Biosafety Risk Assessment of a Routine Diagnostic Laboratory During the Coronavirus Disease 2019 Pandemic

Abstract

Background: In this study, we aimed to perform a biosafety risk assessment to determine measures to be taken against coronavirus disease 2019 (COVID-19) in the routine diagnostic parasitology laboratory of a tertiary health care center.

Methods: The risk assessment template included in the supplement of the interim guidance of “WHO Laboratory Biosafety Guidance Related to COVID-19” was used for the risk assessment. Risk assessments were carried out for the “diagnosis of protozoan diseases in respiratory tract samples” and “diagnosis of intestinal parasitic diseases” processes. Initial risk of the laboratory activities was determined before additional risk control measures and overall initial risk was estimated for each process. Overall residual risk of the laboratory activities after risk control measures was estimated for each process.

Results: Overall initial risk for both processes was “very high.” Fresh microscopic examination steps in both processes and concentration steps for “diagnosis of intestinal parasite diseases” were discontinued. All aerosol-generating steps were moved into a class-IIA biological safety cabinet. Overall residual risk was “medium” for both processes.

Conclusion: This study serves as an example for clinical laboratories regarding how the risk assessment approach in guidelines can be transferred to daily practice.

Introduction

A cluster of acute respiratory disease cases, later named coronavirus disease 2019 (COVID-19), occurred in December 2019 that was linked to a seafood wholesale market in Hubei Province of Wuhan, China.^1–3 The World Health Organization (WHO) declared COVID-19 as a pandemic on March 11, 2020, while 118,319 cases were confirmed globally.⁴ The first laboratory-confirmed COVID-19 case in our country was declared on March 11, 2020, by the Republic of Turkey Ministry of Health.⁵ The number of globally confirmed cases on May 17, 2020, was 4,529,027 and the total global number of deaths was 307,565 according to the WHO situation report.⁶

Besides being a global hazard, COVID-19 has become an important threat to health care workers due to transmission through aerosol. According to the report of the WHO–China Joint Mission on COVID-19 as of February 20, 2020, COVID-19 has been confirmed in 2055 health care workers in China. However, most of the cases (88%) occurred in the Hubei region, where the outbreak was first seen, at the early stage of the outbreak, when resources and experience were insufficient.⁷ The Republic of Turkey Ministry of Health declared the number of confirmed COVID-19 cases in health care workers as 601 on April 1, 2020.⁸ Therefore, it has a great importance of being proactive about taking biosafety measures in health care settings to protect health care workers from being infected.

According to the WHO Laboratory Biosafety Manual, the backbone of the practice of biosafety is risk assessment.⁹ The most important component of risk assessment is the professional judgment by the individuals most familiar with characteristics of the organisms, the equipment, procedures, animal models, the containment equipment, and facilities available. The laboratory director is responsible to ensure that adequate and timely risk assessments are performed, reviewed routinely and revised when necessary, taking into consideration the acquisition of new data having a bearing on the degree of risk and other relevant new information from the scientific literature, according to the WHO Laboratory Biosafety Manual.⁹

Microbiological agent risk groups are the most useful tools to perform a biosafety risk assessment. However, simple risk groups are not sufficient for a particular agent to conduct a risk assessment, especially when working with a recently defined microorganism. Other factors that should be considered that were defined in WHO Laboratory Biosafety Manual include the infectious dose and pathogenicity of the agent, the potential outcome of exposure, natural or other routes of infection that result from laboratory manipulations, the environmental stability of the agent, the concentration of the agent and volume of concentrated material to be manipulated, presence of a suitable host, data available from animal studies and reports of laboratory-acquired infections or clinical reports, laboratory activity planned (sonication, aerosolization, centrifugation, etc.), any genetic manipulation of the organism that may extend the host range of the agent or alter the agent's sensitivity to known availability of effective treatment regimens, and local availability of effective prophylaxis or therapeutic interventions.⁹

Although it has not been revealed with which body fluids new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV-2]) can be transmitted, the presence of SARS-CoV-2 RNA was shown in throat swabs, sputum, feces/anal swabs, whole blood, and serum in various studies to date.^10–13 Therefore, it is important to assess the biosafety risk of procedures other than the diagnosis of COVID-19 in which those samples are used.

