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Tuesday, June 30, 2009

The Promise of a Cure: 20 Years and Counting

Science 19 June 2009:
Vol. 324. no. 5934, pp. 1504 - 1507
DOI: 10.1126/science.324_1504

News Focus


The Promise of a Cure: 20 Years and Counting

Jennifer Couzin-Frankel

The discovery of the cystic fibrosis gene brought big hopes for gene-based medicine; although a lot has been achieved over 2 decades, the payoff remains just around the corner.

Figure 1

All grown up. Danny Bessette, a 24-year-old with CF, was 4 years old when he appeared on the cover of Science announcing the discovery of the CF gene.


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The gene hunt began quietly, with few theatrics and much uncertainty.

For Mitch Drumm, the starting gate lifted in the fall of 1985. He and geneticist Francis Collins met on opposite sides of a volleyball net, during a faculty-student mixer at the University of Michigan, Ann Arbor. Drumm, shorter than the lanky Collins, was outmatched in volleyball. But Collins quickly recruited Drumm to join the lab he was setting up, as its first graduate student. There, Drumm began experimenting with a gene-hunting technique Collins had developed. As a test case, they chose cystic fibrosis (CF), an inherited disease in which sticky mucus accumulates in the lungs and elsewhere, eventually killing the patient. At the time, life expectancy hovered in the early 20s.

Coincidentally, CF had been on Drumm's mind. Just months before, the infant son of his family's next-door neighbors, close friends in New Philadelphia, Ohio, had been diagnosed with the disease. Drumm still recalls the phone call from his mother relaying the devastating news. Like many others studying CF, he became immersed in the field by a personal connection, which carried him through ups and downs in the decades ahead. A big triumph came nearly 4 years after signing up with Collins, in the spring of 1989. In collaboration with a large research group in Toronto, Canada, that had started an aggressive chase for the CF gene years earlier, the team cloned the CF gene—called the cystic fibrosis transmembrane conductance regulator (CFTR)—and nailed a crucial, disease-causing mutation (Science, 8 September 1989, pp. 1059, 1066, 1073).

Figure 2


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Everyone in the CF community recalls the electric moment when they heard the news. "I remember seeing it roll off the fax machine, gathering people in the lab, and thinking, ‘What did we need to know’" now? says Michael Welsh, a pulmonary physician at the University of Iowa, Iowa City. Most believed that the disease had grown vastly less complex overnight and would soon be eliminated, probably by gene therapy.

On the 20th anniversary of the identification of the CF gene, as new gene discoveries pile up weekly and hype over the power of genes to transform medicine flows fast, CF offers an object lesson in how difficult it is, and how long it takes, to convert genetic knowledge into treatments. Every CF expert agrees that the gene discovery transformed their understanding of the disease's pathology. But even after so much hard work, not a single therapy based on the CF gene has reached the market. Some promising treatments, especially gene therapy, have proven bitterly disappointing.

"We were naïve," says Johanna Rommens, who at the time was a postdoc in Lap-Chee Tsui's lab at the Hospital for Sick Children in Toronto, the counterpart to Collins's group in Michigan. In her 20s and relatively new to science back then, Rommens couldn't imagine a problem that defied resolution. "I thought I could do anything," she says. "I sometimes feel discouraged that this was so hard." Keen to experiment with other genetic diseases, Rommens subsequently left the CF field.

Although gene therapy hasn't paid off, prospects have improved for those with CF: Their median life expectancy has stretched almost 10 more years and now exceeds 37. This is thanks not to genetic knowledge, however, but to more aggressive and earlier treatment to keep the lungs clear.

Soaring hopes

In October 1989, a month after the CF gene was published in Science, gene therapist James Wilson strode to the speaker's podium at a Florida cystic fibrosis conference to discuss prospects for gene therapy. Thousands of people—physicians, scientists, families—packed the meeting. "I get shivers talking about it right now," says Wilson, who then worked down the hall from Collins at Michigan and is now at the University of Pennsylvania. "The excitement was palpable. I have never felt energy like that ever before."

There was broad consensus that the time for CF gene therapy had arrived. Two advances buoyed hopes that this new technique, still in its infancy, would eliminate CF. First, scientists had managed to "cure" the disease in test tubes. They introduced a normal version of CFTR into cells from CF patients, compensating for a defective gene that produced no protein, or none the cell could use. In addition another researcher, W. French Anderson, then at the National Institutes of Health, began the first-ever clinical trial of gene therapy to treat an immune deficiency syndrome, demonstrating that gene transfer in people was feasible. By then, in the fall of 1990, says Wilson, "expectations for the success of gene therapy for CF were as high as I've ever seen for any disease, under any circumstances, in the 20 years I've been involved in this."

Figure 3

Then and now. Geneticist Mitch Drumm (inset, right) worked with Francis Collins (inset, left) in the 1980s and helped identify the CF gene. He remains in the CF field today (main picture).


