Abstract
Background
Tests for diagnosing infectious diseases (ID-POCT) play a special role among the available point-of-care testing (POCT) methods.
Methods
A systematic literature search was performed in PubMed. Based on this literature review and our own experience, aspects associated with using molecular biological methods in the diagnostic amplification of pathogen DNA/RNA (nucleic acid testing = NAT) and/or signal amplification were discussed in an interdisciplinary manner. This resulted in the following recommendations for the near-patient use of NAT methods.
Results
Due to the current rise in the use of near-patient NAT testing (e.g., using disposable cartridges), recommendations are made for their implementation and appropriate clinical use in the hospital setting. Particular emphasis is placed on the analytical quality of the results. Furthermore, internal best-practice rules and selection criteria are proposed to ensure rapid diagnosis. Equally important are a medically valid interpretation of results and compliance with hygiene requirements. These recommendations emphasize that near-patient NAT should always be procured in conjunction with a (preferably) multidisciplinary institution responsible for POCT and knowledge of the test specifications and risks, as well as quality assurance need to be in place before they are carried out.
Conclusions
These recommendations are intended to improve patient safety and to avoid economically questionable expenditures.
Introduction
The timeliness of the following recommendations is reflected by the events of the 2020 SARS-CoV-2 pandemic. Specific RNA amplification protocols for the detection of the novel SARS-CoV-2 coronavirus have been in place since January 2020, initially as laboratory developed tests (LDT) and now widely as CE-certified test systems. Compact, automated nucleic acid testing (NAT) systems have also been available since April 2020 for near-patient laboratory testing. The reaction sequences take place as isothermal processes or in accordance with the PCR principle and include extraction, reverse transcription of the viral RNA, amplification and detection [1].
In response to these developments, the POCT Section of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL), in collaboration with the German Society for Hygiene and Microbiology (DGHM), the Society for Virology (GfV), the German Association for the Control of Viral Diseases (VVK), and the Professional Association of Physicians of Microbiology, Virology and Infection Epidemiology (BÄMI e.V.), has developed the following recommendations for implementing near-patient analysis systems for the molecular detection of infectious agents in the clinical setting.
Definition of point-of-care testing
According to the guideline of the German Medical Association (Rili-BÄK) [2], point-of-care testing (POCT) is defined as medical laboratory testing that is performed directly on individual (not serial) samples without sample preparation (volume dosing is permitted). Another important criterion is the ability to derive further diagnostic or therapeutic measures from the test. Rili-BÄK stipulates simplified quality assurance for the utilization of unit-use reagents that are used for and consumed after one test.
Additional characteristics of POCT are listed in Table 1.
Additional characteristics of point-of-care testing (POCT).
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Tests for identifying infectious agents (infectious disease POCT = ID-POCT) play a special role among the near-patient laboratory tests currently on the market. While most systems are based on the immunochromatographic detection of a specific microbial antigen (lateral flow immunoassay = LFIA), a variety of molecular biological methods have been available for some time that amplify the pathogen DNA/RNA. Recommendations for the appropriate implementation and use of these procedures will be made here for hospitals since these point-of-care NAT methods are primarily being used in this setting and because currently only a handful of regulations are in place for comprehensive quality management of NAT conducted at the point of care.
Clinical use of near-patient NAT methods
POCT – including the ID-POCT methods – is performed in almost all German hospitals. Over time, the Rili-BÄK guideline [2] has placed the responsibility for the quality assurance of near-patient testing on central laboratories. However, quality assurance needs to be further developed in light of the advancements, especially in molecular testing for infectious diseases.
Because medical laboratory testing is handled very differently by different hospitals, the authors have arrived at the following recommendations, which are listed in Table 2.
Recommendations for the appropriate use of near-patient nucleic acid testing (NAT).
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These recommendations were formulated jointly by the professional societies and aim to ensure that near-patient NAT testing is only procured in conjunction with the multidisciplinary unit responsible for POCT processes with access to a functioning Laboratory Information System/Hospital Information System (LIS/HIS) network. In-depth knowledge of the test specifications and risks is required, and quality assurance should be in place. Another aim is to ensure that the risk-based quality assurance introduced by the current Rili-BÄK guideline is implemented comprehensively in the interest of patient safety.