In this study, we aimed to perform the biosafety risk assessment to determine the measures to be taken against the COVID-19 in the routine diagnostic parasitology laboratory of a tertiary health care center.

Materials and Methods

The risk assessment template included in the supplement of the interim guidance of “WHO Laboratory Biosafety Guidance Related to Coronavirus Disease 2019 (COVID-19)” was used for the risk assessment.¹⁴ Risk assessments were carried out for the “diagnosis of protozoan diseases in respiratory tract samples” and “diagnosis of intestinal parasitic diseases” processes applied in our parasitological direct diagnostic laboratory. A brief overview of the laboratory activities to be conducted that are included in the scope of the risk assessment was provided from the “standard operating procedure” of each process. The brief overview for the “diagnosis of protozoan diseases in respiratory tract samples” and “diagnosis of intestinal parasite diseases” is shown in Figures 1 and 2. The potential situations in which exposure and/or release could occur were described for each step. As there is no effective therapy or vaccine for COVID-19 to date, the consequences of exposure were assumed as “severe.” The initial risk of the laboratory activities was determined before additional risk control measures have been put in place and the overall initial risk was estimated for each process. The implementation of risk control measures was prioritized according to urgency, feasibility/sustainability, delivery and installation time, and training availability. The requirements that have been prescribed by international and national regulations, legislation, guidelines, policies, and strategies on biosafety were listed.^9,15 The resources available for risk control were described and their applicability, availability, and sustainability in the local context including management support were considered. The risk control measures that are needed and the level of residual risk when these risk control measures are in place were described. The residual risk that remains after risk control measures for each process was evaluated to determine whether that level of risk is below the tolerance level and whether work should proceed. The overall residual risk of the laboratory activities after risk control measures are in place was estimated for each process. Standard operating procedures for the “diagnosis of protozoan diseases in respiratory tract samples” (Figure 3) and “diagnosis of intestinal parasite diseases” (Figure 4) were redetermined due to risk assessment.

Figure 1.

The brief overview of the “diagnosis of protozoan diseases in respiratory tract samples.” Different steps of process are shown schematically in colored boxes according to working place (bench, fume hood, biosafety cabinet) of the steps.

Figure 2.

The brief overview of the “diagnosis of intestinal parasite diseases.” Different steps of process are shown schematically in colored boxes according to working place (bench, fume hood, biosafety cabinet) of the steps.

Figure 3.

The brief overview of the “diagnosis of protozoan diseases in respiratory tract samples” after risk control measures. Different steps of process are shown schematically in colored boxes according to working place (bench, fume hood, biosafety cabinet) of the steps. Background of the discontinued steps are dark-colored.

Figure 4.

The brief overview of the “diagnosis of intestinal parasite diseases” after risk control measures. Different steps of process are shown schematically in colored boxes according to working place (bench, fume hood, biosafety cabinet) of the steps. Background of the discontinued steps are dark-colored.

Results

The Risk and the Measures in the “Diagnosis of Protozoan Diseases in Respiratory Tract Samples” Process

Pipetting of the specimen, preparation of slides for fresh or stained microscopic examination, and cytocentrifuge steps in the “diagnosis of protozoan diseases in respiratory tract samples” process were evaluated as the steps that aerosols that include SARS-CoV-2 can be generated. The initial risk was “very high” for pipetting and cytocentrifuge steps, “high” for fresh microscopic examination and fixation of slides steps, and “medium” for Gram-Weigert and Giemsa staining of fixed slides, and microscopic examination of stained slides steps (Table 1). The overall initial risk for “diagnosis of protozoan diseases in respiratory tract samples” process was “very high.” It was evaluated that the process could not proceed without additional risk control measures.

The fresh microscopic examination step was discontinued (Table 2). All aerosol-generating steps were moved into a class IIA biological safety cabinet (BSC-IIA) from a fume hood or laboratory bench (Table 2). The overall residual risk for the “diagnosis of protozoan diseases in respiratory tract samples” process was “medium” (Table 2). The summary of standard operating procedures for the “diagnosis of protozoan diseases in respiratory tract samples” after risk control measures is shown in Figure 3.