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Looking back, many CF experts consider an excessive focus on gene therapy in the early years to have been a big mistake. Like an investor who gambles much of his or her fortune on a single stock, "people kind of stopped doing the other things they were doing" and turned instead to strategies for getting the gene into lung cells, says Raymond Frizzell, a physiologist at the University of Pittsburgh in Pennsylvania.

Early successes in basic CF research also convinced scientists that new treatments were right around the corner. In addition to correcting the gene defect in cells, having CFTR in hand revealed that the healthy CFTR protein was an ion channel that helped transport chloride and control the movement of water across cell membranes. Researchers determined that several mutations led to a protein-folding defect—one of the first protein-folding diseases identified. Then in 1992, Welsh and his colleagues published a paper in Nature explaining that the protein-folding problem could be corrected by chilling the cells. Although this tactic wasn't useable in patients, it allowed "people to say, ‘It's misfolded but you can overcome that,’" Welsh says.

Even with these heartening advances in the early 1990s, there were hints that choppy waters lay ahead. The CF protein was a bear to work with because it didn't respond well to classic analytical tools like Western blots and antibody assays, recalls Margarida Amaral, now on sabbatical at the European Molecular Biology Laboratory in Heidelberg, Germany, who worked on the protein at the University of Lisbon in Portugal. "Nothing seemed to work."

In Wilson's lab, meanwhile, postdoc John Engelhardt, now at the University of Iowa, was running into difficulties getting gene therapy to work. The gene's expression varied wildly depending on which parts of the lung researchers examined. One great appeal of gene therapy for CF was that a vector carrying a working CF gene could easily be introduced into the lung with aerosols. But Engelhardt found that only about 1% of cells lining the lung's airway—the cells that come into contact with aerosols—boasted high levels of CFTR protein.

Each advance provoked more questions. When Richard Boucher, an adult pulmonologist at the University of North Carolina, Chapel Hill, won a three-way race to create the first mouse model of CF in 1992, he and others were dismayed to find that the rodents didn't mimic human disease. They shared the gut afflictions of CF patients, who must take pancreatic enzymes for life to break down thick secretions. But the lungs of CF mice, unlike those of their human counterparts, were healthy.

Like Drumm, Boucher traces his passion for CF to a personal experience: His daughter suffered several bouts of pneumonia as a baby and was suspected of having CF. Panicked, he read up on the disease; this drew him to a career teasing apart its mysteries. The CF mouse was a big one: Why were its lungs clear? Boucher and others determined that the animals had a second chloride channel that was unaffected by CF. This led to a new understanding of how the airway surfaces stayed healthy: As long as chloride could pass through, the lungs fared well. But in other ways, the mouse proved of virtually no value. It would be 15 years before other researchers found a better animal model.

Meanwhile, gene therapy plowed ahead. In 1993, a 23-year-old became the first CF patient to receive a dose of healthy CFTR by gene transfer. Three small clinical trials began: one led by Wilson, one by Welsh, and one by Ronald Crystal at Weill Cornell Medical College in New York City. "Boy, there were all kinds of issues," Wilson recalls. Among them: potent fears that the virus carrying healthy CFTR into a patient's nasal passages or lungs would recombine with another virus, be shed by that patient, and "create an environmental catastrophe." Volunteers in the trials were put in strict isolation.

The bigger issue, as it turned out, was that gene therapy simply didn't work. Few lung cells took up the gene. There were also concerns about inflammation, as the lung rebelled against a viral intruder. "You had to confront the reality of eons of evolution" that had built barriers against toxins and infections, says Boucher, who also worked in CF gene therapy. Researchers in the United States spent several more years trying to get around this, tinkering with gene therapy in baboons, rhesus macaques, and other animals, before largely giving up.

Shifting gears

Although the trials failed to help CF patients, they mattered to clinical research: For the first time, viral vectors were injected directly into a patient (as opposed to affected cells being removed, modified, and reinfused), and this became the new model for a nascent specialty. The CF trials also underscored the problem of immune reactions to treatment, which hadn't previously been appreciated, says Wilson.

In a funny way, "science has benefited more from the CF gene than CF has benefited from the science," says John Riordan, a biochemist and, with Tsui and Collins, one of the co-discoverers of the CF gene when he worked at the Hospital for Sick Children. Now at the University of North Carolina, Chapel Hill, Riordan never thought he'd still be working on CF 20 years later. But he points out that CFTR, which belongs to a large family of membrane proteins, is unusual, using several different mechanisms to carry out its functions. As the years passed, biologists studying CFTR learned much about how chloride is transported across cells, and that the protein may also influence inflammation, cell signaling, and other processes. They found that cells build complexes of CFTR and other proteins to keep the system humming.

