Mobile diagnostics in outbreak response , not only for Ebola : a blueprint for a modular and robust field laboratory

R Wölfel 1 2 , K Stoecker 1 2 , E Fleischmann 1 2 , B Gramsamer 1 2 , M Wagner 1 2 , P Molkenthin 1 2 , A Di Caro 3 , S Günther 4 2 , S Ibrahim 1 5 , GH Genzel 1 , AJ Ozin-Hofsäss 6 , P Formenty 7 , L Zöller 1 1. Bundeswehr Institute of Microbiology, Munich, Germany 2. European Mobile Lab Consortium 3. Lazzaro Spallanzani National Institute for Infectious Diseases, Rome, Italy 4. Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany 5. Current affiliation: US Army Research Development and Engineering Command, Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, United States 6. European Centre for Disease Prevention and Control, Stockholm, Sweden 7. World Health Organization, Geneva, Switzerland


Background
Since December 2013, West Africa has been facing the deadliest outbreak of Ebola virus disease (EVD), previously known as Ebola haemorrhagic fever.The current epidemic has killed within a year of its onset more than 10,300 people as reported from eight affected countries Liberia, Guinea, Sierra Leone, Mali, Nigeria, Spain, the United Kingdom and the United States [1].
It was as early as March 2014 that two European laboratories identified the causative agent as the Zaire ebolavirus [2], which was immediately followed by a response of the World Health Organisation (WHO) with the support of its Global Outbreak Alert and Response Network (GOARN).
EVD usually begins with fever, chills, and general malaise.These and other symptoms, including fatigue, headache, vomiting, diarrhoea, anorexia, and myalgia [3], can also result from a number of other infectious diseases endemic in Africa, e.g.malaria, dengue, leptospirosis and typhus [4].Therefore, accurate identification of the causative agents is critical for effective containment of the outbreaks and provision of appropriate supportive care to the patients [5][6][7][8][9].In preparedness for rapid, global response to naturally occurring and emerging infectious disease outbreaks, the International Cooperation and Development Office of the European Commission (DG DevCo) launched the European Mobile field laboratory (EMLab, www.emlab.eu) initiative in 2012.Three mobile laboratory units were established in home bases in Europe (Munich, Germany), West Africa (Nigeria) and East Africa (Tanzania).
The concept of a modular, rapidly deployable field laboratory was previously developed at the Bundeswehr Institute of Microbiology in 2008.The primary goal of the Bundeswehr Medical Mobile Laboratory (BML) initiative was to establish field-deployable diagnostic capabilities for medical surveillance and investigation of unusual disease outbreaks in theatres of military operation.The driving design criterion was to enable worldwide deployment within 72 hours with minimal logistical burden (Figure 1).Therefore, modular units with multiple, flexible configurations to accommodate different mission requirements were envisioned.The BML is staffed with a four-person team equipped with the capability to provide confirmatory identification of a variety of bacterial and viral pathogens and biological toxins [10].From 2008 to 2014, the BML has successfully been deployed in several missions to the Balkans, South and Central Asia, West Africa, North America and Europe (data not shown).Based on the BML model, the EMLab units were adopted for civilian multinational outbreak response missions in Africa and Europe.
On 26 March 2014, an EMLab team was deployed to Guinea at the request of the Guinean Ministry of Health and WHO GOARN.Within three days, the first mobile laboratory became operational at the Ebola Treatment Unit (ETU) in Guéckédou, southern Guinea, and its impact was evident as the turnaround time from specimen collection to reporting of test result was reduced from days to hours.Between March 2014 and May 2015, more than 5,800 samples were tested in this field laboratory (Table 1).Further EMLab units were deployed to Nigeria, Liberia and Sierra Leone in the following months of the Ebola outbreak.In addition, the technical concept of the EMLab units served as a blueprint for other mobile Ebola laboratories which have been set up in Mali, Côte d'Ivoire, Sierra Leone and other countries in West Africa.

Key elements of mobile laboratory units
Four elements were identified as essential for effective mobile laboratories: (i) personnel and training, (ii) biosafety and biosecurity management, (iii) methods and equipment and (iv) logistical support.These elements can be adapted to suite specific missions according to the nature of outbreaks, geographic areas, season and climatic conditions, and on-site resources.

