Ebola profoundly elevated the impact of point-of-care testing, now recognized worldwide as essential to detect the disease, reduce risk, monitor patients in isolation, achieve recovery, and importantly, contain outbreaks. The goal is to become resilient – a new and possibly more contagious threat might appear. We must stop it where it starts!

by Prof. G. J. Kost, W. Ferguson, A.-T. Truong, D. Prom, J. Hoe, A. Banpavichit and S. Kongpila

Introduction – the essential role of point-of-care testing
Point-of-care testing (POCT) is propelling the convergence, integration and sustainability of global diagnostics. We should not be caught off guard at points of need! Using fever to screen patients for Ebola virus disease (‘Ebola’) occurs too far downstream in the clinical course, casts an excessively wide net confounded by other febrile illnesses, defeats rapid epidemiological control of outbreaks and inhibits evidence-based karma essential for compatible point of care culture. In fact, poor focus misleads the public, who, once cognizant of the essential role, importance and comprehensiveness of rapid POC diagnosis, will be receptive to containment and disposed to enter treatment centres, if they are more certain they have Ebola.
The Ebola ‘newdemic’ (an unexpected and disruptive problem that affects the health of large numbers of individuals in a crowded world) moved POCT from parochial fiduciaries often stalled by analysis paralysis to action-oriented value generators, that is, inventors and innovators leading the way with next-generation technologies and high stakes strategies, as summarized in this article, which are beneficial for reducing risk and enhancing resilience. It inspired the Ebola Spatial Care Path™ (SCP) and a useful Diagnostic Centre (DC) design equipped with POCT, presented here as well [1].

Rapid evolution of diagnostic tests for Ebola virus disease
Table 1 chronicles the pioneering ongoing efforts of industry, academia and government to produce workable immunoassays and molecular diagnostics for the detection of Ebola. In fact, this research development will spill over to energize POC diagnostics for highly infectious diseases in general. Novel research also is exploring digital detection of Ebola virus and viral load, which is higher in fatal cases and may be related to the development of virus-induced shock. Aside from the logistic challenges of getting assays ready in time, new assays, which might be implemented on instruments like the GenePOC, must be proven to work in clinical studies. As far back as 2006, investigators reviewed laboratory diagnostics for Ebola. Now, nearly a decade later, the FDA is accelerating the ongoing development, validation and approval of new diagnostic tests by issuing emergency use authorizations (EUAs) more or less continuously since autumn 2014 (Table 1).

Ebola-specific challenges for molecular diagnostics include: (a) reduction in initial false negatives (FN), FN = FN(t), as a function of time, to ramp up sensitivity, {TP/[TP + FN(t)]} (where TP=true positive), to ultrahigh levels in infected patients during the first 72 hours when symptoms may be mild or absent, in order to avoid shunting false negative cases to community hospitals ill prepared to receive high-risk patients; (b) automation of totally self-contained and sealable specimen cassettes and cartridges to eliminate need for expensive high-level biosafety cabinets; (c) proof of effectiveness in controlling internal contamination in portable instruments, thereby sustaining high specificity [TN/(TN + FP)] (where TN=true negative) and minimizing false positives (FP), which place people at risk when near infected patients; and as more sophisticated but compact technologies become available in the future, (d) determination of quantitative viral genome titers, which will be useful for early detection of exposure in smaller volumes of specimen and also for de-escalating the level of care and quarantine as the patient improves.

When performed properly with biohazard precautions in the near-patient testing area of a DC, results will be available much more quickly than sending specimens to a public health laboratory or to the Centers for Disease Control and Prevention (CDC). The gain in time can be substantial, just 1 hour or less needed to obtain an answer (see Table 1), which facilitates rapid screening, focused triage, and effective workflow. Self-contained cartridge/cassette-based rapid molecular tests are available on small portable platforms that test for infectious diseases. Development of POC molecular diagnostics for high risk infectious diseases forecasts the feasibility of introducing Ebola assays on light-weight platforms, such as the Alere I (see http://www.alere.com/us/en.html), and the tiny light-weight Roche Diagnostics cobas Liat (see https://usdiagnostics.roche.com/en/instrument/cobas-liat.html); both of these nucleic acid testing devices are Clinical laboratory Improvement Amendments (CLIA)-waived, user-friendly and, therefore, good candidates for point-of-need testing.

