The content of this article does not necessarily reflect the views or policies of the US Department of Health and Human Services or of the institutions affiliated with the authors. This research was supported by Paul G. Allen Family Foundation &#34;Enhanced Zero-Impact, Emergency Smart Pod&#34;. We deeply appreciate all the fruitful discussions and collaboration with the colleagues from Baylor College of Medicine, GSS Health, NASA&#39;s Johnson Space Center. We are sincerely thankful to Thermo Fisher Scientifics and its representatives for a loan of the RT-PCR machine, centrifuge, and automated pipettes to carry out a respiratory virus diagnostic test in the remote laboratory facility. The authors are thankful to Marta Storl-Desmond and Sidney Stephen Sorrell for their assistance in the manuscript preparation and videography.

<ol>
	<li>World Health Organization (WHO) Ebola Response Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ebola+virus+disease+in+West+Africa+-+the+first+9+months+of+the+epidemic+and+forward+projections.">Ebola virus disease in West Africa - the first 9 months of the epidemic and forward projections.</a> New England Journal of Medicine. 371 (2), 1481-1495 (2014).</li><li>World Health Organization (WHO). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Media Center: Ebola Virus Disease Fact Sheet No. 103. , (2014).</li><li>Fauci, A. S., Morens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+in+the+Americas+-+yet+another+arbovirus+threat.">Zika Virus in the Americas - yet another arbovirus threat.</a> New England Journal of Medicine. 374 (7), 601-604 (2016).</li><li>Campos, G. S., Bandeira, A. C., Sardi, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+outbreak,+Bahia,+Brazil.">Zika virus outbreak, Bahia, Brazil.</a> Emerging Infectious Diseases. 21 (10), 1885-1886 (2015).</li><li>Aly, M., Elrobh, M., Alzayer, M., Aljuhani, S., Balkhy, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+the+Middle+East+Respiratory+Syndrome+Coronavirus+(MERS-CoV)+across+the+Gulf+Corporation+Council+countries:+Four+years+update.">Occurrence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) across the Gulf Corporation Council countries: Four years update.</a> PLoS ONE. 12 (10), e0183850 (2017).</li><li>World Health Organization (WHO). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+East+respiratory+syndrome+coronavirus+(MERS-CoV)+-+Oman.">Middle East respiratory syndrome coronavirus (MERS-CoV) - Oman.</a> Media Center: Disease outbreak news. Mar 18. , (2018).</li><li>Ducharme, J., Johnson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=This+flu+map+shows+how+the+biggest+influenza+outbreak+in+years+spread+across+the+U.S.">This flu map shows how the biggest influenza outbreak in years spread across the U.S.</a> Time, Time Health: Public Health. , (2018).</li><li>Gaglani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+vaccine+effectiveness+against+2009+pandemic+influenza+A(H1N1)+virus+differed+by+vaccine+type+during+2013-2014+in+the+United+States.">Influenza vaccine effectiveness against 2009 pandemic influenza A(H1N1) virus differed by vaccine type during 2013-2014 in the United States.</a> Journal of Infectious Diseases. 213 (10), 1546-1556 (2016).</li><li>Gates, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next+epidemic+-+lessons+from+Ebola.">The next epidemic - lessons from Ebola.</a> New England Journal of Medicine. 372 (15), 1381-1384 (2015).</li><li>Mills, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+care+systems+in+low-+and+middle-income+countries.">Health care systems in low- and middle-income countries.</a> New England Journal of Medicine. 370 (15), 552-557 (2014).</li><li>W&#246;lfel, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobile+diagnostics+in+outbreak+response,+not+only+for+Ebola:+a+blueprint+for+a+modular+and+robust+field+laboratory.">Mobile diagnostics in outbreak response, not only for Ebola: a blueprint for a modular and robust field laboratory.</a> Euro Surveillance. 20 (44), 30055 (2015).</li><li>Connelly, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Through+innovation,+education+and+collaboration,+Baylor+Global+Initiatives+seeks+to+transform+health+and+patient+care+worldwide.">Through innovation, education and collaboration, Baylor Global Initiatives seeks to transform health and patient care worldwide.</a> TMC Pulse, Global Perspective. , (2015).</li><li>Hersh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=USAID+funds+Ebola+'smart+pod'+project.">USAID funds Ebola 'smart pod' project.</a> Federal Times. , (2015).</li><li>Waite, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergency+Smart+Pods+-+Transforming+Containers+into+Modern+Medical+Clinics.">Emergency Smart Pods - Transforming Containers into Modern Medical Clinics.</a> DipNote, U.S. Department of State Official Blog. , (2018).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to US Department of Health and Human Services Regulations. , (2015).</li><li>Center for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Website: Clean up after a Disaster. , (2017).</li><li>Flannery, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+genetic+characterization+of+influenza+A(H3N2)+viruses+and+vaccine+effectiveness+by+genetic+group,+2014-2015.">Enhanced genetic characterization of influenza A(H3N2) viruses and vaccine effectiveness by genetic group, 2014-2015.</a> Journal of Infectious Diseases. 214 (7), 1010-1019 (2016).</li><li>Spencer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+associated+with+real-time+RT-PCR+cycle+threshold+values+among+medically+attended+influenza+episodes.">Factors associated with real-time RT-PCR cycle threshold values among medically attended influenza episodes.</a> Journal of Medical Virology. 88 (4), 719-723 (2016).</li><li>Cherpillod, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ebola+virus+disease+diagnosis+by+real-time+RT-PCR:+A+comparative+study+of+11+different+procedures.">Ebola virus disease diagnosis by real-time RT-PCR: A comparative study of 11 different procedures.</a> Journal of Clinical Virology. 77, 9-14 (2016).</li><li>Dedkov, V. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+real-time+RT-PCR+assay+for+the+detection+of+Ebola+virus+(Zaire)+during+an+Ebola+outbreak+in+Guinea+in+2014-2015.">Development and evaluation of a real-time RT-PCR assay for the detection of Ebola virus (Zaire) during an Ebola outbreak in Guinea in 2014-2015.</a> Journal of Virological Methods. 228, 26-30 (2016).</li><li>Cnops, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developement,+integration+of+a+quantitative+reverse-transcription+polymerase+chain+reaction+diagnostic+test+for+Ebola+virus+on+a+molecular+diagnostics+platform.">Developement, integration of a quantitative reverse-transcription polymerase chain reaction diagnostic test for Ebola virus on a molecular diagnostics platform.</a> Journal of Infectious Diseases. 214 (3), S192 (2016).</li><li>Keitel, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+research+response+to+the+2009+A(H1N1)pdm09+influenza+pandemic+(Revised).">Rapid research response to the 2009 A(H1N1)pdm09 influenza pandemic (Revised).</a> BMC Research Notes. 6, 177 (2013).</li><li>Parsons, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+diagnosis+of+tuberculosis+in+resource-poor+countries:+challenges+and+opportunities.">Laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities.</a> Clinical Microbiology Reviews. 24 (6), 314-350 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Expandable Bicon Shelter. Commercial Manual. Sea Box. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Safety.+Operating+the+Glovebox.">Lab Safety. Operating the Glovebox.</a> JoVE Science Education Database. , (2018).</li><li>World Health Organization (WHO). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Collecting, preserving and shipping specimens for the diagnosis of avian influenza A(H5N1) virus infection. , (2006).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li></ol>

