Journal of Infectious Diseases Advance Access published July 30, 2015

SUPPLEMENT ARTICLE

Ebola Virus Diagnostics: The US Centers for

Disease Control and Prevention Laboratory in
Sierra Leone, August 2014 to March 2015
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Mike Flint,1 Christin H. Goodman,14 Scott Bearden,15 Dianna M. Blau,2 Brian R. Amman,1 Alison J. Basile,14
Jessica A. Belser,3 ric Bergeron,1 Michael D. Bowen,4 Aaron C. Brault,14 Shelley Campbell,1 Ayan K. Chakrabarti,1
Kimberly A. Dodd,16 Bobbie R. Erickson,1 Molly M. Freeman,5 Aridth Gibbons,1 Lisa W. Guerrero,1 John D. Klena,17
R. Ryan Lash,6 Michael K. Lo,1 Laura K. McMullan,1 Gbetuwa Momoh,18 James L. Massally,18 Augustine Goba,18
Christopher D. Paddock,7 Rachael A. Priestley,7 Meredith Pyle,8 Mark Rayfield,9 Brandy J. Russell,14 Johanna S. Salzer,10
Angela J. Sanchez,13 Amy J. Schuh,1 Tara K. Sealy,1 Martin Steinau,11 Robyn A. Stoddard,12 Cline Taboy,1
Maryann Turnsek,5 David Wang,3 Galina E. Zemtsova,7 Marko Zivcec,1 Christina F. Spiropoulou,1 Ute Strher,1
Jonathan S. Towner,1 Stuart T. Nichol,1 and Brian H. Bird1
1

Viral Special Pathogens, 2Infectious Diseases Pathology, 3Influenza Division, Immunology and Pathogenesis, 4Gastroenteritis and Respiratory Virus
Laboratory, 5Enteric Diseases Laboratory, 6Travelers Health, 7Rickettsial Zoonoses, 8Laboratory Research, 9Global Disease Detection, 10Poxvirus and
Rabies, 11Chronic Viral Diseases, 12Bacterial Special Pathogens Branches, and 13Office of Technology and Innovation, Centers for Disease Control and
Prevention, Atlanta, Georgia; 14Arboviral Diseases, and 15Bacterial Diseases branches, Centers for Disease Control and Prevention, Fort Collins, Colorado;
16
University of California, Davis, School of Veterinary Medicine; 17Division of Global Health Protection, Centers for Disease Control and Prevention, Beijing,
China; and 18Ministry of Health and Sanitation, Kenema Government Hospital, Sierra Leone

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention
established a eld laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through
March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the
organization and procedures of the laboratory located in Bo, Sierra Leone.
Keywords.

Ebola; diagnostics; eld laboratory.

Since the discovery of Ebola virus (EBOV) in 1976,

there have been >20 outbreaks, but the West African
epidemic of 20132015 is the largest recorded, with
>10 000 fatalities and 25 000 persons infected as of 29
March 2015, surpassing all the other outbreaks combined. EBOV is classied in the family Filoviridae,
genus Ebolavirus, with the Zaire ebolavirus species
being responsible for the 2014 West African outbreak
[1]. The EBOV genome is negative-strand RNA approximately 19 kb long that encodes 7 genes: nucleoprotein
(NP), viral protein (VP) 35, VP40, glycoprotein, VP30,
VP24 and the viral polymerase L [2]. Several tests can be

Correspondence: Stuart T. Nichol, Viral Special Pathogens Branch, Division of

High Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton
Rd, Mail Stop G-14, Atlanta, GA 30333 ([email protected]).
The Journal of Infectious Diseases
Published by Oxford University Press on behalf of the Infectious Diseases Society of
America 2015. This work is written by (a) US Government employee(s) and is in the
public domain in the US.
DOI: 10.1093/infdis/jiv361

