RESEARCH ARTICLE

Rapid deployment of a mobile biosafety level-

3 laboratory in Sierra Leone during the 2014
Ebola virus epidemic
Yi Zhang1, Yan Gong2, Chengyu Wang1, Wensen Liu1, Zhongyi Wang1, Zhiping Xia1,
Zhaoyang Bu1, Huijun Lu1, Yang Sun1, Xiaoguang Zhang3, Yuxi Cao3, Fan Yang4,
Haoxiang Su4, Yi Hu5, Yongqiang Deng5, Bo Zhou1, Zongzheng Zhao1, Yingying Fu1,
David Kargbo6, Foday Dafae6, Brima Kargbo6, Alex Kanu7, Linna Liu1*, Jun Qian1*,
Zhendong Guo1*
a1111111111
1 Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Changchun Veterinary Research
a1111111111 Institute, Chinese Academy of Agriculture Sciences, Changchun, Jilin, China, 2 School of Materials Science
a1111111111 & Engineering, Beijing Institute of Fashion Technology, Beijing, China, 3 National Institute for Viral Disease
a1111111111 Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China, 4 Institute of
a1111111111 Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China,
5 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology,
Beijing, China, 6 Sierra Leone Ministry of Health and Sanitation, Freetown, Sierra Leone, 7 Sierra Leone-
China Friendship Hospital, Freetown, Sierra Leone

* [email protected] (ZDG); [email protected] (JQ); [email protected](LNL)

OPEN ACCESS

Citation: Zhang Y, Gong Y, Wang C, Liu W, Wang

Z, Xia Z, et al. (2017) Rapid deployment of a mobile
biosafety level-3 laboratory in Sierra Leone during
Abstract
the 2014 Ebola virus epidemic. PLoS Negl Trop Dis
11(5): e0005622. https://doi.org/10.1371/journal.
pntd.0005622
Background
Editor: Brian Bird, School of Veterinary Medicine
Ebola virus emerged in West Africa in December 2013. The high population mobility and
University of California Davis, UNITED STATES
poor public health infrastructure in this region led to the development of the largest Ebola
Received: October 27, 2016
virus disease (EVD) outbreak to date.
Accepted: May 4, 2017

Published: May 15, 2017

Methodology/Principal findings
Copyright: © 2017 Zhang et al. This is an open
On September 26, 2014, China dispatched a Mobile Biosafety Level-3 Laboratory (MBSL-3
access article distributed under the terms of the
Creative Commons Attribution License, which Lab) and a well-trained diagnostic team to Sierra Leone to assist in EVD diagnosis using
permits unrestricted use, distribution, and quantitative real-time PCR, which allowed the diagnosis of suspected EVD cases in less than
reproduction in any medium, provided the original
4 hours from the time of sample receiving. This laboratory was composed of three container
author and source are credited.
vehicles equipped with advanced ventilation system, communication system, electricity and
Data Availability Statement: All relevant data are gas supply system. We strictly applied multiple safety precautions to reduce exposure risks.
within the paper and its Supporting Information
files.
Personnel, materials, water and air flow management were the key elements of the biosafety
measures in the MBSL-3 Lab. Air samples were regularly collected from the MBSL-3 Lab,
Funding: This work was funded by the National
Key Technology R&D Program of the Ministry of
but no evidence of Ebola virus infectious aerosols was detected. Potentially contaminated
Science and Technology (No. 1061400100275) objects were also tested by collecting swabs. On one occasion, a pipette tested positive for
and the National Natural Science Foundation of EVD. A total of 1,635 suspected EVD cases (824 positive [50.4%]) were tested from Septem-
China (No.31502077 and No.31402221). The
ber 28 to November 11, 2014, and no member of the diagnostic team was infected with
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of Ebola virus or other pathogens, including Lassa fever. The specimens tested included blood
the manuscript. (69.2%) and oral swabs (30.8%) with positivity rates of 54.2% and 41.9%, respectively. The

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

Competing interests: The authors have declared China mobile laboratory was thus instrumental in the EVD outbreak response by providing
that no competing interests exist. timely and reliable diagnostics.