Baylor College of Medicine holds a U.S. provisional patent application for Mobile Clinics (U.S. Patent Application No. 15/523,126, # 620078924). The authors declare that they have no competing financial interest.

The remote laboratory facility described above is logistically-oriented, expandable, rapidly deployable, multifunctional, and based on human-centered design concepts that have been geared to protect laboratory personnel and workspace efficiency. The detailed protocol for quick laboratory set-up and safe respiratory virus isolation and diagnosis was developed and presented.
For optimal equipment functioning, the following conditions must be maintained in the laboratory units: ambient temperature of 21 &#177; 2 &#176;C, permissible temperature of 5 to 40 &#176;C, humidity of 14 &#177; 5% RH, permissible maximum relative humidity of 80% RH (noncondensing), and an altitude between 0 and 2,000 m above sea level.
Energy consumption is one of the most important parameters for management of an off-grid laboratory. For core laboratory equipment, the power efficiency can differ 15-40%; however, average energy consumption is estimated here to deliver an appropriate service. The highest power rate (1,500-2,000 W) relates to the air conditioner, the glovebox system, the PCR machine, and the autoclave sterilizer. Considering 8 hours of intensive work carrying out the protocol and 16 hours of the laboratory environment control, the daily energy consumption of laboratory units is approximately 36 kWh/day for BSL-2, about 43 kWh/day for BSL-3, and 73 kWh/day for the connected BSL-2/BSL-3+ facilities. For a single unit, we recommend providing a source of electrical power with capacity of running/continuous power &#8805;8 kW, surge/starting power &#8805;10 kW; for the connected facility, running/continuous power &#8805;12 kW, and surge/starting power &#8805;14 kW. Note, in the BSL-3 laboratory facility, a backup energy source is strongly recommended to avoid accidental power outage and guarantee steady work of the glove box and negative pressure system during a diagnostic test.
A gasoline powered electric generator is a cost-effective solution for emergency energy supply. Assume that fuel efficiency of a gasoline generator is approximately 1.5 gallons per hour at 100% load. Then, if the average daily energy consumption is 8 hours of 40% load and 16 hours of 10% load, the laboratory unit BSL-2 or BSL-3 requires 7-9 gallons of fuel per day, correspondingly, and the connected facility needs ~15 gal/day.
The remote laboratory units are designed to fit capabilities of off-grid solar panel systems. Solar panels do not require additional fuel and can be operated with high productivity in the tropical and subtropical regions of Africa, Asia and Latin America due to the high solar irradiation. Currently, one unit of a commercially available solar panel system allows a daily power usage of up to 44 kWh/day.
Regardless of the selected type of alternative electrical energy source, dirty electricity filters are strongly recommended and preinstalled in the laboratory facilities to improve power quality and to protect laboratory equipment. Keep the PCR system away from sources of strong and unshielded electromagnetic radiation because strong electromagnetic radiation may interfere with the proper operation of the device. It is also important do not use the PCR system near strong vibration sources, such as a centrifuge or pump because excessive vibration will affect instrument performance. The laboratory equipment may only be installed in an environment that has nonconductive pollutants, such as dust particles or wood chips. Ensure the room is away from any vents that could expel particulate material onto the instrument components.
The laboratory water usage depends on number of diagnostic tests running daily and number of laboratory technicians working in the facility. Nuclease free water is required for preparation of mixers during diagnostic procedure including extraction and PCR test and must be delivered in advance as other supplies and chemicals. At least 50 mL of nuclease free water is needed to run one diagnostic test; the required volume of nuclease free water depends on work load (i.e., on number of samples). Distilled water is needed to run the autoclave sterilizer. Autoclave water consumption in one cycle is 160-180 mL; the autoclave is recommended for daily use. Most of the plastics (tubes, pipette tips, etc.) are disposable, but some are re-usable and need to be washed (large containers, racks, etc.). Regular running water is used for washing hands between procedures and its minimal volume is estimated to be 15-20 L daily. The water needs to be pumped for pressure; sediment pre-filter system is recommended to protect the water appliances from the damaging effect of sediment and to improve the quality of running water.
