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New DNA tool diagnoses leptospira bacteria infection in brain and saves ill boy

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In numerous areas of science and medicine, researchers are working on a new technique that uses DNA sequencing to diagnose these potentially deadly diseases. Read and listen to this NPR story.

Take a look at this one case, for example. Because of a new type of DNA test to see what species of bacteria is infecting an individual, a 14-year-old boy’s turnaround and quick recovery is the result, according to a June 4, 2014 University of California, San Francisco news release by Jeffrey Norris, "Faster DNA Sleuthing Saves Critically Ill Boy." After mysteriously being stricken by brain-inflaming encephalitis - which led to him being hospitalized for six weeks and put into a medically induced coma after falling critically ill, scientists harnessed the newest generation of DNA analysis tools to reveal the cause of a life-threatening infection even when physicians have no suspects or diagnosis as to what type of bacteria caused the boy's brain to swell, put him in a coma, and make him so ill.

In this recent study, a new tool quickly found the species of bacteria causing the infection. It turned out to be a potentially lethal type of bacteria called Leptospira. and that type of bacteria is readily treated with penicillin. The boy soon received the penicillin, and quickly began to recover as his swelling brain returned to normal. The case, reported on Wednesday in The New England Journal of Medicine, made the New York Times as a news story. Researchers also describe their new strategy in a second paper published on June 5, 2014 in the journal Genome Research.

The quick diagnosis and successful treatment of the adolescent just 48 hours after cerebrospinal spinal fluid and blood were received for analysis portends the broader application of powerful, “next-generation sequencing” (NGS) techniques in solving infectious disease mysteries, not only in cutting-edge research labs, but also in clinical laboratories accessible to hospital physicians everywhere, according to Charles Chiu, MD, PhD, a professor of laboratory medicine at UC San Francisco. Chiu is senior author of the case study, published online in the New England Journal of Medicine (NEJM) on June 4, 2014.

The workflow pipeline developed in Chiu’s UCSF laboratory to streamline genetic sleuthing of disease pathogens with NGS dramatically cut the time between sample collection and actionable diagnosis and helped a medical team at the University of Wisconsin save the young patient’s life.

The New England Journal of Medicine (NEJM) study reflects the convergence of faster DNA sequencing, ever-growing genome databases for identifying pathogens and other organisms, and more sophisticated computational analysis tools to quickly analyze millions of data points. The protocol enabled rapid sequencing and simultaneous identification of all DNA in the patient samples without culturing or targeting for specific infectious disease agents.

“From the perspective of cost and turnaround time, this is a very powerful technology that has become practical to implement routinely in clinical laboratories,” Chiu said, according to the June 4, 2014 news release, "Faster DNA Sleuthing Saves Critically Ill Boy." Some clinical labs now offer NGS testing to identify cancer mutations in clinical trials and to identify mutations underlying birth defects, but until now NGS has been regarded as too slow and laborious to be useful for routine infectious disease diagnosis.

New genomics techniques and tools identify previously unknown pathogens such as the SARS coronavirus

Study co-author Joseph DeRisi, PhD, chair of biochemistry and biophysics at UCSF, a Howard Hughes Medical Institute (HHMI) investigator, and a leader in using new genomics techniques to identify previously unknown pathogens, such as the SARS coronavirus, said that at a cost of a few thousand dollars, essentially any pathogen now can be detected with a single test.

“This is one test to rule them all,” DeRisi said, according to the news release. DeRisi was first contacted by the boy’s physician, James Gern, MD, a professor of pediatrics and medicine at the University of Wisconsin School of Medicine and Public Health in Madison, and a research collaborator on an earlier study. He then enlisted other UCSF scientists working on NGS techniques, including Chiu and a member of DeRisi’s lab, Michael Wilson, MD, an assistant professor of neurology and the first author of the New England Journal of Medicine (NEJM) study.

After the previous weeks of extensive and costly tests and procedures in Wisconsin had failed to point to a cause of illness, application of the UCSF lab group’s powerful NGS protocol finally led to an unmistakable, if unusual, diagnosis

Earlier tests for microbial infection had revealed no culprit. Instead, the boy’s clinical lab results and medical history – he was born with SCID, an immunodeficiency syndrome – had made it appear most likely that an immune response might be responsible for his illness. The Wisconsin physicians had administered corticosteroids, normally an effective treatment for autoimmune encephalitis, but the boy’s condition worsened. He experienced a serious build-up of fluid in the brain and uncontrollable seizures, quelled only after physicians placed him in a coma in an effort to buy time.

