Diagnosis of human enteroviruses that cause hand, foot and mouth disease

KEYWORDS:

• HFMD
• enteroviruses
• diagnosis
• POC diagnostics
• POCT

Hand, foot and mouth disease

Hand, foot and mouth disease (HFMD) is a communicable sub-febrile enanthematous cum exanthematous viral disease commonly targeting young children, elderly and immuno-compromised individuals. It is caused by a myriad of etiological agents under the genus Enterovirus within the Picornaviridae family. Thus far, 12 species of enteroviruses have been documented; with nine species capable of affecting humans [1], of which three species can cause HFMD. At present, there are neither effective anti-viral drugs nor multivalent prophylactic vaccines available to target this multicausative disease. Hence, prompt clinical isolation and management will be the best approach to avert transmission and provide pertinent supportive therapies. However, this requires an early and accurate diagnosis which is unfortunately still lacking at the moment.

Existing HFMD diagnostic approaches and their limitations

The classical laboratory diagnosis of HFMD is based on micro-neutralization assay of cultured patient samples using a variety of commercially available anti-serum pools adapted from the 1960s and 1970s [2–4]. This ‘golden standard’ typing method is extremely challenging and time-consuming as virus isolation is mandatory prior to the assay. In addition, serotype-specific anti-sera are not always available to detect all the possible perpetrators, especially due to evolutionarily driven antigenic drifts and shifts.

The subsequent introduction of molecular testing, which is based on the principle of reverse-transcription polymerase chain reaction (RT-PCR), reduces the arduous diagnostic process by omitting the obligatory virus isolation step [5,6]. Detection of enteroviruses is achieved either by universal amplification of their highly conserved 5ʹ-untranslated region [5], VP1 region amplification coupled with sequencing [7,8], or direct amplification of their unique serotype-specific regions [9,10]. Since then, incessant efforts have been devoted to develop fast and sensitive molecular diagnostic methods to shorten the laboratory turnaround time (TAT) further, and this is especially evident following the invention of real-time quantitative RT-PCR (qRT-PCR) [11]. The problem with pan-enterovirus primers used for universal targeting of enteroviruses is the risk of non-specific binding and amplification of other closely-related viruses due to compromised stringency in primer design and qRT-PCR reaction conditions. However, the feasibility of using serotype-specific primers remains questionable, due to the non-exhaustive list of HFMD-causing viruses. Nevertheless, molecular diagnostics is a better alternative to the ‘golden standard’, with a shortened TAT from a matter of days and weeks to hours and days. Unfortunately, the lack of commercially available point-of-care (POC) testing (POCT) translates to a need for laboratory infrastructure and support which are not readily available on-site in day-care centers, schools, communities and outpatient settings thereby resulting in a delayed diagnostic process.

The aforementioned circumstances often result in a compromised rudimentary diagnosis crudely based on the characteristic clinical presentation of HFMD, including fever, rashes and blisters in the limbs, and ulcers in the oral cavities. This, however, is accompanied with an inevitably high false discovery rates. The non-distinctive signs and symptoms associated with HFMD overlap with the clinical features of other viral infections such as cytomegalovirus, herpes simplex virus and varicella-zoster virus (chicken pox) infections [12–17]. This often makes diagnostic discrimination tricky, risking false positives, inappropriate therapeutic judgments and delayed treatment for the actual cause. Some HFMD cases are accompanied with atypical clinical features, sometimes resembling other conditions or diseases such as herpes infections (cold sores) and allergic dermatitis (eczema) [12–17]. This gives rise to the risks of false negatives, resulting in persisted transmission due to delayed isolation. While false positives result in unwarranted concerns and/or inappropriate treatment, false negatives are also of great concerns due to untraceable transmissible cases.

