Novel lab-on-a-disk platforms: a powerful tool for molecular fingerprinting of oral and respiratory tract infections

1. Introduction

Respiratory tract infections (RTIs) are communicable, bacteria- or virus-borne diseases [1] which, especially in acute phase, can progress rapidly and lead to hospitalizations, complications or even death, while some of them lead to epidemic outbreaks. In any case, they require urgent handling. Oral infections, in particular referring to the bacteria-borne caries and periodontitis, are non-communicable diseases. Caries affects the hard tissue of the teeth causing tooth decay, while periodontitis affects the tissues that surround and support the teeth (gums), leading to tooth loss [2]. Both caries and periodontitis are treatable, though not life-threatening to the degree of RTIs. However, especially periodontitis has been shown to be related to systemic disorders such as diabetes and cardiovascular diseases [3].

2. Translation of medical needs into engineering innovations: the lab-on-a-disk platform

At a first glance, there is not much in common between oral diseases and RTIs. However, they do share fundamental unmet medical needs (Table 1), some of them being likely applicable to other infectious disease areas too, and the question is: can these needs be addressed for both examined disease areas by diagnostic tools? To answer this question, we refer to a novel lab-on-a-disk microfluidic platform as, among other similar/competitive technologies that are reported [4], it has demonstrated to cover the needs outlined in Table 1 by means of: (i) Point-of-Care (POC) compatibility, (ii) capability to detect multiple nucleic acid (NA) targets and protein biomarkers on the same instrument, (iii) interfaceability with clinical algorithms and (iv) all the above, for both RTIs and oral diseases [5]. Figure 1 shows the whole value chain of processes and components for a diagnostic workflow, elaborated in the following sections.

Figure 1. Value chain of components and processes that are common in the diagnostic workflows of oral and respiratory tract infections based on the lab-on-a-disk platform. (Figure reprinted from [5], Copyright (2016), with permission from IOS press. The publication is available at IOS press through http://dx.doi.org/10.3233/978-1-61499-653-8-61)

Table 1. Converging unmet medical needs between oral diseases and RTIs

Challenges	Features of lab-on-a-disk platforms that can assist to overcome the challenges
Both, RTIs and oral diseases are a heavy burden to health systems and especially at hospitals/clinics [6,7]	Decentralization of diagnosis to primary care (dentists, general practitioners, pulmonary experts): (i) Automation capability (minimum hands-on steps); (ii) Point-of-Care compatibility
Clinical (symptom-based) examination is insufficient, leading to false management: (i) For RTIs: several bacteria/virus-borne diseases exhibit the same symptoms but require different treatment; (ii) For oral: when visual inspection identifies the diseases it is too late as they have already progressed	Application of molecular-based analysis for diagnosis: (i) Detection of multiple microorganisms with nucleic acid amplification technologies (NAATs): DNA of oral bacteria and DNA/RNA of RTI bacteria/viruses; (ii) Detection of host response protein biomarkers with immunoassays: Systemic inflammation and antibiotic stewardship [8], as well as periodontal inflammation [9]
Combined microbiological and immunological analysis of several targets/biomarkers generates a high load of analytical data	Use of clinical decision support (CDS) for management of multiple data: (i) Conversion of analytical parameters (Cq values from NAAT, biomarker concentrations, viral/bacterial load) into clinically meaningful parameters; (ii) Interface of analytical output with algorithms and conversion into clinical output for improved diagnostic precision and reliability
Adoption of new, even excellent-performing diagnostic technologies by end-users may face obstacles/delays due to distrust	Development and application of disease area-specific ‘implementation package’ [10] by the stakeholders: (i) Education and training of end-users for the trust, uptake and sustainability of new diagnostics; (ii) Consideration of end-users’ opinions and requirements, while ensuring technological flexibility to meet future (even currently unknown) demands

2.1. Sampling

Upper and lower RTIs require nasal or naso/oropharyngeal swab, and sputum or bronchoalveolar lavage (BAL) specimens, respectively. It has been reported that the viral or bacterial load of the same pathogens found in the different specimens can be different and consequently, the sensitivity and specificity of a test might be different according to the specimen [1,11]. This is why the selection of the proper specimen is of utmost importance for the reliability of the results. In case of oral diseases, one sampling method is the periodontal probing but this is invasive and not recommended for diabetics due to the risk of bacteremia [12]. Saliva is easier to collect, but also challenging to handle due to its viscosity. The lab-on-a-disk platform has the intrinsic advantage of centrifugation and, therefore, separation of the bacteria pellet from the supernatant in the saliva (the former for microbiological and the latter for immunological analysis). For example, magnet beating-based homogenization of fresh whole saliva has been reported on this platform and compared against the gold standard freezing/thawing/centrifuging, with a very high correlation of the immunological results [13].

2.2. Bioassays

Several nucleic acid amplification technologies (NAATs) have been developed, some of them isothermal [14], while real-time PCR is still a powerful and well-established one that enables the simultaneous detection of multiple genes per reaction (including controls). POC- and lab-on-a-disk-compatible TaqMan probe duplex assays have been developed, with lyophilized amplification reagents for the scope of oral bacteria detection [15]. Quadruplex TaqMan probe assays have also been used for the detection or External Quality Assessment respiratory panels [16]. At the immunoassay level and for RTIs, the simultaneous detection of C-reactive protein (CRP) and/or procalcitonin (PCT) supports a more accurate prescription of antibiotic or antiviral treatments, especially if a NAAT result is negative but the symptoms indicate a bacterial/viral infection [17]. In oral diseases, host response biomarkers such as matrix metalloproteinases 8, 9 (MMP-8, MMP-9), tissue inhibitor of metalloproteinases (TIMP-1), hepatocyte growth factor (HGF) and others can differentiate between healthy, gingivitis and periodontitis disease states [18]. Bead-based immunoassays using fluorescent nanoparticles for detection can ensure multiplexing and render the immunoassay compatible with the TaqMan probe PCR detection during the integration in automated systems. Furthermore, the flexibility and adaptability of such assays with diverse media (blood and saliva in case of our two examined disease cases) is important [19].