Personnel and training
The human factor is the most critical element for a successful mobile field laboratory mission.Modern laboratory equipment and sophisticated diagnostic methods have to be handled by highly skilled laboratory personnel in the environment of a field laboratory.Previous experience in biosafety level (BSL) 3 or BSL-4 laboratory work can facilitate, but cannot replace, the adaption of the laboratory personnel to the biosafety and biosecurity procedures of a field laboratory.We consider a four-person team as the ideal size for fieldwork over a period of three to six weeks to allow for rest and rotation during peak operations.Handover from one team to the next should consider an overlap of at least two days to ensure seamless transition and continuation of the operation.
Before deployment, all team members should be trained under realistic field conditions.Based on the experience of several laboratory deployments, we developed a training curriculum consisting of 25 modules.It covers realistic scientific, medical, technical and operational challenges that could be encountered in a field situation.A shortened version of this training enables participants to conduct Ebola diagnostics in one of the already established EMLab units in West Africa during the latest outbreak (Table 2).

Biosafety and biosecurity management
The difficulty of operating and relocating BSL-3 or BSL-4 laboratories in the field environment is well understood [11].Several container-based or truck-mounted laboratory units have been purported to meet most BSL-3 regulatory requirements.However, those have rarely been deployed worldwide due to their large footprint, heavy weight and high logistical burden.
These constraints often interfere with rapid and timely response because sophisticated air, maritime and land transport infrastructure are needed as well as significant and continuous technical maintenance.Most of these requirements cannot be met in rural areas of developing countries, such as Guinea, Sierra Leone or Liberia.
In general, biosafety in a mobile laboratory is achieved by optimising laboratory practices, equipment safety and qualification of the laboratory staff [12][13][14].For example, neither cultivation nor enrichment of infectious material are foreseen in the mobile laboratory as this would dramatically increase biosafety risks as well as biosecurity risks [15].The diagnostic procedures used, e.g.qPCR or RT-qPCR, immunoassays or light microscopy, do not depend on living organisms, and the specimens can easily be inactivated by validated chemical or heat treatment methods before further analysis [16][17][18].
For the protection of laboratory personnel during sample inactivation, two options can be employed: One option is wearing personal protective equipment (PPE), e.g.coverall suits, goggles and FFP-3 face masks within a designated 'hot zone' [6].However, this option has a number of disadvantages: (i) the time laboratory personnel can work in full protective gear is very limited especially in tropical climate, (ii) a designated separate 'hot zone' is needed, adding another factor to the already complex task of setting up a field laboratory, and (iii) this 'hot zone' has to be considered as contaminated until it has been completely disinfected.The second option is the use of a hermetically sealed glovebox system.Gloveboxes are regarded as the best way to minimise the risk of exposure for workers especially in hot climate.At the same time, heat stress is reduced for the operator since the glovebox renders the PPE dispensable.Only in case of failure of the glovebox, the laboratories teams would rely on PPE (coveralls and powered air-purifying respirators with hoods) for sample inactivation.In addition, the PPE is to be used to safely unpack samples that are too large for the glovebox or in the unlikely event of contamination inside the mobile laboratory environment.
Several flexible film isolator models have previously been used as gloveboxes in field laboratory missions [5,19].Although designated as portable, we found that those units are either too large and heavy to be easily deployed, or, because non-solid walls are used, only minimal negative pressure can be applied to the interior of these gloveboxes.Negative pressure of more than −1 mbar (−2 mbar is the minimum requirement for stationary BSL-3 glovebox systems [20]) would lead to the collapse of the flexible film walls.
We bridged the gap between biosafety needs and mobility by developing a foldable glovebox system with solid polycarbonate walls (Figure 1C).Its design was based on the basic technical requirements for microbiological safety cabinets described in European Standard EN 12469 [20].With a negative internal pressure up to −2.5 mbar that can be monitored and a leak rate less than 10% per 30 min at an overpressure of 5 mbar, this glovebox combines the safe working environment of a stationary glovebox with minimised packing size.Uncomplicated and safe handling was the main objective for the design of this foldable glovebox system.In the course of the Ebola missions in West Africa, the glovebox design was further optimised for durability and resistance in hot climate.The gloves can be adapted to the individual hand size of the operator to allow comfortable and safe performance of the inactivation protocols.Damaged gloves or sleeves can easily be replaced aseptically during glovebox operation.Two rapid transfer ports of different size, a well-established docking system in the pharmaceutical industry, allow contamination-free, safe and easy transfer of samples, supplies and waste into and out of the glovebox without breaking the containment.
All patient samples are stored in secondary packaging (plastic tubes or bags labelled with bleach resistant markers), floating in buckets filled with hypochlorite until further processing.This ensures thorough surface decontamination.For protection of the laboratory staff, all patient samples are finally unpacked inside the glovebox only.Two field laboratory team members operate the foldable glovebox: One operator is conducting all processing steps in the glovebox while another team member crosschecks and documents all steps with biosafety or diagnostic relevance on the respective laboratory request form.To prevent cross-contamination, samples are inactivated one after another, not simultaneously.