If tests satisfy certain conditions, they can be ‘waived’. In other words, the tests are cleared by the US Food and Drug Administration (FDA) to be performed in clinics and possibly even at home. Testing is simple to carry out and the instruments are operator-friendly, which make chances of an inaccuracy less likely. Such tests are referred to as a CLIA-waived. We will see facilitated-access, self-testing (FAST) POC solutions emerge as industry moves forward in the chronological evolution of Ebola EUAs in Table 1, some of which will be appearing commercially as inexpensive, portable, safe, and appropriate for detection of virus in the early stages of clinical illness. True, we are behind on the timeline. However, the good news is that everyone recognizes the need, the problem has been defined, POCT is part of the solution, and the feasibility of immediate testing at points is proven, as summarized in Table 2.

The Ebola Spatial Care Path
We define a Spatial Care Path (SCP) as the most efficient route taken by the patient when receiving definitive care in a small-world network (SWN). SCP principles include: (a) start diagnosis immediately wherever the patient is located; (b) implement POC technologies according to needs in the home, ambulance, primary care, SWN hubs, and at the bedside in critical care; (c) thereby achieve timely evidence-based decision making based on POC test results as the patient progresses through the SWN of healthcare; (d) coordinate access to the most critical elements and scarce specialists of the SWN to achieve a continuum of care; and (e) optimize the use of medical resources for the problem at hand, especially when the SWN becomes compromised or patients are selectively quarantined.

Spatial in this definition refers to shrewd positioning of POCT, elimination of unnecessary process steps, use of geographic information systems (GISs) to identify effective and efficient routes from population clusters to the nearest medical care, and in the case of Ebola, consolidation of SWN dispersion into one or more community alternative care facilities (ACFs) and DCs in which the useable space and workflow are optimized. Figure 1 illustrates the Ebola SCP with ACF and embedded POCT (on the left) integratively connected to a current expedient solution (on the right) of an individual hospital isolation area with a limited number of beds. A strategic Ebola SCP will deploy the best available molecular diagnostic testing at the point of initial patient contact and eliminate time-consuming steps in the sequence of care, such as transporting high risk Ebola patients from one community to another or sending hazardous samples to reference laboratories in heavily populated cities. Designing SCPs will facilitate prevention, intervention, and resilience in the event of wider presence of Ebola and simultaneously, will fulfill community recommendations of the CDC. We propose that each regional SWN analyse and ready its own SCP with POCT.

The Diagnostic Centre and interpretation of test results
Figure 2 shows the DC designed for Ebola care in Southeast Asia. POCT within the biosafety cabinet (top left) comprises: (a) the Spotchem EZ (Arkray, http://www.arkrayusa.com/) for determination of glucose, total protein, albumin, ALT, AST, alkaline phosphatase, cholesterol, triglycerides, HDL, urea nitrogen, creatinine, calcium, and total bilirubin, or combinations thereof (this instrument has been used for support of patients with viral hemorrhagic fever in Ghana); (b) the Opti CCA-TS2 whole blood analyser  (http://www.optimedical.com/products-services/opti-CCA-TS2.html) for measurements of pH, pCO₂, pO₂, total hemoglobin, oxygen saturation, Na⁺, K⁺, Ca⁺⁺ (ionized or free calcium), Cl⁻, glucose, urea nitrogen, and lactate, but only eight of these analytes at one time using a directly loading syringe cartridge that minimizes contamination; (c) a hematology instrument (optional), such as the QBC Star (http://www.druckerdiagnostics.com/hematology/qbc-star/qbc-star-centrifugal-hematology-analyzer.html), a dry reagent analyser that produces a nine-component complete blood count [hematocrit, hemoglobin, MCHC (mean corpuscular hemoglobin concentration), platelet count, white blood cell count, granulocyte count and percentage, and lymphocyte/monocyte count and percentage] from a specialized sample tube with stains and float separator inside, or the HemoCue CBC-DIFF (http://www.hemocue.com/en/products/white-blood-cell-count/wbc-diff); and within the isolation confines, (d) a vital signs monitor (e.g. VTrust TD-2300).