But what about a cure for this genetic disease, for which there'd been such high hopes? By 1998 or so, researchers knew far more about CF than they had 10 years before. They knew, for example, that hundreds of different mutations in CFTR could cause the disease and that not all disabled the protein in the same way—suggesting that different treatments might be needed for different patients. They knew, too, that CFTR couldn't explain everything. Some severely affected 12-year-olds needed lung transplants, and some 28-year-olds were running marathons—even when the quirk in their CFTR gene was identical. This led researchers to consider that other genes also play a role in CF, as do environmental factors.

None of this was quickly leading to new treatments. "1997, 1998 was really the point where we said, ‘Academics are great, but if we really want to discover drugs, we've got to become more businesslike,’" says Robert Beall, president and CEO of the Cystic Fibrosis Foundation. The CF Foundation had been instrumental in funding the gene hunt and subsequent CF research, raising and investing tens of millions of dollars. In the late 1990s, Beall began shopping around plans to develop small-molecule, traditional drugs—back to basics after gene therapy had failed.

"A lot of people thought that Bob Beall was going far out on a limb to put a lot of money into a strategy that was clearly risky," says Collins. Multiple drugs might be needed to tackle different CFTR defects. Companies apparently were wary, too. Beall telephoned seven; two called back. One was Aurora Biosciences in San Diego, California, which was bought by Vertex Pharmaceuticals in 2001. It agreed, with generous support from the CF Foundation, to see what it could do.

Progress at last

More than $75 million and another 10 years later, two Vertex drugs are taking the CF world by storm. One, VX-809, is designed for the most common CF mutation and helps CFTR get to the surface of the cell. Only safety data are available on VX-809 so far.

The other Vertex drug, VX-770, aims to boost the function of CFTR protein that's already made its way to the cell surface—which would help in at least one of the CF mutations, accounting for a few percent of cases of the disease. Last October, Vertex reported that in a phase II trial, lung function of volunteers improved by 12% in 4 weeks of treatment. "That's more than any drug ever improved the disease" in any span of time, says Beall. The excitement around Vertex is so great that at a recent CF fundraising walk, organizers gave to Vertex employees the bib numbers 770 and 809, says Paul Negulescu, a vice president of research at the company's San Diego office. And everyone knew what those numbers meant.

The CF field has enjoyed other recent breakthroughs. In September 2008, Welsh and his colleagues described a CF pig model in Science—the first animal model that closely resembles human CF. More recently at Iowa, Engelhardt, who worked in Wilson's lab in the old days and also collaborated on the pig, has developed a CF ferret, the culmination of 10 years' work. (The group spent more than 2 years just trying to understand ferret lung biology.) "Not having good animal models has really slowed the field down," says Engelhardt. Therapies can have harmful side effects, so "when you think about treating a kid before they have overt disease, you've got to be pretty sure you've got a great treatment." Testing in animals offers some reassurance.

Gene therapy, too, is experiencing a resurgence. In the United Kingdom, a team of 80 investigators is launching a 100-person trial using fat particles—unlike the viral vectors in earlier U.S. studies—to carry CFTR to cells. The U.K.'s Cystic Fibrosis Trust has dedicated considerable effort, and $50 million, to gene therapy. "Someone needs to find out" if this works, says Eric Alton, a gene therapist at Imperial College London who's heading up the trial, which he hopes will reveal how distant the goal is. "We're either sitting on the therapy, or we're a million miles away from it." Despite earlier setbacks, Alton still feels that gene therapy offers more hope than drugs, because in theory at least, it's more comprehensive. Researchers have found at least 20 functions for the CFTR protein. A drug can correct only one or two at once—whereas gene transfer, if it works, can do it all. Results fromAlton's trial, which is just beginning, will come in 2012.

There have been other developments: Prenatal testing is increasingly offered to couples contemplating pregnancy, potentially reducing the number of babies born with CF, although figures are difficult to come by. Forty-seven states and many countries now test newborns for CF, enabling treatment to start right away rather than months or years later, when a child fails to thrive.

Humbling science

Many CF experts say that, after 20 long, frustrating years, it's possible now, finally, to look patients in the eye and assure them that in a few years, treatment will be vastly improved. And patients are optimistic, too. "I can't imagine where we're going to be in another 25 years if it's not cured," says Ryan Ress, a 24-year-old with CF. Ress was Drumm's infant next-door neighbor who inspired the geneticist, now at Case Western Reserve University in Cleveland, Ohio, to stick with CF, and the two remain in touch. Ress majored in biochemistry in college and spent a summer working in a CF lab next door to Drumm's. He's now studying to be a neonatal nurse practitioner. Ress is convinced that CF will be conquered, based on his reading of the disease literature and the belief that "there will be a reward" for CF researchers for their backbreaking years of work.

Figure 4

Needle in a haystack. Capsules, each containing DNA that might harbor the CF gene, filled the freezer in Lap-Chee Tsui's lab in Toronto.


