Guidelines for Specimen Collection, Storage and Transport for OPS ...

Department of Health 

Research Institute for Tropical Medicine 

National Reference Laboratories for Emerging Infectious Diseases 

Filinvest Corporate City, Alabang, Muntinlupa City 1781 

GUIDELINES FOR SPECIMEN COLLECTION AND LABORATORY TESTING 

FOR CASE FINDING AND INVESTIGATION OF HUMAN INFECTION CAUSED BY 

NOVEL RESPIRATORY PATHOGENS 

Version 3 | 2 July 2013 

1. BACKGROUND 

Several novel respiratory pathogens (NRPs) have recently emerged and have continued 

to pose global public health threats with their potential to cause outbreaks or pandemics of 

severe acute respiratory infections: 

 

 

 

Avian Influenza A (H5N1) Virus. Since the occurrence of large epizootics of the 

avian Influenza A (H5N1) virus in Southeast Asia in 2004 and 2005, the virus 

continued to spread among avian populations geographically [1]. In the past year, 

the World Health Organization (WHO) reported sporadic human cases of H5N1 in 

Cambodia, Vietnam, Indonesia, Egypt and Bangladesh. [2]. Recent sporadic human 

cases in Egypt, Bangladesh, Cambodia, Vietnam and China indicate that 

appearance of other cases are expected and will likely occur in the future [3]. 

Middle East Respiratory Syndrome Coronavirus (MERS-CoV). On September 

2012, the United Kingdom Health Protection Agency (UK HPA) informed the WHO of 

a novel Coronavirus from a patient with acute respiratory syndrome, with travel 

history to Saudi Arabia and Qatar. The virus is similar to an earlier (July 2012) isolate 

from a fatal case in Saudi Arabia [4]. To date, WHO reports of laboratory-confirmed 

cases originating in the following countries: Jordan, Qatar, Saudi Arabia, and the 

United Arab Emirates. Furthermore, WHO encourages “all Member States to 

continue their surveillance for severe acute respiratory infections and to carefully 

review any unusual patterns” [5]. 

Avian Influenza A (H7N2) Virus. WHO has been reporting cases of human infection 

with Avian Influenza A H7N9 viruses, with the source of infection and the mode of 

transmission currently unknown [6]. As of 29 May 2013, WHO has been informed of 

a total of 132 laboratory-confirmed cases, including 37 deaths. It is expected that 

there will be further cases of human infection until the source of infection has been 

identified and controlled [7].

GUIDELINES FOR SPECIMEN COLLECTION AND LABORATORY TESTING FOR 

CASE FINDING AND INVESTIGATION OF HUMAN INFECTION CAUSED BY NOVEL RESPIRATORY PATHOGENS 

Laboratory confirmation is needed to classify a patient as a confirmed case of any of these 

NRPs and initiate clinical and epidemiological investigations. The Research Institute for 

Tropical Medicine (RITM), the National Reference Laboratory for Emerging and Reemerging 

Infections, performs WHO-recommended methods of molecular detection by 

polymerase chain reaction (PCR) in the rapid detection of these NRPs. Furthermore, there 

may be other potential primary etiologies of community-acquired pneumonia aside from 

NRPs like Streptococcus pneumoniae, Hemophilus influenza type B, Legionella 

pneumophila, other recognized primary bacterial pneumonias, influenza, and respiratory 

syncytial virus. Testing for these other microorganisms is needed to identify the etiologic 

agent from a patient presenting with community-acquired pneumonia. RITM will be testing 

for specific and differential diagnosis for these NRPs, as indicated by the attending 

physician in the laboratory request. 

This document provides the interim guidelines for sample collection, storage and transport 

and laboratory testing for specific detection and differential diagnosis of NRPs. This 

document supersedes earlier and similar versions of guidelines release by RITM. 

2. SAMPLE TYPES FOR COLLECTION 

2.1. For patients falling under the DOH Case Definition for Case Under Observation 

(CUO) for the NRP being investigated, collect the following priority specimens for the 

specific detection of the NRP and for differential diagnosis of other possible causative 

agents: 

 

For MERS-CoV: 

Priority specimens: Lower respiratory specimens such as sputum, 

endotracheal aspirate, broncheoalveolar lavage or pleural fluid collected within 

14 days after the onset of symptoms. 

 

Alternative specimens: Nasopharyngeal swab AND oropharyngeal swab 

combined in Virus Transport Medium 1 (VTM), if patients do not have signs and 

symptoms of lower respiratory tract infection and lower tract specimens are not 

possible or clinically indicated. 

1 VTM collection kits are available by request from the RITM Clinical Laboratory, 

Telephone Nos. (02) 8422442 or (02) 8072628 to 32 local 201. Refer to Section 7 

of this document for the prices. 

