key: cord-0838983-klkd8v9x
authors: Xing, Wanli; Wang, Jiadao; Zhao, Chao; Wang, Han; Bai, Liang; Pan, Liangbin; Li, Hang; Wang, Huili; Zhang, Zhi; Lu, Ying; Chen, Xiang; Shan, Sisi; Wang, Dong; Pan, Yifei; Weng, Ding; Zhou, Xinying; Huang, Rudan; He, Jianxing; Jin, Ronghua; Li, Weimin; Shang, Hong; Zhong, Nanshan; Cheng, Jing
title: A Highly Automated Mobile Laboratory for On-site Molecular Diagnostics in the COVID-19 Pandemic
date: 2021-03-31
journal: Clin Chem
DOI: 10.1093/clinchem/hvab027
sha: 312353ea0e2bb53783ce59a4a5ddbed441dfbad2
doc_id: 838983
cord_uid: klkd8v9x

BACKGROUND: Infectious disease outbreaks such as the COVID-19 (coronavirus disease 2019) pandemic call for rapid response and complete screening of the suspected community population to identify potential carriers of pathogens. Central laboratories rely on time-consuming sample collection methods that are rarely available in resource-limited settings. METHODS: We present a highly automated and fully integrated mobile laboratory for fast deployment in response to infectious disease outbreaks. The mobile laboratory was equipped with a 6-axis robot arm for automated oropharyngeal swab specimen collection; virus in the collected specimen was inactivated rapidly using an infrared heating module. Nucleic acid extraction and nested isothermal amplification were performed by a “sample in, answer out” laboratory-on-a-chip system, and the result was automatically reported by the onboard information platform. Each module was evaluated using pseudovirus or clinical samples. RESULTS: The mobile laboratory was stand-alone and self-sustaining and capable of on-site specimen collection, inactivation, analysis, and reporting. The automated sampling robot arm achieved sampling efficiency comparable to manual collection. The collected samples were inactivated in as short as 12 min with efficiency comparable to a water bath without damage to nucleic acid integrity. The limit of detection of the integrated microfluidic nucleic acid analyzer reached 150 copies/mL within 45 min. Clinical evaluation of the onboard microfluidic nucleic acid analyzer demonstrated good consistency with reverse transcription quantitative PCR with a κ coefficient of 0.979. CONCLUSIONS: The mobile laboratory provides a promising solution for fast deployment of medical diagnostic resources at critical junctions of infectious disease outbreaks and facilitates local containment of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) transmission.

The coronavirus disease 2019 (COVID-19) pandemic has posed major challenges to medical care globally (1) (2) (3) . Timely diagnostics and treatment are critical in response to the COVID-19 pandemic (4). Among various diagnostic techniques, nucleic acid-based tests (NATs) of the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (5, 6), including reverse transcription quantitative PCR (RT-qPCR) (7) , reverse transcription loop-mediated isothermal amplification (LAMP) (8, 9) , and reverse transcription recombinase polymerase amplification (10) , have been vital in identifying potential virus carriers (11) .

As large numbers of patients with respiratory infection go to medical centers for NATs, there is an accompanying high risk of cross-infection by SARS-CoV-2. For community screening, medical personnel are dispatched to collect specimens and send them to central laboratories for testing; such screening involves both tedious work and high risk of exposure. The inability to inactivate the virus on-site may lead to biohazards due to unexpected leakage and contamination. For massive screening of travelers within a limited time at transportation hubs, on-site NATs are required. However, countries affected by the COVID-19 pandemic have had vast disparities in medical resources (12, 13) . Especially in remote areas, NATs of the virus are largely unavailable given scarcity of medical centers with molecular diagnostics capability. The cost and time to build new stationary laboratories in the event of an outbreak are unsustainable. There is an urgent need to develop fielddeployable fast-response mobile laboratories to provide on-site specimen collection and diagnostics.

Several field-deployable laboratories have been developed for on-site diagnostics. Early mobile laboratories adapted a room-based structure with specially designed equipment to improve portability (14) (15) (16) (17) (18) (19) . These laboratories required local housing and facilities such as water and electricity supply; owing to the need to establish separated working areas for nucleic acid amplification, their deployment suffered from poor mobility and dependence on local facilities. Later, truck-based mobile laboratories were developed with their own stand-alone power generators and ventilation systems that eliminated the need for local facilities; however, the large footprint restricted their use (20, 21) .

