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02 Nov 2017

Towards a urine or whole blood test for Japanese encephalitis

Doherty institute researchers  hope to have kick-started the process to more easily diagnose Japanese encephalitis by demonstrating that the virus can be detected in the urine or whole blood. The mosquito-borne virus – related to dengue and yellow fever – is endemic in the rice paddies and marshes of regional and remote areas of South East Asia, where it is the most common cause of vaccine-preventable encephalitis, causing 68,000 new cases a year. 

There is no cure. Most infections are mild or even symptom-free. But some people are unlucky enough to have the virus enter their nervous system. Death strikes a third of this group, while survivors may be left brain damaged and often severely disabled. 

“You have to be desperately unlucky, but obviously it’s devastating,” says infectious diseases physician and researcher Associate Professor Steven Tong from the Victorian Infectious Diseases Service, based at the Peter Doherty Institute.

Earlier this year, Dr Tong was the treating physician for a man who died of the disease soon after returning from a brief holiday in Thailand. After coming down with what he initially thought was a bad cold, the man rapidly deteriorated on returning to Australia. He became unconscious, needed to be placed on a ventilator to support his breathing, and eventually died one month after falling ill. 

He was bombarded with broad-spectrum antibiotics and anti-convulsant medication during his illness. Doctors ran tests for bacteria and viruses including measles, HIV, herpes, influenza, meningococcus, syphilis and flaviviruses like dengue, Zika, Yellow Fever and West Nile virus.

 “We didn’t know what the diagnosis was when he first presented,” Dr Tong said. “We look for everything and treat broadly when someone presents as very unwell.” 

The grail of Japanese encephalitis: detecting directly from a patient sample

An initial breakthrough came when antibodies to JEV were detected, pointing to the diagnosis. However, confirmation of a JEV diagnosis requires antibodies to be found in multiple samples. Additionally, false positives due to antibodies reacting against other flaviviruses can also occur.

“It would be much easier, faster and more accurate if we could directly detect the JE virus from a patient sample,” Dr Tong said. 

Associate Professor Tong then spoke with Dr Julian Druce, lead scientist at the Victorian Infectious Diseases Reference Laboratory molecular diagnostic laboratory, also located at the Doherty Institute. Dr Druce had noted that during the recent Zika outbreak in the Americas, the virus had been detected in patients’ urine. It was unclear whether this would also be the case with JE virus. Because these are blood-borne viruses, urine detection had not previously been considered seriously. 

“We then obtained urine and whole blood samples for testing and to our surprise, the virus could be detected – all the way out to day 28 of the illness. This was the first time the prolonged detection of JE virus in urine and blood had been demonstrated,” Dr Druce said.

“It’s only the second time the virus has been detected in urine, and the first time it’s been done proactively, in a clinically relevant timeframe, rather than retrospectively,” Dr Druce said. 

Traditionally, detecting flavivirus nucleic acid happens in a very narrow window of about a week, and antibodies are very unreliable and cross reactive. Indeed, the cerebrospinal fluid for this patient taken around day seven tested negative. 

Throwing away the good part

“Because of zika and this incident we’ve realised we’ve been throwing away the good part – red blood cells.  But the virus is detectable for longer  and at higher levels in the red blood cells. And the mosquitoes that spread these viruses take up whole blood, not plasma. Mosquitoes risk being infectious for way longer than we’d thought." 

Mike Catton, Deputy Director of the Doherty Institute, said this work allows for earlier, easier and more accurate diagnosis of Japanese encephalitis. "Although Japanese encephalitis is vanishingly rare in Australian returned travellers, our work will be of benefit to those living in high-risk areas where Japanese encephalitis is common. The findings are a wonderful example of collaborative research between astute clinicians and cutting-edge scientists that is facilitated by their co-location within the Doherty Institute.”

The team comprised researchers from the Victorian Infectious Diseases Reference Laboratory and the Victorian Infectious Disease Service, both based at the Doherty Institute; and the Menzies School of Health Research in Darwin. There was also assistance from the Royal Melbourne Hospital Neurology Unit, and ICU, as Micromon at Monash University for library preparation and next generation sequencing. The patient’s family agreed to the team publishing its results in the Open Forum for Infectious Diseases. 

Although the risk of getting JEV is very low – one in 500,000 – the vaccine used nowadays is vastly improved, with fewer side effects, and confers lifelong immunity with one shot. 

“It’s something you should at least discuss at least with your doctor if you’re travelling to an endemic area.” 

Read the paper here.

- Elisabeth Lopez