Conclusions/Significance
The MBSL-3 Lab significantly contributed to establishing a suitable laboratory response
capacity during the emergence of EVD in Sierra Leone.

Author summary
A Mobile Biosafety Level-3 Laboratory (MBSL-3 Lab) and a well-trained diagnostic team
were dispatched to Sierra Leone to assist in Ebola virus disease (EVD) diagnosis when the
largest outbreak of EVD to date emerged in West Africa in 2014. This setup allowed for
the diagnosis of suspected EVD cases in less than 4 hours from the time of sample receiv-
ing. The laboratory was composed of three container vehicles and was equipped with
advanced ventilation system, communication system, electricity and gas supply system.
Multiple safety precautions were strictly applied to reduce exposure risks. A total of 1,635
suspected EVD cases were evaluated from September 28 to November 11, 2014, and none
of the staff members was infected with Ebola virus or other pathogens. The China mobile
laboratory was thus instrumental in the EVD outbreak response by providing timely and
accurate diagnostics. Therefore, the MBSL-3 Lab played a significant role in establishing a
suitable laboratory response capacity during the emergence of EVD in Sierra Leone.

Introduction
Ebola virus belongs to the Filoviridae family of enveloped viruses and contains a non-seg-
mented negative-strand RNA genome [1,2]. Infection in humans can cause Ebola hemorrhagic
fever, with exceptionally high case-fatality rates of more than 50% [3,4]. The incubation period
of Ebola virus disease (EVD) is 2 to 21 days [5]. The clinical signs and symptoms are extremely
similar to those of the Marburg virus and include fever, body aches, vomiting, diarrhea, rash
and, in some cases, both internal and external bleeding [5]. Patients usually die of multiple-
organ failure or hypovolemic shock. No licensed therapeutic or prophylactic treatments are
currently available.
The largest outbreak of EVD has been ongoing in West Africa since December 2013. As of
April 15, 2015, 25,826 cases (10,704 deaths [41.4%]) had been reported by the World Health
Organization (WHO) [6]. Although direct contact is the main route of transmission [7–10],
EVD is still easily contagious, and healthcare workers have constituted a considerable propor-
tion of all cases. In particular, by April 11, 2015, 864 healthcare workers (503 deaths [58.2%])
had been infected [6].
Ebola virus is classified as a biosafety level-4 agent. Clinical specimen inactivation should be
performed in a biosafety level-3 laboratory, and subsequent to this step, routine testing can be
performed in a biosafety level-2 laboratory. However, at the time of the outbreak, West Africa
had few high-level biosafety facilities, so scientists had to work under difficult and dangerous
conditions associated with potential exposure risks [11]. It would take a fairly long time, a
large staff and many resources to construct a new fixed biosafety facility, thus delaying preven-
tion and control of the epidemic. Therefore, a mobile unit [12,13] with both biosafety and

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

flexibility was urgently needed to manage epidemics and emergent public health incidents
such as the EVD outbreak.
In September 2014, China responded to the appeal made by the United Nations and WHO
and offered assistance to the government of Sierra Leone. A truck-based mobile biosafety
level-3 laboratory (MBSL-3 Lab) and a well-trained diagnostic team were then dispatched and
deployed to the Sierra Leone-China Friendship Hospital, in one of the hardest-hit areas, near
Freetown, to assist in EVD diagnosis. The team members and aid supplies arrived on Septem-
ber 17, 2014. It took approximately one week to rebuild part of the hospital into multiple func-
tional regions to meet the specimen testing requirements, including a specimen-receiving
region, a supply-storage region, a waste-incineration region, a nucleic-acid-detection region,
and a staff-rest area, among others. The MBSL-3 Lab was transported by an airlift jet aircraft
(Antonov An-124 Ruslan, Russia) from Beijing Capital International Airport on September
24, 2014, at 03:00 (Beijing time) to Freetown International Airport on September 25, 2014, at
14:00 (Freetown time), with a flight duration of 43 h. It took another three and a half hours to
drive the MBSL-3 Lab to the Sierra Leone-China Friendship Hospital. With strict training and
standard operating procedures (SOPs), clinical specimen testing began within 60 h after the
arrival of the MBSL-3 Lab, enabling the diagnosis of suspected EVD cases in less than 4 hours
from the time of sample receiving. In total, 1,635 suspected EVD cases (824 positive [50.4%])
were tested from September 28 to November 11, 2014, and none of the staff members was
infected with Ebola virus or other pathogens. Here, we provide a brief overview of the MBSL-3
Lab and the biosafety precautions applied to manage the EVD outbreak.