Premonitory POC test results, such as initial leukopenia, suppressed lymphocyte count on the differential, increased percentage of granulocytes and thrombocytopenia help confirm the diagnosis of Ebola. Later, patients have increased white blood cells (WBC), immature granulocytes and atypical lymphocytes. West Africa should be replete with POCT and DCs, but is not, thereby handicapping expeditious detection of premonitory signs and evidence-based critical care support in treatment centers. Striking electrolyte changes need monitoring to support repletion. Unfortunately, there is no small FDA-cleared handheld device for monitoring of coagulation (except PT/INR when adjusting warfarin anticoagulant, where PT is prothrombin time and INR is international normalized ratio). Filoviral hemorrhagic fever is accompanied by prolonged PT, activated PTT and bleeding time, potentially progressing to DIC with elevated D-dimer. D-dimer is available on the handheld cobas h232 (Roche Diagnostics, http://www.cobas.com/home/product/point-of-care-testing/cobas-h-232.html) available outside the U.S. As demonstrated by the two recent U.S. Ebola patients, platelets are consumed rapidly early in the course of the infection, and should be trend mapped to see recovery, possibly along with assessment of platelet function. Note that fatally infected patients fail to develop an antibody response. Thus, the detection of virus-specific IgM and IgG is a good prognostic sign. In critically ill Ebola patients, plasma loss and bleeding affect hemoglobin and the hematocrit, both of which should be monitored at the point of care.

Conclusions
POCT is facilitating global health. Now, global health problems are elevating POCT to new levels of importance for accelerating diagnosis and evidence-based decision making during disease outbreaks. Authorities concur that rapid diagnosis has potential to stop disease spread. New technologies offer minimally significant risks for personnel and can be used in conjunction with risk prediction scores for patients. With embedded POCT, strategic SCPs planned by communities fulfill CDC recommendations. POC devices should consolidate multiplex test clusters supporting Ebola patients in isolation. The ultimate future solution is FAST POC. DCs in ACFs and transportable formats also will optimize Ebola SCPs. In short, POCT can help stop outbreaks.

Acknowledgements and disclaimer
_{Spatial Care Path™ is a trademark by William Ferguson and Gerald Kost, Knowledge Optimization®, Davis, CA. Figures and tables were provided courtesy and permission of Knowledge Optimization®, Davis, California, and Visual Logistics, a division of Knowledge Optimization®. Figure 2 was created by Lab Leader Company, Ltd., Bangkok, Thailand. Devices must comply with jurisdictional regulations in specific countries, operator use limitations based on patient conditions, federal and state legal statutes, and hospital accreditation requirements. Not all POC devices presented in this paper are cleared by the FDA for use in the U.S.A. FDA emergency use authorization is limited in scope and term. Please check with manufacturers for the current status of Ebola diagnostics and POC tests within the relevant domain of use.}