used to diagnose EBOV infection, including reversetranscription polymerase chain reaction (RT-PCR) to
detect the virus genome [36], enzyme-linked immunosorbent assay to measure virus-specic immunoglobulin (Ig) M or IgG [7, 8], or assays for viral antigen, either
in blood (with enzyme-linked immunosorbent assay)
[7] or in skin or liver biopsy specimens (with immunohistochemistry) [9, 10]. RT-PCR has been the most
common assay used in the current outbreak, detecting
EBOV RNA in whole blood samples obtained from living patients or oral swab samples collected from dead
bodies.
Laboratories have been established in many parts of
the outbreak region, often associated with an EBOV
treatment unit (ETU), to provide testing for diagnostic
purposes, aiding admittance, treatment, and discharge
decisions. For living patients, RT-PCR may give a negative result if performed <72 hours after onset of clinical
symptoms, apparently owing to low viremia during the
early stages of infection [11, 12]. Thus, a negative test
result may be valid only if the blood sample is obtained
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deployment of EBOV diagnostic eld laboratories for this and

future outbreaks.
MATERIALS AND METHODS
Biosafety

It was not possible to establish a biosafety level 4 (BSL-4) containment laboratory in Sierra Leone. To allow CDC scientists to
work safely, a hot laboratory was set up. This was the only area
where patient samples were handled. All persons entering the
hot laboratory were trained by VSPB scientists experienced in
working with EBOV under BSL-4 conditions. The personal protective equipment (PPE) worn in the hot laboratory included
scrubs and Croc clog shoes (Uniform Advantage), level 3 surgical gowns (Proxima Sirus; Medline Industries), gloves (High
Five Products), extended cuff gloves (Purple Nitrile-Xtra; Kimberly-Clark), and shoe covers (Proshield 3; DuPont). Powered
air purifying respirators (PAPRs) and accessories were manufactured by 3M (Air-Mate). Potentially infected materials
were decontaminated using 5% (vol/vol) Micro-Chem Plus disinfectant: a mixture of dimethyl benzyl ammonium chloride,
dimethyl ethyl benzyl ammonium chloride, and polyethylene
mono-ether glycols (National Chemical Laboratories). Extensive
safety testing conrmed that this concentration of disinfectant
was virucidal for EBOV (P. Jahrling, personal communication).
Specimen transport containers were Air Sea BioJar (code 500;
Biopack 2; 1.5-L UN combination packaging 4G/class 6.2)
from Air Sea Containers, and absorbent paper pads were from
Saf-T-Pak.
RNA Extraction and RT-PCR Assays

Figure 1. Map of Sierra Leone, with the different districts shown. District names are in black text, city names in white. The Centers for Disease
Control and Prevention laboratory was initially established in Kenema but
was relocated to Bo in September 2014.

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RNA was extracted from patient specimens using the MagMAX

magnetic bead system (Life Technologies), according to the
manufacturers instructions. In this process, 100 L of each
specimen was mixed with 400 L of lysis buffer supplemented
with 2 L of carrier RNA solution. RNA was extracted, using
either a MagMAX Express-96 Deep-Well magnetic particle extractor or a BeadRetriever automated magnetic bead separation
system, and then eluted in 90 L of elution buffer. Full details of
the RT-PCR assays will be described elsewhere, but are available
on request. Briey, 3 tests were performed concurrently: 2 specic for EBOV NP and VP40 genes and 1 for the human B2M
gene as a control for sample integrity. RT-PCR assays were performed on a CFX96 Real-Time System (BioRad). Samples for
use as positive controls were generated in the BSL-4 laboratory
at the CDC in Atlanta, Georgia; Z. ebolavirus (Mayinga variant
from the 1976 outbreak) from the VSPB collection was grown in
Vero E6 cells, and viral RNA was extracted from the culture
medium.
Results from the quantitative RT-PCR (qRT-PCR) assays
were evaluated comprehensively with attention paid to each
of the 3 targets qRT-PCR test results, the date of onset of clinical

