key: cord-0908696-1qsdhlt2
authors: Mishra, Deepak Kumar; Goel, Gaurav; Arora, Neeraj; Krishnan, Shekhar; Bhattacharya, Sanjay; Mathur, Purva; Walia, Kamini; Chandy, Mammen
title: The Importance of Intra- and Inter-Institutional Networks for Capacity Building in Severe Acute Respiratory Syndrome Coronavirus 2 Reverse Transcription Polymerase Chain Reaction Services: Experience from an Oncology Centre in Eastern India
date: 2020-07-31
journal: Indian Journal of Medical Microbiology
DOI: 10.4103/ijmm.ijmm_20_255
sha: f42ccc92de1099ef031e52d70711b2a210f44e9c
doc_id: 908696
cord_uid: 1qsdhlt2

Abstract High-throughput, accurate, cost-effective and rapid testing for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is the need of the hour in face of the global coronavirus disease pandemic. This target is achievable, within a relatively short time through capacity building of reverse transcription polymerase chain reaction (RT-PCR) tests by utilising the strengths of intra and inter institutional networks. These networks act as force multiplier for vital resources which are required for capacity building, namely, leadership, expertise, equipment, space, infection control inputs and human resources. In this article, we report the experience of capacity building for delivery of RT-PCR tests for SARS CoV-2 from a cancer hospital in Eastern India. The relevance, mode of operation and value addition of this essential public health service are discussed in the context of inter departmental collaboration and interaction with other institutes through the existing diagnostic, surveillance and infection control networks. This networking model for service development and delivery could be used by other centres.

transcription polymerase chain reaction (RT-PCR)-based testing for SARS-CoV-2. Measures were taken later to increase the number of testing laboratories within India in view of its large population and wide geographic expanse. As of 19 May 2020, 555 operational laboratories were reporting to the Indian Council of Medical Research (ICMR) on testing for SARS-CoV-2, including 391 government laboratories and 164 private laboratories. This included conventional RT-PCR testing in 431 laboratories, (government, 293; private, 138), the True Natmicro RT-PCR test system in 77 laboratories (government, 73; private 4) and the cartridge based nucleic acid amplification test (CBNAAT) system in 47 laboratories (government, 25; private 22). As of 18 May 2020, the government of India reported that 108,233 samples were tested for SARS-CoV-2. [1] [2] [3] [4] the requIrement and challengeS oF capacIty buIldIng For Severe acute reSpIratory Syndrome coronavIruS 2 teStIng