References and notes
1. Kost GJ, Ferguson WJ, Hoe J, Truong A-T, Banpavichit A, Kongpila S. The Ebola Spatial Care Path™: accelerating point-of-care diagnosis, decision making, and community resilience in outbreaks. American Journal of Disaster Medicine 2015 [accepted for publication].
2. The FDA Emergency Use Authorization (EUA) status can be found at: http://www.fda.gov/EmergencyPreparedness/Counterterrorism/MedicalCountermeasures/MCMLegalRegulatoryandPolicyFramework/ucm182568.htm#current.
3. See WHO Emergency Quality Assessment Mechanism for EVD IVDs Public Report. Product: RealStar® Filovirus Screen RT-PCR Kit 1.0 Number: EA 0002-002-00. http://www.who.int/diagnostics_laboratory/procurement/141125_evd_public_report_altona_v1.pdf?ua=1.
4. FierceMedicalDevices. One-hour Ebola test receives FDA emergency use authorization.  http://www.fiercemedicaldevices.com/story/one-hour-ebola-test-biom-rieux-receives-fda-emergency-use-authorization/2014-10-27.
5. Jones A, Boisen M, Radkey R, Bidner R, Goba A, Pitts K. Development of a multiplex point of care diagnostic for differentiation of Lassa fever, Dengue fever and Ebola hemorrhagic fever. American Association for Clinical Chemistry Poster. http://www.nano.com/downloads/Ebola%20testing_PCR%20vs%20Immunoassay.pdf.
6. Instrumentation and corporate/academic relationships may have changed. See ‘Letters of Authorization’ on the FDA EUA webpage for details. Contact company and investigator sources for updates.
7. Benzine J, Manna D, Mire C, Geisbert T, Bergeron E, Mead D, Chander Y. Rapid point of care molecular diagnostic test for Ebola virus. Poster at ASM-Biodefense 2015. http://www.douglasscientific.com/NewsEvents/News/2014-10-21%20Lucigen%20to%20Seek%20FDA%20Emergency%20Use%20Approval%20for%20Isothermal%20Point-of-Care%20Ebola%20Test.pdf
8. See Piccolo xpress for test clusters. http://www.piccoloxpress.com/products/panels/menu/.
9. See Siemens website for details. clinitekhttp://www.healthcare.siemens.com/point-of-care/urinalysis/clinitek-status-analyzer/technical-specifications.
10. FDA-cleared for warfarin monitoring only.
11. See Sysmex website for list of variables and parameters. https://www.sysmex.com/us/en/Brochures/Brochure_pocH-100i_MKT-10-1025.pdf for list of variables and parameters.
12. Ebola assay FDA-cleared for emergency use only.
13. Beckman-Coulter, La Brea, California, manufactures the DxI800 and DXC800i.
14. Walker NF, Brown CS, Youkee D, Baker P, Williams N, Kalawa A, et al. Evaluation of a point-of-care blood test for identification of Ebola virus disease at Ebola holding units, Western Area, Sierra Leone, January to February 2015. Euro Surveillance 2015; 20(12): pii=21073.
15. Owen WE, Caron JE, Genzen JR. Clin Chim Acta 2015; 446: 119-127.
16. Nicholson-Roberts T, Fletcher T, Rees P, Dickson S, Hinsley D, Bailey M, et al. Ebola virus disease managed with blood product replacement and point of care tests in Sierra Leon. QJM 2015; pii: hcv092 [advance access publication]. http://qjmed.oxfordjournals.org/content/qjmed/early/2015/05/07/qjmed.hcv092.full.pdf.

The authors
Gerald J. Kost* MD, PhD, MS, FACB (emeritus); William Ferguson BS, MS; Anh-Thu Truong; Daisy Prom; Jackie Hoe; Arirat Banpavichit MS, MBA; Surin Kongpila MS
Point-of-Care Center for Teaching and Research (POCT•CTR), School of Medicine, University of California, Davis, CA, USA

*Corresponding author
E-mail: gjkost@ucdavis.edu

Point-of-care testing (POCT or POC testing) describes diagnostic tests which are performed at or physically close to a patient. This distinguishes POCT from traditional testing, which involves extracting specimens from a patient and transporting them to a laboratory for analysis. Settings for POC tests range from in-hospital bed sites and primary care offices to patient homes.

Over a half century of use
The POCT era is considered to have begun in 1962, after development of a system that measured blood glucose levels during cardiovascular surgery. The year 1977 saw the US launch of the first POC test for application wholly outside a hospital – the so-called ‘epf’ rapid pregnancy test.

POCT is increasingly used to diagnose and manage a range of diseases, from chronic conditions such as diabetes to acute coronary syndrome. One of the latest additions is a genetic test – CYP2C 19*2 allele for anti-platelet therapy.
Common POC tests includes “blood glucose testing, blood gas and electrolytes analysis, rapid coagulation testing, rapid cardiac markers diagnostics, drugs of abuse screening, urine strips testing, pregnancy testing, fecal occult blood analysis, food pathogens screening, hemoglobin diagnostics, infectious disease testing and cholesterol screening.” Nevertheless, just three tests – urinalysis by dipstick, blood glucose and urine pregnancy – are believed to account for the majority of POCT.

Turnaround time key to POCT appeal
The principal objective of POC testing is to reduce turnaround time (TAT) – a reference to the duration between a test and the obtaining of results which aid in making clinical decisions. In the past, such a process was unavoidable because of the sophistication and size of equipment required for the vast majority of medical diagnostic tests. However, technology developments have since made it possible to perform a growing number of tests outside of the laboratory.

Product miniaturization
Since the late 1980s, one of the key drivers of POCT has been product miniaturization with dedicated onboard integrated circuits. As described in a recent book on biomedical engineering, increasingly sophisticated microdevices have made it feasible to diagnose disease at point-of-care. These include “microfilters, microchannels, microarrays, micropumps, microvalves and microelectronics”, with their mechanical and electrical components “integrated onto chips to analyse and control biological objects at the microscale.” The authors list the key advantages offered by miniaturizing diagnostic tests as compared to centralized laboratory testing:  portability, small size and low power consumption, simpler operation, smaller reagent volumes, faster analysis, parallel analysis, and functional integration of multiple devices.