The Risk and the Measures in the “Diagnosis of Intestinal Parasite Diseases” Process

Stirring and pipetting of the specimen, preparation of slides for fresh or stained microscopic examination, concentration, and centrifuge steps in the “diagnosis of intestinal parasite diseases” process were evaluated as the steps in which the aerosols that include SARS-CoV-2 can be generated. The initial risk was “very high” for stirring, concentration, centrifuge, and pipetting steps; “high” for fresh microscopic examination and fixation of slides steps; and “medium” for acid-fast, trichrome, modified trichrome, acid-fast-trichrome staining of fixed slides and microscopic examination of stained slides steps (Table 3). The overall initial risk for the “diagnosis of intestinal parasite diseases” process was “very high.” It was evaluated that the process could not proceed without additional risk control measures.

The fresh microscopic examination and modified formol–ethyl acetate concentration steps were discontinued (Table 4). All aerosol-generating steps were moved into a BSC-IIA from a fume hood or laboratory bench (Table 4). The overall residual risk for the “diagnosis of intestinal parasite diseases” process was “medium” (Table 4). The summary of standard operating procedures for the “diagnosis of intestinal parasite diseases” after risk control measures is shown in Figure 4.

Discussion

Laboratory staff, like all health care workers, are at risk for infectious diseases because of working with infectious material. Clinical laboratories are an intersection point for infected materials. For this reason, biosafety should be considered in all laboratory processes. The biosafety measures that clinical laboratories should take are outlined according to the risk groups of microorganisms likely to be encountered, in the biosafety guidelines.^9,15 Human coronaviruses other than SARS-CoV and the Middle East respiratory syndrome coronavirus (MERS-CoV) are classified in “Risk Group 2” according to appendix B of National Institute of Health Guideline for Research Involving Recombinant or Synthetic Nucleic Acid Molecules.¹⁶ Although these coronaviruses cause common colds, SARS-CoV and MERS-CoV cause severe lower respiratory tract disease and are classified in “Risk Group 3.” Recently, SARS-CoV-2 has been classified in “Risk Group 3.” To date there is no effective therapy for COVID-19 and a proper risk assessment is vital for clinical laboratories.

The laboratory where we perform the biosafety risk assessment is a routine direct parasitological diagnostic laboratory of a university hospital with 1809 beds. Izmir, the city where our hospital is located, ranks second in the ranking of COVID-19 cases in Turkey. Our hospital served only to COVID-19 suspected or diagnosed patients during the COVID-19 pandemic. During the pandemic, our laboratory continued routine service. Although tests for the diagnosis of COVID-19 are not carried out in our laboratory, tests for the diagnosis of diseases other than COVID-19 are carried out in the samples of patients with confirmed COVID-19. Considering the situations already listed, the biosafety risk assessment for COVID-19 was performed in our laboratory on March 11, 2020, the day when the first case of COVID-19 was seen in our country and all laboratory processes stopped until finishing the risk assessment.

Although the risk assessment process is an application that follows certain steps, it is a subjective application and the result obtained in a process is not a single recipe that can be used in each process. For COVID-19 risk assessment, although the consequences of exposure are “severe” because there is no effective therapy or vaccine for COVID-19 to date, the likelihood of exposure has become a key point in determining the level of risk (Tables 1–4).

We found the overall initial risk “very high” for the “diagnosis of protozoan diseases in respiratory tract samples” process. We started to perform all the steps from opening the sample container to fixing the preparation in the BSC-IIA (Figure 3). Thus, we reduced the “very high” risk steps to “medium” risk (Table 2). In daily practice, accidents such as leakage between the coverslip and cracking of the coverslip during fresh microscopic examination are encountered at a considerable frequency. In such incidents, there will be ∼15–20 cm between the sample containing inactivated virus and the nose of the examiner. The aim of the fresh microscopic examination in the “diagnosis of protozoan diseases in respiratory tract samples” process is to find motile trophozoites of Lophomonas blattarum. The trophozoites of Lophomonas blattarum can also be seen in Giemsa-stained preparations. Although we have no opportunity to perform the fresh microscopic examination in BSC-IIA and considering the aforementioned situations, the fresh microscopic examination step was discontinued in the “diagnosis of protozoan diseases in respiratory tract samples” process (Table 2 and Figure 3).

