Together we stop the pandemic #COVID-19
Together we stop the pandemic #COVID-19

COVID-19 Pandemic: Impacts on Specimen Collection and Processing for Viral Respiratory Culture

Zeti Norfidiyati Salmuna @ Ayub 1,2, Siti Asma’ Hasan1,2, Rafidah Hanim Shueb1,2, Nur Fazlin Akmal Muhammad Faizal1
1Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, MALAYSIA.
2Hospital Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, MALAYSIA.
Pengarang Koresponden:
Dr Zeti Norfidiyati Salmuna @ Ayub
Clinical Microbiologist
Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.


SARS-CoV-2 is the causative agent of the novel COVID-19, which was first detected in December 2019 in Wuhan province, China. It is an enveloped positive single stranded RNA virus belonging to the Coronaviridae family. It has been detected in blood, respiratory specimen and stool of infected patients.(1)(2) Prior to the COVID-19 outbreak, our laboratory received quite a number of respiratory specimens for influenza-like illness (ILI ) and severe acute respiratory infections (SARI) for detection of eight common viral respiratory pathogens, influenza A and B, parainfluenza virus 1,2 and 3, adenovirus, respiratory syncytial virus (RSV) and human metapneumovirus. Nasopharyngeal aspirate (NPA) are the most common type of samples received in our laboratory followed by endotracheal tube secretion (ETT), nasal secretion and sputum. The aim of this writing is to discuss the impact of COVID-19 outbreak on specimen collection, handling and processing for viral respiratory culture in virology laboratory.

Before viral culture could be run for these eight respiratory viruses during current or post-pandemic COVID-19 situation, laboratories should do risk assessment which includes a site-specific and activity-specific risk assessment to identify and mitigate the risks of possibility of culturing SARS-CoV-2.(3) Risk assessments and mitigation measures are dependent on:

  • The procedures performed
  • Identification of the hazards involved in the process and/or procedures
  • The competency level of the personnel who perform the procedures
  • The laboratory equipment and facility
  • The resources available

Standard Precautions when handling clinical specimen must be followed as all specimen may contain potentially infectious materials. Standard Precautions include hand hygiene and the use of personal protective equipment (PPE), such as laboratory coats or gowns, gloves, and goggles for eye protection.

Types of respiratory specimens received for viral respiratory cultures in hospital USM

Based on unpublished data in 2019, Virology Laboratory Hospital USM received a total of 955 respiratory specimens for direct immunofluorescence antibody assay (DFA) and cultures for respiratory viruses. This finding is shown in the bar graph below (Figure 1). Most of the samples were nasopharyngeal aspirates (NPA) (97.8%).


Figure 1. Percentage of respiratory specimens sent for viral culture to Virology Laboratory USM in 2019

NPA, endotracheal tube (ETT) secretion and nasal secretion are usually obtained from aerosol-generating procedures (AGPs). Sputum sometimes are also obtained via AGP by hypertonic saline nebulization. World Health Organization (WHO) advises healthcare workers involved in AGPs to practice ‘airborne precautions’ as safety measures against COVID-19.(4) Airborne refers to droplet nuclei with the size of less than 5 µm.

Respiratory viruses isolated from viral culture in Hospital USM

The data from Hospital USM laboratory showed that the commonest respiratory virus isolated from the respiratory specimens was respiratory syncytial virus (RSV) followed by parainfluenza viruses. Data for 2019 obtained from laboratory is presented in Figure 2.


Figure 2 Respiratory viruses isolated in Virology Laboratory Hospital USM from January to December 2019

COVID-19 co-infection with other respiratory pathogens such as bacteria or viruses has been recently reported in the literatures. A real-time reverse transcriptase PCR tests for SARS-CoV-2 and other respiratory pathogens on nasopharyngeal swabs of symptomatic patients was done in a study. Out of 116 samples that were positive for SARS-CoV-2, 23 (19.8%) patients had co-infection with influenza A, RSV, parainfluenza viruses, metapneumoviruses, rhinoviruses/ enteroviruses and other coronaviruses.(5) A case report had also reported a co-infection of SARS-CoV-2 with Influenza A in a patient with pneumonia.(6)

Hence, to reduce potential exposure and the ensuing infection among healthcare workers due to possible co-infection of SARS-CoV-2 with other viral or bacterial respiratory infections, certain procedures in virology lab handling respiratory specimens need to be amended, as discussed below.