For cold storage, at least one 5.1 cubic feet refrigerator (+4 &#176;C) and one 4.9 cubic feet (-20 &#176;C to -30 &#176;C) freezer are required in each laboratory unit to store samples/ RNA.
Laboratory decontamination includes several levels: cleaning &#62; antisepsis &#62; disinfection &#62; sterilization. Simple cleaning can be performed using soap and water while scrubbing with a gloved hand or brush. Antisepsis includes washing with liquid antimicrobial chemical in order to inhibit the growth and multiplication of germs. Alcohol solutions (70%) can be used as an antiseptic liquid. Disinfection is the application of a liquid chemical to eliminate nearly all pathogenic microorganisms (except bacterial spores) on work surfaces and equipment. Chemical exposure time, temperature, and concentration of disinfectant are important. Sodium hypochlorite solution (0.5%), or bleach, is an effective disinfectant on a large scale for surface purification and water purification. Ultraviolet germicidal irradiation is another method of disinfection. A germicidal lamp produces UV-C light and leads to the inactivation of bacteria and viruses. Sterilization employs a physical or chemical procedure to destroy all microbial life -- including highly resistant bacterial spores. Sterilization can be performed with an autoclave sterilizer.
All laboratory waste must be segregated at the point of generation. Place solid, non-sharp, infectious waste in leak-proof waste bags marked as biohazard. If generated waste is sharp, it must be placed in puncture-resistant containers. Collect potentially infectious liquid waste in properly labeled biohazard containers for liquids. Containers and bags should not be filled more than 2/3 the volume. The disposal of all bleach products must be sorted into their own designated waste bin. Laboratory waste must be handled gently to avoid generating aerosols and breakage of bags/containers. Collection bags/bins with biohazard waste must be sealed and external surfaces decontaminated after use with 0.5% sodium hypochlorite solution. Sterilize all laboratory waste in autoclave at 121 &#176;C for 30 minutes prior to incineration. Refer to functioning manual for the proper use of an autoclave. If possible, add a chemical or biological indicator to the autoclave to ensure proper sterilization. All autoclaved solid and liquid waste must be clearly labeled as sterilized with the setting, date, time, and operator. The labeled waste must then be placed in a secure, separate area prior to incineration.
As expected, workflow of diagnostic test depends on the disease and specimen. If it is recommended for virus identification to collect blood samples (e.g., Ebola19), sample aliquots can be stored at -20 &#176;C instead of -80 &#176;C (necessary for respiratory viruses). It is always better to take more than one specimen when sampling from a patient than to subdivide specimens later. If possible, for each type of specimen at least two specimens must be taken in separate specimen tubes. Specimens must be sub-divided if additional sampling is not possible.
If alternative specimens cannot be stored at appropriate temperatures (e.g., no freezers are available), swabs should be stored in pure (100%) ethanol or 99% methylated spirit (methanol additives only). In this case, the swab tip must be put into a vial with 1-2 mL of ethanol. Note that such specimens are suitable only for PCR. Also, note that a well-established assay is necessary for each particular virus diagnosis8,23, and unknown virus samples must be sent to assigned laboratories for further investigations19,20,21.
Mandatory and recommended requirements to the list of laboratory equipment for respiratory virus diagnostic PCR tests must be recognized. Table 3 underscores basic and minimally advanced (recommended) equipment and requirements for the RT-PCR diagnostic test. For BSL-3 practice, extra negative pressure protection (e.g., glove box) of personnel is crucial and necessary.
The connected laboratory modules are preferable to increase the number of personnel involved in laboratory testing and speed up the time required for a single test. Replacing the time-consuming manual RNA extraction is possible with automated qPCR (e.g., QiaCube). While this instrument is cumbersome (width 65 cm, length 62 cm, height 86 cm), it can fit the mobile laboratory workspace after rearrangement of furniture in BSL-2 or BSL-3 units.
Future work will be focused on the development of augmented reality (AR) and virtual reality (VR) trainings. The AR/VR glasses will be used to provide an interactive platform to teach requisite skills needed to become a well-trained laboratory worker. Helpful tips to perform some of the difficult, multistep procedures in laboratory diagnostic tests will be included in the software guide. This approach to personnel training should improve the quality of diagnostic test performance and management in remote laboratory facilities, especially remote and resource constrained areas.