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For MERS-CoV (continued): 

The WHO recommends that “[i]f initial testing of a nasopharyngeal swab is 

negative in a patient who is strongly suspected to have MERS-CoV infection, 

patients should be retested using a lower respiratory specimen or a repeat 

nasopharyngeal specimen with additional oropharyngeal specimen if lower 

respiratory specimens are not possible” [8]. RITM may for request additional 

specimens depending on initial testing results. 

 

For Novel Influenza Viruses A(H5N1) and A(H7N9): 

 

Oropharyngeal swab (OPS) AND Nasopharyngeal swab (NPS) combined in 

Virus Transport Medium 1 (VTM) and collected within 14 days after the onset of 

symptoms 

2.2. Other specimens which may be tested for the molecular detection of the NRPs and for 

differential diagnosis are as follows: 

 

 

 

 

Fresh tissue samples (e.g., lung tissues), collected as clinically indicated 

Serum sample, collected within seven days after the onset of symptoms 

Blood sample in BHI broth for patients with signs and symptoms of septicemia 

Nasopharyngeal swab (NPS) in Atypical Pneumonia Transport Medium 1 (APTM), 

collected within 14 days after the onset of symptoms, for detection of causative 

agents of atypical pneumonia 

2.3. Refer to the document on the RITM Website for the laboratory investigation by electron 

microscopy and histopathology. 

2.4. Other specimen types may be collected as indicated by the attending physician. 

1 VTM/APTM collection kits are available by request from the RITM Clinical Laboratory, 

Telephone Nos. (02) 8422442 or (02) 8072628 to 32 local 201. Refer to Section 7 of 

this document for the prices. 

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3. BIOSAFETY GUIDELINES DURING SAMPLE COLLECTION 

During the collection of specimens from patients suspected with any of the NRPs, isolate 

patient to minimise contact/exposure to staff and other patients. Wear personal protective 

equipment – correctly-fitted N95 respirator, gown, gloves and eye protection. 

Good Laboratory Practices and standard precautions for Biosafety Level 2 laboratories must 

be strictly observed in handling specimens suspected of containing NRPs. Refer to the 

following document (WHO Laboratory Biosafety Guidelines for Handling Specimens 

Suspected of Containing Avian Influenza A Virus) for more information: 

http://www.who.int/influenza/resources/documents/guidelines_handling_specimens/en/. 

Furthermore, laboratories are advised to avoid performing the following procedures with 

samples from suspected cases. Performance of these procedures could put the 

laboratory staff at risk for laboratory-acquired infections. These procedures may only be 

performed at RITM, using Biosafety Level 3 practices. 

 

 

 

 

aliquoting and/or diluting specimens 

performing diagnostic testing that involves propagation of viral agents in vitro or in 

vivo 

performing nucleic acid extractions that involve untreated specimens 

preparing smears using heat or chemical fixation 

4. GUIDELINES FOR SPECIMEN COLLECTION 

Effective and accurate diagnosis is highly dependent on the timing of specimen collection, 

appropriate clinical sample, and the condition during transport to the laboratory. Refer to the 

instructions below for specimen collection. 

4.1. General Guidelines 

Label each specimen container with the Patient’s Name, Age and Sex, Specimen Type 

and Date of Specimen Collection. The label should remain attached to the specimen 

container under all conditions of storage and transport. The information on the label 

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must be legible and should match the information on the RITM Laboratory Test 

Request Form, downloadable from the RITM website. Please fill out all the indicated 

fields in the form. Failure to do so could result in delays in the processing of the 

specimen. 

After collection, transport the specimens immediately to RITM. Refer to Section 5 of this 

document for proper transport of specimens. If delays between collection and transport 

are expected, refrigerate specimen at 4°C and ship within 48 hours of collection. 

4.2. Sputum 

Have the patient rinse the mouth with water. Let the patient expectorate deep cough 

sputum directly into a sterile screw-capped sputum collection cup or a sterile dry 

container. 

4.3. Broncheoalveolar lavage, tracheal aspirate, pleural fluid and nasal aspirate 

Collect 2 - 3 mL into a leak-proof, screw-capped sputum collection cup or sterile dry 

container. 

4.4. Nasopharyngeal and oropharyngeal swabs 

Refer to the document: Guidelines for Specimen Collection, Storage and Transport 

for OPS and NPS in the RITM website: 

www.ritm.gov.ph/Announcements/Guidelines%20for%20Specimen%20Collection,%20S 

torage%20and%20Transport%20for%20OPS%20and%20NPS.pdf 

Collect nasopharyngeal swabs for VTM and APTM media separately. 