We have previously developed a wide range of biochips and molecular diagnostic technologies based on laboratory-on-a-chip systems, including the pioneering bioelectronic chip that performed on-chip isolation, lysis, and hybridization of bacteria (22) . More recently, we have focused on detection of infectious pathogens such as SARS-CoV-2 and Mycobacterium tuberculosis (23) (24) (25) (26) . In this article, we present a highly automated and fully integrated van-based mobile laboratory for rapid response to infectious disease outbreaks. The modular mobile laboratory is equipped with a 6-axis robot arm for automated oropharyngeal swab specimen collection, a heating module for quick pathogen inactivation, and a self-contained "sample in, answer out"type microfluidic analyzer for nested isothermal amplification of pathogen nucleic acids. The results and laboratory status are monitored and automatically reported using an onboard information platform. Evaluation of the van-based mobile laboratory demonstrated excellent performance in specimen collection, inactivation, analysis, and reporting.

The mobile laboratory was constructed by remodeling a midsized van to achieve compact size and full functionality (see Fig. 1 and the online Data Supplement). Five discrete modules were developed and integrated to form the mobile laboratory, including automated specimen collection, quick specimen inactivation, pathogen NATs, a mobile laboratory information system, and a supporting system.

Manual collection of swab specimens from test participants is standard procedure; however, prolonged exposure to potential virus carriers and intensive labor have posed substantial risk to medical personnel (27) . In the current work, a 6-axis robot arm was developed for automated specimen collection (Fig. 2, A) . The specimen collection workflow is shown in Fig. 2 , B. The robot arm was guided by streamed video images from a CCD camera with predetermined check positions for autocorrection. A tension and compression force sensor at 0.4-N resolution was integrated to provide force feedback and to reduce discomfort and potential harm to the test participant. Once the swab reached the oropharynx, the robot arm moved twice up and down and twice left and right, collecting a sufficient amount of oropharyngeal specimen for nucleic acid analysis.

To evaluate the efficiency of specimen collection, human cellular materials in oropharynx from healthy participants were collected and compared between automated collection by the robot arm and manual collection by trained medical personnel. The oropharyngeal cell samples were analyzed by LAMP using primers targeting human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To assess the collected specimen, the time to positivity (Tp) value was determined as the second derivative inflection of amplification results, and lower Tp values indicated higher concentrations of the test sample (28) . The Tp values of GAPDH curves were thus analyzed and compared between automated and manual collection.

The collected specimen should be contained in a biosafety cabinet and inactivated before the NAT (29) . Conventional water bath heating is time consuming and difficult to automate. A quick inactivation device was developed so that collected specimens were automatically loaded to the inactivation device inside the biosafety cabinet by the robot arm. The inactivation device utilized differential infrared radiation and thermoelectric effect to control the temperature in the upper and lower parts of the collection tubes, respectively (Fig. 3, A) .

To measure temperature uniformity, a set of thermocouples were placed in the upper part of the collection tubes in each of the 16 positions of the holder. Temperature profiles by infrared radiation were captured in real time for the duration of 12 min at an interval of 10 s by a data-acquisition module (34972 A, Data Acquisition/Switch Unit; Keysight).

The quick inactivation device satisfied the needs of 2 working scenarios. On one hand, the collected specimen was maintained in preservation buffer, where both infrared bulbs and the Peltier element functioned as heaters for complete thermal inactivation. On the other hand, the collected specimen was maintained in lysis buffer, where infrared bulbs provided thermal inactivation of pathogen residues on the sidewalls and caps in the upper part, and the Peltier element remained idle for chemical inactivation of pathogens (30) .

The inactivation efficiency was tested using the pseudoviruses Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2. First, pseudovirus solutions were added to collection tubes at 200 lL and heated at 56 C for 30 min or 70 C for 12 min, respectively, with the Peltier element. Infrared radiation was applied simultaneously to the set temperature of 95 C in the upper part of collection tubes. Viral activity of inactivated samples was examined using a luciferase assay (31) . Second, smears of pseudovirus MERS-CoV were applied to upper sidewalls and caps of collection tubes and heated by infrared radiation to set temperatures of 90 C, 95 C, and 100 C, respectively, whereas the lower part filled with 60 lL diethyl pyrocarbonatetreated water was not heated. For each temperature, infrared heating was tested for the duration of 5 min and 12 min, respectively. After quick inactivation, the tubes were vigorously shaken so that smears were remixed The mobile laboratory consists of the 6-axis robot arm; the quick pathogen-inactivation device; microfluidic nucleic acid analyzers; the information platform that automatically reports experimental results and laboratory status; the biosafety cabinet; and the supporting system, including separate ventilation systems, the electrical cable wheels, a backup generator, and the waterway system.