Rapid diagnostics is a critical instrument in timely viral infection control, especially if early symptomatology is indistinguishable to a variety of infection diseases. The recent Ebola outbreak (2014-2015) in West Africa1,2, Zika virus epidemics (2015-2016) in Asia and Latin America3,4, the emergence of the Middle East Respiratory Syndrome (MERS) coronavirus infections5,6, and the unusually deadly flu (influenza) epidemics (2017-2018) in the U.S.7,8 uncovered the need for rapidly-deployable, laboratory facilities that address a multitude of issues from transportation, access, facilities, equipment, and communication.
Off-grid capability (autonomous power and water supply, etc.) is crucial in rural, resource-constrained global settings9,10,11. Our experience in clinical operations and global programs at Baylor College of Medicine was used to design and build a container-based mobile laboratory with capabilities for easy deployment, set-up and multifunctional usage (BSL-2 and BSL-3). Images of this versatile, logistically-enhanced laboratory facility is shown in Figure 1.
This rapidly-deployable, laboratory facility has an expandable design similar to the previously described container clinic (The &#39;Emergency Smart Pod&#39;)12,13,14, developed by Baylor College of Medicine and sponsored by USAID. A single packed unit (in Transport Mode) has the dimensions of 9 feet 9 inches x 8 feet x 8 feet (Figure 1A, B), and expands to an area of 170 square feet (15.75 m2) (Figure 1C, D). The unit can be deployed by two to four people in less than ten minutes.
The remote laboratory is built for a BSL-2 lab facility (Figure 2A) with a separate, modular, attachable, BSL-3 unit (Figure 2B) designed for work with infectious agents that may cause serious or potentially lethal disease through inhalation15. The connectivity of the two laboratory modules enables optimization of experimentation workflows, sharing of resources, and cost savings (Figure 2C-E).
The modules are air-tight and water-tight to create a comfortable, energy efficient mobile shelter. Heating, ventilation, and air conditioning (HVAC) system is used for centralized and temperature-controlled units. In general, the design of the laboratory units minimizes power consumption by usage of their own alternate power sources such as solar panels and/or an independent electrical generator. Each unit includes a sink and eyewash station, electrical power and water connectors (Figure 3A-C). The ICT platform delivers an optional, tablet-based (Android phone/Tablet or iPhone/iPad) documentation app for supply tracking and laboratory result documentation (Figure 3D) developed in partnership with Baylor&#39;s Information Technology (IT) research group that is well-experienced in working in remote environments with limited connectivity. The system can function using cellular or wireless signals, and allows documentation without connectivity, with immediate backup or transmission to a secure-cloud based server when connectivity is re-established.
The laboratory has several key infection-control features including: (a)&#160;negative pressure air flow, (b) a glove box or biosafety cabinet, (c) a health risk management system: a germicidal ultraviolet (UV-C) lighting system using 4 hierarchies of defense proven to eliminate 99.7% of pathogens that cause healthcare-related infections. The facility is easily disinfected using hydrogen peroxide or sodium hypochlorite (bleach) systems for efficient and effective decontamination.16
The assurance of quality laboratory results depends on a commitment to assess all aspects of the entirety diagnostic testing process. Here, we present a checklist for the BSL-2 and BSL-3 laboratory workflow, and a protocol for rapid respiratory virus diagnostic test. The proposed diagnosis of viral diseases relies on the detection of viral RNA or DNA in specimen (nasal wash, blood, stool, and urine, etc.) through real-time reverse-transcription polymerase chain reaction (RT-PCR). The ability to rapidly estimate viral loads in a specimen makes PCR an efficient tool for viral disease screening17,18. The implementation of novel, molecular diagnostic assays allows expansion of diagnostic capabilities for viruses such as Ebola19,20,21, influenza8,22, and tuberculosis (TB)23.
The goal of this work is to validate a novel modular and rapidly-deployable laboratory facility and provide a training guide for laboratory personnel working in remote, low-resource environments during an epidemic, natural disaster or other emergency relief situation. Here, we present a protocol for respiratory influenza diagnosis in this innovative, portable laboratory.