HFMD disease burdens; the importance of point-of-care testing

Over the past two decades, the escalating cases of HFMD has brought the once intermittent childhood disease to the attention of public healthcare authorities worldwide. Despite being a generally mild and self-limiting disease with low and infrequent complications and fatalities, HFMD is highly transmissible through infected bodily fluids from the oral-fecal and respiratory routes, affecting millions of young pre-adolescence children each year. Consequently, unjustified and potentially exorbitant socio-economical, financial and/or psychological disease burdens ensue, presenting HFMD as a perpetual public health threat; caregivers have to face obligatory closure orders of care-centers; parents have to leave their work aside or make alternative arrangements to look after their unsupervised or sick child; healthcare systems are subjected to the risk of becoming unnecessarily bogged down by sudden outbreaks and epidemics; badly-affected countries are slapped with international travel advisory alert labels; etc.

Hence it is imperative that more attention and resources are channeled to establish a framework which can position researchers to quickly translate basic research findings to clinically valuable diagnostic applications to ease the anticipated disease burdens, at least to tide us through till effective anti-virals and/or broad-spectrum prophylactics are in place. Due to the nature of the disease, a rapid POC diagnostic detection tool that is user-friendly and affordable yet sensitive and specific is crucial for combating HFMD. The feasibility of POC diagnostic strategies has certainly been proven through successful inventions such as the classic blood glucose meters and pregnancy test kits, and rapidly evolving lab-on-a-chip micro-technologies [18,19]. In fact, several preliminary studies on HFMD POCT have been conceptualized, including virus detection using magnetic nanobeads coupled with fluorescence technology [20] and antisera-based indirect immunofluorescence assay [21], as well as antibody detection using colloidal-gold immunographic assay strips [22].

Combating HFMD demands collective efforts

While the battle with HFMD persists, much more can be done. An easily accessible common communication platform can be made available for constant exchanges of information, experiences and opinions between healthcare professionals and researchers in order to facilitate the smooth transition from clinical observations to laboratory research to industrial applications. Healthcare professionals can also keep in mind that HFMD cases are not always presented with the conventional clinical manifestations, and be critical with diagnosis. In addition, efforts can be made to ensure that parents and caregivers are adequately educated on HFMD and actively involved in the watch for and isolation of HFMD cases.

Concluding remarks

Diagnosis dependent on clinical presentation is often inaccurate and subjective due to the lack of standardized case definitions and guidelines. Even if these are implemented, atypical cases can still be overlooked. Diagnosis based on molecular detection of viruses may also not be the most ideal as virus is usually detectable within a narrow window period, and may be cleared from the common sites of sample collection before the onset of visible clinical indications. Moreover, adequate experience is required for the appropriate collection of clinical specimens for successful virus isolation. More importantly, virus dynamics can become a bottleneck in full diagnostic coverage of the broad spectrum of HFMD-causing viruses. Thus, alternative approaches based on biomarker discovery of virus-specific systemic host signatures may be the way forward for the development of HFMD diagnostic strategies, at least to serve as a complement to the current diagnostic options. This concept is not new, as many independent studies have reported the use of host signatures to determine disease status [23–26]. Two very recent and successful examples of using host signatures to aid diagnosis are the studies by Oved et al. (2015) and Tsalik et al. (2016), who suggested the use of a combination of independent host-protein identifiers for differential diagnosis of viral and bacterial infections thereby addressing inappropriate use of antibiotics and its subsequent resistance [27,28]. It will be a major leap forward if differential HFMD diagnosis can be achieved using combinational host signatures, as current diagnostic options can be ambiguous in distinguishing HFMD from other virus infections.

While unique molecular biomarkers may open avenues to the development of POC diagnostics, further research is necessary before POCT for HFMD can be realized; the biological changes have to persist in the body for an extended period to encompass varying stages of HFMD; the biomarker must be easily monitored in a regular basis to promptly isolate and manage HFMD cases; clinical research methods need to be made more robust to have higher success rates of clinical validations; and clinical findings need to be translated quickly and successfully to clinically useful technology.

Financial & competing interests disclosure

This study was supported by the Biomedical Research Council, A*STAR. FMS Teo is doing her PhD in the National University of Singapore (NUS) Graduate School for Integrative Sciences and Engineering (NGS) under the support of the A*STAR Graduate Scholarship (Singapore). The authors have no other 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 apart from those disclosed.