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72 hours after onset. Because reverse-transcriptase inhibitors

in the sample can cause false-negative results, the laboratory will
usually include a control for this, either through amplication
of a human transcript such as 2-microglobulin (B2M; which
also acts as a check for sample integrity), or by addition of an
exogenous RNA, which is then also detected with RT-PCR.
The Viral Special Pathogens Branch (VSPB) of the US Centers for Disease Control and Prevention (CDC) has many decades of experience in establishing and operating diagnostic
laboratories in low-resource settings, in support of public health
efforts to control EBOV and Marburg virus disease outbreaks.
In August 2014, in response to an ongoing EBOV outbreak in
West Africa, the Sierra Leone Ministry of Health and Sanitation
invited the CDC to establish an EBOV diagnostic laboratory in
Sierra Leone. The laboratory was initially situated at Kenema
Government Hospital, but owing to westward movement of
cases, it was moved in late September to the Bandajuma region
of Bo within the Mdicins Sans Frontires (MSF) case management center (Figure 1). When it opened, the CDC laboratory
was one of only 3 EBOV diagnostic laboratories operating in Sierra Leone, and during the next 7 months it received samples
from 12 of Sierra Leones 14 districts. Here we describe the organization and work ow of the Bo laboratory. We hope this information will facilitate the establishment and aid the rapid

RESULTS
Establishment of the CDC Field Laboratory in Sierra Leone for
EBOV Diagnostics

MSF provided a building on the grounds of their Bo case management center for CDC use; this space was converted into a
eld laboratory. Running water was provided by the MSF
water and sanitation engineers, and electric power was supplied
by medium capacity generators on the MSF compound and
with small portable high quality backup generators (HondaEU2000i) for sensitive electronic equipment. Three rooms
within the house were designated as work areas: a clean room
to set up PCR and RNA extraction reagents, a room for extracting RNA and adding it to PCR plates, and a room for performing the PCR assays (Figure 2A). The third room also served as
an ofce area for laboratory workers during their deployment.
Although many eld laboratories opt to use class III biological safety cabinets (glove boxes), we established a separate hot
laboratory. Glove boxes require electrical power, can be uncomfortable to work in for extended periods, and the glove ports can
block the scientists eld of view while manipulating samples.
The gloves may be corroded and weakened by repeated exposure to the decontaminating solution. However, they can be

used with a more minimal PPE and in the presence of air conditioning. In contrast, a hot laboratory, with a larger working
area and less restricted movement, allows multiple scientists
to work on the same specimen, in a production line with different scientists performing individual steps of the processing
sequence.
For the hot laboratory, MSF constructed a wood-framed scaffold with corrugated iron roong and plastic sheeting walls
about 20 yards from the main laboratory building (Figure 2B).
An anteroom was used as a changing area where scrubs, PPE,
and PAPRs were donned and doffed. To allow transfer of materials out of the hot laboratory, a storage trunk was lled with
disinfectant (approximately 40 L) and situated with one end accessible from the hot area and the other end in the changing
room. This was used as a dunk tank, in which items from
the hot area could be submerged and the exterior surfaces disinfected before retrieval in the changing room. The disinfectant
solution in the dunk tank was changed once every 34 weeks.
Only a single area (room 1 inside the house) was air conditioned, and temperatures in the other rooms, especially the hot
lab, frequently exceeded 32C. Consequently, when workers
were inside the hot laboratory, those outside would check on
their status every 510 minutes. In addition, a wireless doorbell
system was arranged to allow signaling from inside the hot laboratory to the outside, to indicate that assistance was required.
The hot laboratory was not a closed structure, having an opening between the walls and the roong all around its perimeter
(Figure 2B), so it was possible to communicate with those inside
from the anteroom area. For routine communications with scientists working in the hot laboratory no additional PPE was required for those in the anteroom.
The CDC laboratory teams routinely consisted of 4 members:
3 scientists to perform sample testing and a team leader responsible for data entry, quality control, and reporting of results. On
days with high sample loads, 2 scientists processed samples in
the hot laboratory, while the other scientist performed RNA extraction and PCR in the main laboratory building. On days with
fewer samples, a single person was required for the hot laboratory, and the other 2 implemented RNA extraction and PCR.
On most days, a morning and an afternoon extraction and
RT-PCR assay were performed. Generally, the laboratory
teams were deployed for 28 days, with 13 days of overlap
with the previous team. Each team worked 1014 hours a
day, 7 days a week, with no days off.
Daily Work Flow and Specimen Processing

