By Associate Professor Justin Jang Hann Chu,
Parveen Kaur and Nyo Min 

Hand, foot and mouth disease (HFMD) is an illness commonly reported in children and endemic to many parts of Asia. Symptoms include fever and blisters or rashes on the hands and legs, as well as ulcers in the mouth. HFMD is caused by a group of viruses from the genus Enterovirus, the most common of which, are coxsackievirus A16 (CV-A16), coxsackievirus A6 (CV-A6) and enterovirus 71 (EV-A71). While most cases of HFMD are often mild and self-limiting, neurological complications and fatalities have been associated with EV-A71 infections in particular1. Viruses causing HFMD are transmitted via direct contact with body fluids including saliva, nasal discharge, fluid from blisters, as well as faeces of an infected patient.


There is currently no antiviral treatment or vaccine against HFMD. Treatment is limited to supportive care and medications to alleviate symptoms. HFMD became a legally notifiable disease in Singapore following an outbreak in 2000 when several deaths were reported among children with EV-A71 infections. This allows monitoring of HFMD cases over time and this information is used to determine the implementation of control measures in an effort to limit the spread of HFMD. Many schools and childcare facilities conduct daily checks for HFMD symptoms in children, encourage frequent hand-washing and perform regular cleaning of shared items like toys with bleach-based disinfectants. A comprehensive public education programme also ensures that parents and caregivers are able to play their part in mitigating risks associated with HFMD.


Symptomatic diagnosis and lab tests

To diagnose HFMD, clinicians rely primarily on physical symptoms2. Diagnosing based on symptoms is often tricky and carries risks of misdiagnosis, since the symptoms of HFMD overlap with many other illnesses, including chicken pox, oral herpes (cold sores) and eczema2. This may lead to patients being administered incorrect treatment and further delays in recovery. It may also increase the likelihood of transmission to uninfected individuals when HFMD cases are mistaken for another infection and patients are not promptly isolated2.


Laboratory-based confirmation of infection with HFMD-causing viruses can be carried out by reverse-transcription polymerase-chain reaction (RT-PCR), which detects for the presence of the viral RNA in throat swabs, blood and stool samples2. There are, however, several limitations of this method. Detection via RT-PCR requires laboratory facilities, expensive equipment and specific technical expertise, which means that patients have to wait several days after the sample is collected before results are known. Waiting for test results before making a diagnosis would increase the risk of patients spreading HFMD to others, especially if they continue attending school or their childcare centres. Due to the wide number of viruses causing HFMD, there remain risks of false negative and false positive results2. Using RT-PCR for diagnosis during a major epidemic is also not feasible, since the large number of samples collected might lead to an even longer turnaround time. As a result, the examination of physical symptoms continues to be the most widely-practised method of diagnosis for HFMD, while laboratory testing is considered unnecessary for mild cases2,3.


Point of care testing

The limitations of the current methods of diagnosis underlie the necessity for more effective diagnostics. An ideal diagnostic method would be reliable, affordable and allow rapid retrieval of results without the need for specialised facilities or expertise. In addition, having a diagnostic method that can be utilised in GP clinics (point-of-care testing, or POCT) or even in childcare centres and schools is likely to reduce delays in diagnosis, resulting in quicker management of symptoms and more effective control of disease spread.

Several approaches to POCT have been explored in preliminary studies involving direct detection of HFMD-causing viruses in patient samples. For instance, a surface-enhanced Raman spectroscopy (SERS)-based technique involving colloidal gold nanostars conjugated to the EV-A71 cellular receptor has been recently reported4. In the absence of EV-A71, the nanostars were observed to aggregate, while the presence of EV-A71 disrupted the aggregation. A similar approach using magnetic nanobeads with EV-A71 or coxsackievirus B3 (CV-B3) receptors, which emit fluorescence in the presence of these two viruses, has also been explored and validated with a small number of clinical throat swab samples from HFMD patients5. A limitation of these methods is that they detect only for specific HFMD-causing viruses and require additional technology (e.g. portable Raman device or fluorescence spectrometer) for detection, a necessity that may be logistically problematic in some instances4,5. Simplifying the detection process, a separate group of researchers has also proposed a POC test using strips coated with antibodies against EV-A71 and CV-A16. Due to the presence of colloidal gold attached to the antibodies in the assay strips, a dark band can be visually observed when the strips are incubated with samples containing EV-A71 and CV-A166. This proposed POC test has been evaluated using blood samples from children with HFMD6.