The COVID-19 pandemic has resulted in an unprecedented need for large-scale testing for SARS-CoV-2 infection. A number of laboratories have been identified by the government in both the public and private sector to manage this diagnostic challenge. There is an urgent need for capacity building in both sectors for the testing and timely reporting of results. In this context, a European consortium reported in January 2020 that the common problems associated with capacity building with regard to SARS-CoV-2 molecular testing included lack of adequate positive controls; lack of personnel trained in molecular biology, lack of primers/ probes; lack of appropriate quality control panels to test specificity of primers/probes against other corona viruses; difficulty in implementing standards as per ISO15189; lack of adequate number of commercial tests and difficulty in their procurement; lack of adequate training; equipment shortage and inadequacy of adequate level of biosafety laboratories. [5] In the case of RT-PCR testing, Corman et al. discussed candidate SARS-CoV-2 gene targets, including genes encoding the envelope (E), membrane (M), nucleocapsid (N), open reading frame, RNA-dependent RNA polymerase (RdRp) and spike (S) proteins. [6] With RT-PCR testing, a challenge highlighted by Konrad et al. is the uncertain significance of amplification curves around the cycle threshold of 40. [7] RT-PCR on nose and throat swab samples is not the only method for detection of SARS-CoV-2 infections. As reported by Carter et al., diagnostic approaches for COVID-19 include (a) integration of computed tomography imaging of chest with results of RT-PCR testing (b) rapid IgM-IgG combined antibody testing for SARS-CoV-2 (c) diagnosis from faecal specimens in patients with the characteristic respiratory syndrome (d) digital droplet PCR for more sensitive and accurate detection of SARS-CoV-2 in low viral load specimens (e) rapid SARS-CoV-2 detection using a CRISPR-Cas12-based lateral flow assay. [8] Peter et al. had reported previously from Botswana and Ethiopia along with the US Centers for Disease Control (CDC) the importance of capacity building to combat Ebola outbreaks in Africa. [9] Similar were the findings of Phommasack et al. from Laos in the case of influenza. [10] The African Field Epidemiology Network demonstrated similarly the importance of networking in building laboratory capacity among resource-poor African countries. [11] In a detailed analysis, Gupta et al. from the ICMR elaborated on various methods for laboratory capacity building with India for COVID. These include (a) increase in working hours, (b) redeployment of RT-PCR machines in research institutes for COVID-19 diagnostic work, (c) use of RT-PCR machines from the National AIDS Control Organization centres, (d) redeployment of nucleic acid amplification testing (NAAT) equipment of the National Tuberculosis Elimination Programs, (e) procurement of automated high-throughput RT-PCR testing platforms and nucleic acid extraction systems and (f) using a combination of testing kits. [12] Similarly, Mourya et al. in another publication from ICMR detailed the various basic biosafety requirements for the laboratories. [13] capacIty buIldIng meaSureS For reverSe tranScrIptIon polymeraSe chaIn reactIon teStIng For Severe acute reSpIratory Syndrome coronavIruS 2 In IndIa For each laboratory, the average number of samples that require testing may vary from a handful to 1000 each day. [2, 4] At the beginning of the COVID-19 pandemic, clinical samples in each state were sent to designated Government laboratories/ hospitals with RT-PCR testing capability. Soon, it become clear that centralised testing at a state/district level is not feasible on a large scale, given the size of the Indian population, its density in select areas, the wide geographical spread and the related logistical challenges. Thus, both the Central and State governments have enabled additional laboratories in both the public and private sectors to perform RT-PCR based testing for SARS-CoV-2.

This in turn raises the question of total quality management of SARS-CoV-2 testing facilities [ Figure 1 ]. In the case of private laboratories, the essential requirement to carry out viral RT-PCR testing is to possess accreditation for such testing with the National Accreditation Board for Testing and Calibration Laboratories. [3] This however is not a mandatory requirement for government laboratories. In Figures 2 and 3 we have depicted the number of samples tested at Tata Medical Center, Kolkata, and in the state of West Bengal [ Figure 3 ] while capacity building was in progress. Additionally, capacity building for SARS-CoV-2 testing involves developing and sustaining facilities in the following sectors: [1] [2] [3] 1. Sample collection, transport, packaging 2. Molecular Laboratory set up 3. Equipment 4. Test kits and reagents 5. Storage facility for samples, kits and consumables 6. Human resources 7. Data entry and information technology (IT) infrastructure 8. Total quality management 9. Administration, management and leadership.

Essential components to deliver and expand the capacity for sample collection include well-trained staff, robust systems for sample identification including use of a standardised sample requisition form and the ICMR-approved RT-PCR app developed by the National Informatics Centre and using designated areas for sample collection to ensure safe and efficient collection of respiratory samples. Adequate supply of viral transport medium (VTM) and appropriate swabs to manage the expected sampling load, along with logistical planning for the labelling, packaging, storage and transport of the collected samples is critical. Multiple collection points, including field-based collection using the RT-PCR app will help in enhancing sample collection capacity.

The minimum requirement to initiate molecular biology-based diagnostics for SARS-CoV-2 testing is a biosafety cabinet class II type A2 and PCR machines. The lab outlay and the type of PCR machine will depend on the chosen methodology of testing. In the case of conventional RT-PCR, organisation of the laboratory will involve having separate areas for RNA extraction, PCR master mix preparation, PCR set-up and nucleic acid amplification.