<table><tbody><tr><td>Autoclave Sterilizer &#039;BioClave&#039;</td><td>Benchmark Scientific, Edison, NJ, USA</td><td>B4000-16</td><td>16 liter, Benchtop, Dims: 22x17.5x15.7 in, Fully automatic, Extremely Compact</td></tr><tr><td>Barcode Scanner</td><td>Zebra Technologies ZIH Corp., Lincolnshire, IL, USA</td><td>Symbol LS2208</td><td>Handheld, lightweight</td></tr><tr><td>Breaker Box Panelboard Enclosure</td><td>Square D (Schneider Electric), France&amp;nbsp;</td><td>MH62WP&amp;nbsp;</td><td>NEMA 3R/5/12, Dims: 20 W x 62 H x 6-1/2 in. D, Electrical distribution board</td></tr><tr><td>Centrifuge - Microcentrifuge 17,000 x g</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>75002440</td><td>Holds 24 x1.5 or 2 ml tubes, Dims: 8.9x9.6x13.8 in</td></tr><tr><td>Class II Biological Safety Cabinet</td><td>NuAire, Inc., Plymouth, MN, USA</td><td>NU-602-400</td><td>4 Ft. Class II Type A2 Cage Changing Biological Safety Cabinet, 12&quot; Access Opening, HEPEX Pressure Duct&amp;nbsp;</td></tr><tr><td>Class III Biological Safety Cabinet (Glove box)</td><td>Germfree Laboratories, Ormond Beach, FL, USA</td><td>Model #PGB-36, Serial #C-2937</td><td>Glove box, Portable, 36&quot;, Class III BSC. Dims: 36x20x23.75 in, Includes 2 interior outlets</td></tr><tr><td>Cryo Coolers</td><td>VWR, Radnor, PA, USA</td><td>414004-286</td><td>0.5 or 1.5 ml tube benchtop coolers</td></tr><tr><td>Freezer (30&amp;deg;C freezer)</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>Model ULT430A</td><td>To occupy 4.9 Cubic feet</td></tr><tr><td>Laminar Flow Cabinet</td><td>NuAire, Inc., Plymouth, MN, USA</td><td>NU-126-300</td><td>3 Ft. Vertical Laminar Airflow Cabinet, 8&quot; Access Opening, HEPA filter supply, 99.99%</td></tr><tr><td>Mini Centrifuge</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>75004061</td><td>Dims: 4.1x5.0x6.0 in</td></tr><tr><td>Pipettes automated</td><td>VWR, Radnor, PA, USA</td><td>05-403-151</td><td>Pipet 4-pack (2.5,10, 100 and 1,000&amp;mu;L volume)</td></tr><tr><td>Pipettes automated &#039;Finnpipette&#039;</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>4700880</td><td>Pipet 4-pack (2, 20, 200 and 1,000&amp;mu;L volume), Advanced Volume Gearing(AVG), Ultra durable</td></tr><tr><td>Power Generator</td><td>Cummins Power Generation, Minneapolis, MN, USA</td><td>C60 D6</td><td>60 kW, 60 Hz, 1 Phase, 120/240V, Diesel</td></tr><tr><td>Refrigerator</td><td>BioMedical Solutions, Inc., Stafford, TX, USA</td><td>BSI-HC-UCFS-0504W</td><td>Standard Undercounter Refrigerators &amp;amp; Freezers</td></tr><tr><td>Refrigerator</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>05LRAETSA&amp;nbsp;</td><td>To occupy&amp;nbsp; 5.1 Cubic feet</td></tr><tr><td>RT-PCR machine &#039;Step-one plus&#039;</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>4376598</td><td>Holds 96 samples, Dims: 9.7x16.8x20.2 in&amp;nbsp;</td></tr><tr><td>Vortex Mix</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>88880017TS</td><td>Dims: 6.1x8.3x3.3 in</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AgPath-ID One-Step RT-PCR Reagents</td><td>Applied Biosystems, Foster City, CA, USA</td><td>4387391</td><td></td></tr><tr><td>Ethanol Koptec Pure 200 Proof</td><td>Decon Labs, Inc., King of Prussia, PA, USA</td><td>V1001</td><td></td></tr><tr><td>Nuclease-free Water</td><td>Ambion, Inc., Carlsbad, CA, USA</td><td>AM9906</td><td></td></tr><tr><td>QIAamp Viral RNA Mini Kit</td><td>Qiagen, Hilden, Germany</td><td>52906</td><td></td></tr><tr><td>SuperScript III Platinum One-Step qRT- PCR Kit</td><td>Invitrogen, Carlsbad, CA, USA</td><td>11732-088</td><td></td></tr><tr><td>&lt;strong&gt;Disposable&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 mL cryogenic tubes</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>03-337-7X&amp;nbsp;</td><td></td></tr><tr><td>1.5 mL tubes</td><td>VWR, Radnor, PA, USA</td><td>10025-726</td><td></td></tr><tr><td>10 &amp;micro;L Filter Tips</td><td>Neptune, VWR, Radnor, PA, USA</td><td>Neptune, BT10XLS3</td><td></td></tr><tr><td>20 &amp;micro;L Filter Tips</td><td>Multimax, BioExpress, VWR, Radnor, PA, USA</td><td>MultiMax, P-3243-30X</td><td></td></tr><tr><td>200 &amp;micro;L Filter Tips</td><td>ART, Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>ART, 2770</td><td></td></tr><tr><td>1000 &amp;micro;L Filter Tips</td><td>Phenix Research Products, Candler, NC, USA</td><td>TS-059BR</td><td></td></tr><tr><td>AB custom probes</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>N/A</td><td>Custom probes</td></tr><tr><td>Combitips</td><td>Eppendorf, Hauppauge, NY, USA</td><td>89232-972</td><td></td></tr><tr><td>Integrated DNA Technology (IDT) custom probes and primer</td><td>IDT</td><td>N/A</td><td>Custom probes</td></tr><tr><td>MicroAmp Fast Optical 96-Well Reaction Plate</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>490003-978 CS</td><td></td></tr><tr><td>MicroAmp Fast Reaction Tubes (8 tubes/strip)</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>4358293</td><td></td></tr><tr><td>MicroAmp Optical 8-Cap Strip</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>4323032</td><td></td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Thermo Fisher Scientific, Carlsbad, CA, USA</td><td>4311971</td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biohazard waste bags</td><td>VWR, Radnor, PA, USA</td><td>14220-046</td><td>20.3 x 30.5 cm Biohazard bags</td></tr><tr><td>Gloves</td><td>Denville Scientific, Holliston, MA, USA</td><td>G4162-250</td><td>Small, meduim or large Nitrile or latex gloves</td></tr><tr><td>Lab coat</td><td>N/A</td><td>N/A</td><td>Customizable</td></tr><tr><td>Masks</td><td>VWR, Radnor, PA, USA</td><td>414004-663</td><td>Advanced protection mask</td></tr><tr><td>Protective shoes</td><td>N/A</td><td>N/A</td><td>Customizable</td></tr></tbody></table>

remote laboratory management: respiratory virus diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