2.3. Analysis all-in-one

The automation and rapid analysis is achieved using the cartridge in which the (bio)chemical components are integrated and the assays are performed. The selection of centrifugal microfluidics [20] is based on some comparative advantages: (i) the modular nature of the unit operations [21] that can be interfaced like ‘puzzle pieces’ for nucleic acid and immunoassay analysis; (ii) the combination of the centrifugal forces with other physical principles for liquid handing, e.g., capillary forces, (centrifugo-, thermo-)pneumatic, local heating and magnetic forces deriving from instrument built-in magnets. The relatively large footprint of a disk-shaped cartridge may comprise some inherent limitation for simultaneous multi-cartridge utilization. Therefore, such a platform would not be suitable in cases where high throughput or massive screening is required, e.g., for epidemic surveillance. Nevertheless, smart engineering solutions offer at least a small to medium throughput by testing several samples on the same disk [22].

The lab-on-a-disk as a platform technology provides the necessary universality for being applicable in both fields of oral diseases and RTIs: for the nucleic acid analysis the same cartridge design is applied in both applications. Biochemical components such as the amplification enzymes and reagents for nucleic acid extraction and amplification (magnetic beads and buffers) are also the same. Such high degree of universality pays off at the level of scalable manufacturing. The cartridge remains easily adaptable to the detection of diverse targets by exchanging the primers/probes in the geometrically discrete reaction chambers, instead of re-engineering the entire cartridge [16,23].

2.4. Readout and detection

The lab-on-a-disk processing instrument provides the necessary automation and portability. Another important parameter for an instrument is the interoperability. Not only should the same instrument be applicable on different disease area diagnostics, but importantly, it should be compatible with both, nucleic acid- and immunoassay-detection cartridges. From landscape reports [4] there are some instruments targeting RTIs but barely any for the oral diseases. Even among the ones for RTIs, there is no reference that one can detect nucleic acids and proteins on the same instrument. The lab-on-a-disk processing device has demonstrated such interoperability capacity [5,16,24]. This is an attractive business model for the technology innovators, and clinical diagnostic model for the end-users, as they may use the same instrument for performing microbiological and immunological examinations.

2.5. Data interpretation for clinical decision support (CDS)

Addressing the need for multiple target detection and for combined microbiological and immunological analysis generates a data-challenge. Can the clinicians by themselves co-assess and interpret several Cq values, concentrations of protein biomarkers and bacterial/viral load values? In such cases, digital (information and communication) technologies need to be interfaced with the diagnostic system. For RTIs, clinical decision support algorithms [25] can co-assess results from molecular analyses (biomarkers, multiplexed PCR) and POC systems [26,27], and, together with the history and clinical symptoms of the patient, propose one or more treatment options [28]. For oral diseases, where the number of biomarkers is broad, the use of bioinformatics is of key importance for the correct interpretation of the analytical data, and especially in the course of time when a patient is followed upon treatment [29].

3. Conclusion and outlook

Despite their seemingly different fields, oral diseases and RTIs share common needs and challenges, which are addressed with novel lab-on-a-disk platforms that can be installed and used at the clinical settings. Advantages of lab-on-a-disk microfluidic platforms compared with traditional detection methodologies for the examined diagnostic applications can be summarized in (i) compatibility with diverse complex sample matrices, (ii) modularity and adaptability in microfluidic integration with pre-stored reagents enabling minimum hands-on steps, (iii) interoperability between nucleic acid and protein analysis, (iv) provision of quantitative results and (v) scalable manufacturability. Nevertheless, even the best-performing new technology may remain unexploited if the actual end-users are not convinced for its adoption. Therefore, in parallel to the efforts for achieving excellent technical performance, suitable and tailored implementation strategies need to run, in order to convince the end-users that the novel technologies will act in a complementary, rather than competitive way to the current standards of care, and always for the benefit of the patient.

4. Expert opinion

The disease areas of oral and respiratory tract infections (RTIs), despite their differences in clinical manifestation, severity and urgency of treatment, pose common technological and innovation challenges for their diagnosis, for example in (i) sample uptake and processing, (ii) early and accurate diagnosis using combined molecular-based microbiological and immunological testing and (iii) transfer of the diagnosis procedure from central laboratories to the point of care. Although some marketed technologies address one or more of these challenges in the field of RTIs, not all the above requirements are met, and especially not for oral infections, which is still an underexplored area in terms of point-of-care diagnosis, despite the tremendous relevant global costs and disease cases.

The technological developments on the lab-on-a-disk platforms have indicated that this approach has the potential to address all mentioned challenges due to its modular, adaptable and interoperable nature, as it covers all components and processes of the diagnostic value chain for both areas of RTIs and oral infections. Future developments on the lab-on-a-disk platforms are expected to be towards (i) increasing the throughput of sample analysis per run and (ii) interfacing with digital (information and communication) technologies that will enable the platform to be more easily adopted by end-users when acting as clinical decision support system.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This paper was not funded.