CBNAAT for SARS-CoV-2 may be carried usingCepheid's GeneXpert, Qiagen's QIAstat-Dx and Biomeriéux's Biofire FilmArray systems. CBNAAT systems have minimal footprints, requiring only a table-top to accommodate the instrument and the accompanying laptop. These systems will need to be positioned adjacent to a BSC-IIA2 and require suitable electrical supply. By contrast, conventional RT-PCR-based diagnostics requires at least 3 separate work areas, one each for nucleic acid extraction, PCR master mix preparation and viral nucleic acid template addition. At least one BSC-IIA2 and a refrigerated centrifuge is required in the nucleic acid extraction room, while a dead air box and/or laminar flow box may be sufficient in the other 2 rooms. The choice between CBNAAT and conventional RT-PCR testing is usually based on the test needs of the centre. CBNAAT will obviously need fewer personnel, occupies less space and is less labour-intensive, but carries the major disadvantage of low throughput. When large-scale or field testing of samples is the need of the day, testing by conventional RT-PCR is the more appropriate option.

The list of equipment for conventional RT-PCR is extensive and includes: 1. BSC-IIA2, dead air boxes and laminar flow cabinets for sampling handling . For instance, the Rotor Gene Q peak ramp rates, in air, >15ºC/s heating, >20ºC/s cooling The Quant Studio 3 has a ramp rate between 6.5 to 9ºC/s depending on the reaction volume. Other approaches toenhanceinstrumentcapacity is to use automated nucleic acid extraction machines with batch systems and use of liquid handling systems for automated mixing of template and PCR master mix (e.g., Qiagen's QIAgility).

Selection of appropriate systems, equipment and processes is critical for capacity building. This may be done using checklists so that essential points for consideration are not missed during system selection [ Tables 1-3] . Some examples are as follows:

Checklist for the selection of an automated nucleic acid extraction system Checklist for the selection of a reverse transcription polymerase chain reaction system 1. Open or closed system (CBNAAT is a closed system) 2. Random access or batched testing (CBNAAT is a random-access system) 3. Reaction volume range (this will determine size of PCR tubes) 4. Number and type of optical filters (this will determine compatibility with probe and other dyes used in RT-PCR) 5. Ramp rate (this will determine run time) 6. Heat flux (Peltier heat block or convection heating) 7. Throughput (number of reactions per run) 8. Run time (time required for PCR)-determined by ramp rate, assay type 9. IT Software and hardware support-program and laptop 10. Cost and type of PCR plastic consumables required.

Checklist for the selection of an reverse transcription polymerase chain reaction kit for severe acute respiratory syndrome coronavirus 2 detection 1. Open versus closed system kits (CBNAAT cartridges are closed system kits) 2. Cost per reaction and Cost per sample tested 3. SARS-CoV-2 genes detected: E gene/S gene/RDRP gene 4. TaqMan versus SYBR chemistry for detection of PCR products and the availability of suitable optical filters for fluorescence detection in the RT-PCR instrument 5. Multiplex, duplex or monoplex format of the PCR (E gene only in a reaction tube; E gene plus internal control in a reaction tube; E gene, S gene and internal control in a reaction tube) 6. Endogenous or exogenous internal control 

A key bottleneck regarding molecular diagnostics is the availability and access to indigenous PCR kits and technologies. Currently, Germany and the USA are the leading producers of PCR equipment and reagents. Although domestic kits such as TRUPCR have been marketed by Blackbio, the number of such manufacturers are limited, leading to a wide gap between supply and local demand. Furthermore, critical consumables such as personal protective equipment (PPE), PCR plastics (e.g., pipette tips, columns, microtubes) and VTM may not meet expected quality requirements due to poor manufacturing and quality assurance standards. Therefore, our main goal through capacity building in kits and reagents should be to develop sufficient inventory via imports and stockpiling and expanding local manufacturing of essential consumables to deliver high-quality diagnostic kits in adequate quantity.

Human resource requirements in a molecular biology lab will also depend on the test systems in place. CBNAAT systems are less demanding and require fewer skilled staff. Automation of sections of the conventional RT-PCR process will also reduce the staff numbers. Use of conventional manual intensive systems requires the maximum number of trained personnel. Hence, a good understanding of the lab workflow and systems is vital for staff planning. All staff must be trained in Good Laboratory Practice (GLP) and laboratory safety including biosafety, while prior specific training in molecular techniques would be desirable. To augment staff capacity, regular training modules in GLP, molecular diagnostics and biomedical waste management, coupled with periodic assessments are necessary. Suitable staff is required in all components of testing from sample collection, through nucleic acid extraction, PCR testing and data management. As example, personnel specification for RT-PCR testing will include: 1. Good knowledge and understanding on:

• GLP • Biosafety • Molecular biology.