Methods
Ethical statement
This Ebola outbreak response was a humanitarian aid mission. The SOPs used were approved
by the WHO and the Sierra Leone Ministry of Health and Sanitation (MoHS). The diagnostic
results were released immediately after the specimen analyses were completed.

Specimen collection
Specimens were delivered to our worksite daily from two sources: the emergency operations
center jointly established by the Sierra Leone MoHS and the China medical aid team who
accompanied us and was also deployed to the Sierra Leone-China Friendship Hospital.
When picking up the specimens, the staff wore one layer of personal protective equipment
(PPE), including a protective suit (Lakeland INC or DuPont, USA), an N95 mask (3M, USA),
an anti-impact goggle (3M, USA), two pairs of latex gloves with the inner pair taped to the pro-
tective suit and a pair of dedicated shoes and waterproof shoe covers (S1 Fig). The surface of
the specimen bucket and the packing bag were disinfected by spraying with 0.25% chlorine-
containing disinfectants.

Specimen inactivation and RNA extraction

The staff extracted RNA in the BSL-3 Lab wearing two layers of PPE. The inner PPE included
a protective suit, an N95 mask, a pair of inner gloves and a pair of dedicated shoes and water-
proof shoe covers (S1 Fig). The external PPE included a HEPA filter-equipped powered air
purifying respirator (3M, USA), a disposable sterilized surgical gown, a pair of external gloves
and waterproof shoe covers (S1 Fig). The specimen bucket was opened within the biosafety
cabinet. As Buffer AVL in the QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD,
USA) was insufficient to inactivate samples [14], a combination of physical and chemical

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

inactivation was performed to enhance the inactivation efficiency. The specimens were first
inactivated by incubation in a water bath at 62˚C for 1h before opening the tube cap to pipette
the samples and were then further inactivated by the addition of Buffer AVL to the samples.
RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD,
USA) according to the manufacturer’s protocol. All waste was first chemically inactivated
(with 0.25% chlorine-containing disinfectant), then sterilized using a double-leaf autoclave
and finally incinerated.

Q-RT-PCR diagnostic assays

Quantitative real-time PCR (Q-RT-PCR) assays were performed using a set of published prim-
ers and probes [15], targeting regions of the glycoprotein gene (F: 5’-TGGGCTGAAAAYTGC
TACAATC-3’; R: 5’-CTTTGTGMACATASCGGCAC-3’; Probe: FAM-50 -CTACCAGCAGCG
CCAGACGG-30 -TAMRA). RNA was amplified using the One Step PrimeScript RT-PCR Kit
(TaKaRa, Japan), and 40-cycle Q-RT-PCR assays were run on the LightCycle 96 System
(Roche, Switzerland). Melt curve analysis was performed to confirm the identity of the amplifi-
cation products. The specimens were considered positive if there was an apparent logarithmic
phase in the amplification curve, with melting point confirmed amplification products and the
Ct value36 (Ct value<26, intense positive; 26Ct value 36, weak positive). In contrast, the
specimens were considered negative if there was no apparent logarithmic phase, with the Ct
value undetermined, and they were considered suspect when 36<Ct value40.

Specimen storage
The MBSL-3 Lab was equipped with a -20˚C freezer and a -80˚C freezer, and there was another
-80˚C freezer outside the MBSL-3 Lab. As a result, we could store a total of 1500–2000 specimens.
For short-term storage, namely, within 1 day, we stored the specimens at -20˚C. For long-term
storage, we stored the specimens at -80˚C. The specimens were well packed and surface disinfected
with 0.25% chlorine-containing disinfectant before storage. The Sierra Leone-China Friendship
Hospital was guarded by the military guard of Sierra Leone, and the freezers were well locked.