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But with lessons learned the hard way, caution abounds, too: "We have miles to go before we sleep," says Paul Quinton, a physiologist at the University of California, San Diego. Quinton is a rare bird. At 20, in college and thinking about his own mortality, he says, he began combing through textbooks in his campus library, hunting for an explanation for the abdominal troubles, chronic cough, and lung problems that had plagued him for years. In books he found an answer: CF. Soon after, Quinton abandoned his dream to become a poet and turned to understanding his disease. In 1983, he determined that chloride transport was the fundamental defect in CF, one of the biggest breakthroughs in the field.

Quinton learned via genetic testing that he harbors one severe mutation and one that's milder, a combination that may explain why he's survived as long as he has. Now 64, he admits that he was as optimistic as the next person when the CF gene was found, even declaring in an editorial in Nature that the chance to cure CF had become reality. These days, though, he sees questions everywhere. How, exactly, does normal CFTR function? How does the absence of CFTR lead to the thick mucus of CF? Will even today's most promising drugs work in more than a very narrow slice of patients?

The case of CF, agrees Amaral, is "a lesson in being humble in science."

What does this mean for the flood of genes identified in the years since—both for single-gene diseases and more complex ailments? One shouldn't generalize from the CF story, says the irrepressibly optimistic Collins, former director of the National Human Genome Research Institute, because every disease is different. He knows of at least one—progeria, which causes accelerated aging—in which a gene he helped identify led to a drug within 5 years that's now being tested in nearly every child with the disease.

Collins's early competitor and later collaborator in the CF gene hunt, Tsui, treads more carefully. "Because of the excitement, some scientists, perhaps even disease funding agencies, ... wanted to give people hope, or give themselves hope," says Tsui, who in 2002 left Toronto to become vice-chancellor and president of the University of Hong Kong. "They were a little bit optimistic at predicting when a cure would be there. ... [It] taught a lesson to other gene researchers." Namely: don't spin prophecies, don't assume that the gene is the end of the story. Rather, it's just the end of the beginning, with a long road still ahead.

Exercise programs for children with cystic fibrosis: A systematic review of randomized controlled trials.

Exercise programs for children with cystic fibrosis: A systematic review of randomized controlled trials.

van Doorn N.

Department of Exercise and Sports Science, University of Sydney, Sydney, Australia.

Purpose. The aim of this article was to review the evidence for using exercise programs to improve pulmonary function and fitness in children with cystic fibrosis (CF). Method. Electronic databases - Medline, SPORTDiscus, CINAHL, AMED, EMBASE, and Cochrane Central Register of Controlled Trials were searched with terms 'CF' and keywords related to 'exercise'. These papers were analysed for study quality, participant details, exercise intervention details, and outcomes on pulmonary function and fitness components. Results. Only four randomized controlled trials (RCTs) met the eligibility criteria for review. These detailed exercise interventions cover short- and long-term duration. Modalities consisted of aerobic, strength, and anaerobic interventions. Severity of CF ranged from mild to severe. Significant improvement in pulmonary function was seen from short-term in-hospital aerobic or strength interventions. Significant strength gains were seen from strength training interventions. Aerobic fitness was shown to improve with short-term aerobic training. Conclusions. There is some evidence to support that both aerobic and strength training can impact positively on pulmonary function, aerobic fitness, and strength. More RCTs in this area would be welcomed. There is potential for future research into designing exercise programs for children with CF using a combination of modalities.

PMID: 19562582 [PubMed - as supplied by publisher]

CFF Shout Out

Funding From an Unlikely Place

The credit crunch and bear market have made it tough on drug developers. Gone is the easy money of secondary offerings and loans using the company's intellectual property as collateral. But the sources of cash aren't completely dried up; nonprofits still have some, and they're willing to fork it over to for-profit drug companies.

Earlier this month, the Global Alliance for TB Drug Development agreed to share development costs of Johnson & Johnson's (NYSE: JNJ) multidrug-resistant tuberculosis drug, TMC207. Early indications from a phase 2 trial suggest that the drug is working well, and it could become the first drug in 40 years to be developed for TB using a new method of action.

In exchange for its investment, the TB Alliance gets a royalty-free license to TMC207 in developing countries. Johnson & Johnson probably wouldn't be able to make much money selling the drug in developing countries where the incidence of TB is the highest, so the health-care giant isn't giving up much in exchange for a little help with development.

Vertex Pharmaceuticals (Nasdaq: VRTX) took a slightly different approach with its partnerships with the Cystic Fibrosis Foundation. In exchange for $79 million, the company gave the nonprofit royalties on VX-770, should it make it to market. Vertex is almost certainly spending a lot more of its own money on the development of the drug, but I imagine the royalties are in the low single digits for that kind of cash.

