By Tiffany O'Callaghan

Invasive biopsy is currently the only sure way to diagnose the degenerative neurological condition Creutzfeldt-Jakob Disease (CJD). But a highly sensitive assay could change that, providing a fast, accurate alternative for early diagnosis of this rare but deadly condition.

In its most common form, known as sporadic CJD, the disease affects roughly one in a million people. Beginning in the 1990s, several cases of a variation of CJD known as vCJD were reported among people who had consumed beef from cows infected with another disease, bovine spongiform encephalopathy (BSE).

The findings, published online in Nature Medicine, also suggest that the assay--developed by microbiologist Ryuichiro Atarashi of Nagasaki University, Japan, and his team--could pave the way for the screening of broad sectors of the population.

CJD is a prion disease, in which an isomer of a common protein known as the prion protein (PrP) takes on an abnormal shape and becomes an infectious variant called PrP^Sc. This variant is thought to trigger the subsequent malformation of other PrP proteins. Unlike their normal counterparts, PrP^Sc prions cannot be broken down, and instead accumulate--often clustering in brain tissue.

The pockets of abnormal tissue that result cause brain tissue to develop a sponge-like appearance, and because prion conditions can be spread by affected humans or animals, the diseases are often referred to as transmissible spongiform encephalopathies (TSEs). Humans can be affected by several such conditions, while in addition to BSE in cows, there are several other such disorders among animals, including a condition called scrapie in sheep and hamsters.

No false positives

One problem that has plagued developers of non-biopsy diagnostic techniques is that it is often difficult to avoid false positives among samples taken from patients with neurodegenerative disorders other than CJD.

So Atarashi and his colleagues used a new assay known as a real-time quaking-induced conversion (RT-QUIC) assay. "Quaking-induced" refers to in vitro shaking, which researchers believe helps to accelerate the reactions, enabling the assay to produce results more quickly.

The team tested cerebrospinal fluid samples from 18 people with CJD and 35 people with other neurodegenerative diseases. This pilot group produced no false positives, and CJD was correctly diagnosed more than 83 percent of the time.

The researchers compared these results with those obtained using an existing assay that tests for levels of a protein known as 14-3-3, which is a marker for sporadic CJD. When tested on patient samples, the accuracy of 14-3-3 was 72.2 percent, whereas the specificity was 85.7 percent.

In a subsequent blind trial on 30 cerebrospinal fluid samples from Australia, RT-QUIC showed 100 percent specificity, resulting in no false positives among the 14 control samples, and correct diagnoses of 87.5 percent. 14-3-3 was equally accurate, but the rate of false positives was much higher.

"This technique allows definitive ante-mortem confirmation of CJD," says Atarashi, adding that this is currently difficult because it demands the detection of PrP^Sc in patients' biopsy specimens.


The RT-QUIC assay is also extraordinarily sensitive--detecting the presence of harmful prions at very high dilutions--and speedy, yielding results within 48 hours.

Atarashi first began developing RT-QUIC as a researcher at the National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, in Montana. In 2008, he and his team successfully used it to screen cerebrospinal fluid taken from scrapie-infected hamsters.

In December, Atarashi co-authored a paper reporting the efficacy of the RT-QUIC assay on nasal secretions and cerebrospinal fluid from hamsters with prion disorders.

Byron Caughey, chief of prion/TSE research at the Rocky Mountain lab and a co-author of the two hamster studies, is encouraged by the application of the assay to human cerebrospinal fluid samples. "Of course it will also be important to detect prion diseases in other species, but human diagnosis is of pre-eminent importance," Caughey says.

And although the next step is to replicate the findings in a much larger sample, the promise shown by RT-QUIC in analyzing substances other than spinal fluid in hamsters suggests a potentially fertile area for future research in humans. If RT-QUIC could be used to screen blood samples, or cheek or nasal swabs, for example, it could open up the possibilities of much earlier diagnosis and more widespread screening of donated blood.

"The earlier you're able to detect the presence of an infection in humans or animals, the more chance you have of preventing transmission to others and treating the disease in those who are infected," Caughey says.