Meanwhile, at UCSF, Wilson worked with Chiu’s lab team to generate a library of 10 million distinct DNA sequences from samples of the patient’s cerebrospinal fluid and blood, and from a control sample. The medical researchers used a MiSeq DNA sequencer made by Illumina -- recently approved for use in clinical diagnostic labs by the U.S. Food and Drug Administration -- and sequencing was completed overnight.

The analysis tool is the SURPI

An analysis tool, called SURPI, developed by Chiu’s team and described in a simultaneously published article in Genome Research, allowed the researchers to more quickly compare DNA from patient samples to the GenBank database maintained by the National Center for Biotechnology Information, and to discover that 475 distinct DNA sequences among the 3 million DNA sequences from the patient’s cerebrospinal fluid came from a type of bacteria called Leptospira. Although Leptospira was detected in cerebrospinal fluid, there was no evidence of Leptospira DNA in the patient’s blood or in a negative control sample.

This analysis of DNA sequences required just 96 minutes

A similar analysis conducted with the use of previous generations of computational software on the same hardware platform would have taken 24 hours or more to complete, Chiu said, according to the news release. More specifically, the scientific investigators determined that the boy had been infected by the bacterium Leptospira santarosai, a pathogen native to the Caribbean and warmer climes.

The Wisconsin and UCSF physicians consulted, and based on the strength of the finding and the low risk of antibiotic treatment – penicillin -- the boy’s medical team decided to treat without confirming the diagnosis with a clinically approved test. The treatment eliminated the infection, and the patient was discharged from the intensive care unit three weeks after treatment began. He returned home after inpatient rehabilitation therapy, 76 days after entering the hospital.

No standard clinical test could immediately confirm the diagnosis after treatment began

Staff at the U.S. Centers for Disease Control and Prevention ultimately confirmed the UCSF diagnosis five months after treatment, by modifying their standard tests for Leptospira. In the United States about 20,000 people, mostly children, are hospitalized each year with brain-swelling encephalitis, and more than 4,000 more are hospitalized with meningitis, a swelling of the brain covering.

No cause can be identified in almost half of encephalitis cases, yet as the New England Journal of Medicine (NEJM) case demonstrates, the consequences of being unable to distinguish infectious from autoimmune causes can be serious. The UCSF investigators aim to establish a meningitis and encephalitis center at UCSF Medical Center and UCSF Benioff Children’s Hospital San Francisco, in part to use NGS techniques to more quickly and accurately diagnose potential causes of encephalitis, meningitis and other neuroinflammatory conditions.

Leptospira

In retrospect, it appears that the teenager might have encountered the bacterium during a family stay in Puerto Rico nearly a year before he was hospitalized. He experienced bouts of headache, fever and other symptoms only later. These initially improved, but eventually the boy’s symptoms became more serious, and he was hospitalized three times in four months before the Leptospira discovery.

No NGS test for infectious disease has been validated yet for use in commercial clinical labs or approved for routine diagnosis by regulatory agencies such as the Centers for Medicare & Medicare Services (CMS) or the Food and Drug Administration (FDA), according to Chiu. The experimental diagnostic protocol used in the New England Journal of Medicine (NEJM) case was carried out in a clinical lab according to standard operating procedures common to all regulated laboratories, Chiu said, adding that he expects to obtain approval within a few months to offer an NGS diagnostic test at the UCSF clinical microbiology laboratory for diagnosis of certain types of infectious diseases.

“This case was a test run for how you would validate and deploy a clinically-approved test,” Chiu said, according to the news release. Funding for work described in the New England Journal of Medicine (NEJM) case study was provided by the American Brain Foundation, the National Institutes of Health, HHMI, Abbott Laboratories, Amazon Web Services and the University of California.

UC San Francisco (UCSF), now celebrating the 150th anniversary of its founding, is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care.

It includes top-ranked graduate schools of dentistry, medicine, nursing and pharmacy, a graduate division with nationally renowned programs in basic, biomedical, translational and population sciences, as well as a preeminent biomedical research enterprise and two top-ranked hospitals, UCSF Medical Center and UCSF Benioff Children’s Hospital San Francisco. You also may wish to read more news about this research, in the June 4, 2014 New York Times article by Carl Zimmer, "In a First, Test of DNA Finds Root of Illness."

Another noteworthy study is how lifestyle changes may lengthen telomeres, a measure of cell aging

In 2013, scientists did a small pilot study shows for the first time that changes in diet, exercise, stress management and social support may result in longer telomeres, the parts of chromosomes that affect aging, according to the news release, "Lifestyle Changes May Lengthen Telomeres, A Measure of Cell Aging." It's the first controlled trial to show that any intervention might lengthen telomeres over time. The study appeared online September 16, 2013 in The Lancet Oncology.