Generally speaking, the funding comes for disease, where nonprofit money is needed to help make the drug research a financial success. There is, however, some money available for drugs that could become blockbusters, but that need a helping hand because they might be scientific long shots. For example, the Juvenile Diabetes Research Foundation has funded programs at Biogen Idec (Nasdaq: BIIB) and others that seek to reverse the effects of diabetes by regenerating insulin-producing cells.

Seal of approval?
If a nonprofit is willing to throw its hard-earned donations to fund a drug's development, it's easy to think that the potential for success is higher, but investors need to be careful not to read too much into these partnerships.

Sure, the nonprofits have likely had experts look at the data before committing money to the drug development, but that doesn't ensure that the drug will succeed. The partnership is an endorsement of the potential for the drug, not much different than GlaxoSmithKline (NYSE: GSK), sanofi-aventis (NYSE: SNY), or Bristol-Myers Squibb (NYSE: BMY) licensing a drug from Exelixis (Nasdaq: EXEL); the company or nonprofit is saying that the drug is worth some amount of money at this point in development, but sharing development risk doesn't make it more likely that the drug will get through the clinical-trial marathon.

Foolish takeaway
There are two investment takeaways from nonprofits funding drug development. The first is that big pharma may have lots of cash, but they don't have all of it. While the credit crunch over the past year has taken its toll on development-stage drugmakers, there's an alternative source of funds available. For fledgling companies that might be lucky enough to be developing a drug for a disease with an associated nonprofit to support it, those funds could help the company fight off a takeover by a bigger company at fire-sale prices.

The second related takeaway is that investors shouldn't take the funding too seriously. It's a nice input of funds that takes some of the development risk away from the for-profit company, but investors should treat these cash infusions the same way they would a drug development deal with another for-profit company.


Saturday, June 27, 2009

Glutathione exhibits antibacterial activity and increases tetracycline efficacy against Pseudomonas aeruginosa

Thanks so much to dasjsmum for providing us with the link to the full article! :)


YaNi Zhang1 and KangMin Duan1 Contact Information

(1) Molecular Microbiology Laboratory, the College of Life Sciences, Northwest University, Xi’an, 710069, China

Received: 5 February 2009 Accepted: 5 March 2009 Published online: 26 June 2009

Abstract Glutathione (GSH) plays important roles in pulmonary diseases, and inhaled GSH therapy has been used to treat cystic fibrosis (CF) patients in clinical trials. The results in this report revealed that GSH altered the sensitivity of Pseudomonas aeruginosa to different antibiotics through pathways unrelated to the oxidative stress as generally perceived. In addition, GSH and its oxidized form inhibited the growth of P. aeruginosa.

Keywords glutathione - antibiotics - Pseudomonas aeruginosa - tetracycline - cystic fibrosis

Supported by the National Natural Science Foundation of China (Grant Nos. 30870097 and 30611120520)


*I wish I had the full text of this article. Contact your CF Center or Pulmo to see if they subscribe to this journal*

Friday, June 26, 2009

Transave Receives $2.2 Million Cystic Fibrosis Foundation Award for Development of ARIKACE

MONMOUTH JUNCTION, N.J., June 25 /PRNewswire/ -- Cystic Fibrosis Foundation Therapeutics Inc., the nonprofit affiliate of the Cystic Fibrosis Foundation, has provided a $2.2 million award to Transave Inc., for the continued development of ARIKACE(TM) (liposomal amikacin for inhalation), an antibiotic that aims to treat Pseudomonas lung infections in cystic fibrosis (CF) and non-CF bronchiectasis patients. Transave announced today that the award will be used to support an ongoing Phase II clinical trial in U.S. CF patients with Pseudomonas lung infections and a preclinical study. The company also reported today completion of enrollment for its second CF Phase II clinical trial; results are expected to be available in July. The first CF Phase II trial, completed in June 2008, achieved positive results.

In other developments, the company announced that it was granted a patent for ARIKACE by the U.S. Patent and Trademark Office. U.S. Patent No. 7,544,369 entitled "Sustained Release of Antiinfectives" covers ARIKACE for the once-daily treatment of Pseudomonas lung infections.

"We have followed and supported Transave's work in this area from the beginning, and we are excited to play a role in potentially bringing another new treatment option to CF patients," said Robert J. Beall, Ph.D., President and CEO of the Cystic Fibrosis Foundation. "The potential for ARIKACE is encouraging, both in terms of possible lung function improvements and quality of life outcomes."

Under the terms of the agreement, Transave stands to receive $2.2 million upon achievement of certain milestones. Cystic Fibrosis Foundation Therapeutics provided a previous award of $1.7 million to support the development of ARIKACE.

"We believe ARIKACE -- a once-a-day treatment with sustained effect -- can be a real breakthrough in CF patients' arduous treatment burden , and are looking forward to seeing the Phase II results and moving to Phase III soon afterward," said Tim Whitten, Transave's President and Chief Executive Officer. "We are grateful for the continued support of the CF Foundation as we strive to improve treatment options for CF patients."