Testing report release

Every patient was assigned a unique Outbreak Case ID by the emergency operations center
jointly established by the MoHS. Each time a sample was collected, the patient was asked to
complete a “VIRAL HEMORRHAGIC FEVER CASE INVESTIGATION FORM”. The sample
tube and the investigation form were marked with the Outbreak Case ID and patient name
and were then delivered to us. Therefore, the Outbreak Case ID provided a unique number for
tracking the patient, the specimen and the test result. The information in our testing report
included the Outbreak Case ID, the Ct value yielded by Q-RT-PCR and the confirmed result
(Yes/No/Suspect).
According to an agreement with the MoHS, we usually did not contact hospitals directly.
Instead, we submitted the testing report to the WHO and the MoHS, which was in charge of
delivering the results to hospitals. In particular, the China medical aid team who came with us
and was also deployed to the Sierra Leone-China Friendship Hospital could get testing results
from us directly.

Biosafety risk assessment

Air sample collection. To assess the aerosol exposure risk of the working environment,
we used the SASS 4100 air sampler (3,500L/min) and SASS 3010 filter extractor (Research

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

International, Inc., USA) to collect air samples from the MBSL-3 Lab approximately every 15
days (for a total of 3 times).
The virus was then concentrated by polyethylene glycol (PEG) precipitation. In brief, the
pH of the virus-containing supernatant was adjusted to 7.2–7.5, after which PEG (MW 6,000)
was added to a final concentration of 8%. The samples were stirred at 4˚C for 2 hours and then
centrifuged at 10,000 g for 2 hours. Finally, the pellet was resuspended in 400 μl of RNase-free
ddH2O for virus RNA preparation.
Swab collection. To assess the contamination risk during the experimental operations, we
collected swabs from the surfaces of potentially contaminated objects, one or two sites per
workday, including experimental gloves, pipettes, workbenches, doorknobs, centrifuges and
specimen buckets. The swabs were placed in phosphate-buffered saline (PBS)-containing peni-
cillin (100U/ml) and streptomycin (100μg/ml).

Results
Staff composition and worksite layout
The China MBSL-3 Lab arrived in Sierra Leone on September 25, 2014, and specimen tests
were carried out within 60 h of its arrival. The worksite layout was shown in Fig 1. After receiv-
ing specimens, scientists sent them to the MBSL-3 Lab, where RNA was extracted. One room
in the hospital was rebuilt and used for subsequent Q-RT-PCR analysis. The MBSL-3 Lab was
powered by alternate use of 200kW diesel generators. Lab and household trash was incinerated
away from the lab or structures in a pit. There were surveillance cameras all around the work-
site and inside every experimental room, and scientists could watch real-time surveillance
video and communicate with the experimenters in the laboratory.

Fig 1. Worksite layout for the China mobile laboratory diagnostic team.
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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

Table 1. Overview of the China mobile laboratory diagnostic team’s composition and their tasks.
Task Staff member(s)
External contact 1×Scientist
Virus diagnosis • Sample receiving 8×Scientists
• RNA extraction
• Q-RT-PCR analysis
• Info check and data control
Technical support 3×Scientists, 1×Assistant
Health care 2×Medical doctors
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An overview of the composition of the China mobile laboratory diagnostic team and the
team members’ tasks was shown in Table 1. One scientist was in charge of contacting the
MoHS to coordinate issues such as sending specimens and releasing analysis results. In addi-
tion, eight scientists engaged in virus detection. Technical support personnel were in charge of
the operation of the MBSL-3 Lab, including overseeing the water and electricity supply, main-
tenance and repair of equipment, sterilization and incineration of lab trash as well as watching
and recording the daily experimental process. Two medical doctors monitored the health con-
ditions of every staff member.