The coordination unit defines the test specifications, monitors equipment maintenance and performance, and assures the quality of the analysis. It also monitors the workplace-related hygiene requirements and produces medically sound findings from reports sent via the LIS/HIS network. Additional equipment and test data are taken into account when producing the findings (e.g., Cp/Cq/Ct values, curve progression).
Treatment decisions can be made directly at the hospital after interpretation of the NAT results. In addition, hospital hygiene measures, such as isolation measures, can be derived immediately on the basis of the results. The coordination unit also fulfills the obligation to report in accordance with Section 7 of the German Protection Against Infection Act (obligation of laboratories to report). Another important task is the continuous training (in particular with regard to preanalytical issues) and certification of the clinical staff involved in point-of-care laboratory analyses.
In Germany, the requirements of the Rili-BÄK guideline are binding for laboratory testing and have a legal character. In terms of certification, quality assurance of decentralized POCT systems falls under the provisions of DIN EN ISO 22870:2017-04. In contrast, DIN EN ISO 15189:2014 is applied to ID-POCT support, provided by the microbiology/virology lab under its sole responsibility.
In the past, LFIA showed a limited analytical sensitivity for detecting bacterial, viral, mycological, or parasitic antigens [3]. This led to the recent development of highly sensitive molecular genetic assays. POCT-NAT methods were also developed with the aim of speeding up diagnosis in comparison to traditional microbiological/virological analysis methods. This improves treatment options and enables a more rapid and targeted use of anti-infectives. Devices with PCR cartridges have been developed that allow rapid, contamination-free, single-test analysis performed by appropriately trained personnel. Ready-to-use cartridges pre-filled with reagents – some in blister packs – are used for this purpose. Here all PCR process steps (sample digestion with nucleic acid release, amplification and detection) are fully mechanized. A large selection of such NAT cartridge devices, including multiplex PCR systems, are available on the In-Vitro Diagnostics (IVD) market.
Unlike the above-mentioned immunological test systems, for the majority of NAT methods, the test samples (e.g., swabs with liquid medium) must be placed or filled into a disposable cartridge either immediately or after mixing them with elution solution. This is then inserted into the NAT system which performs the sample preparation process. This preanalytical step is critical as errors can occur at this point. The type and quantity of the sample material used in the analysis must be selected according to the manufacturer’s specifications. The swab and transport systems (e.g., swabs and liquid media) must also be compatible with the device being used. It should be noted that NAT pathogen detection only makes sense when the sample material is highly likely to contain the pathogen in detectable quantities and is validated accordingly. Special attention may need to be paid to ensuring that samples are taken correctly from the right location (e.g., deep nasopharyngeal swab) to avoid deficient samples that could lead to false analytical results. In this case, the personnel taking the sample and the personnel using POCT-NAT must undergo intensive training in order to ensure that a preanalytical control of the sample material, which is otherwise carried out in the microbiology/virology lab, is also performed as part of routine point-of-care testing.
Some newer systems enable several pathogens to be detected at once in a single assay run (so-called multiplex PCR). Such an approach provides rapid and sensitive pathogen detection when there are symptoms that can be assigned to several pathogens. The panels of various devices for “syndromic testing” allow for the detection of a wide range of pathogens. These approaches should only be used if there is a strong clinical indication. In addition to the costs, the multiplex approach often requires a higher level of interpretation when evaluating results and knowledge is needed of the detection limits for the pathogen composition.
The assurance of high-quality results is important for POCT-NAT methods that use closed (e.g., cartridge) systems. In Germany, this has been incorporated in the latest version of the Rili-BÄK guideline published in 2019 [2]. More specifically, and as explicitly defined in this guideline, these cartridges already contain all the necessary reagents and no further reagents or patient material are required and/or can penetrate the closed cartridge once the patient material has been manually or automatically added. This significantly minimizes the risk of contamination. It is also not possible to open the system, preventing the release of nucleic acid amplificates.
Implementing near-patient NAT testing in a hospital setting
There are undeniable advantages of using POCT in hospitals. These include the elimination of long transport times for samples and a rapid availability of the results which could have immediate treatment implications for the patient. Studies have shown [4] that POCT reduces the length of patient stays in emergency rooms. However, this time savings is not always accompanied by medically verifiable benefits [5]. An ill-considered and hasty introduction of such tests, especially NAT, can lead to diagnostic uncertainties and the inability to conduct follow-up tests using the same sample material. Nevertheless, there is an increasing demand for the clinical use of such analytical systems in the hospital sector. For example, NAT testing for influenza A, B and Respiratory Syncytial Virus (RSV) is already being performed in some large emergency rooms. In addition to obtaining fast results, it has resulted in a reduction in “bed blocking” which has proven beneficial. Thus, the near-patient use of NAT to detect the influenza virus can also be cost-effective [6]. The same applies to the NAT-based detection of SARS-CoV-2 RNA.