Samples were received from referring facilities, most frequently

delivered by a motorcycle courier, but sometimes delivered by
ambulance or by a United Nationscoordinated helicopter.
Most deliveries, but not all, were accompanied by the appropriate case investigation forms. On receipt of the sample, the transport boxes, the outside of the triple-package Air Sea shipping
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illness, the date of collection of the specimen, and the case history of the patient (if known). Specimens were considered negative if 3 criteria were met: (1) both EBOV NP and VP40 were
undetected, (2) the internal extraction control B2M was detected in an acceptable range of cycle threshold values (blood specimens, approximately 1830; oral swab samples, approximately
2839), and (3) the specimen was collected >72 hours after
onset of clinical illness. Specimens were considered positive regardless of the timing of collection relative to onset of clinical
illness if (1) either of the NP or VP40 assays were detected
(cycle threshold, <39) and (2) the B2M internal control was detected in the acceptable ranges. In instances where results were
discordant between the NP and VP40 assays (typically in the
rst 13 days after onset of illness when viremia levels were
lower) a follow-up specimen was requested for nal denitive
conrmation.
Results were considered pending if (1) the NP and VP40 assays were not detected and the B2M internal control also failed
detection or (2) the specimen was collected <72 hours after
onset of clinical illness. In these pending cases, a subsequent
(>72 hours) follow-up specimen was requested for denitive diagnosis of EBOV status. A small minority of specimens were rejected as nondiagnostic if the B2M internal control failed to be
detected after 2 separate RNA extractions and qRT-PCR test
runs. The overwhelming majority of the rejected specimens
were surveillance oral swab samples obtained from dead bodies
whose poor quality was probably due either to improper collection technique or to excessive transit time to the laboratory.

containers, and each sheet of paperwork were decontaminated

by misting with disinfectant. The sheets of paper were allowed
to air dry before examination. The containers were stored, unopened, at 4C before processing and were never opened outside
the hot laboratory. For each sample listed on the case investigation form, the time of delivery, the patients name, and the referring facilitys identication number was recorded, and a
unique laboratory identication number was assigned. A 2mL Wheaton cryogenic tube and a screw-cap Sarstedt tube
were labeled with the same unique laboratory identier, the former for an aliquot of the sample material (for freezing, in case a
repeat of the assay was required), the latter tube with 400 L of
lysis buffer supplemented with carrier RNA for sample inactivation before RNA extraction.
Owing to differences in work requirements, the PPE used in
the hot laboratory differed signicantly from that used by MSF
clinicians for patient care in treatment centers (Figure 3). The
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hot laboratory workers used lightweight level 3 laboratory

gowns, which are much cooler than a full-Tyvek suit, an important consideration given the ambient environmental conditions.
For face and respiratory protection instead of N-95 respirators
and goggles, the hot laboratory workers wore a full-face shield
and hooded PAPR, which, in addition to respiratory protection,
provided some air ow and relief from the heat. The lter unit
of the PAPR was worn underneath the gowns and was fully protected from contamination, thus allowing for easier surface decontamination without harming the internal high-efciency
particulate air lter and electronics.
In the changing room of the hot laboratory structure, scientists removed all jewelry and clothing, including underwear, and
put on scrubs, socks, and lightweight plastic footwear (Crocs or
equivalent) and shoe covers. The scrub pants were tucked into
the socks and taped in place to prevent them from falling and
revealing the skin. A PAPR with full hood was donned next,

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Figure 2. A, Floor plan of the Centers for Disease Control and Prevention (CDC) laboratory at Bo. Room 1 is the clean room, used for preparation of
polymerase chain reaction (PCR) master mix and reagents for RNA extraction, room 2 is used for RNA extraction and the addition of RNA to PCR plates, and
room 3 is an office area with thermocyclers, used for performing the PCR reactions and for data analysis. Work is undertaken in a unidirectional flow, from
room 1 to room 2 to room 3.