Healthcare reforms drive POCT
Healthcare reforms have also driven POCT demand.
Spending controls and hospital mergers have led to shorter stays and faster patient turnaround. There have been growing demand for tests in outpatient clinics and patient homes. Test results have been needed quickly, not only for reasons of clinical urgency but also to ease patient waiting lists and reduce backlogs in emergency departments. Accompanying this has been the closure of several large central laboratories, which have further enhanced demand for POCT.

Making a case
The case for POCT has grown with time. In 2004, it was associated with a significant reduction in the time to treatment initiation and a shorter length of stay. More recently, a POCT cardiac marker screening stage at six UK hospitals led to a marked increase in the percentage of successful home discharges.
Such breakthroughs will increase as POCT use grows further, and as the tests become more sophisticated.

Early POC tests were based on the simple transfer of traditional methods from a central laboratory, accompanied by their downscaling to smaller platforms.
Subsequently, unique and innovative assays were designed specifically for POCT (such as the rapid streptococcal antigen test). Wide arrays of POCT-specific analytic methods have also been developed, ranging from simple (such as pH paper for assessing amniotic fluid) to the ultra-sophisticated (for example, thromboelastogram for intraoperative coagulation assessment).

Contemporary POCT systems are usually based on test kits and portable, often handheld, instruments. Many tests are realized as easy-to-use membrane-based trips, often enclosed by a plastic cassette. This requires only a single drop of whole blood, urine or saliva, and they can be performed and interpreted by any general physician within minutes.

Hospital emergency departments
Given its time-sensitive relevance, one of the fastest growing users of POCT have been hospital emergency departments (EDs).
In 2008, a study in  ‘Academic Emergency Medicine’ simulated the impact of reduced turnaround times and established grounds for a “compelling improvement in ED efficiency.” Though its authors concluded that specific outcomes such as the length of stay and throughput in the emergency department warranted further investigation, they categorically recommended POCTs as a means to improve turnaround time.
Over recent years, favourable perspectives on POC tests in the ED have strengthened. At the end of last year, a study in ‘Critical Care’ found POCT increased the number of patients discharged in a timely manner, expedited triage of urgent but non-emergency patients, and decrease delays to treatment initiation. The study quantitatively assessed several conditions such as acute coronary syndrome, venous thromboembolic disease, severe sepsis and stroke, and concluded that POCT, when used effectively, “may alleviate the negative impacts of overcrowding on the safety, effectiveness, and person-centeredness of care in the ED.”
Other POCT users include ICUs as well as endocrinology, cardiology, gastroenterology and hematology.

Primary care remains principal user
The bulk of POC tests are however conducted by primary care physicians. 
In 2014, the ‘British Medical Journal’ published the findings of the first-ever survey of POCT use by primary care physicians in five countries (Australia, Belgium, the Netherlands, the UK and the USA). The study found that blood glucose, urine pregnancy and urine leukocytes or nitrite were the most frequently used POC tests. Overall, more respondents in the UK and the USA reported using POC tests than respondents in the other countries. The widest gap in use of POC test was for fecal occult blood, used by 83% of US doctors against only 2–18% of primary care clinicians in the other countries.
One of the key findings of the ‘British Medical Journal’ study, however, was that there was an unmet need for new POC tests. Included here were tests for D-dimer, troponin, chlamydia, gonorrhea, B-type natriuretic peptide, CRP, glycated hemoglobin, white cell count and hemoglobin, which were desired by more than half of respondents across all the five countries.

Fast growing market
Over the past two-and-a-half decades, the availability and use of POCT has steadily increased. By 2012, nearly 100 companies worldwide were developing, manufacturing or marketing POC tests. One study, cited by the National Institutes of Health in the US, places POCT sales in 2011 at about $15 billion (€13.5 billiion). Of this figure, the US accounted for a share of 55%, Europe for 30% and Asia for 12%. The market is projected to show compound annual growth of 4% to reach $18 billion (€16.2 billion) by 2016.
Further growth in the use of POCT is expected to be driven by increases in accuracy, reliability and convenience. Alongside, one of the biggest catalysts for increased POCT use may consist of quality standards.