with diethyl pyrocarbonate-treated water for the luciferase assay. Considering that these pseudoviruses could infect host cells for only a single time and that the poor capability of infection may lead to false-negative results, the replicable vesicular stomatitis virus was utilized to simulate inactivation of real pathogens. , the negative control refers to cell-plated microwells that do not contain virus samples, and the noninactivated groups refer to cell-plated microwells that contain virus samples that did not go through the inactivation process. The numbers and color codes in the charts represent the magnitude of fluorescent signals in luciferase assay. a.u., arbitrary units. (E), RT-qPCR amplification results of pseudovirus FNV-2019-ncov-abEN at the concentrations of 5E3 copies/ mL after inactivation at different conditions (n ¼ 3). Noninactivated pseudovirus FNV-2019-ncov-abEN was used as a control to check the integrity of treated nucleic acids. In group 1, the collection tubes containing pseudovirus samples were heated in the water bath at 56 C for 30 min, whereas in groups 2, 3, and 4, the upper part of tubes was heated at 95 C while the lower part was heated at 56 C for 30 min, 70 C for 12 min, and 95 C for 12 min, respectively. Ct, threshold cycle.

A possible risk factor for false-negative results by NATs (30, 32, 33) with thermal inactivation of pathogens is compromised nucleic acid integrity due to inappropriate heating. The integrity of heatinactivated specimen was evaluated using RT-qPCR with pseudovirus FNV-2019-ncov-abEN (Fubio Biological Technology Co., Ltd.). After inactivation, the RNAs of the heat-inactivated samples were released by mixing with sample release reagent (10-lL sample to 10 lL reagent; Sansure Biotech) for 10 min at room temperature. Viral RNAs were quantified by calculating quantification cycle (Cq) 

The microfluidic nucleic acid analyzer is comprises a fully integrated centrifugal microfluidic chip and associated machinery such as spinning and heating modules and the optical detection module. The centrifugal microfluidic chip hosts 2 parallel reactions for analyzing 2 samples simultaneously. Each consists of 3 main modules including nucleic acid extraction, preamplification by reverse-transcription recombinase polymerase amplification, and amplification by LAMP. Inactivated specimen was directly dispensed into the chip by pipetting, and the following liquid-handling and amplification procedures were automatically driven by the centrifugal force to achieve "sample in, answer out"-type NATs of pathogens.

To evaluate the sensitivity of the analyzer, pseudovirus FNV-2019-ncov-abEN was diluted to a wide range of concentration gradients of 100, 150, 300, 1000, 5000, and 10 000 copies/mL and added to the microchip. Repeatability of the analyzer was assessed by testing the pseudovirus FNV-2019-ncov-abEN at the concentration of 1000 copies/mL using 5 batches (6 replicates in each batch) of centrifugal microchips.

Robustness of the analyzer was tested by adding a variety of interferents to the pseudovirus FNV-2019-ncov-abEN specimen prepared at the concentration of 150 copies/mL, including azithromycin at 5 mg/L, oseltamivir at 12 mg/L, budesonide at 10 mg/L, meropenem at 100 mg/L, mometasone at 100 mg/L, mucin at 10 mg/ L, ceftriaxone at 40 mg/L, tobramycin at 10 mg/L, zanamivir at 10 mg/L, and levofloxacin at 10 mg/L. Final results were processed and reported automatically by the information platform.

To evaluate the performance of the mobile laboratory in a complete workflow of molecular diagnostics, oropharyngeal swab specimens were collected by the automated robot arm from healthy volunteers, spiked with pseudovirus FNV-2019-ncov-abEN to the concentration of 150 copies/mL, inactivated, and analyzed by the microfluidic nucleic acid analyzer. The control group differed by manual collection of oropharyngeal swab samples (n ¼ 10).

Clinical samples were obtained with informed consent from patients. A total of 736 clinical samples (377 samples from male patients and 359 from female patients; average age: 43.11 years) were collected from 723 patients (370 male patients and 353 female patients; average age: 43.17 years). The diagnostic results of clinical samples using the onboard microfluidic nucleic acid analyzer were compared with those of the reference RT-qPCR method using j consistency analysis, and the j coefficient was determined. The diagnostic results of patients using the microfluidic analyzer were also compared with those of the reference standard by clinical assessment.

The air ventilation system provided negative pressure at À15 Pa to the whole cabin of the mobile laboratory, and the biosafety level 2 cabinet used a stand-alone airventing system. Because the sampling window remained open during specimen collection to prevent crosscontamination with the outside environment, a specially designed air curtain was used through which outside air was drawn into the gratings along the sides of the sampling window by negative pressure. Simulation of the air-flow profile in the biosafety cabinet and in close proximity to the sampling window was performed using COMSOL Multiphysics. Details on the stand-alone waterway and power supply systems are given in the online Data Supplement.