Methods and equipment
The BML system offers a range of diagnostic technologies, including qPCR, ELISA, immunofluorescence assays (IFA), immunochromatographic tests and microscopy.It is important to emphasise that all assays have to be adapted to and validated on the laboratory equipment before being included in the diagnostic portfolio of the mobile laboratory.Over the past seven years, we established more than 50 assays for 33 different pathogens and toxins that can be used under field conditions with the BML system.Performance of all PCR assays is controlled by the use of either competitive or non-competitive internal controls (IC), as well as extraction, negative and positive controls.Generally, IC are added to lysis buffer and patient serum samples before nucleic acid extraction in order to validate the complete diagnostic process.
For the ongoing Ebola outbreak in West Africa, we decided to focus on the detection of Ebola virus in different body fluids using RealStar Filovirus Screen RT-qPCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) and of Malaria parasites in the blood of In contrast to other qPCR instruments, the optical system has no moving parts or bulbs that could be damaged during transport.In case of technical failure of one of the 16 PCR modules the remaining modules are still operational.Furthermore, laboratory team members can easily replace the defective modules in the field.
Given the fact that in remote regions, replacement of broken or lost equipment can be difficult or impossible within a reasonable time frame, a concept of fallback options for essential parts of the field laboratory has been implemented: Key components such as centrifuges, power sources and IT equipment are safeguarded against failure by duplication.If the qPCR cycler would be irrevocably damaged, small conventional thermal cyclers and gel electrophoresis systems are available.
The infrastructural requirements for setting up the mobile laboratory are minimal.Generally, almost every local building can be used as long as it provides at least 28 m 2 of space.In several overseas deployments, the rapidly inflatable BML tent system with prefabricated interior room separations (ADS 1611 B-LABORATORY, OWR, Elztal-Rittersbach, Germany) was successfully used even in remote areas.Without the need for a dedicated 'hot room' for sample inactivation, the mobile laboratory requires a minimum of two rooms, to separate the pre-and post-PCR area (Figure 2 A/B and C/D).However, subdivision of the laboratory into four rooms (Figure 2 A-D) is preferred and can be prepared by the team using construction foil, foil doors and duct tape.The complete set-up of the field laboratory takes 4-5 h to operational readiness.The laboratory design adopts

Technical instruction on handling of mobile laboratory equipment 2 h
Immunofluorescence and light microscopy protocols 2 h

Glovebox maintenance 1 h
SmartCycler maintenance and repair 1 h

Personnel protective equipment 2 h
Power generator operation and maintenance 1 h

Scenario-based laboratory team training 24 h
Additional scenario-based team training 8 h

Documentation, statistics and reporting of data 2 h qPCR results interpretation and troubleshooting 2 h
Gel electrophoresis 2 h

Storage of samples 1 h
Preparation of samples for IATA shipment 2 h

VHF radio communication 2 h
Map and GPS training 2 h

Disinfection regime and waste management 2 h
Additional laboratory waste management in the field 1 h Field laboratory dismantling and packaging    2A), to sample inactivation and nucleic acid isolation (B), ELISA and microscopy (C) and ending in the PCR analysis room (D) to avoid cross-contamination of the samples.In order to reduce the number of separated rooms, the preparation of the PCR master mix is located inside a flexible film isolator designed for material protection.Tables and chairs are not part of the mobile laboratory equipment and have to be acquired locally.They are also covered with construction foil in order to facilitate easy surface decontamination.If electrical power is available locally (from portable generators or power line), power inverters are used as back-up systems and interruption-free power supplies (Figure 2).Each laboratory is equipped with two 1,500 W power inverters that provide 230 V AC power from a car battery.