The quality challenge
Issues about POCT quality continue to vex experts. Variability in the interpretation of POC test results is a widespread concern, given differences in the education and experience of staff who conduct the tests. In addition, POCT results may also not be comparable across sites (e.g. when patients travel) and differences in specimen types (serum, plasma or whole blood) can impact on results – as compared to those from a traditional central laboratory.
In a laboratory setting, analytical quality is usually assessed by QC (quality control) and QA (quality assurance) procedures. Their aim is “to monitor the stability of the analytical measurement system and to alert the operator to a change in stability”… “that may lead to a medically important error.” While these processes serve a laboratory well, it is unclear whether these processes are relevant, transferable and practical for monitoring quality on POCT devices.

Regulators and POCT in the US and the EU
Future developments are expected to be driven by regulatory bodies.
In the US, CLIA88 (Clinical Laboratory Improvement Amendments of 1988) provided a major impetus for growth in POCT. The rules, published in 1992, expanded the definition of ‘laboratory’ to include any site where a clinical laboratory test occurred (including a patient’s bedside or clinic) and specified quality standards for personnel, patient test management and quality.
One of CLIA88’s biggest contributions to POCT growth was to define tests by complexity (waived, moderate complexity and high complexity control), with minimal quality assurance for the waived category.
CLIA88 has been followed by US federal and state regulations, along with accreditation standards developed by the College of American Pathologists and The Joint Commission. These have established POCT performance guidelines and provided strong incentives to ensure the quality of testing.

In Europe, POCT devices are regulated under the 1998 European Directive 98/79/EC on in vitro diagnostic medical devices, although the term itself is not specifically mentioned. There have since been several amendments, most recently in 2011 (2011/100/EU), as well as standards based on the Directive’s framework.
However, at the European level, specific coverage of POCT is referred by international standard ISO 22870:2006, used in conjunction with ISO 15189 which covers competence and quality in medical laboratories. It is important to note that patient self-testing in a home or community setting is not covered by ISO standards.

The role of ICT
The role of ICT in driving the growth of POCT is also likely to become crucial. In the late 1990s, there were concerns that POCT implementation, especially in the real-time critical care context, was accompanied by little understanding of its information technology requirements.
However, the situation has since changed dramatically, especially as ICT is seen as the only appropriate interface between POC test results and computerized patient records – seen as the means to restructure clinical care pathways.
ICT is also accepted as the best means to standardize care protocols. In 2012, a study found that the impact of point-of-care panel assessment on successful discharge and costs varied markedly from one hospital to another and that outcomes depended on local protocols, staff practices and available facilities. In effect, the study highlighted the importance of optimizing clinical pathways to derive maximum benefit from the reduced turnaround times provided by POCT.

Head and neck cancers (HNC) are a globally prevalent malignancy. Despite the efforts of reducing several known etiological factors such as smoking and drinking to lower the incidence of HNC at the population level, the incidence of oropharyngeal cancers (OPC) is on the rise. OPC is caused by human papillomavirus (HPV) and most prevalent in a younger age group. This review critically examines the epidemiology, biology and laboratory detection of OPC and provides future insights into combating this debilitating disease.

by X. C. Sun, P. Tran and Dr C. Punyadeera

Introduction
Head and neck cancers (HNC) are the sixth most prevalent neoplasm in the world with approximately 650 000 cases diagnosed each year [1–5]. Oral and oropharyngeal squamous cell carcinomas (OSCC & OPSCC) together constitute 90% of malignancies in the head and neck region. Several known traditional etiological factors such as tobacco and alcohol use are recognized in the development of these cancers. More recently, human papillomavirus (HPV) infection is recognized as an additional risk factor for the development of a subset of HNCs, mainly OPSCC [6].

In recent decades, the overall incidence of HNC caused by smoking and alcohol is on the decline. In contrast, HPV^+ve OPSCC is on the rise. In developed countries such as the United States of America, the incidence of HPV+ve OPSCC is escalating, with predictions that more than 50% of patients will be HPV^+ve by 2030 [7]. Interestingly, patients who are HPV^+ve OPSCC are relatively younger than HPV^-ve HNC patients and are therefore less likely to have any history of chronic or excessive alcohol or tobacco use but are more likely to engage in social habits that increase the likelihood of HPV transmission (oral sex). The clear distinction between HPV^+ve OPSCC and HPV^-ve cases provides multiple downstream inputs that can be applied into clinical treatment modalities. Conversely, it provides an exciting opportunity for the development of early diagnostic and screening methods to combat HNC at a population level through prevention strategies.