Scientists at UC San Francisco and the Preventive Medicine Research Institute, conducted the study. the Preventive Medicine Research institute is a nonprofit public research institute in Sausalito, California that investigates the effect of diet and lifestyle choices on health and disease. The researchers say they hope the results will inspire larger trials to test the validity of the findings.

“Our genes, and our telomeres, are not necessarily our fate,” said lead author Dean Ornish, MD, UCSF clinical professor of medicine, and founder and president of the Preventive Medicine Research Institute, according to the news release. “So often people think ‘Oh, I have bad genes, there’s nothing I can do about it,’” Ornish said in the news release. “But these findings indicate that telomeres may lengthen to the degree that people change how they live. Research indicates that longer telomeres are associated with fewer illnesses and longer life.”

Study of Early-Stage Prostate Cancer Patients

Telomeres are the protective caps on the ends of chromosomes that affect how quickly cells age. They are combinations of DNA and protein that protect the ends of chromosomes and help them remain stable. As they become shorter, and as their structural integrity weakens, the cells age and die quicker.

In recent years, shorter telomeres have become associated with a broad range of aging-related diseases, including many forms of cancer, stroke, vascular dementia, cardiovascular disease, obesity, osteoporosis and diabetes. For five years, the researchers followed 35 men with localized, early-stage prostate cancer to explore the relationship between comprehensive lifestyle changes, and telomere length and telomerase activity. All the men were engaged in active surveillance, which involves closely monitoring a patient’s condition through screening and biopsies.

Ten of the patients embarked on lifestyle changes that included: a plant-based diet (high in fruits, vegetables and unrefined grains, and low in fat and refined carbohydrates); moderate exercise (walking 30 minutes a day, six days a week); stress reduction (gentle yoga-based stretching, breathing, meditation). They also participated in weekly group support.

Lifestyle changes made

They were compared to the other 25 study participants who were not asked to make major lifestyle changes. The group that made the lifestyle changes experienced a “significant” increase in telomere length of approximately 10 percent. Further, the more people changed their behavior by adhering to the recommended lifestyle program, the more dramatic their improvements in telomere length, the scientists learned.

By contrast, the men in the control group who were not asked to alter their lifestyle had measurably shorter telomeres – nearly 3 percent shorter – when the five-year study ended. Telomere length usually decreases over time.

Possibilities for General Population

The researchers say the findings may not be limited to men with prostate cancer, and are likely to be relevant to the general population.“We looked at telomeres in the participants’ blood, not their prostate tissue,” said Ornish, according to the news release.

The 2013 study is a follow up to a similar, three-month pilot investigation in 2008 in which the same participants were asked to follow the same lifestyle program. After three months, the men in the initial study exhibited significantly increased telomerase activity. Telomerase is an enzyme that repairs and lengthens telomeres.

The 2013 study was designed to determine if the lifestyle changes would affect telomere length and telomerase activity in these men over a longer time period.

“This was a breakthrough finding that needs to be confirmed by larger studies,” said co-senior author Peter R. Carroll, MD, MPH, according to the news release. Carroll is a professor and chair of the UCSF Department of Urology. “Telomere shortening increases the risk of a wide variety of chronic diseases,” Carroll said, according to the news release. “We believe that increases in telomere length may help to prevent these conditions and perhaps even lengthen lifespan.”

Other co-authors from UCSF include senior author and Nobel laureate Elizabeth H. Blackburn, PhD, professor of biochemistry and biophysics; Jue Lin, PhD, associate research biochemist; June M. Chan, DSc, associate professor of epidemiology & biostatistics; Elissa Epel, PhD, associate professor of psychiatry; Mark Jesus M. Magbanua, associate specialist; Jennifer Daubenmier and Nancy K. Hills, PhD, associate adjunct professors; and Nita Chainani-Wu, DMD, MPH, PhD, assistant clinical professor.

The research was supported by the U.S. Department of Defense; the National Institute of Health and National Cancer Institute grant number RO1 R01CA101042; Furlotti Family Foundation; Bahna Foundation; DeJoria Foundation; Walton Family Foundation; Resnick Foundation; Greenbaum Foundation; Natwin Foundation; Safeway Foundation; and the Prostate Cancer Foundation.

Jue Lin, Elissa Epel and Elizabeth Blackburn were co-founders of Telome Health Inc., a diagnostic company that assess telomere biology – THI had no relationship to this study. Dean Ornish works with Healthways, Inc. to educate and support people in making healthier behaviors. The other authors declared no conflicts of interest. You may wish to check out the study's abstract that appeared on September 16, 2013 in The Lancet Oncology.

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