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Sequencing goes deep to find rubella in uveitis patient

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William Check, PhD

November 2016—Metagenomic deep sequencing, or MDS, has scored another coup in the diagnosis of an unexplained disease in a patient who had already had extensive workup with all other available tools. MDS had been used in 2014 to detect unsuspected leptospirosis in a critically ill encephalitis patient, enabling appropriate treatment and full recovery (Wilson MR, et al. N Engl J Med. 2014; 370[25]:2408–2417). Now some of the same clinical scientists at the University of California San Francisco Medical Center who helped diagnose that patient have identified rubella virus infection in the eyes of a patient with bilateral chronic intraocular uveitis that had been misdiagnosed as idiopathic inflammation for 16 years (Doan T, et al. Genome Med. 2016;8[1]:90).

More broadly, the UCSF clinical investigators reported “a case series of patients in which MDS was used for the first time to identify a range of infectious agents in intraocular fluid from patients with uveitis or intraocular inflammation,” neurologist Michael Wilson, MD, MAS, who worked on the encephalitis and uveitis cases, tells CAP TODAY. In addition to rubella virus, organisms included Toxoplasma gondii, herpes simplex virus type 1, and Cryptococcus neoformans. In two cases of noninfectious inflammatory ocular conditions, metagenomic deep sequencing ruled out infections.

“To our knowledge, this is the first ever report of MDS being used for infectious disease diagnostics on intraocular fluid, which is a very challenging specimen type to work with, given the small volumes that are typically available,” Dr. Wilson says. Having a laboratory method to identify infectious causes of uveitis is important, he notes, because of the frequent co-occurrence of eye inflammation with many systemic diseases.

Dr. Doan

Uveitis is difficult to diagnose clinically, says Thuy Doan, MD, PhD, a specialist in uveitis and an assistant professor at the UCSF Proctor Foundation for Research in Ophthalmology, who saw the rubella patient and initiated the use of MDS in the diagnosis. “It will be completely a game changer for ophthalmology to be able to offer MDS instead of a panel of assays for infectious agents,” Dr. Doan says. In addition to being limited to very few pathogens, a PCR panel requires much greater volume than MDS, which can be done on 20 to 30 µL of intraocular fluid. “The entire volume of eye fluid is about 3 mL,” Dr. Doan says. Only about 300 µL can be safely removed in the clinic.

The MDS method used was developed in the laboratory of Joseph DeRisi, PhD, professor and chair of biochemistry and biophysics at UCSF. MDS on the uveitis patient was performed in his laboratory. “It is absolutely clear to us where we stand with metagenomic next-generation sequencing done in a completely unbiased fashion,” Dr. DeRisi says. “I foresee that this technology will likely replace or overtake many of the traditional sendout assays for individual agents.

“Because this technology operates without bias,” Dr. DeRisi adds, “it is likely that many more either obscure or unanticipated infectious agents will be captured in a wide range of infectious disease indications.”

Making advances toward this goal is possible because of the philanthropic support from the Sandler, William K. Bowes Jr., and Charles and Helen Schwab foundations, which support the UCSF Center for Next-Gen Precision Medicine Diagnostics. Dr. DeRisi is the principal investigator. A grant of $600 million from the Chan Zuckerberg Initiative to establish a Biohub facility at UCSF, which Dr. DeRisi and Stephen Quake, DPhil, of Stanford University will co-direct, will also greatly aid these efforts. One major project in the Biohub will focus on infectious diseases, including new ways to detect and identify infectious agents (www.bit.ly/ucsf-biohub).

As Dr. Doan recalls it, her first encounter with the uveitis patient was “almost like a CSI case.

“In 2015, when I first started as new faculty here, he came to my uveitis clinic,” says Dr. Doan, director of the Ralph and Sophie Heintz laboratory at the Proctor Foundation. The man moved to Southern California from Germany in 2006. In Germany, he was diagnosed in 1999 with ocular inflammation of one eye that was treated with topical steroid drops. Two years later he had inflammation of the other eye that was treated similarly.

At an institution in Southern California, where he was seen initially, he was exhaustively worked up for an infectious etiology, but all test results were negative. “They thought his symptoms were autoimmune related,” Dr. Doan says, “so he was put on methotrexate and high-dose prednisone for a year.” This regimen didn’t help.
In 2012 the man moved to San Francisco and was examined at UCSF, where the clinicians obtained intraocular fluid for testing. Fuchs uveitis syndrome is known to be due to rubella, Dr. Doan notes. However, the majority of the patients with Fuchs present with inflammation in the front of the eye and in only one eye. “For this patient, the presentation was different,” she explains. “He had inflammation in the front and middle of the eye—both aqueous and vitreous compartments—and in both eyes.” Nonetheless, a viral cause was sought given his characteristic clinical findings (Fig. 1).

Fig. 1. Ocular findings of a 40-year-old man with bilateral, idiopathic chronic anterior and intermediate uveitis. The top panels show different colored irises (heterochromia) between the right and left eyes and transillumination defects that are prominent in the left eye because of iris atrophy (lower panels). These findings are suggestive of viral-related uveitis. (From Doan T, et al. Genome Med. 2016;8[1]:90. Original publisher: BioMed Central.)

At one point, the patient had a diagnostic and therapeutic vitrectomy—removal of all vitreous fluid—and the fluid was sent for testing. “In ophthalmology, we are very elementary in microbiology diagnosis compared to neurology and other fields,” Dr. Doan says. “Here at the Proctor Foundation we have one of the biggest panels available in the U.S., and we only do PCR for four organisms—herpes simplex virus, Toxoplasma, varicella-zoster virus, and cytomegalovirus—as well as bacterial and fungal cultures.” Again, all tests were negative.

“Without an infectious cause, I was about to go to immunosuppression again,” Dr. Doan says.

She had one last recourse. She knew about Dr. DeRisi and Dr. Wilson’s work with MDS in the encephalitis case. So she sought their help in using MDS on the uveitis patient. “We were able to adapt the technology to analyze the patient’s aqueous fluid,” Dr. Doan says. When she saw the result, she says, “I almost fell off my chair. It clearly showed rubella virus. There was no doubt that it was the organism.” Later, the patient reported he had a full body rash and fever that resolved quickly when he was 17 years old.

The sequencing was done in Dr. DeRisi’s research laboratory. To verify the result in a CLIA-certified facility for clinical diagnostic purposes, they had the California Department of Public Health do PCR for rubella, which validated the MDS finding. Rubella was confirmed also at the Centers for Disease Control and Prevention.
“In neurology and ophthalmology pretty much the first thing to do is to determine whether the patient has an infectious or autoimmune etiology,” Dr. Doan says. “If you make a mistake, the patient can potentially die. This technology is perfect for that situation.” It can not only give a positive diagnosis but also rule out infection. “If we can’t find a pathogen [with MDS], it’s probably not there,” she says. “Then you can feel comfortable to give the patient steroids.”

On the other hand, Dr. Doan calls giving steroids in the face of an undiagnosed infection “a futile treatment.

“It makes inflammation worse. Now I know how to manage this patient. It doesn’t have to be anti-inflammatory drugs or immunosuppression.”

Unfortunately, there is no specific antiviral agent for rubella, as there is for herpes. Supportive treatment would be to control high eye pressure and, most important, to do no harm. The latter principle is not simply anodyne. “This patient was on high-dose prednisone for many months and now has osteoporosis as a result,” Dr. Doan says. Perhaps future patients with uveitis from an infectious agent will not have to be subject to futile therapy.

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