HPV^+ve OPSCC are both clinically and biologically distinct tumour entities compared with HPV^-ve counterparts. Classically, HPV^+ve OPSCC patients present with a molecular profile that includes retinoblastoma (pRB) pathway inactivation, p53 degradation and p16 upregulation. Clinically, HPV^+ve OPSCC patients often present with smaller primary tumours but more advanced nodal disease, similar rates of metastasis and differing patterns of metastasis [8, 9]. In addition, patients with HPV^+ve tumours have better prognosis with 5-year survival at 75% (c.f. 25% for HPV^-ve patients). There are a number of techniques for the diagnosis and detection of HPV^+ve OPSCC, including histopathology, polymerase chain reaction (PCR) and immunohistochemistry (IHC).

Biology
Upon integration of HPV DNA into the host genome, E6 and E7 viral oncoproteins activate a number of pathways within the host cell. The primary molecular target of E7 is the Rb protein and the E7 viral oncoprotein reprogrammes terminally differentiated epithelial cells to re-enter the cell cycle. E7 disrupts the Rb–E2F complex leading to the release of E2F, subsequently resulting in cyclin A and E activation and entry of the cell into S phase. As a consequence p16 is overexpressed [10, 11]. The E6-associated protein (E6-AP) is a specific ubiquitin-ligase that binds to the viral E6 oncoprotein, resulting in p53 degradation. E6 and E7 have also been shown to interfere with growth inhibitory cytokines [such as tumour necrosis factor-α (TNFα)] and to disrupt the mitochondrial apoptotic pathway by interfering with pro-apoptotic BAK and BAX [10]. E6 and E7 alone are insufficient to cause malignant cell transformation; however, due to their interference with proliferation checkpoints and apoptotic pathways, it is certain that the accumulation and damaged DNA, mitotic defects and integration of foreign DNA substantially increase the risk of malignant progression [10].

Detection methods
A number of diagnostic methods are currently available to evaluate whether a tumour is HPV^+ve. These methods include both indirect as well as the direct methods; i.e. the latter includes the detection of HPV genomic DNA (gDNA). Besides clinical examination, current methods for the diagnosis of HPV status include tissue biopsy staining for p16 (indirect method). Biopsies may fail when tumours are too small to access or when they are located in hidden anatomical sites [10]. Other methods include the detection of HPV gDNA using PCR and in situ hybridization (ISH) as well as the detection of HPV viral transcripts E6 and E7 by PCR.

p16 detection by IHC is widely used in cervical cancer cases for the detection of HPV and it is being studied extensively in the field of HNC [12]. During immortalization of host cells, the E7 protein binds to Rb, resulting in the compensatory overexpression of the tumour suppressor gene p16 in HPV-infected tumour cells. Therefore, IHC detection of p16 is considered as an indirect surrogate marker to determine the presence of HPV [11]. However, there are pitfalls associated with p16 IHC detection. A number of studies have shown suboptimal specificity of IHC [10, 11]. As a consequence of the extreme anatomical and biological heterogeneity in HNC, elevation of p16 by non-viral materials may contribute to a considerable false positive rate [11]. Although it has been reported that p16-positive patients have a better prognosis and increased radiosensitivity, it has been advised that p16 detection by IHC alone cannot accurately identify HPV infection in HNC [12].

Detection of HPV gDNA is a widely used method because of its high sensitivity and cost-effectiveness. Common primers (MY09/MY11 and GP5/GP6) that target the L1 open reading frame are used to detect wide-spectrum HPV genotypes [11]. However, standard PCR primers do not allow detection of specific HPV genotypes [10]. In addition, the target L1 region could also be deleted upon viral integration, which may affect sensitivity of the test [10, 11]. Although, specific E6 and E7 primers have been designed and used to overcome L1 deletion, this method still lacks the ability to distinguish stromal/tumour and episomal/integrated DNA materials and is prone to contamination interference, which undermines the clinical usefulness [11].