In the constructed, van-based mobile laboratory, a 6axis robot arm with built-in artificial vision and force feedback was used for oropharyngeal swab sampling. The collected specimens were transferred directly to a heating module for rapid inactivation of pathogens, followed by onboard NAT using a fully integrated microfluidic nucleic acid analyzer. The mobile laboratory was equipped with 8 microfluidic nucleic acid analyzers, which appreciably increased the throughput of the mobile laboratory to 150 samples in 8 hours. The onboard information platform possesses a data-driven pivot capable of real-time and multidimensional perception, surveillance, and intelligent decision-making for response to COVID-19 outbreaks (see online Supplemental Fig. S4 ).

The robot arm demonstrated good repeatability of 60.1 mm, a small footprint of 128 mm in diameter, and low power consumption of 100 W under typical settings. Movement of the robot arm was precisely controlled, and the status was monitored at multiple checkpoints in a single run, during which autocorrection of the robot arm was applied to ensure safe and seamless operation.

The specimen collection efficiency was evaluated by comparing the Tp values of the GAPDH gene in LAMP reactions from human cellular materials of healthy participants that was collected by automated and manual collection (Fig. 2, C) . The mean (SD) Tp values of the GAPDH gene of automated and manual collections were 13.7 (0.6) min and 14.1 (1.2) min, respectively (n ¼ 8). These results showed no significant difference of collected oropharyngeal swab specimens between automated and manual collection. Fig. 2 , D and E, show that the air curtain efficiently takes in air flow from both inside and outside of the biosafety cabinet without noticeable leakage through the sampling window. These results demonstrate successful isolation of the biosafety cabinet from both the outside environment and the laboratory cabin during specimen collection.

As shown in Fig. 3 , B, the temperature profiles of the upper part of collection tubes in all positions of the holder demonstrate uniform heating by infrared radiation. It took <5 min to reach the set temperature at 95 C in the upper part of the collection tubes, with maximum overshooting of <2.0 C. The SD of stabilized temperatures after heating for 5 min was 2.4 C. Figure 3 , C, provides data showing that in thermoelectric heating inactivation, the pseudoviruses could be successfully inactivated by heating at both 56 C for 30 min and 70 C for 12 min. Fig. 3 , D, shows that no viral activity was detected, and the residual viruses left in the upper sidewalls and caps were successfully inactivated. Complete inactivation of the replicable vesicular stomatitis virus can be achieved after infrared heating of the upper part at 95 C and 97 C for 12 min (see online Supplemental Fig. S5) . Overall, these results demonstrate successful inactivation of pathogens in both solutions and sidewalls within 12 min at the optimized temperature of 95 C. Figure 3 , F, shows that the Cq values of the RT-qPCR tests of pseudovirus FNV-2019-ncov-abEN increased by >2 when the lower part of the collection tubes was heated at 95 C compared with other conditions. This result suggests that high-temperature heating in the lower part of the collection tubes damages specimen nucleic acids, leading to substantial loss of detectable nucleic acids in the inactivated samples. In addition, when the temperature of lower part of the collection tubes was <70 C, the Cq values of inactivated groups remained similar to the control (DCq <1), demonstrating the wide applicability of the quick inactivation device to achieve efficient inactivation without causing damage to pathogen nucleic acids.

The developed microfluidic nucleic acid analyzer is shown in Fig. 4 , A, and the centrifugal disk microfluidic chip is shown in Fig. 4 , B. Automated operation brought unprecedented speed; nucleic acid extraction, preamplification, and amplification took <10 min, <15 min, and <20 min, respectively, accounting for a total of <45 min in analysis. Moreover, because of the high sensitivity of the nested isothermal amplification system, preliminary tests with pseudovirus FNV-2019ncov-abEN frequently produced positive results in as short a time as 35 min. Because all of the nucleic acid amplification operations of inactivated specimens were carried out automatically inside the sealed microchip, physical isolation of operation space as in PCR laboratories was not necessary, greatly alleviating the space restriction of the mobile laboratory.