The double-blind, 3 cohort, placebo-controlled study is designed to evaluate ARIKACE in the treatment of CF patients with Pseudomonas lung infections. A total of 46 patients have been randomized. ARIKACE or placebo are administered once daily for 28 days using an eFlow(R) Nebulizer System (PARI Pharma GmbH), a novel, highly efficient, portable aerosol delivery system. Eighteen clinical sites throughout the United States are participating in the study.

Thursday, June 25, 2009

Aquagenic palmar wrinkling as a presenting feature of cystic fibrosis gene dysfunction.

Aquagenic palmar wrinkling as a presenting feature of cystic fibrosis gene dysfunction.

Stewart LC, Doe SJ, Bourke SJ, Leech S.

Department of Dermatology, Royal Victoria Infirmary, Newcastle upon Tyne, Newcastle, UK.

Aquagenic palmar wrinkling (APW) is characterized by the rapid and transient oedematous wrinkling of the palms after brief immersion in water. APW has been associated with cystic fibrosis (CF). Since the discovery of the CF gene, the clinical spectrum of CF has broadened from classic severe CF to include milder 'atypical CF' and 'CF-related disorders'. We report an unusual case in which APW occurred in a patient with no lung disease, and in whom investigations showed evidence of CF gene dysfunction. APW may be a presenting feature of a CF-related disorder and should prompt investigation of CF gene dysfunction.

PMID: 19548945 [PubMed - as supplied by publisher]

Cancer risk among patients with cystic fibrosis and their first degree relatives.

Cancer risk among patients with cystic fibrosis and their first degree relatives.

Johannesson M, Askling J, Montgomery SM, Ekbom A, Bahmanyar S.

Department of Public Health and Caring Sciences; Family Medicine and Clinical Epidemiology Section, Uppsala University, Uppsala, Sweden.

Patients with cystic fibrosis (CF) are at increased risk of some cancers. Little is known about the cancer risks among carriers heterozygous for the CF mutation and it is hypothesized this may be associated with reduced cancer risk.

Using Swedish general population-based registers, we identified 884 patients with CF from 1968 to 2003 and 3,033 of their first degree relatives The subjects were followed from birth of index persons or 1958, whichever came later, until death, emigration or 2003, whichever came first. Cancer risks were compared with the general Swedish population using standardized incidence ratios (SIR) with 95% confidence intervals (CI). Patients, followed for an average of 21 years, were at a higher overall risk of cancer. Some 26 cancer diagnoses, after excluding multiple diagnoses of non-melanoma skin cancer in one man, produced an overall SIR of 3.2 (95% CI 2.1-4.6).

We found statistically significantly increased risks for kidney, thyroid, endocrine, lymphoma and non-melanoma skin cancer. There was no modification of cancer risk among parents and siblings, with an average of 21 years of follow-up. This study did not identify a heterozygote advantage for CF gene mutations in relation to cancer risk. (c) 2009 UICC.

PMID: 19551859 [PubMed - as supplied by publisher]

Saturday, June 20, 2009

DHA Clinical Trial

Oral Docosahexanoic Acid Supplementation in Cystic Fibrosis
This study is not yet open for participant recruitment.
Verified by Vanderbilt University, June 2009
First Received: June 17, 2009 No Changes Posted
Sponsored by: Vanderbilt University
Information provided by: Vanderbilt University
ClinicalTrials.gov Identifier: NCT00924547

Oral supplementation of patients affected by cystic fibrosis with docosahexanoic acid (DHA) will result in normalization of the known fatty acid derangements in these patients and will diminish the production of proinflammatory isoprostanes such as 8-isoprostane-PGF2α.

Condition Intervention Phase
Cystic Fibrosis
Dietary Supplement: Docosahexanoic Acid Supplement
Dietary Supplement: Placebo
Phase II

Study Type: Interventional
Study Design: Treatment, Randomized, Double Blind (Subject, Caregiver, Investigator, Outcomes Assessor), Placebo Control, Crossover Assignment, Safety/Efficacy Study
Official Title: Oral Docosahexanoic Acid Supplementation in Cystic Fibrosis: Effects on Exhaled Pro-Inflammatory Isoprostanes and Analysis of Its Esterification Sites in Plasma

Resource links provided by NLM:

Further study details as provided by Vanderbilt University:

Primary Outcome Measures:
  • Exhaled breath 8-isoprostane-PGFα [ Time Frame: 4 measurements: baseline, 4 weeks, after a 2-4 week washout, and 4 weeks later ] [ Designated as safety issue: No ]

Secondary Outcome Measures:
  • Fatty acid profile analysis including esterification sites in plasma [ Time Frame: 4 measurements - Baseline, 4 weeks, 2-4 weeks later, and 4 weeks after that ] [ Designated as safety issue: No ]