with a level 3 closed-front gown over the scrubs with the upper
portion of the gown tting between the inner and outer folds of
the PAPR hood. An inner pair of gloves was taped to the sleeves
of the gown; a middle pair, with extended cuffs, went over the
rst pair and was taped also. A third pair of gloves went over the
second pair, the third pair being light colored to make any
splashes on the gloves more visible. Once donned, the PPE
was checked by another scientist, with special attention to the
rear of the gown to ensure that the PAPR and the scientists
back were not exposed.
The objective of the hot laboratory work was to ensure that
the received samples were correctly documented, to transfer an
aliquot of the specimen to a cryogenic tube for freezing, and to
place 100 L of the specimen into lysis buffer for RNA extraction. Scientists therefore entered the hot laboratory with the
shipping containers, a list of the samples that were documented
as being inside the containers, cryovials for specimen storage,
and tubes containing lysis buffer (each tube labeled with a
unique VSPB identifying number). The rst task inside the
hot laboratory was to open the shipping containers and to
sort the specimens for processing, identify any that might be
missing or unlisted, and record any information present on

the specimen containers that differed from that on the case investigation form. A scientist outside the hot laboratory took
notes of discrepancies, passed in additional tubes, and corrected
the log books if necessary.
Once sample identication and documentation was complete, the specimen tubes were opened one at a time at arms
length over a bucket of disinfectant. When possible, a disposable transfer pipette was used to transfer up to 1.5 mL of the
sample to the 2-mL cryogenic tube. Specimens that were inaccessible by transfer pipette were retrieved using a regular micropipette with an aerosol barrier tip. Then, from the cryogenic
tube, 100 L of specimen was transferred to a screw-cap tube
containing lysis buffer and carrier RNA. The latter tube was
shaken to ensure complete mixing, and each tube was transferred to a plastic, lidded box with holes in the bottom (so
that the box would ood with disinfectant when submerged
in the dunk tank, without the tubes oating out). After use,
each pipette tip or transfer pipette was used to aspirate disinfectant to decontaminate its interior, and then it was discarded
into a disinfectant waste bucket, along with any excess specimen
and the now empty specimen tubes. After processing of each
sample, the scientists gloves were sprayed with disinfectant. If
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Figure 2 continued. B, Hot laboratory at the CDC Bo laboratory. Constructed by Mdicins Sans Frontires, this is a temporary wooden structure with
corrugated metal roofing and plastic sheeting walls. An anteroom serves as a changing area, separated from the exterior by a plastic sheet that can be
raised or lowered for privacy. Abbreviation: PPE, personal protective equipment.

bag, which was in turn decontaminated. For liquid waste, the

lid, handle, surfaces and sides of the waste bucket were sprayed
with disinfectant, and the bottom of the bucket was dipped into
the dunk tank. The bucket could then be passed out of the hot
laboratory. Solid waste was incinerated by MSF workers, and
liquid waste was disposed in the compound septic tank.
Challenges and Solutions

pipettors were used, they were wiped down with a paper towel
soaked in disinfectant after each sample.
Once all samples were processed, the plastic boxes were immersed in the dunk tank, and weighed down with a rock, for at
least a 3-minute contact time. The reusable shipping containers
were dunked out in the same fashion. To leave the hot laboratory, the scientist opened the door back to the changing room
and, while standing in the doorway, was sprayed from head to
toe with disinfectant by another scientist wearing a face shield,
gown, and gloves. Once sprayed, the hot laboratory scientist removed and discarded his or her shoe covers and outer gloves.
The shoes, middle gloves, and tape attaching the gloves to the
gown were also then sprayed with disinfectant, and the middle
gloves removed. The inner gloves were then sprayed with disinfectant, and the scientists stepped out of the hot area. The
tape, the gown, and the inner gloves were removed and safely
discarded. The PAPR hood was removed, with only the interior
surface touched. Tubes were retrieved from the dunk tank,
specimen aliquots were frozen at 20C, and RNA was extracted from the samples in lysis buffer.
Discarded specimen tubes, transfer pipettes, and tips were
soaked in disinfectant overnight, before disposal the next morning. A colander set over a bucket was used to lter them from
the liquid, and they were then disposed along with other solid
waste, double bagged in biohazard autoclave bags. The exterior
of the double-bagged trash was sprayed with disinfectant, transferred out of the hot laboratory, and placed in a third biohazard
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Figure 3. Personal protective equipment used within the hot laboratory

at the Centers for Disease Control and Prevention laboratory in Sierra Leone.