The goal of this study is to demonstrate that the proposed BSL-2 and BSL-3 mobile laboratory facilities provide an adequate environment allowing respiratory virus diagnostic tests with representative results identical to tests performed in high-quality stationary laboratories. The laboratory facilities are designed to comply with the test requirements given in Occupational Health and Safety (OHS) recommendations. As soon as the remote laboratory facility is deployed (Figure 4) and all equipment and supplies are installed (Figure 5), laboratory tests can be run.
In accordance with laboratory standard operating procedures, PPE (lab coats, protective shoes, gloves, advanced mask, protective eyewear, etc.) appropriate for BSL-2 practice is required. For BSL-3 practice, the PCR laboratory module of negative pressure is equipped with a certified glove box. The laboratory units are upgraded by external pass-through windows to protect personnel at the step of sample receiving. The registration process can be simplified with previously developed tablet-based application (Figure 3D). Other acceptable applications that run on a laptop can be used as well.
This particular respiratory virus diagnostic test can be performed in the connected laboratory modules to separate steps of the diagnostic procedure on purpose to avoid contamination or potential interference between biochemical reagents, which may affect the testing results. To maximize the quality of diagnosis, the rapid diagnostic test practice utilizes (i) both the basic laboratory BSL-2 and the traverse connected PCR room (Section 3) or (ii) the GB and PCR rooms connected by pass-through window (Section 4). The diagram of the proposed laboratory workflow is presented on Figure 6 and emphasizes personal protection. The diagram recognizes the importance of each indicated step for personnel protection, especially if laboratory staff in remote areas is minimally trained.
The rapid diagnostic test of influenza is accomplished via the RT-PCR technique. The procedure contains four main steps. Note that individual workspaces are assigned for each stage of the protocol.
The first step is to obtain a sample and sub-divide it into several aliquots. The aliquots can then be marked with barcodes to improve effectiveness of data control and stored in the freezer for further investigations. The second step is to inactivate a sample in lysis buffer by centrifuging and heating. The first and second steps must be carried out in biosafety cabinets. Utilize individual pipette sets and equipment. A PCR test is proposed to be performed in the PCR room, if available. The third step is the documentation of results. Step four is the maintenance after the equipment usage, and reminder of personnel protection at the end of experiment.
If the specimen is expected to be classified as BSL-3+ (e.g.,Ebola, Zika, MERS, TB) the glove box facility must be used. In the remote laboratory, the GB room has its own pass-through window to receive specimens and a laptop or tablet for sample registration. The sample aliquot and virus inactivation must all be performed in the glove box chamber. UV-C lighting is recommended to avoid contamination during procedure. After inactivation of a sample, further steps for protocol are similar to the basic laboratory BSL-2 and BSL-3 test and follows Checklist Part III (Table 1, Figure 6). 
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59188/59188fig1.jpg" /> 
Figure 1.&#160;Laboratory facility prototype. (A, B) Transport mode; (C) Deployed mode: outside; (D) Deployed mode: interior. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59188/59188fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59188/59188fig2.jpg" /> 
Figure 2.&#160;Schematics. (A) The basic laboratory BSL-2; (B) The BSL-3 module includes the glove box and PCR laboratories, which have a common pass-through window for protected specimen transfer; (C) Connected laboratory facilities (A) and (B) with shared utilities. (D,E) Photographs of the connected units from opposite sides. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59188/59188fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59188/59188fig3.jpg" /> 
Figure 3. (A) Interior of the BSL-3 facility has (1) a pass-through window, a sink and (2) an eyewash station at the inlet; (B) Electrical power connectors, (C) Water connectors; (D) Tablet-based software for supply tracking and laboratory result documentations. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59188/59188fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59188/59188fig4v2.jpg" /> 
Figure 4.&#160;Deployment of the laboratory facility. Instruction for panels unfolding on one side of the unit as illustrated (A-D). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59188/59188fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59188/59188fig5.jpg" /> 
Figure 5.&#160;Schematics of the connectable laboratory: (A) BSL-2 module 1; (B) Glove Box and PCR module 2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59188/59188fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59188/59188fig6.jpg" /> 
Figure 6. Flow chart for a respiratory virus diagnostic RT-PCR test in the remote laboratory facility. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59188/59188fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Remote Laboratory BSL-2</td>
			<td>Remote Laboratory BSL-3</td>
		</tr>
		<tr>
			<td>Part I</td>
			<td>Part I</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160; i.&#160; Lab tech to enter through door labeled entrance and put on lab coat, which is hanging on rack next to entrance door. Open shoes are prohibited, advanced mask and protective eyewear are encouraged.</td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; i.&#160;&#160;&#160; Lab tech to look into Glove Box window from outside of unit to insure negative pressure is activated. (Pink Ball should be visible in the unit to show the negative pressure is working).&#160;</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; ii.&#160; Lab tech to wash hands in sink, put on disposable gloves and begin with intake of samples.</td>
			<td>&#160;&#160;&#160;&#160;&#160; ii.&#160;&#160;&#160; If negative pressure is working, lab tech to enter through only door and put on lab coat, which is hanging on rack next to entrance door.&#160; Open shoes are prohibited, advanced mask and protective eyewear are desirable.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; iii.&#160; Samples that were dipped in hypochlorite bath prior to being dropped at pass-through window are sitting in pass through for lab tech.</td>