• Nucleic acid extraction • PCR Master mix preparation and PCR set up • Post-PCR analysis.

• Rigorous documentation of experimental work, ability to work as part of a team, readiness to work flexibly, equipped with troubleshooting and problem-solving abilities.

Increasing storage capacity in lab needs sufficient space for two types of storage: 1. Dry storage for PCR plastics, extraction kits, PPE, unused VTMs and the paperwork related to testing (sample requisition form, PCR worksheets etc.) 2. Cold storage for samples, templates and reagents.

Short-term storage of samples at 2°C-8°C in a standard refrigerator is feasible while long-term storage of samples and viral nucleic acid templates require a -80°C freezer. Test reagents require -20°C for optimal storage. Large-volume short-term storage may be done using walk-in coolers, thus increasing the available space for cold storage. However, indexing and cataloguing of samples, reagents and consumables is vital to ensure efficient storage and retrieval of material when needed. Therefore, proper labelling and archiving (using paper or electronic means) is paramount to ensure efficient storage and retrieval capacity. 

Building up capacity in the earlier sections is impractical without having an efficient and reliable system for sharing results and notifying the authorities. App-based platforms such as RT-PCR App from ICMR make this accessible and easy. This may be enhanced by linking the app to Lab Information Systems that are integrated with the lab instruments. [14] Importance oF qualIty control In capacIty buIldIng

The importance of quality control in capacity building can hardly be overemphasised. For example, human resource capacity requires careful selection of personnel, training, competence assessment and periodic appraisals. Biomedical equipment used for sample storage, nucleic acid extraction and PCR require regular preventive maintenance, calibration, maintenance contracts (annual or comprehensive). Kits and reagents need to be checked through daily internal quality controls and assessments of precision accuracy. New batches of test kits will need to be reassessed for performance. All pre-analytical factors with regard to sample collection transport and storage will need to be standardised and optimised. Finally, all laboratory techniques will need to be linked to external quality assurance programs.

Leadership, administrative and managerial support at various levels (test registration, logistics, inventory management, laboratory administration, quality control, biomedical engineering and IT) is critical for capacity building. Effective coordination between various sections is important. Administrative decisions on the suitable laboratory techniques and processes that best meet requirements for throughput, efficiency and cost are crucial for long-term service viability. These decisions will reflect on other variables such as consumables and reagents inventory, staff and equipment requirements. An effective leader is not just a mentor and a visionary but also a manager of man, machine material, method, money, time. Administrative action is critical for capacity building. The medical and laboratory administration decides what is to be done and when it is to be done, whereas the institutional management decides how best to implement the administrative decisions. Administrators have a thinking function, whereas managers have an operational function. The leadership asks questions and mentors provide strategic directions and address roadblocks in implementation. Coordination and integration is required at various levels and sectors: (a) Vertical integration: Establishing synergy within defined areas (e.g., sample collection; inventory management; maintaining IT networks); (b) Horizontal integration: establishing synergy across several functional domains e.g., (sample collection, nucleic acid extraction, RT-PCR, test result reporting); (c) Resource and performance management through management principles such as Lean and Six Sigma which aims at waste elimination and speed (the focus of Lean); customer satisfaction and accuracy (the focus of Six Sigma whose methodology involves Defining, Measuring, Analyzing, Improving and Controlling). [15] [16] [17] 

Exaggerated fear of acquiring COVID-19 at the workplace can be a significant hurdle in the path to build capacity. Danger is a real phenomenon, whereas; fear is a psychological phenomenon which may be unrelated to the degree of risk. In order to build capacity for testing SARS-CoV-2 addressing staff concerns about contracting infection while working in diagnostic laboratories or in the clinical areas is important. This can be achieved through induction programs, periodic and regular training, staff briefing about best practices, monitoring biosafety and infection trends and making available adequate PPE for all levels of laboratory operations. In select cases, staff may be referred to a psychologist or provided professional counselling about risks, infection prevention measures and risk management.