4.5. Serum 

Collect 5mL to 10mL of whole blood in a serum separator tube. After clotting, centrifuge 

the tube and collect the resulting serum in polypropylene, external screw-capped vials 

with internal O-ring seals. If the vial has no internal O-ring seal, seal the vial tightly with 

the available cap and seal with Parafilm to prevent leakage. RITM requires a 

minimum amount of 500µL serum for testing and banking. 

Page 5 of 12



5. SPECIMEN STORAGE AND TRANSPORT 

The sending laboratory is responsible for the proper packaging of the specimens and in 

assuring that the specimen reaches RITM in good condition. 

5.1. For NPS and OPS samples: Wrap the specimen tubes in tissue paper or any 

absorbent material; place upright in a separate 50 mL centrifuge tube or any 

leak/puncture proof container. Place the 50 mL tube in a resealable plastic bag (e.g., 

Ziplock). The materials needed are provided in the VTM or APTM collection kit 

available from RITM (See Section 2.1 above). 

5.2. For serum, sputum and other samples: Use screw-capped, polypropylene tubes or 

containers for transport. Do not transport the samples in glass containers. Individually 

place each sample container in a resealable plastic bag (e.g., Ziplock). 

5.3. Place all the specimen containers except BHI in a shipment/carrier box with at least four 

(4) frozen ice packs inside to maintain prescribed temperature. Put the frozen ice packs 

in first, at the bottom and at the sides of the carrier box; then place specimens at the 

middle so that they are surrounded by the ice packs. Cover the carrier box. 

5.4. BHI samples must be stored and transported in room temperature. Do not include BHI 

samples inside the box with samples to be transported in cold chain. 

5.5. Place the completely filled-up RITM Laboratory Test Request Form in a separate ziplocked 

plastic bag and put on top of the box and secure with tape. 

5.6. Notify RITM before sending the specimen. RITM receives samples 24 hours a day, 

seven days a week. 

5.7. Send to the Research Institute for Tropical Medicine (RITM) using the following contact 

information: 

RESEARCH INSTITUTE FOR TROPICAL MEDICINE 

CLINICAL LABORATORY 

Filinvest Corporate City, Alabang 

Muntinlupa City 1781 

Telephone Numbers: (02) 842 2442 

(02) 807 2628 to 32 local 201 

Page 6 of 12



5.8. If delays between collection and transport are expected, refrigerate specimen at 4°C 

and ship within 48 hours of collection. 

5.9. All polystyrene foam (“styrofoam”) boxes used for specimen transport will be 

decontaminated and disposed of by RITM as infectious waste. 

Page 7 of 12

6. LABORATORY TESTING AT RITM 

Available Tests as of 2 July 2013 

Samples referred as Case Under Observation (CUO) by the National Epidemiology Center are FREE of testing charges. 

Samples referred by other institutions will be charged by RITM with the indicated testing fees. Testing fees indicated in this 

section supersede the testing fees for similar tests indicated in earlier documents released by RITM. 

Tests Available 

Specimen 

Type 

Appropriate Collection 

Time; Days After Onset 

of Symptoms 

Turnaround 

Time 

Testing Fees 

Specific detection of NRPs 

Influenza A screening and subtyping for Novel 

Influenza viruses by PCR 

- Avian Influenza H5N1 

- Avian Influenza H7N9 

NPS and OPS 

in VTM 

other respiratory 

samples 

within 14 days 

within 7 days 

4 working days 

single subtype: 

PhP 4,000.00 

package: 

PhP 5,000.00 

MERS-CoV detection by PCR Refer to Section 2.1 within 14 days 4 working days PhP 5,000.00





Type 



of Symptoms 

Turnaround 

Time 

Testing Fees 

Differential diagnosis 

Influenza A screening and subtyping for 

currently circulating Influenza viruses by PCR 

- Seasonal Influenza H1N1 

- Seasonal Influenza H3N2 

- Pandemic Influenza 2009 H1N1 

NPS and/or OPS 

in VTM 


samples 


within 7 days 


screening and 

subtyping 

package: 

PhP 6,000.00 

Influenza B PCR detection 


in VTM 


samples 


within 7 days 

4 working days PhP 4,000.00 

Respiratory Syncytial Virus PCR detection 


in VTM 


samples 


within 7 days 


panCoronavirus detection by PCR 

(Detects all human Coronaviruses) 

NPS and OPS in 

VTM 


samples 


within 7 days 


Page 9 of 12





Type 



of Symptoms 

Turnaround 

Time 

Testing Fees 

Atypical pneumonia PCR panel 

(Mycoplasma, Legionella and Chlamydia) 

NPS in APTM 

as clinically indicated, 

before antibiotic 

treatment 


single pathogen: 

PhP 3,500.00 

package: 

PhP 5,000.00 

Invasive bacterial diseases PCR panel 

(H. influenzae, S. pneumonia, N. meningitidis) 

serum 



treatment 


single pathogen: 

PhP 3,500.00 

package: 

PhP 5,000.00 

Aerobic culture 

sputum and/or 

blood in BHI 



treatment 


Electron microscopy 

Histopathological examination 

fresh tissues 

and secretions 

fresh tissues 

and secretions 

as clinically indicated Please refer to a separate 

document in the RITM website 

for specimen collection, cost and 

turn-around time for electron 

as clinically indicated microscopy and histopathology 

diagnostics. 