Quality assurance – new Rili-BÄK requirements for NAT testing using closed test systems (e.g., disposable cartridges)
The latest version of the Rili-BÄK guideline from December 2019 [2] has defined in Chapter 2.1.2.3. the measures for implementing quality assurance for closed, fully mechanized molecular genetic test systems (e.g., cartridge systems) to detect pathogen-specific DNA/RNA. In addition, internal quality assurance for molecular biological procedures is listed in Tables B 3-1 and B 3-1a. Point 5 of this chapter is particularly relevant for near-patient NAT, an excerpt of which is presented below:
Quotation: (5) For closed test systems (e.g., cartridge systems) for the qualitative or quantitative detection of pathogen-specific nucleic acid, there is no need for additional control samples for each test procedure if sufficient procedural controls are in place to ensure reagent functionality, including extraction, purification, amplification and inhibition.
In the absence of a manufacturer’s specification or if no positive and/or no negative control sample is deemed necessary by the manufacturer, the laboratory must substantiate the frequency of positive and/or negative control samples by means of specifications adapted to the respective procedure (risk-based quality assurance). The frequency and results of these control sample measurements must be documented.
Internal standards for best practice
When implementing near-patient NAT testing in hospitals, various preparatory measures must be made on site by the clinical departments involved (e.g., emergency department) in close cooperation with the team of microbiology/virology experts in the unit responsible for POCT (often also referred to as the POCT coordination unit, see also “Clinical use”). Primary aspects include the selection of a suitable system, training of the primary clinical staff in the correct indications, preanalytical steps and the conducting of the testing, a suitable IT connection to the LIS, a clinical validation of the results, correct interpretation of the results, a comprehensive cost/benefit analysis and adequate therapy consultation. In addition, quality assurance measures (internal and external quality controls) should be in place alongside algorithms for mandatory reporting in accordance with the German Protection Against Infection Act (IfSG) and/or internal reporting to those responsible for hospital hygiene. Particular attention should be paid to the correct use of point-of-care NAT. When indications are too broadly defined, the tests are often used incorrectly, resulting in unnecessary costs. On the other hand, well defined indications improve process flows, which can lead to corresponding cost savings.
Selection criteria
The NAT systems should be selected based on the conditions of the respective clinical setting. This assessment must be carried out by persons with microbiology/virology expertise together with the unit responsible for the POCT processes (see section “Clinical use”). In addition to the analytical aspects, requirements for the preanalytical process must also be considered. Table 3 lists potential selection criteria that should be taken into account and reviewed prior to performing point-of-care NAT (modified from van der Eijk et al. [7]).
Selection criteria for using near-patient nucleic acid testing (NAT).
| NAT technology: extent to which the technology |
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| Implementation: checking whether |
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| Use and alternatives: checking and/or estimating |
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| Operation: clarification and/or taking into account |
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| Strategy: clarifying how the new NAT service contributes to |
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The test method needs to be evaluated based on the conditions present in the respective facility. It is important to take into account the patient population in the facility (proportion of patients primarily receiving emergency care, proportion of pediatric or geriatric patients, ratio of surgical to non-surgical cases, proportion of intensive care patients, etc.). The specificity, sensitivity as well as the positive and negative predictive values of the assay must be evaluated in detail as information has been published on false-positive and false-negative results of some commercially available POCT systems for various forms of pathogen detection [8], [9].
Preanalytical requirements
As with any other laboratory procedure, including microbiological procedures, conducting proper preanalytics plays a decisive role in diagnostic testing. Four important aspects of this are explained in Table 4.
Preanalytical aspects of near-patient nucleic acid testing (NAT).
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Interpreting results
The issuance and interpretation of the results should be clear and unambiguous, i.e., without the need for further interpretation, e.g., “pathogen detected/not detected” or “mutation present/not present”. Depending on the selected POCT approach, once the POCT-NAT results have been transferred to the LIS, they are verified for the final written report using microbiology/virology expertise and, if necessary, interpreted further. This ensures that immediate decisions can be made at the point of care based on the results. The findings are prepared by a competent party and, if necessary, reported to the health authorities in line with the IfSG. These aspects are covered in detail in the document “MM03: Molecular Diagnostic Methods for Infectious Diseases” published by the Clinical & Laboratory Standards Institute (CLSI) [10] (Table 5).