Besides access to tangible resources, capacity building requires the development of networks-both intra-and inter-institutional. The cumulative value addition generated by the network is often greater than the sum of its constituent parts [ Table 4 ]. In 2011, ICMR invested in creating a Viral Research and Diagnostics Lab network. [18] This network was instrumental in providing initial COVID testing support in the country and later worked with the other labs in the country to bring them to speed. The mutually complementary data, information, knowledge, skills, mentorship, leadership and wisdom offered by network members often creates innovative approaches for addressing challenges and a better appreciation of the bigger picture. It is critical in this context of interdependence that we support and reinforce the existing laboratory and infection prevention networks within India. [19, 20] The existing network of hospitals working on Infection Control and Healthcare Associated Infection (HCAI) surveillance under the aegis of ICMR-AIIMS and CDC are facilitating trainings of several hospitals. Through these weekly trainings, the hospitals are remotely trained on COVID-related infection control measures, safe handling of specimens and other frequently asked questions (FAQs) [ Table 5 ]. [21] As resources get prioritised to meet the challenges of pandemic microbiology it is also important that COVD diagnosis capacity building should not compromise the diagnostic systems in bacterial and fungal infections for COVID or non COVID patients. India faces formidable challenges and remarkable opportunities in the domains of Antimicrobial Resistance, Infection Prevention and Control (IPC), Antimicrobial Stewardship Program (AMSP) and Diagnostic Stewardship. The solution requires an integrated approach. If laboratory diagnosis of infections, IPC, non-antibiotic AMSP and non-bacterial or non-infective diagnostic stewardship works together-the problem of multidrug-resistant bacteria and drug resistant fungi can be handled more effectively. This would require capacity building in viral diagnostics, similar to capacity building in fungal diagnostics, HCAI surveillance and antifungal stewardship. Viral pandemic management also benefit from capacity building in bacterial diagnostics and IPC. It may be more resource intensive in the short term, but an integrated approach eventually is likely to be more rewarding and efficient by reducing redundancy of precious resources.

Creation of sustainable laboratory networks within a country as vast and varied as India takes time, resources and persistence besides a sustained degree of leadership commitment. The COVID-19 pandemic and its ripple effect within India teaches us how existing networks created by ICMR with the help of public and private sector partnership can be put to good use during times of national, regional and international health emergencies. With the SARS-CoV-2 RT-PCR capacity building exercise within India, we are probably steps closer to realising the larger vision of establishing an integrated high-quality molecular microbiology service, research and surveillance network within the country.

Nil.

There are no conflicts of interest. Table 5 : Frequently asked questions Is a BSL-3 lab required for SARS CoV-2 RT-PCR?

No: A BSL-2 lab with a certified biosafety cabinet of IIA2 variety is sufficient BSL-3 labs are essential if viral culture work is done. None of the labs should attempt viral culture (ICMR guideline) Can lower respiratory samples be used for SARS CoV-2 RT-PCR?

Yes: Sputum or BAL or endotracheal secretion may be used For patients with a productive cough, sputum should be collected. The induction of sputum is not recommended Where should specimens be collected?

Specimen collection should be performed in a single patient room with the door closed What are the Aerosol generating procedures in the laboratory?

These include, but are not limited to: Pipetting, sonication, vortexing, centrifugation, preparation of smear, streak inoculation of culture, inserting hot loop into a culture How to prevent aerosol generation and lab staff exposure?

All manipulations should be performed in appropriately maintained and validated biosafety cabinet Laboratory personnel performing procedures must demonstrate capability While centrifuging Use sealed safety cups and sealed rotors Open cups inside a biosafety cabinet Allow cups to sit prior to opening to allow aerosols to settle if no biosafety cabinet available What disinfectants can be used on smart phones?

Lab staff should avoid using mobile phones during lab work. If used an alcohol wipe with 70% isopropyl can be used by gently wiping the exterior surfaces of the phone SARS CoV-2: Severe acute respiratory syndrome coronavirus 2, RT-PCR: Reverse transcriptase-polymerase chain reaction, ICMR: Indian Council of Medical Research, BSL: Bio-Safety Level

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