Estimated Enrollment: 18
Study Start Date: July 2009
Estimated Study Completion Date: April 2010
Estimated Primary Completion Date: April 2010 (Final data collection date for primary outcome measure)
Arms Assigned Interventions
Docosahexanoic Acid Supplement: Experimental Dietary Supplement: Docosahexanoic Acid Supplement

The active treatment will consist of Martek's chewable DHA capsules containing 200mg in each capsule. The treatment will be provided as approximately 35mg/kg/day. The dosing is as follows:

Patient weight (kg) 35mg/kg/day dose 20-24kg: 800mg 25-34kg: 1000mg 35-44kg: 1400mg 45-54kg: 1800mg 55-64kg: 2200mg 65-74kg: 2400mg 75+kg: 2600mg

These dosages will be divided BID-TID and will be given for 4 weeks.

Placebo: Placebo Comparator Dietary Supplement: Placebo
Placebo identical to active treatment.

Detailed Description:

The study design will be a single-center, randomized, placebo-controlled, cross-over trial. After informed consent has been obtained, 18 eligible subjects with pancreatic insufficient cystic fibrosis will be enrolled in the study. Participants will take part in two 4 week study sessions, each separated by a 2-4 week washout period. One session will involve treatment with placebo and one will provide treatment with approximately 35 mg of DHA/kg of body weight (maximum dose 3 g/day. The patients will be assigned to each of the treatment sessions in random order, as described above. The DHA source will be provided by Martek Biosciences Corporation, Columbia, MD, USA in the form of a chewable capsule containing 200 mg of DHA. The placebos will be identical to the DHA supplement but will not contain the active ingredient, DHA. Subjects will be instructed to take the study capsules in addition to their normal doses of pancreatic enzymes with meals and to maintain their usual diets. Blood, urine, and exhaled breath condensate samples will be collected at baseline, after completion of the first session, after the washout period, and after the completion of the second session. Patients will be screened and enrolled when they present to clinic for their routine check-up. The subjects have routine blood work at their annual check-ups, and when possible will have an additional tube of blood saved for the baseline fatty acid profiles so as to avoid unnecessary blood draws. The patients will be scheduled for a visit to the CRC to complete the baseline data gathering, submit a urine sample, and complete an exhaled breath collection (EBC).

The subjects will complete a clinical questionnaire administered by the study PI to evaluate their health and diet. The patients will also be given the supply of DHA of placebo for their first session at that Visit 1. The order in which they take the supplement or the placebo will be determined using a randomization table provided by Li Wang, the biostatistician working with this study.


Ages Eligible for Study: 6 Years and older
Genders Eligible for Study: Both
Accepts Healthy Volunteers: No

Inclusion Criteria:

  • Diagnosis of Cystic Fibrosis based on sweat chloride value > 60 mEq/L or genotyping
  • Pancreatic insufficiency, defined by requirement for treatment with exogenous pancreatic enzymes
  • FEV 1 > 40
  • Less than 3 pulmonary exacerbations in the last year (as diagnosed by pulmonary attending physician)
  • Age greater than 6 years
  • Capability of performing pulmonary function tests
  • Ability to swallow gel capsule
  • Ability to comply with medication use, study visits, and study procedures
  • Written informed consent obtained from subject or study subject's legal representative

Exclusion Criteria:

  • Presence of severe CF-related liver disease, including SGOT or SGPT>3 times the normal limits, history of biliary cirrhosis, or portal hypertension
  • Severe pulmonary disease, as defined by FEV1 <>
  • Elevated serum creatinine or BUN
  • Pregnancy
  • PT >1.5 time normal
  • Diabetes mellitus
  • Daily use of NSAIDs or other anticoagulants
  • History of fish allergy
  • Use of ticlopidine, clopidogrel, dipyridamole
  • Use of glucocorticoids
  • History of lung transplant or currently on lung transplantation list
  • Presence of a condition or abnormality that in the opinion of the investigator would compromise the safety of the subject or the quality of the data
Contacts and Locations
Please refer to this study by its ClinicalTrials.gov identifier: NCT00924547

Contact: Kelly F King, MD 615 322 7449 kelly.king@vanderbilt.edu

United States, Tennessee
Vanderbilt University Medical Center
Nashville, Tennessee, United States, 37232
Sponsors and Collaborators
Vanderbilt University
Principal Investigator: Kelly F King, MD Vanderbilt University
More Information
No publications provided

Responsible Party: Vanderbilt University Medical Center ( Kelly Fair King, MD )
Study ID Numbers: 081363
Study First Received: June 17, 2009
Last Updated: June 17, 2009
ClinicalTrials.gov Identifier: NCT00924547 History of Changes
Health Authority: United States: Institutional Review Board

Keywords provided by Vanderbilt University:
Cystic Fibrosis
Docosahexanoic Acid
fatty acid

Study placed in the following topic categories:
Digestive System Diseases
Genetic Diseases, Inborn
Respiratory Tract Diseases
Cystic Fibrosis
Lung Diseases
Infant, Newborn, Diseases
Pancreatic Diseases

Additional relevant MeSH terms:
Pathologic Processes
Digestive System Diseases
Genetic Diseases, Inborn
Respiratory Tract Diseases
Lung Diseases
Infant, Newborn, Diseases
Pancreatic Diseases
Cystic Fibrosis

ClinicalTrials.gov processed this record on June 19, 2009


Hepatobiliary Abnormalities and Disease in Cystic Fibrosis: Epidemiology and Outcomes Through Adulthood.