Issuance and interpretation of results as per the Clinical & Laboratory Standards Institute (CLSI) document [10].
| Reporting results | NAT results should be primarily communicated to the clinician as “positive”, “negative”, “ambiguous”, or “uninterpretable” – with further interpretation as needed. |
| Critical results | Critical results should be defined for tests that significantly influence treatment decisions. The attending physician is to be informed immediately. Users of point-of-care NAT should understand the significance of critical results for the test formats they perform themselves. |
| Notification of clinically significant test limitations | This should only be briefly stated in the report to avoid distracting from the primary finding. Examples include cross-reactions, systematic errors caused by genome changes in the pathogen, and the presence of interfering substances. In some cases, these limitations are already stated in the test manufacturer’s package insert. The use of laboratory developed tests and the use of NAT methods for other types of sample material or for previously undefined purposes (intended use) is only permitted as an exception. |
Hygiene requirements
Hygiene aspects must also be taken into account. As part of a risk assessment conducted in accordance with Section 4 of the Ordinance on Biological Substances (BioStoffV) and Section 3 of the German Labor Protection Act (ArbStättV), users of POCT-NAT must take suitable protective measures in line with the current hygiene plan before working with the potentially infectious (swab) materials and, if necessary, develop operating instructions. Particular attention must be paid to hand hygiene, personal protective equipment, disinfection of surfaces and waste treatment. The relevant regulations (e.g., TRBA 100 and 250, and the specifications of the Committee on Biological Agents [ABAS]) must be observed [11], [12].
Surfaces on which NAT is performed may be contaminated with pathogens or, even after proper disinfection/decontamination, with their nucleic acids. Therefore, in order to control infection and to avoid cross-contamination, the basic rules listed in Table 6 must be observed.
Hygiene requirements – basic rules.
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When performing near-patient NAT to detect pathogens, aerosols can be generated during various steps, e.g., when opening sealed lids on sample containers, mixing samples, and when pipetting and emptying syringes and sample containers. Therefore, depending on the NAT method, a microbiological safety workbench is required. If this is not possible due to space limitations, personal protective equipment (PPE) in accordance with TRBA 100 must be worn when pipetting the sample into the test cartridge (e.g., safety goggles, protective gown, mouth/nose protection/FFP mask) depending on the pathogen to be detected. The PPE must be disposed of properly afterwards. Further precautions must be taken where necessary (e.g., when testing for SARS-CoV-2 [12]). The area around the testing device must be decontaminated regularly with a suitable surface disinfectant.
Further consequences
Further clinical and molecular biological consequences are summarized in Table 7.
Clinical management and further tasks of the unit responsible for point-of-care testing (POCT).
| Clinical management of the NAT results is supported by intensive collaboration between the clinician and the unit responsible for POCT. |
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| Additional tasks as part of POCT molecular genetic testing: |
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Examples of successful realization of POCT-NAT include the implementation of rapid influenza or SARS-CoV-2 NAT in the emergency rooms of university hospitals in Germany. Here, collaboration with microbiology/virology departments has resulted in the quality control of testing through the networking of POCT devices, fast support in the event of technical problems, and, in selected cases, confirmatory and comparative testing at the virology laboratories [13].
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Research funding: None declared.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Professor Luppa has received consulting fees from the company Lumira DX, UK. He has been paid a speaking fee by the companies Werfen and Nova Biomedical. He has received re-search support (indirectly to the employer) from GNA Biosolutions, Martinsried. Professor Rabenau declares no conflict of interest. Dr. Huzly declares no conflict of interest. Professor Nauck declares no conflict of interest. Dr. Schoerner declares no conflict of interest. Professor Zeichhardt is the owner and CEO of IQVD GmbH – Institut für Qualitätssicherung in der Virusdiagnostik, Berlin, as well as a shareholder and CEO of GBD – Gesellschaft für Biotechnologische Diagnostik mbH, Berlin; with no influence on this article.
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Informed consent: Not applicable.
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Ethical approval: Not applicable.
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© 2021 Peter B. Luppa et al., published by De Gruyter, Berlin/Boston
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