Specimens transportation

Respiratory specimens for viral respiratory culture must be sent in triple layer packaging. The triple layer packaging from the laboratory has been prepared according to WHO recommendation (Figure 3). Request form should be placed outside of polystyrene box. Sanitization of the polystyrene box with 70% alcohol solution is a must.


Figure 3 Triple layer packaging for sample transportation


Specimen processing, direct immunofluorescence assay and viral culture

In terms of specimen processing, the personnel handling the respiratory specimens for viral culture should wear full personal protective equipment (PPE) gear. This is in accordance with CDC guideline in 2009 and 2020 for handling specimens suspected to have coronavirus. CDC recommends that manipulation of untreated specimen must be  performed in BSL-2 facilities following BSL-3 practices.(7) However, culture of SARS-CoV-2 must be performed in BSL-3 facilities with BSL-3 practices. SARS-CoV-2 is not suitable for culture in BSL-2 facilities available, therefore all measures must be taken to avoid accidental culture of this virus while doing the culture for these eight respiratory viruses. 

Tubes containing the specimens should only be opened in a biosafety cabinet (BSC). Later, the specimen needs vortexing and centrifugation to disrupt cells and release viral particles. The supernatant is removed and  the sediment is used for direct immunofluorescence antibody assay (DFA). However, it is important to note that these procedures could generate infectious aerosol. Hence, the usage of sealed centrifuge rotors or gasketed safety carriers. Besides, the process of loading and unloading of the rotors and safety carriers should be done in the BSC.(7)

Regarding viral respiratory culture, the National Public Health Laboratory (NPHL) Sungai Buloh had attempted to culture SARS-CoV-2 on different cell lines including MDCK, MK2, A549 and Vero E6 although the virus was shown to grow only in Vero E6 cells.(8) The cytopathic effect (CPE) exhibited by infected cells was described as focal, with cell rounding and refractivity followed by cell detachment.(8)

Another study  reported similar CPE was observed  on culturing SARS-CoV-2 on Vero E6 cell line after 3 days of inoculation.(9) In laboratory, 0.5mls of fresh specimen was taken prior to centrifugation for viral respiratory culture. Hep2 and MDCK cell lines are used  for viral respiratory culture of these eight viruses. Therefore, currently it is safe to use these cell lines (Hep2 and MDCK), unless SARS-CoV-2 acquires mutation in future and able to replicate in the cell lines that we use.

In general, human coronaviruses are known to persist on inanimate objects such as metal, plastic or glass up to 9 days.(10) Alcohol-based wipes or spray containing at least 70% alcohol must be use to disinfect working area before and after procedures.(3) Surfaces need to be dried thoroughly to avoid pooling of liquids. Any breaks during laboratory procedures (i.e: spillage of specimens), exposure to either symptomatic or asymptomatic patients or non-exposure but with symptoms of ILI and  acute respiratory infection (ARI) must be notified and precautionary measures should be taken including assessment of risk, quarantine and environmental decontamination.(7)

Disposal of specimens

Decontamination of waste is mandatory prior to disposal. Before decontamination of the waste, the tubes that contain liquid need to be tightened to avoid spillage. All waste should be kept inside an autoclave bag and autoclaved before being disposed as clinical waste.


In conclusion, strict infection control practices are important for specimen collection, handling and processing for viral respiratory culture to avoid exposure of SARS-CoV-2  to healthcare workers, laboratory personnel and person handling the waste in case patients are having co-infection of SARS-CoV-2 with these eight respiratory viruses. Standard operating procedures of the laboratory need to be changed to avoid accidental culture of this virus in a BSL-2 facility.


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