The packaging of samples delivered to the laboratory was frequently challenging. Samples were generally either whole blood
obtained from living patients, into purple-top tubes containing
anticoagulant ethylenediaminetetraacetic acid or red-top tubes
with no anticoagulant, or oral swab samples obtained from
dead bodies (in a transport medium). The supply chain to the
districts was inconsistent for several months; initially, samples
were received in a variety of containers, including coffee pots
(possibly intended to insulate the samples), bloodied urine containers wrapped in gloves, lled syringes with needles attached,
and sample tubes in plastic shopping bags. Occasionally samples
were received many days after collection, with the clotted blood
stuck to the stopper lids. The referring facilities, when it was
possible to contact them, were responsive to feedback and were
generally able to work toward xing these biosafety issues. To improve sample transport safety, materials for a triple-layer packing
system were purchased by CDC and provided to couriers on each
sample delivery for return to the referring facilities. The packing
system consisted of a watertight, leak-proof, shatter-resistant
shipping container lled with soft-paper tissues for padding
and reclosable bags containing absorbent pads into which specimen tubes could be placed. Each container was inserted in a corrugated cardboard box to protect it during transport.
The triple packing system became widely adopted for sample
transportation, but the quality of the samples themselves remained inconsistent. For example, no standardized swabbing
system was in use, and persistent problems were associated
with these specimens. Often the swab samples were dry and required rehydration with lysis buffer. The wooden shafts of
swabs were sometimes broken off, leaving the sharp, splintered
ends exposed when the specimen tube was opened; sometimes
the shafts were too long for the lid to be tted back on to the
tube, causing the lid to be loose and the tube contents to leak
inside the shipping container. One district repeatedly sent
swab samples in tubes wedged inside Vacutainer tubes, which
could not be retrieved. Two districts consistently sent swab samples in bacterial agar transport medium. The CDC attempted to
provide swabs and tubes of virus transport medium to various
districts, but these did not always make it to the intended recipients, and specimens prepared with these materials were only
rarely received by the laboratory.
It was sometimes difcult to link patients with their results;
multiple patients with identical names could be present in an
ETU simultaneously. On occasion, one identier was assigned

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Figure 4. A, Cumulative number of samples tested for Ebola virus (EBOV) RNA by the Centers for Disease Control and Prevention laboratory in Sierra
Leone, 22 August 2014 to 22 March 2015. B, Number of samples tested per day.

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Laboratory Throughput

The laboratory received and processed specimens daily from 22

August 2014 through 22 March 2015, including the day of relocation from Kenema to Bo, Thanksgiving, Christmas Day, and
New Years Day. More than 12 000 specimens were tested in
total (Figure 4A). (Note added in proof: at the time of manuscript acceptance, more than 20 000 specimens had been tested.) The mean number of samples tested per day was 63, with
the highest number tested being 178 (Figure 4B). Samples transported from far aeld sometimes arrived too late in the day for
processing and were tested the next morning, but on average,
71% of samples were processed on the day they arrived at the
laboratory, with the results distributed that evening. Samples
were held over for next-day processing for several reasons: to reduce fatigue on the team, for safety concerns due to low light in
the hot laboratory after dark, and, most importantly, if patients
would not be moved after dark and the laboratory results
would not be acted on until the next day. In all, 99.9% of samples
were tested either on the day of receipt or the next day. Full details
of the assay performance, the laboratory results, and their impact
on the outbreak response will be published elsewhere.
DISCUSSION
The role of the US CDC laboratory in Bo was to provide EBOV
diagnostics for 3 purposes: (1) testing blood drawn from patients in holding centers to guide their transfer to an ETU or
otherwise, (2) testing patients in the ETU to help with decisions
regarding their discharge, and (3) testing oral swab samples collected from dead bodies to facilitate contact tracing and the implementation of safe burial protocols. Although we believe that
the CDC laboratory was an important resource for the clinicians caring for EBOV patients across much of Sierra Leone,
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improvements to the laboratory services were possible. Owing