The HPV DNA ISH method is unique because of its high specificity and the ability to be evaluated microscopically. The visible hybridization signals that precipitate within the nuclei help distinguish integrated and episomal DNA [11]. It is noteworthy that the presence of HPV DNA detected by ISH significantly correlates with p16 detection by IHC. [10]. However, ISH methods carry lower sensitivity compared to its excellent specificity [11]. The detection of HPV-16 viral transcripts E6 and E7 can highlight whether a patient is suffering from persistent infection – information that is clinically more valuable [12]. However, because of the fragile nature of mRNA, formalin-fixed paraffin-embedded (FFPE) specimens are often not ideal for RNA analysis and frozen fresh specimen are required [12].

The detection of HPV-specific IgG in serum is a useful biomarker to determine previous and current HPV infection status [13]. Serological biomarkers are not site-specific, and can arise due to HPV infections at sites other than the oral cavity, hence potentially affecting the specificity of the assay.

The effect of Gardasil™
From treatment and management of HPV-related diseases, the paradigm of HPV care has shifted to a preventative approach since the breakthrough introduction of the HPV vaccine, Gardasil™ (Merck & Co.). The biologic basis of HPV vaccines relies on the mechanism of neutralizing antibodies generated against virus-like particles (VLP), which consist of the major capsid protein HPV L1 [10]. The quadrivalent HPV 6/11/16/18 vaccine Gardasil™ was licensed by the FDA to prevent cervical, vaginal and vulvar infections in women in 2006 and genital warts in men in 2009, followed by the licensing of bivalent HPV 16/18 vaccine Cervarix™ (GlaxoSmithKline), in women in 2009 [14].

The benefits of HPV vaccination for the oral cavity include not only the biologically-plausible direct effect on oral infections, but also the sequential oral infection reduction following genital infection reduction, due to the sexually transmitted nature of HPV. To date, there are only a few studies examining the effect of Gardasil on oral infection; however, the results showed a promising outlook with high vaccine efficacy (as high as 93% in 4 years time, as recorded by randomized controlled trial in Costa Rica) [15] and reduced viral prevalence (oral prevalence dropped to 1.4% from 9.3% in the 15–23 age group in youth clinics in Sweden) [16].

Future outlook
As previously mentioned, HPV-related OPSCC involve a new segment of the population, which is distinctively different to the traditional HNC patient cohort caused by excessive smoking and drinking. This requires clinicians to conduct thorough cancer screening of at-risk groups. Such screening programmes should pay particular attention to cervical lymph nodes as some subtypes of HNC, especially OPSCC, involve hard-to-examine areas for clinical visual examination.

As a result of the sexually transmitted nature of HPV, some studies have advocated routine sexual behaviour education in clinical practice. However, this practice carries inherent controversies of sexual harassment and confidentiality [17]. Public awareness campaigns have been argued to be a more efficient preventative means in altering patients’ behaviour. Studies have shown that many oral health practitioners have limited knowledge with regards to HPV-related HNC and HPV vaccinations [17]. Professional bodies and health authorities are required to address this knowledge gap by establishing new clinical guidelines and using continuing educational methods, in order to effectively control the rising trend of HPV-related HNC.

Conclusion
All current diagnostic methods require excision of tumour tissue and this can be challenging when they are located in hidden sites. Efforts have been made globally to develop a less invasive, more cost-effective and clinically-relevant test. Serology tests that detect HPV-specific IgG have been shown to indicate viral presence and are linked with prognostication; however, this method inherently lacks site specificity [10]. Oral specimens, more specifically oral rinse, have shown promise in this field. Oral rinse samples not only are non-invasive and cost-effective, the proximity of collection to the area of interest ensures the localized sampling field. It is also important to note that shedding of normal cells into the oral cavity/oral pharynx may interfere with and/or decrease the HPV detection level [10]. OraRisk^® HPV test, uses oral rinses for HPV detection [10]. Translational collaborations between scientists and clinicians have resulted in an assortment of tumour markers and diagnostic techniques for OPSCC. However, these need to be tested in clinical trials to determine the cost-effectiveness.

Acknowledgments
This work is supported by Garnett Passé & Rodney Williams Memorial Foundation and the Queensland Centre for Head and Neck Cancer funded by Atlantic Philanthropies, the Queensland Government and the Princess Alexandra Hospital.

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The authors
Xiaohang Charles Sun¹, Peter Tran¹ and Chamindie Punyadeera*² MSc, PhD
¹School of Dentistry, The University of Queensland, Brisbane, Australia
²The Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.

*Corresponding author
E-mail: chamindie.punyadeera@qut.edu.au