Performance of the MBSL-3 Lab

The MBSL-3 Lab was composed of three container vehicles. The container encompassing
the BSL-3 laboratory was called the main container (L×W×H: 9125×2438×2896mm); the
second container, of the same size, was used for personnel cleaning and technology support
and was called the auxiliary container; and the third container was the command container
(L×W×H: 6300×2460×2100mm). As shown in Fig 2, the main and auxiliary containers were
connected by an airtight soft connection and together formed a complete BSL-3 Lab. From
the entrance to the inside, in order, there was the outside locker room (0-5Pa), the inside
locker room (Buffer room-2, -10Pa), the semi-contaminated channel (-20±5Pa), the air lock
room (Buffer room-1, -45±5Pa) and the BSL-3 laboratory (-70±10Pa). The doors were
interlocking.
The checklist for the different workplaces and instruments in the MBSL-3 Lab was listed in
S1 Table. The MBSL-3 Lab provided triple protection for humans, specimens and the environ-
ment. The main performance of the MBSL-3 Lab was detailed as follows.
• Power system. The power supply for the MBSL-3 Lab could be provided by two means:
the diesel generator and the mains supply, which could switch automatically to ensure a timely
and reliable supply. In the case of sudden power loss, the Uninterruptible Power Supply (UPS)
could power the ventilation, illumination, auto-control system and experimental equipments
for at least 45 min. Due to the lack of a stable mains supply in Sierra Leone, we employed two
diesel-generating sets (200kw) for this mission.
• Ventilation system. A cascade of low pressure with steps of 0 Pa, -10 Pa, -20 Pa, -45 Pa
and -70 Pa was realized with a ventilation system using 100% fresh filtrated air, allowing an air
renewal rate of up to 20 times per hour. The air supply was purified by three-grade-filtration,
namely, a primary efficiency filter (G4, plate-type), a medium efficiency filter (F8, pocket-type)
and a high efficiency filter (H14, HEPA). The exhaust was purified by two-grade-filtration,
namely, a high efficiency filter and a BAG-IN/BAG-OUT Filter Housing. The static cleanliness
of the laboratory was up to International Standard Organization (ISO) Class 8 [16]. The
MBSL-3 Lab was also equipped with an air conditioning system, which was a big selling point

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

Fig 2. Layout of the mobile biosafety level-3 laboratory. The main and auxiliary containers were connected by an airtight soft connection and
together formed a complete biosafety level-3 (BSL-3) lab. The instruments represented by letters were listed in S1 Table.
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for laboratory personnel working in tropical climates. The temperature regulation range was
from 16˚C to 30˚C.
• Waterway system. The water storage tank in the auxiliary container could store 2 tons
of water. The water supply for the autoclave, vapor generator and air conditioning humidifier
was softened using a water softener. Wastewater from hand washing and the shower was
stored in the wastewater tank directly under the shower cubicle and could be pumped into the
sewage treatment tank by the vacuum pump for sterilization. The sewage treatment tank had
two sterilization methods: high-temperature vapor and chemical sanitizer.
• Gas supply system. Compressed air generated by the air compressor served the double-
leaf autoclave (STERIS, Amsco Century) and expanded-metal door. There were two 8-Liter
CO2 gas bombs that provided continuous CO2 to the incubator.
• Communication system. Instruments, logic controllers and industrial control comput-
ers together formed a data acquisition and monitoring system. The status and data of the
MBSL-3 Lab, including the pressure, temperature, humidity, resistance of filter, working state
of the blower, biosafety cabinet and glove box, as well as fault inquiries, could be transmitted
to the command container though signal lines and was monitored using “King View” industry
control software. In addition, our MBSL-3 Lab was equipped with an internal communications
network and a video surveillance system. Telephone calls, data transfer and faxes between the
BSL-3 laboratory and the command container could be completed via a local area network.

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

Fig 3. Mobile biosafety level-3 laboratory at its mission in Sierra Leone. (A) Exterior of the mobile
biosafety level-3 laboratory. (B) View to the biosafety level-3 laboratory. Two different perspectives (B1 and
B2) were shown. (C) View to the auxiliary container. C1) Pass box (left) and expanded-metal door (middle).
C2) Monitoring unit and table for experimental preparation. C3) Shower cubicle. C4) Waste treatment room.
(D) View to the command container. D1) Room for meeting or for watching monitoring videos. D2) “King View”
industry control software. D3) Real-time surveillance video.
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• Modes of transportation. The MBSL-3 Lab can be transported by air, land and sea. It
can be operated on trucks, or can be dismounted to be operated on the ground. In the latter
case, hoisting with a crane and self-lifting by four elevating motors are two methods that can be
used for dismounting to the ground (S2 Fig). When the MBSL-3 Lab is needed in a remote set-
ting that cannot be reached by land transportation due to impassable roads, it could be trans-
ported by hoisting with a helicopter, similar to how it is lifted by a crane, as shown in S2C Fig.
The MBSL-3 Lab at its mission in Sierra Leone was shown in Fig 3.