Page 10 of 12

7. COLLECTION KITS 

VTM and APTM collection kits are available by request from the RITM Clinical Laboratory, 

Telephone Nos. (02) 8422442 or (02) 8072628 to 32 local 201. Cost of the kits are indicated 

below. 

Collection Kit 

VTM Collection kit (contains VTM tube, OPS, NPS, tongue 

depressor, ziplock bag, absorbent material and 50mL tube as 

secondary container) 

Cost 

P 300.00 

APTM Collection kit (contains APTM tube, NPS, , ziplock bag, 

absorbent material and 50mL tube as secondary container) 

P 250.00 

8. FOLLOW-UP OF RESULTS 

Testing results may be followed-up after the indicated turnaround time at this number: (02) 

425 8910. RITM will be releasing additional contact information as soon as these are 

available. 

Results will be faxed to the official fax number of the referring institution or emailed as a pdf 

copy to the official email address of the referring institution, as indicated in the RITM 

Laboratory Test Request Form. Alternately, results may be picked-up at RITM-DOH. 

As an institutional policy, RITM does not release Official Results by phone. 

9. REFERENCES 

[1] The Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza 

A/H5, "Avian Influenza A (H5N1) Infection in Humans," The New England Journal of Medicine, vol. 

353, pp. 1374-1385, 2005. 

[2] World Health Organization, "Situation Updates - Avian influenza," 2013. [Online]. Available: 

http://www.who.int/influenza/human_animal_interface/avian_influenza/archive/en/index.html. 

[Accessed 22 April 2013]. 

[3] World Heath Organization, "Influenza at the human-animal interface: Summary and assessment as 

of 4 June 2013," 4 June 2013. [Online]. Available:



http://www.who.int/influenza/human_animal_interface/Influenza_Summary_IRA_HA_interface_04Ju 

n13.pdf. [Accessed 2 July 2013]. 

[4] United Kingdom Health Protection Agency, "Questions and Answers - novel coronavirus September 

2012," 24 September 2012. [Online]. Available: 

http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/RespiratoryViruses/NovelCoronavirus 

/respqandanovelcoronavirusspet2012/. [Accessed 27 September 2012]. 

[5] World Health Organization - Global Alert and Response, "Middle East respiratory syndrome 

coronavirus (MERS-CoV) - update," 26 June 2013. [Online]. Available: 

http://www.who.int/csr/don/2013_06_26/en/index.html. [Accessed 2 July 2013]. 

[6] World Health Organization, "Background and Summary of Human Infection with Influenza A(H7N9) 

Virus– As of 5 April 2013," [Online]. Available: 

http://www.who.int/influenza/human_animal_interface/latest_update_h7n9/en/index.html. [Accessed 

22 April 2013]. 

[7] World Health Organization - Global Alert and Response, "Human Infection with Avian Influenza 

A(H7N9) Virus in China - 19 April 2013 Update," [Online]. Available: 

http://www.who.int/csr/don/2013_04_19/en/index.html. [Accessed 22 April 2013]. 

[8] World Health Organization, "Novel coronavirus infection - update," 25 September 2012. [Online]. 

Available: http://www.who.int/csr/don/2012_09_25/en/index.html. [Accessed 27 September 2012]. 

[9] United Kingdom Health Protection Agency, "Infection Control Advice - Suspected or Confirmed 

Novel Coronavirus Cases," 26 September 2012. [Online]. Available: 

http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1317136232722. [Accessed 27 September 

2012]. 

[10] World Health Organization, "Revised interim case definition – novel coronavirus," 29 September 

2012. [Online]. Available: 

http://www.who.int/csr/disease/coronavirus_infections/case_definition/en/index.html. [Accessed 3 

October 2012]. 

[11] World Health Organization, "Novel coronavirus infection - update," 25 September 2012. [Online]. 

Available: http://www.who.int/csr/don/2012_09_25/en/index.html. [Accessed 27 September 2012]. 

[12] World Health Organization - Global Alert and Response, "Middle East respiratory syndrome 

coronavirus (MERS-CoV)," 26 June 2013. [Online]. Available: 

http://www.who.int/csr/don/2013_06_26/en/index.html. [Accessed 2 July 2013]. 

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