCR (qPCR) 2,800.00 

Microscopic Agglutination Test 1,120.00 

Culture (no sensitivity test) 2,800.00 

Turn-around Time: Real-Time PCR (qPCR) 2-3 days 

(working days) Microscopic Agglutination Test 7 days 

Culture (no sensitivity test) 

6-8 weeks 

Ver. 1.5 2012 EMS 

Page 1 of 5




Recommended Specimen: (Collection and Transport) 

 

For Real-Time PCR (qPCR) 

1. BLOOD (1 to 10 days after onset of symptoms) 

a. Serum is the preferred sample. Obtain 0.5 to 2 ml of serum and transfer it into a 

sterile 2ml screw capped tube. Freeze sample (-10±5°C) 

b. Venous blood in EDTA tube (do not use citrate and heparin anticoagulants) 

2. CSF (5 to 10 days after onset of symptoms) 

a. Aseptically collect CSF (0.5-1ml)and store in a sterile leak-proof container. 

Preferably a sterile 2 ml screw cap tube. Freeze sample (-10±5°C) 

3. URINE (2 nd week of illness up to 30 days after onset of symptoms) 

a. Collect sample of midstream urine (10-20ml) in a leak proof container. Freeze 

sample (-10±5°C) 

 

For MAT (Serum only) 

1. Preferably, paired sera must be submitted. Obtain 0.5 to 1 ml of serum and transfer it 

into a sterile 2ml screw capped tube. (Label tubes if acute or convalescent phase sample. 

Freeze sample (-10±5°C) 

a. Acute phase sample (collected 5-10 days after onset of symptoms) 

b. Convalescent phase sample (collected 3-20 days after acute phase) 

2. If only one serum is submitted. Obtain 0.5 to 1 ml of serum and transfer it into a sterile 

2ml screw capped tube. Freeze sample (-10±5°C) 

 

For Culture 

1. BLOOD (1 to 10 days after onset of symptoms) 

a. Bedside collection and inoculation only. Obtain 2 EMJH or Fletcher’s media from 

the clinical laboratory before collection. Label tubes A and B. Aseptically extract 

1-2 ml of venous and aseptically add 2 drops of blood to tube A and 4 drops of 

blood to tube B. Wrap tubes with opaque material and store in room 

temperature away from light. Store at room temperature (30±2°C) 

2. CSF (5 to 10 days after onset of symptoms) 

a. Bedside collection and inoculation only. Obtain 2 EMJH or Fletcher’s media from 

the clinical laboratory before collection. Add 0.5 ml of CSF per tube. Wrap tubes 

with opaque material and store in room temperature away from light. Store at 

room temperature (30±2°C) 

b. If quantity is not sufficient, inoculate into one tube only. 

Ver. 1.5 2012 EMS 





3. URINE (2 nd week of illness up to 30 days after onset of symptoms) 

a. Collect sample of midstream urine (10-20ml) in a leak proof container and 

transport immediately to RITM within 2 hours. Refrigerate sample (6±2°C) 

Transport of specimen: 

 

All sample containers must have tight-sealing caps or lids and secure with paper tape 

(micropore) or parafilm whenever available before packing for transport. 

1. Check the following: 

a. The request form which should contain at least: 

i. Patient’s complete name (with middle initial) 

ii. Date of birth 

iii. Age and Sex 

iv. Date of onset of illness 

v. Referring Institution 

vi. Test requested 

b. All samples should be labeled on the container body with the: 

i. Patient’s complete name 

ii. Age 

iii. Sex 

iv. Date of specimen collection 

2. Packaging should follow the triple-packing system wherein: 

a. Primary container - sample container (ex. screw cap tube, red top tube.) 

b. Secondary container - plastic bag for samples and can be sealed (ziplock) 

c. Tertiary container - sample packaging (ex. transport box, ice chest, styro box) 

3. Pack sample containers in an upright position inside the “Ziplock” bag and add enough 

absorbent material (ex. tissue paper, paper towel) inside and seal the “Ziplock” bag with 

tape ensuring that samples will stay upright. 

4. Place the “Ziplock” bag inside the tertiary container and tape the bag to the wall of the 

tertiary container or position the sample in the center and secure in position with filling 

material. 

5. Place 2 ice packs/cool dogs beside or on top of the sample. If travel time is expected to 

exceed 1 day, add 2 ice packs/cold dogs per additional day of transport. Add filling material 

to secure all contents inside from moving during transport. 

Ver. 1.5 2012 EMS 





6. Seal box with packing tape and indicate outside the following: 

a. Sender’s name 

b. Sender’s address 

c. Sender’s contact information 

d. Destination’s contact person 

e. Destination’s address 

f. Destination’s contact number 

7. Insert in a separate “Ziplock” bag all documents (ex. Request form, PIDSR form, RITM outbreak 

referral form, Line list) and securely attach it preferably on top of the transport box. 

Criteria for Rejection: 

1. Improper sample collected for test requested 

(Please refer to the recommended specimen section of this document) 

2. Inadequate sample quantity. 

(Please refer to the recommended specimen section of this document) 

3. Samples without request forms. 

4. Incomplete data on request form. 

(must contain at least the following: complete name of patient, date of birth, age and sex, 

referring institution, and test requested) 

5. Improperly labeled or unlabeled sample 

(must indicate at least the following: complete name of patient, age and sex, and date of sample 

collection.) 

6. Samples with visible contamination. 

7. Spillage or breakage in transit. 

Contact Persons: 

Office hours (Monday to Friday, 8am to 5pm) 

Department of Microbiology: 807-2628 to 32 local 604 

Lydia T. Sombrero, RMT 

Melisa U. Mondoy, RMT MPH 

Edson Michael M. Simon, RMT 

Weekends and Holidays 

Clinical Laboratory: 807-2628 to 32 local 201 

Ver. 1.5 2012 EMS 





Flowchart from Specimen Submission to Reporting: (without PIDSR form) 

STEP 1: Sample will be submitted by the Clinical Laboratory to the Microbiology 

Laboratory for testing. 

STEP 2: Microbiology Laboratory will release verified result to the Clinical Laboratory. 

STEP 3: CLinical Laboratory will release result afer presenting the OR. 

i. Flowchart from Specimen Submission to Reporting: (with PIDSR form) 

STEP 1: Sample will be submitted or forwarded by the Clinical Laboratory to the 

Microbiology Laboratory for testing. 

STEP 2: Microbiology Laboratory will forward verified result, signed by the 

Microbiology Department head to the Director's Office with the cover letter to be 

signed by the Director. 

Copy furnished the Laboratory Division Chief 

STEP 3: Director's Office will fax the result to respective CHD and to the NEC. 

Ver. 1.5 2012 EMS

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