Biosafety risk management

“Four Flows” management were the key elements of biosafety measures in the MBSL-3 Lab
(Fig 4).
Personnel flow. No more than 2 experimenters were allowed inside the BSL-3 Lab to
avoid influencing the air-flow. After completing an experiment, a step-reducing contamina-
tion process was carried out for the experimenters to exit the BSL-3 Lab. More specifically, the
experimenters disinfected the surface of their PPE with chlorine-containing disinfectants in
the BSL-3 lab and waited in Buffer room-1 for 10 min to ensure adequate disinfection, after
which they doffed their external PPE. Then, in Buffer room-2, they removed the inner PPE
and took a shower.

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

Fig 4. Schematic diagram of the “Four Flows”. Personnel, materials, water and air flow management were the key elements of biosafety measures in
the MBSL-3 Lab.
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Materials flow. Lab supplies were brought in sterilely though pass boxes to avoid contam-
inating experimental objects. Lab trash was chemically inactivated and removed through the
double-leaf autoclave after sterilization. The sterilization effect was monitored using autoclave
indicator tape (3M, USA). RNA samples were well packed and then surface disinfected and
taken out through pass boxes.
Water flow. Water was supplied after purification, and one-way flow was guaranteed by
pressure pumps and check valves. Waste water was discharged after steam sterilization and
chemical disinfection.
Air flow. Intake and exhaust air was HEPA filtered. The cascade of low pressure formed
directional airflow from the outside to the inside. The diagonal ventilation of up-supply and
down-discharge ensured uniform airflow and no dead corners. The air supply was controlled
by a constant air volume (CAV) system, and air exhaust was controlled by a variable air vol-
ume (VAV) system, which reduced the possibility of instantaneous positive pressure.

Risk assessment of the working environment and experimental

operation
To assess the aerosol exposure risk when working in or around the MBSL-3 Lab, air samples
were collected from the BSL-3 lab, locker rooms, water treatment room, equipment room,

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

exhaust outlet and command container and were concentrated for EVD detection every 15
days (S1 Fig). Fortunately, all results were negative.
We also collected swabs from the surfaces of potentially contaminated objects to determine
whether there was an existing exposure risk (S2 Table). On one occasion, the pipette used to
pipette samples from the blood-collection tubes tested positive for EVD, with a Ct value of
27.75.

Other protective measures

Disinfection. Approximately every ten days, on October 5, 15, and 24 and November 5,
2014, we maintained the MBSL-3 Lab and did not perform Q-RT-PCR assays. On these four
days, a thorough disinfection of the MBSL-3 Lab was performed to maintain a safe working
environment. The BSL-3 Lab was disinfected by fumigation with peroxide hydrogen and the
Geobacillus stearothermophilus spores were used as biological indicator (Mesa Labs, USA) to
monitor the disinfection effect. The buffer rooms were disinfected by spraying with peracetic
acid.
Health care. Each staff member had his or her own personal space in which to rest
between shifts. The work arrangement also ensured that the staff had sufficient rest time and
that their health conditions were carefully tended to.
Disease prevention and control. Each staff member carried an alcohol sprayer to disin-
fect potentially contaminated sites at any moment. To keep the staff members healthy and free
from other infectious diseases, such as malaria, yellow fever, Lassa fever and typhoid fever, the
worksite was sanitized using the DEMAND capsule suspension to control mosquitoes, and
mousetraps were used to control rodents (S1 Fig).

Workflow for laboratory testing and test results

The diagnostic algorithm for laboratory testing and the rationale for positive/negative/suspect
test results were presented in Fig 5. We repeated the testing of the suspect and negative cases
and strongly recommended collecting specimens again if collection was performed <3 days
post onset of symptoms. We found no evidence of RNA contamination during the entire oper-
ation. We added positive and negative controls to every experiment, and all controls produced
the expected results.
Overall, 1,635 suspected EVD specimens were tested from September 28 to November 11,
2014, primarily blood/serum samples (69.2%) and oral swabs (30.8%). The sample sources and
test results were presented in Table 2. In total, 824 cases (50.4%) were identified as positive,
and the positive rate of the swab samples (41.9%) was slightly lower than that of the blood sam-
ples (54.2%).