 

And Biomarker detection

 Aside from direct detection of viruses, the use of biomarkers is another strategy that can be explored for POCT. As HFMD patients present with similar symptoms regardless of the virus they are infected with, it is possible that a universal biomarker exists for all HFMD-causing viruses. Biomarkers may also be utilised as predictors of more severe disease (e.g. neurological complications following HFMD), to allow earlier intervention. For instance, severe HFMD induced by EV-A71 infection has been associated with increased levels of HMGB1 (high mobility group box 1), clusterin A and serum amyloid A, all of which are proteins which play important roles in the immune response during infection or injury7,8. In addition, elevated N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels in patients with severe EV-A71-associated HFMD has been reported to be a predictor for many HFMD-associated complications, including cardiopulmonary failure, pulmonary edema, pulmonary haemorrhage and death9. Most of the research on HFMD-associated biomarkers has been performed with blood samples from patients. Since most patients of HFMD are young children, having a biomarker kit which requires blood collection may not be entirely suitable. Furthermore, the studies reported above focused on EV-71-associated severe HFMD, and may not be suitable for the testing of mild cases of HFMD caused by other enteroviruses.

 

Enable testing for MicroRNA in saliva

In our recent study, we have identified several microRNAs that were differentially regulated in the saliva of HFMD patients3. To collect saliva samples, we utilised a saliva-collection kit which was simple enough to be used by caregivers, teachers and parents. Having a collection kit which does not need to be administered by a trained healthcare professional is likely to reduce the distress of saliva collection from young HFMD patients, who may be more comfortable around people they recognise. We developed a diagnostic model which tested for the expression levels of 6 microRNAs in saliva collection samples. In a blinded test comprising 69 saliva samples from HFMD patients and healthy children, this model was able to detect HFMD with an accuracy of 85-93%. The successful detection of HFMD-associated salivary biomarkers is an important step forward towards the development of a less invasive POC test kit which can detect HFMD regardless of the enterovirus causing it.

 

As a paediatric illness, HFMD causes a significant socio-economic burden in the many areas where it is endemic. Current diagnostic methods which rely on physical symptoms are unreliable, and may result in unnecessary distress to affected parties, as well as higher transmission rates in the event of a misdiagnosis. Moving forward, research into HFMD diagnostic strategies, along with innovative technologies are essential in order to develop reliable and affordable POC test kits, as well as identify predictors for severe disease. It is hoped that these diagnostic strategies will aid in better disease management and reduce the socio-economic burdens posed by HFMD.

 

About the authors
Justin Chu is currently the Director of NUS Medicine BSL3 Laboratory and Associate Professor in the Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, NUS. He is also a Joint Principal Investigator in IMCB A*STAR. Parveen is a research fellow and Nyo Min is a research assistant in the Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, NUS.


References

 

1. Lim, C. T., Jiang, L., Ma, S., James, L. & Ang, L. W. Basic reproduction number of coxsackievirus type A6 and A16 and enterovirus 71: estimates from outbreaks of hand, foot and mouth disease in Singapore, a tropical city-state. Epidemiol. Infect. 144, 1028-1034


2. Teo, F. M. & Chu, J. J. Diagnosis of human enteroviruses that cause hand, foot and mouth disease. Expert Rev. Anti Infect. Ther. 14, 443-445, doi:10.1586/14787210.2016.1173543 (2016).


3. Min, N. et al. Circulating Salivary miRNA hsa-miR-221as Clinically Validated Diagnostic Marker for Hand, Foot, and Mouth Disease in Pediatric Patients. EBioMedicine 31, 299-306, doi:10.1016/j. ebiom.2018.05.006 (2018).


4. Reyes, M. et al. Exploiting the Anti-Aggregation of Gold Nanostars for Rapid Detection of Hand, Foot, and Mouth Disease Causing Enterovirus 71 Using Surface-Enhanced Raman Spectroscopy. Anal. Chem. 89, 5373-5381, doi:10.1021/acs.analchem.7b00066 (2017).


5. Wang, J. J. et al. Simultaneous Point-of-Care Detection of Enterovirus 71 and Coxsackievirus B3. Anal. Chem. 87, 11105-11112, doi:10.1021/acs.analchem.5b03247 (2015).


6. Zhang, J. et al. Development and evaluation of rapid point-of-care tests for detection of Enterovirus 71 and Coxsackievirus A16 specific immunoglublin M antibodies. J. Virol. Methods 231, 44-47, doi:10.1016/j.jviromet.2016.01.015 (2016).


7. Zheng, W. et al. Alteration of serum high-mobility group protein 1 (HMGB1) levels in children with enterovirus 71-induced hand, foot, and mouth disease. Medicine (Baltimore) 96, e6764, doi:10.1097/MD.0000000000006764 (2017).


8. Liu, J. et al. Serum amyloid A and clusterin as potential predictive biomarkers for severe hand, foot and mouth disease by 2D-DIGE proteomics analysis. PLoS One 9, e108816, doi:10.1371/journal. pone.0108816 (2014).


9. Qiu, J. et al. N-terminal pro-B-type natriuretic peptide for the prognostic prediction of severe enterovirus 71-associated hand, foot, and mouth disease. Int. J. Infect. Dis. 54, 64-71, doi:10.1016/j. ijid.2016.10.014 (2017).