According to a study conducted in Wuhan, cytopathic effect was shown in Vero E cells 2 days after a second-round passage of the feces of a severe COVID-19 patient. The viral nucleic acid from virus culture was sequenced by using next-generation sequencing (GenBank accession no. MT123292) and full-length viral genome sequence was obtained. The sequence showed 5 nt substitutions compared with the original Wuhan strain (GenBank accession no. NC045512.2). Culture supernatant was negatively stained and visualized by transmission electron microscopy, viral particles were spherical and had distinct surface spike protein projections, consistent with a previously published SARS-CoV2 image.¹⁷ In the same study, 12 of the feces specimens collected from 28 patients were positive for viral RNA and they attempted to isolate SARS-CoV-2 virus from 3 of the viral RNA-positive patients. Results were successful for two of three patients, indicating that infectious virus in feces is a common manifestation of COVID-19.¹⁷ Therefore, we considered the stool sample as potentially infectious material for SARS-CoV-2.

We found the overall initial risk “very high” for the “diagnosis of intestinal parasite diseases” process. The fresh microscopic examination has a preliminary diagnostic value for intestinal protozoan parasites. The microscopic examination of trichrome-stained and acid-fast-stained preparations is the minimum requirement for the definitive diagnosis of intestinal protozoan parasites.¹⁸ Because of the low number of helminth eggs in feces, fresh microscopic examination is not sufficient for helminth diagnosis. Concentration methods such as flotation or centrifugation are needed to diagnose intestinal helminths. The frequencies of intestinal helminths in our laboratory between the years 2008 and 2017 are 1.4% for Enterobius vermicularis, 0.09% for Taenia saginata, and 0.02–0.003% for other helminths.¹⁹ The most frequent intestinal helminth, Enterobius vermicularis, is especially common in school children in our country.^20–24 Because our hospital is a pandemic hospital and symptomatic COVID-19 is not common in childhood, we predicted that the number of pediatric patients will decrease during the COVID-19 pandemic. Considering the aforementioned situations, fresh microscopic examination, cellophane tape investigation, and modified formol ethyl acetate concentration steps that can generate aerosols were discontinued in the “diagnosis of intestinal parasite diseases” process (Table 4 and Figure 4).

Another vital point in risk assessment is risk communication. The measures to be taken can only be as effective as the employees' awareness of risk assessment. Two technicians and two parasitology specialists work full time in our laboratory. In addition, two microscopists serve as consultants. Patient samples are accepted and processed by two technicians working in the same laboratory for >10 years. Technicians have no experience working in a laboratory for high-risk pathogens such as Mycobacterium or Brucella species. Therefore, their awareness of the use of personal protective equipment is not at a desirable level. After finishing the risk assessment, detailed in-service training to all laboratory staff was organized about our risk assessment. The training was structured into two parts, theoretical and practical. Theoretical part included three separate presentations about the basic principles of biosafety in medical laboratories, general information on COVID-19, and risk assessment approach, respectively. In the practical training section, all changes made to our standard application procedures in accordance with our risk assessment were demonstrated to the employees by the trainer. The class IIA biosafety cabinet available in our research laboratory was transferred to our diagnostic laboratory. The appropriate use of biosafety cabinet and personal protective equipment was described, according to guidelines.^7,9,15 We made it mandatory to wear surgical mask, surgical cap, laboratory glasses, and disposable laboratory coat during all laboratory processes for all laboratory staff according to the “Report of the WHO–China Joint Mission on Coronavirus Disease 2019.”⁷ At the end of the training, the participants were asked to do all the processes taught under the supervision of the trainer. Processes restarted with the revised standard operating procedures (Figures 3 and 4) after all measures were taken and the training was carried out. To date, no COVID-19 infection or suspicion has been observed in our laboratory staff. We think that being proactive about risk assessment and taking measures contributed to this situation. According to the data announced by the ministry of health, the highest number of new cases in our country was encountered on April 11, 2020, with 5138 cases, and the number of new cases has been decreasing relatively since this date.²⁵ However, it is important to assess the risk proactively against COVID-19 until a vaccine or effective drug is developed.

To our knowledge, this is the first study that applies the risk assessment approach in routine laboratories during the COVID-19 pandemic. The data obtained by our risk assessment cannot be used directly in other clinical laboratories as a matter of course, but it will serve as an example for clinical laboratories regarding how the risk assessment approach in the guidelines will be transferred to daily practice.

Author Disclosure Statement

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding Information

The author(s) received no financial support for the research, authorship, and/or publication of this article.