Discussion
The number of various paroxysmal public health events has been growing, and most have
occurred in poverty-stricken areas. However, the resources for medical treatment, outbreak
management and laboratory research are concentrated in developed regions, and substantial
expenditure would be required to build new medical systems in these areas. Because epidemic
situations are always urgent, scientists thus work under inadequate conditions and face expo-
sure risks. Therefore, rapid, safe and flexible outbreak response capacity is urgently needed
[17]. A mobile laboratory unit can easily be promptly deployed when needed and can provide
a safe working environment, which will be a vital part of the outbreak response to emerging
public health events or bioterrorism acts and will make great contributions to lessening and
controlling epidemics. Several mobile units have previously been used in natural disaster

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

Fig 5. Diagnostic algorithm for laboratory testing.

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scenarios [18,19], in health surveys [20,21], during the outbreak of severe infectious diseases
[22–24] and in military campaigns [25].
Our MBSL-3 Lab meets the requirements of on-site collection, isolation, cultivation and
detection of emergent infectious pathogens. This laboratory also protects humans as well as
the environment and specimens, and it was designed to be functional in a field setting, even
without logistical support. The major challenges in a remote location may be power supply
and water supply, but there are ways to overcome them. There was an 80kVA (70kW) diesel
generating set in the auxiliary container of the MBSL-3 Lab. Full fuel in the oil box can power
the MBSL-3 Lab in continuous operation for 24h. We can bring as much fuel with us as possi-
ble using oil tanks, and wherever the MBSL-3 Lab can arrive, a refueling truck could also
arrive. The MBSL-3 Lab is also equipped with a water storage tank and a water softener, and
water can be re-supplied with water from a well or clear stream. If the experimenters could do

Table 2. Samples and test results from September 28 to November 11, 2014.
Samples Total samples tested Positive/Negative/Suspect Positive rate
Blood samples 1,131 613/446/72 54.2%
Swab samples 504 211/280/13 41.9%
Total 1,635 824/726/85 50.4%

The positive rate was defined as the number of positive cases divided by the total number of samples.