			<td>&#160;&#160;&#160;&#160; iii.&#160;&#160;&#160; Lab tech to wash hands in sink, put on disposable gloves, PPE and begin with intake of samples.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; iv.&#160; Received in sample reception.</td>
			<td>&#160;&#160;&#160;&#160; iv.&#160;&#160;&#160; Samples that were previously dipped in hypochlorite bath prior to being dropped at pass through window are sitting in pass through for lab tech.</td>
		</tr>
		<tr>
			<td>Part II</td>
			<td>&#160;&#160;&#160;&#160;&#160; v.&#160;&#160;&#160;&#160; Received in sample reception.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; v.&#160; Depending on diagnostic procedure, specimens moved to biosafety cabinet and inactivated.</td>
			<td>Part II</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; vi.&#160; Specimens prepped for microscopy, centrifuge, or ROTs.</td>
			<td>&#160;&#160;&#160;&#160; vi.&#160;&#160;&#160; Specimens inactivated in Glove Box.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; vii.&#160; Appropriate diagnostic tests run.&#160;</td>
			<td>&#160;&#160; vii.&#160;&#160;&#160; Specimens executed for nucleic acid isolation.</td>
		</tr>
		<tr>
			<td>&#160;&#160; viii.&#160; Store specimens in 4&#176;C refrigerator or appropriate freezer.</td>
			<td>&#160; viii.&#160;&#160;&#160; After extraction, specimens moved to pass-through window.</td>
		</tr>
		<tr>
			<td>Part III</td>
			<td>&#160;&#160;&#160;&#160; ix.&#160;&#160;&#160; Lab tech enters through entrance in PCR side of unit (positive pressure).</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; ix.&#160; Use sink for staining &#38; washing of items.</td>
			<td>&#160;&#160;&#160;&#160;&#160; x.&#160;&#160;&#160;&#160; Lab tech to put on lab coat from rack next to entrance, wash hands in sink, put on gloves.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160; x.&#160; Use laptop &#38; counterpace to perform analyses and documentation.&#160;</td>
			<td>&#160;&#160;&#160;&#160; xi.&#160;&#160;&#160; Receive samples from Glove Box room in pass-through window.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; xi.&#160; Sterilize equipment by running autoclave.</td>
			<td>&#160;&#160; xii.&#160;&#160;&#160; If necessary samples prepped in Laminar flow cabinet.</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; xii.&#160; Dispose of any biohazardous waste in biohazard waste container.</td>
			<td>&#160; xiii.&#160;&#160;&#160; Appropriate diagnostics tests run.</td>
		</tr>
		<tr>
			<td>&#160;&#160; xiii.&#160; Wash hands in sink.</td>
			<td>&#160; xiv.&#160;&#160;&#160; Store specimens in 4&#176;C refrigerator or appropriate freezer.</td>
		</tr>
		<tr>
			<td>&#160; xiv.&#160; Hang lab coat back up on rack.</td>
			<td>Part III</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; xv.&#160; Exit through&#160; same door.</td>
			<td>&#160;&#160; xv.&#160;&#160;&#160;&#160; Use sink for staining &#38; washing of items.</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160; xvi.&#160;&#160;&#160; Use laptop &#38; counterpace to perform analyses and documentation.&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160;xvii.&#160;&#160;&#160; Transfer vials into pass-through window to PCR room and sterilize equipment by running autoclave.</td>
		</tr>
		<tr>
			<td></td>
			<td>xviii.&#160;&#160;&#160; Dispose of any biohazardous waste in biohazard waste container.</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160; xix.&#160;&#160;&#160; Wash hands in sink.</td>
		</tr>
		<tr>
			<td></td>
			<td>&#160;&#160; xx.&#160;&#160;&#160;&#160; Exit through same entrance door.</td>
		</tr>
	</tbody>
</table>
Table 1. Checklist for the PCR diagnostics workflow.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Maintenance and calibrations</td>
		</tr>
		<tr>
			<td rowspan="2">Real-time PCR systems&#160;</td>
			<td>Monthly</td>
			<td>Perform background calibrations every month</td>
		</tr>
		<tr>
			<td>18 months</td>
			<td>Perform background, spatial and dye calibrations every 18th months</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>1 year</td>
			<td>Calibrate for revolutions per minute and temperature through external or internal calibration services</td>
		</tr>
		<tr>
			<td rowspan="3">Glove Box</td>
			<td>Daily</td>
			<td>Visually inspect elements, particularly for damage to the exposed surfaces of the HEPA filters, gloves, o-rings and hoses. Make sure duct clamps are tight and in place. Perform leak pressure test. Test the pressure alarm.</td>
		</tr>
		<tr>
			<td>6 months</td>
			<td>Change the HEPA filter</td>
		</tr>
		<tr>
			<td>1 year&#160;</td>
			<td>Calibrate the system</td>
		</tr>
		<tr>
			<td rowspan="3">Autoclave</td>
			<td>Weekly</td>
			<td>Clean the water tank and racks using a mild non-abrasive detergent</td>
		</tr>
		<tr>
			<td>3 months</td>
			<td>Calibrate timer and gauges</td>
		</tr>
		<tr>
			<td>1 year or every 50 cycles</td>
			<td>Inspect, clean thoroughly, test and calibrate</td>
		</tr>
		<tr>
			<td rowspan="2">Refrigerator and Freeezer</td>
			<td>6 months</td>
			<td>Check&#160; fan motor, evaporator coils, vacuuming condensing coils and condensor filters and replace batteries as needed</td>
		</tr>
		<tr>
			<td>1 year</td>
			<td>Calibrate freezer through internal or external calibration services</td>
		</tr>
	</tbody>
</table>
Table 2. Real-time PCR equipment maintenance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mandatory</td>
			<td>Recommended</td>
		</tr>
		<tr>
			<td>Lab coat, protective shoes, gloves</td>
			<td>Lab coat, protective shoes, gloves, masks, eyewear</td>
		</tr>
		<tr>
			<td>Refrigerator 4 &#176;C, freezer -20 &#176;C</td>
			<td>Refrigerator 4 &#176;C, freezer -20 &#176;C, freezer -80 &#176;C</td>
		</tr>
		<tr>
			<td>One set of automated&#160; pipettes&#160;</td>
			<td>Three sets of automated pipettes</td>
		</tr>
		<tr>
			<td>Centrifuge, shaker, thermocycler</td>
			<td>Robotic system</td>
		</tr>
		<tr>
			<td>RT-PCR machine, ice bath</td>
			<td>RT-PCR with temperature control, ice-free cooler</td>
		</tr>
		<tr>
			<td>Biohazard waste bags</td>
			<td>Autoclave to dispose biohazard waste</td>
		</tr>
	</tbody>
</table>
Table 3. Minimum requirements for the RT-PCR respiratory virus diagnostic test BSL-2.