Logistical support
Robustness and transportability are key features of our mobile laboratory design.Therefore, all laboratory instruments are selected for size, weight and durability, even under extreme field conditions.The complete laboratory equipment is packed in wheeled, ruggedised, water-and dustproof boxes (Figure 1B).More than 400 items are allocated to 27 boxes (Table 3).Box weights ranging from 20 kg to a maximum of 31 kg allow not only their transport as passenger luggage by all international airlines, but also ease handling by the laboratory team during reloading and land transport.
Although customs-related import problems are likely to be specific to every deployment, meticulous preparation of foreseeable transport documents (pro-forma invoice, import/export declaration forms, tax exemption certificates and multilingual packing lists) facilitates customs clearance significantly [23].
To guarantee a high degree of flexibility, different sets of equipment are packed for specific deployment missions.If not needed for a certain mission, individual boxes (e.g. for ELISA or microscopy) can be omitted from the deployment without affecting the performance of the other laboratory functions.To ensure the cleanliness of the consumables, such as gloves and reaction tubes, and minimise their space requirement in the boxes, they are packed in daily consumption quantities and vacuum-sealed in multilayer laminated aluminium foil pouches.Consumables and reagents are apportioned to allow continuous operation of a single laboratory for up to 50 days (based on the assumption of handling 25 samples per day) without logistic support.In the current EMLab missions, replacement teams replenish stocks of consumables.Since the active cooling or freezing devices of the laboratory cannot be operated during air transport, vacuum insulated boxes are used to bring temperature-sensitive reagents into the field.

Conclusions
The latest Ebola outbreak support mission, conducted by the EMLab consortium in several West African countries, has confirmed that rapid turnaround times are critical for surveillance and patient management, and at the same time facilitated unique scientific studies.Besides human diagnostics, the EMLabs provided high-quality laboratory expertise to support research and development activities for the assessment of new Ebola rapid diagnostics, for Ebola treatment trials and for Ebola vaccine research [24][25][26].They were also engaged with other partners in operational research to review or confirm public health prevention and control strategies [27].From March 2014 to October 2015, more than 19,000 samples were tested in the EMLab units.The overall costs for the complete field laboratory equipment described here are ca EUR 150,000, compared with the more than 10 times higher costs of other container-or truck-based solutions (DG DevCo, personal communication, 23 April 2015).Even though some security, safety and health risks are always present in mobile missions, they can be mitigated by careful planning, mission preparation and team training.With these measures, a safe working environment was ensured during more than 1,300 Ebola laboratory mission days in West Africa during the rainy season and the dry season.
The mobile laboratory concept described here is a forward-looking solution for outbreak response in remote areas where emerging infectious diseases might occur.It offers rapidly deployable, state-of-the-art technology for effective field diagnostics capabilities.Our field laboratory concept has served as a blueprint for similar projects and may continue to provide a model for future mobile laboratory projects.
first draft of the paper, and their comments were incorporated.All authors read and approved the final manuscript.

Figure 1
Figure 1Mobile Field Laboratory equipment in different phases of deployment

Figure 2 D
Figure 2Schematic setup of the European Mobile Field Laboratory equipment and layout of two independent electrical supply lines in a tent or a fixed building

Table 1
European Mobile Laboratory mission statistics, as per 31 October 2015 Turnaround time is an important indicator of mobile laboratory performance, and can be influenced by many factors, including sample collection, transportation, preparation, analysis, result interpretation and reporting.Time for transportation of samples to a laboratory is the factor contributing most to the total turnaround time.Deployment of the first EMLab unit in close proximity to the ETU in Guéckédou decreased the turnaround time to an average of 4 h instead of several days in many cases.A typical 12 h workload for a fourperson laboratory team was 40 samples.The gloveboxbased sample inactivation may present a bottleneck.However, well-trained laboratory teams should be able to process up to 90 samples within 12 hours as demonstrated by several EMLab teams during the peak of the Ebola outbreak in Guinea.
graphic tests, Ebola IgG IFA and Malaria microscopy.In the course of the Ebola outbreak in West Africa, EMLab teams have successfully participated in two independent laboratory quality control trials conducted by WHO and the European Network for Diagnostics of Imported Viral Diseases (ENIVD).

Table 2
Modular (71 h) training modules for preparation of mobile laboratory teams

Table 3 A
Complete list of components of the European Mobile Field Laboratory www.eurosurveillance.orgTotal number of packages (each max.31 kg weight) for complete mobile field laboratory deployment: 22 packages, 527 kg

Table 3 B
Complete list of components of the European Mobile Field Laboratory a strict one-way rule for processing of the samples, starting from the sample reception area (Figure