https://doi.org/10.1371/journal.pntd.0005622.t002

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

not take a shower in the MBSL-3 Lab, the water requirement is not large, approximately 200L
per day. In addition, the MBSL-3 Lab is equipped with a leveling system, but it still needs a
20m×8m level ground. This was the first time that we executed a mission in Africa. In total,
1,635 specimens were tested from September 28 to November 11, 2014, accounting for more
than one quarter of the nation’s specimen volume during the same period. In all, 824 (50.4%)
specimens were EVD-positive, representing 33.3% of the total number of confirmed cases
reported in Sierra Leone during the same period. The maximum number of specimens that we
could reasonably process in one day is approximately 120–150.
We developed strict SOPs, adopted comprehensive protective measures and used compre-
hensive medical and logistical support systems to ensure safe and orderly performance of the
virus diagnosis task. In particular, the “Four Flows” biosafety protocol was strictly followed.
We monitored the exposure risk during clinical specimen testing. Air samples were collected
from every workspace, and the test results were all negative, indicating that the working envi-
ronment was relatively safe. The surfaces of potentially contaminated objects were also
swabbed. On one occasion, the pipette used to pipette samples from blood-collection tubes
tested positive. Given that a portion of the specimens contained only a small sample volume,
the pipette had to be placed deep into the tubes and was easily contaminated by touching the
inner wall. Therefore, it was suggested that the barrel of the pipette should be disinfected with
disinfectant-containing gauze after pipetting each sample to avoid personnel infection and
cross-contamination of samples.
The test results played an important role in the disposal of symptomatic individuals and
might, in a sense, determine their fates. For positive cases, the patients would be properly iso-
lated and treated without visiting family members, and traditional religious funerals for the
dead were forbidden. For negative cases, the patients would be separated from the positive
cases and kept in an observation ward for follow-up testing or discharge to relieve the limited
wards. Hence, the accuracy of the test results was crucial. False-positive results might lead to
the individual being infected by positive patients, whereas false-negative results might lead to
the spread of EVD to families and even the community. Our diagnostic algorithm suggested a
suspect conclusion when 36<Ct value40 and strongly recommended resampling and consid-
ering clinical information and epidemiological links. Q-RT-PCR is now a preferred method
for pathogen diagnosis due to its rapid and sensitive features [26], but it is prone to contamina-
tion and may result in false-positive results. Therefore, we conducted every experiment in the
biosafety cabinet. The cabinet and PCR room were exposed periodically to ultraviolet radiation
to eliminate nucleic acid contamination. Additionally, PCR tubes were never opened. Every
control included in the PCR assays produced the expected result, indicating high experimental
accuracy. Moreover, the MoHS was in charge of retrospective look at the disease progresses of
the patients, and to date, we have not received any feedback regarding a false diagnostic case
from the MoHS.
We have shown that the positive rate of oral swabs was lower than that of blood samples.
The technique and efficiency of swabbing might be one of the most important factors. Swab
samples should be obtained by vigorous sampling to acquire sufficient biologic material for
testing [27]. A quality-control PCR target (housekeeping gene target), such as Beta 2 Microglo-
bulin (B2M), should be added for sample integrity assessment in the future.
Our MBSL-3 Lab continuously worked for six months and managed 4,867 specimens for
EVD diagnostics. During that time, the China CDC established a fixed BSL-3 Lab near the
Sierra Leone-China Friendship Hospital for long-term surveillance and to serve as the public
health system for future outbreaks and epidemics. Currently, the EVD epidemic situation is
under effective control, and our MBSL-3 Lab has been proven to be an important force for dis-
ease control and emergency disposal.

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 MBSL-3 lab response to the 2014 Ebola virus outbreak in Sierra Leone

Supporting information
S1 Fig. Biosafety risk management. (A) Personal protective equipment (PPE) used when
receiving specimens. (B) Inner PPE (B1) and external PPE (B2) used when extracting RNA.
(C) Air samples were collected from every working room, including the biosafety level-3 labo-
ratory (C1), equipment room (C2) and wastewater treatment room (C3). (D)The worksite
(D1) and personal space in which to rest between shifts (D2) were completely sanitized using
the DEMAND capsule suspension.
(TIF)
S2 Fig. Working modes of the mobile biosafety level-3 laboratory. The mobile biosafety
level-3 laboratory can be operated on trucks (A) or can be dismounted to be operated on the
ground (B). Hoisting with a crane (C) and self-lifting by four elevating motors (D) are two
methods that can be used for dismounting to the ground.
(TIF)
S1 Table. Checklist for the workplaces and instruments of the mobile biosafety level-3 lab-
oratory.
(DOCX)
S2 Table. Swab sampling sites among potentially contaminated objects and analysis
results.
(DOCX)

Acknowledgments
Authors Chengyu Wang, Wensen Liu, Zhaoyang Bu, Huijun Lu, Yang Sun, Xiaoguang Zhang,
Yuxi Cao, Fan Yang, Haoxiang Su, Yi Hu, Yongqiang Deng, Jun Qian, and Zhendong Guo
were members of the China Mobile Laboratory Diagnostic Team in Sierra Leone. We thank
Xianzhu Xia, Songtao Yang, Xuexing Zheng and Hualei Wang at the Key Laboratory of Jilin
Province for Zoonosis Prevention and Control for their testing technical support and Abdul
Kamara, Samileu Kamara, Aminate Lawa, Alie Tie and Bobson Mansaray at the Office of the
Sierra Leone Ministry of Health and Sanitation for their efforts in collecting and transporting
samples.

Author Contributions
Conceptualization: ZG LL JQ.
Formal analysis: BZ ZZ YZ YF.
Investigation: HL YS XZ YC FY HS YH YD.
Methodology: ZG CW WL ZB.
Resources: DK FD BK AK.
Writing – original draft: ZG YG ZW ZX LL JQ.
Writing – review & editing: YZ.

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