Performance of the microfluidic nucleic acid analyzer was evaluated in multiple ways. First, the linearity of fluorescent signal detection was tested with FAM (fluorescein amidite) within the concentration range of 0.0625-1.000 lmol/L, and a linearity of 0.9975 was achieved (Fig. 4, D) . Second, the sensitivity of the analyzer was evaluated as capable of detecting pseudovirus at a 100% positive detection rate with 20 samples at concentrations of 150 and 300 copies/mL, and showed 90% at the concentration of 100 copies/mL. The limit of detection of the analyzer was thus determined to be 150 copies/mL. Successful detection of both Open Reading Frame 1ab (ORF1ab) region and N gene suggests robust and unbiased testing of nucleic acids of pathogens. The typical amplification curves of pseudovirus FNV-2019-ncov-abEN at a wide range of different concentrations are demonstrated in Fig. 4 Mobile Laboratory for On-site Molecular Diagnosis interferents may exist in the collected specimen that would inhibit NATs such as those for antiviral drugs, microorganisms, and so forth. In this regard, the Tp values for the ORF1ab region and N gene of pseudovirus solutions spiked with interferents were close to those without interferents, demonstrating the high specificity of the microfluidic analyzer (see online Supplemental  Fig. S6 ). In particular, even at low concentrations of pseudovirus solutions (e.g., limit of detection of 150 copies/mL), the addition of interferents showed no impact on the detection of target pseudovirus samples. Overall, the microfluidic nucleic acid analyzer demonstrated superior sensitivity, repeatability, and robustness for pathogen NATs, and the compact size and integrated functionality facilitated contamination-free and flexible deployment with the mobile laboratory for onsite diagnostics. Table 1 , in all 10 test groups, the specific ORF1ab region and N gene of the FNV-2019-ncov-abEN virus were reported positive from specimens collected by the robot arm, consistent with the results of manually collected specimens. The mean (SD) of the Tp values of the GAPDH gene of specimens from automated and manual collections were 12.4 (1.4) min and 12.5 (1.1) min, respectively, demonstrating successful detection of human genetic materials from oropharyngeal swab specimens. The Tp values for the ORF1ab region and N gene were very close, demonstrating excellent sensitivity and unbiased detection in pathogenic diagnosis.

A summary of consistency test results of clinical samples analyzed by the microfluidic nucleic acid analyzer compared with the reference RT-qPCR method is provided in Table 2 

Infectious disease outbreaks such as the COVID-19 pandemic pose profound challenges worldwide. Limitations of available medical resources and unmet demand of local community-level NAT screening have hindered implementation of timely and effective responses to potential infectious disease outbreaks. The need for mobile laboratories capable of molecular diagnostics applies not only to COVID-19 but to a wide variety of infectious diseases such as viral hemorrhagic fevers including Ebola, Marburg, and Lassa (34) (35) (36) (37) . Virological evidence has demonstrated that a substantial viral load is present in infected individuals in the early stages of COVID-19 (38) (39) (40) ; therefore, early diagnostics and treatment are the key for outbreak control. The mobile laboratory facilitates rapid and flexible deployment of diagnostic support to disease transmission hotspots such as local communities, airports, and train stations.

The discrete modules of the mobile laboratory have demonstrated superior performance separately and as a whole by successful evaluation with pseudovirus spiked oropharyngeal swab specimens and clinical samples. The mobile laboratory minimized biohazard by using automated specimen collection and inactivation inside the biosafety cabinet and utilizing a fully integrated microfluidic nucleic acid analyzer. Consequently, physical isolation of working areas was not necessary, solving the conflict between biosafety risk management and limited working space onboard. The information platform controls experimental and laboratory information flow and enables every individual mobile laboratory to be a "mobile sentinel" in the infectious disease outbreak, continuously feeding diagnostic results to the real-time monitoring network.

Given regulatory requirements for the van-based mobile laboratory and the limited numbers of patients with COVID-19 in China, testing with clinical samples onboard is currently restricted. However, we completed evaluation of the microfluidic nucleic acid analyzer using 736 clinical samples taken from 723 patients and demonstrated good consistency with RT-qPCR (j ¼ 0.979). The high efficiency of specimen collection by the robot arm and high sensitivity of the nucleic acid analyzer facilitated highly sensitive testing of oropharyngeal swab specimens, which was demonstrated by the evaluation of combined automated sampling with nucleic acid analysis. In addition, by using the specially designed diaphragm, oropharyngeal swab specimen collection could be readily integrated with the robot arm for establishment of a stable sampling path. This approach significantly improved the safety and comfort of patients as well as the quality of the acquired specimens. 

Supplemental material is available at Clinical Chemistry online.

Nonstandard Abbreviations: COVID-19, coronavirus disease

nucleic acid-based test; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; RT-qPCR, reverse transcription quantitative PCR; LAMP, loop-mediated isothermal amplification; Tp, time to positivity

Human Gene: GAPDH, glyceraldehyde-3-phosphate dehydrogenase References

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Acknowledgments: We thank Professor Linqi Zhang at Tsinghua University for providing pseudoviruses and test facility.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.