to the excessive workloads, assays other than the EBOV RTPCR tests were not performed until early February 2015,
when daily sample numbers had dropped signicantly. At this
stage it proved possible to implement blood chemistry tests and
a rapid diagnostic assay for malaria.
Other tests that could have helped guide patient care include
testing for other infectious diseases, such as Lassa fever, shigellosis, and typhoid fever. Serological assays for anti-EBOV IgG and
IgM were occasionally requested by ETU clinicians. The CDC
laboratory did not have the capacity to perform these assays in
Sierra Leone. The IgM assay would have been useful to inform
contact tracing efforts. The IgG assay could have been used to assess titers in the blood of EBOV disease survivors, before its transfusion as an emergency experimental treatment. In these cases,
testing for human immunodeciency, hepatitis B, and hepatitis
C viruses would also have been useful. Such an application was
not a common occurrence, however.
Although the assays described here have been successfully
used under low-resource conditions, they are relatively expensive and require some laboratory infrastructure and trained
staff. A simple, rapid, point-of-care diagnostic test that could
detect EBOV in blood from a nger prick would be invaluable
for testing in community settings. Such a test would need to be
sensitive, with a clear algorithm for retesting negative patients.
Several platforms are currently under evaluation, and will probably be deployed as a presumptive diagnostic test designed to
complement the existing RT-PCR assays.
In summary, we have described the organization and procedures of the US CDC EBOV diagnostic laboratory located in Bo,
Sierra Leone. Visitors to this facility have commented on the
simplicity of the laboratory setup, and expressed surprise at
its throughput, diagnostic accuracy, and rapid turn-around in
support of the EBOV outbreak response in Sierra Leone. We
hope that this article aids the deployment and establishment
of other laboratories, to serve the patients of this EBOV outbreak and any future ones.
Notes
Acknowledgments. This article is dedicated to all those working to
combat the EBOV outbreak, especially the medical technicians and phlebotomists who have risked their lives drawing blood and delivering it to the laboratory. We thank Issah French, Will Pooley, Mambu Momoh, Ian Crozier,
Kaci Hickox, Frederique Jacquerioz, Suzanne Donovan, Lewis Rubinson,
Darrio Gramuglia, Henry Kyobe, and Monia Sayah; your courage and devotion to helping those aficted has awed and inspired us. We thank Tanya
Klimova for assistance with editing this manuscript. We thank the Sierra
Leone Ministry of Health and Sanitation, the World Health Organization
and the Global Outbreak Alert and Response Network for organizing the
deployment of Centers for Disease Control and Prevention (CDC) scientists
to Sierra Leone. We thank the nurses, physicians, and other employees of
Kenema Government Hospital and MSF Bo. We also thank the International Rescue Committee for their support of Bo Government Hospital. The laboratory scientist pictured in Figure 3 provided explicit written consent for
use of the image.

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at a holding facility and a different identier was given at the

ETU. The use of unique patient identiers eventually solved
these issues, but this did not become standard practice until
many months into the outbreak.
Another challenge was the supply of electricity to the laboratory. The Bo laboratory received electrical power from a generator operated by MSF, which was reliable, for the most part.
Power failures did occasionally occur, and the ability of the BioRad real-time system to restart an assay at the point that it had
stopped, once the power supply resumed, proved extremely useful; brief power outages did not affect the data quality. Another
useful feature of this instrument was that it does not require an
internal control uorophore, such as Rox, which simplied reaction setup. To avoid the loss of power during RNA extractions, the instruments were generally powered by a portable
gasoline-powered generator (HondaEU2000i), though overheating or possibly the quality of the locally sourced fuel was
sometimes responsible for problems with these generators.

Disclaimer. The ndings and conclusions in this report are those of the
authors and do not necessarily represent the ofcial position of the CDC.
Potential conicts of interest. All authors: No reported conicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conicts of Interest. Conicts that the editors consider relevant to the content of the manuscript have been disclosed.

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