Watch this Scientific Journal Video about Remote Laboratory Management: Respiratory Virus Diagnostics at JoVE.com

Remote Laboratory Management: Respiratory Virus Diagnostics

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

remote-laboratory-management-respiratory-virus-diagnostics

Universidad San Sebastian

Research

JoVE Journal

Medicine

33.0K Views.  Baylor College of Medicine. A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

Video: Remote Laboratory Management: Respiratory Virus Diagnostics

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Immunology and Infection

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.

An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...

Efficient SARS-CoV-2 Quantitative Reverse Transcriptase PCR Saliva Diagnostic Strategy utilizing Open-Source Pipetting Robots

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by a high rate of transmission and low mortality, the COVID-19 pandemic necessitated accurate and efficient diagnostic testing, particularly in closed populations such as residential universities. Initial availability of nucleic acid testing, like nasopharyngeal swabs, was limited due to supply chain pressure which also delayed reporting of test results. Saliva-based reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) testing has shown to be comparable in sensitivity and specificity to other testing methods, and saliva collection is less physically invasive to participants. Consequently, we developed a multiplex RT-qPCR diagnostic assay for population surveillance of Clemson University and the surrounding community. The assay utilized open-source liquid handling robots and thermocyclers instead of complex clinical automation systems to optimize workflow and system flexibility. Automation of saliva-based RT-qPCR enables rapid and accurate detection of a wide range of viral RNA concentrations for both large- and small-scale testing demands. The average turnaround for the automated system was &#60; 9 h for 95% of samples and &#60; 24 h for 99% of samples. The cost for a single test was $2.80 when all reagents were purchased in bulk quantities.

The protocol describes a SARS-CoV-2 diagnostic method that utilizes open-source automation to perform RT-qPCR molecular testing of saliva samples. This scalable approach can be applied to clinical public health surveillance as well as to increase the capacity of smaller university laboratories.

The emergence of the recent SARS-CoV-2 global health crisis introduced key challenges for epidemiological research and clinical testing. Characterized by ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Enhancing Pulmonary Infection Treatment Using M-ROSE

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging acquisition is relatively straightforward in clinical practice, its lack of specificity often necessitates additional time-consuming tests such as sputum culture, bronchoalveolar-lavage fluid culture, or genetic sequencing to identify the underlying etiology of the disease accurately. Moreover, the limited efficacy of empirical anti-infective treatment may contribute to antibiotic misuse. Recent advancements in interpreting microbial background on rapid on-site evaluation (ROSE) slides have enabled clinicians to promptly obtain samples through bronchoscopy (e.g., alveolar lavage, mucosal brushing, tissue clamp), facilitating bedside staining and interpretation that provides essential microbial background information. Consequently, this establishes a foundation for developing targeted anti-infection treatment and individualized drug therapy plans. With a better understanding of which pathogens are causing infections in real-time, physicians can avoid unnecessary broad-spectrum antibiotics contributing to antibiotic resistance. Establishing a rapid and standardized M-ROSE workflow within respiratory medicine departments or intensive care units will greatly assist physicians in formulating accurate treatment strategies for patients, which holds significant clinical implications.

Author Spotlight: Advancing Pathogen Detection and Disease Assessment in Real-Time Using M-ROSE

The protocol here demonstrates a fast and standardized microbiological rapid on-site evaluation (M-ROSE) workflow, including three steps: slide making, staining, and interpretation. This protocol will help physicians make rapid clinical decisions.

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging ...

Using Colorimetric and RT-LAMP Analysis to Detect SARS-CoV-2 

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP

Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern ...

Biology

Multiplex RT-qPCR Assay for Respiratory Virus Detection

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...