Hepatobiliary Abnormalities and Disease in Cystic Fibrosis: Epidemiology and Outcomes Through Adulthood.

Bhardwaj S, Canlas K, Kahi C, Temkit M, Molleston J, Ober M, Howenstine M, Kwo PY.

*Division of Gastroenterology/Hepatology, Departments of Medicine daggerBiostatistics double daggerDivision of Pediatric Gastroenterology section signDivision of Pulmonary Medicine and Critical Care parallelDivision of Pediatric Pulmonary Medicine, Indiana University Medical Center, Indianapolis, Indiana.

OBJECTIVES: There is limited data regarding the prevalence of hepatobiliary disease in North American patients with cystic fibrosis (CF) through adulthood. Our aim was to determine the prevalence of, and risk factors for, CF-related hepatobiliary abnormalities and determine factors that predict the development of CF-related hepatobiliary disease.

METHODS: We performed a retrospective cohort study of all CF patients who presented to a UnitedStates tertiary care referral academic center over a 32-year period. "CF-related hepatobiliary abnormality" was defined as the presence of abnormal liver chemistries on one or more occasion and "CF-related hepatobiliary disease" was defined as biochemical, physical examination, or ultrasonographic abnormalities on at least 2 consecutive examinations spanning a 1-year period.

RESULTS: Two-hundred eighty-three CF patients who presented between the years 1970 and 2002 were identified, with an age range of 2 months to 63 years. Sixty-five percent had CF-related hepatobiliary abnormalities with a higher prevalence seen in CF patients <18 years of age (84% vs. 16%, P<0.01). Fifteen percent of our cohort had CF-related hepatobiliary disease with 93% of cases occurring in individuals before age 18. One quarter of individuals with CF-related hepatobiliary abnormalities developed hepatobiliary disease.

CONCLUSIONS: Abnormal liver chemistries in CF are common though most of CF patients lack clinical evidence of liver disease and the severe complications of fibrosis/cirrhosis are rare. The risk of liver involvement decreases significantly with age, falling by 10% per annum for those described as having CF-related hepatobiliary disease. CF-related hepatobiliary disease is a rare occurrence after age 18.

PMID: 19525864 [PubMed - as supplied by publisher]

Thursday, June 18, 2009

Scientists after Chopin's heart

DNA debate: The famous Polish composer may have died from cystic fibrosis, not tuberculosis.

Associated Press
Published on: 07/26/08

Warsaw, Poland —- Like a religious relic, the heart of composer Frederic Chopin rests in a Warsaw church, untouched since it was preserved in alcohol after his death in 1849 at age 39.

And that's how the Polish government wants to keep it.

Scientists want to remove the heart for DNA tests to see whether Chopin actually died from cystic fibrosis and not tuberculosis as his death certificate stated.

The heart lies in a jar sealed inside a pillar at Warsaw's Holy Cross Church —- and the only time it has been removed was for safekeeping during World War II.

Before it was returned in 1951, a doctor examined the heart and found it perfectly preserved in an alcohol that many think is cognac. Chopin died in France, where his body is buried, but he asked that his heart be sent to his homeland.

Cystic fibrosis, an incurable genetic disease, was not discovered until decades after Chopin's death, and the scientists who want to examine the heart say many of his symptoms match that illness.

A spokeswoman for the Culture Ministry, Iwona Radziszewska, said ministry officials consulted experts and decided "this was neither the time to give approval, nor was it justified by the potential knowledge to be gained."

Grzegorz Michalski, head of the National Frederic Chopin Institute, argued the scientists failed to demonstrate that they had sufficient expertise carrying out such DNA tests or that the chances of success were high.

Geneticist Michal Witt acknowledged that DNA testing might not prove whether Chopin was afflicted with cystic fibrosis, because it's uncertain what condition the heart is in after so many years in alcohol.

Chopin from an early age suffered frail health and nasal and lung infections typical of cystic fibrosis.

In a paper in the Journal of Applied Genetics, Witt cited other symptoms: At age 22, Chopin complained facial hair wouldn't grow on one side of his face, a sign of delayed puberty. He never fathered any children despite sexual relations with several women.

Though Chopin's death certificate says he died of "tuberculosis of the lungs and the larynx," the doctor who treated him, Jean Cruveilhier, said the death was caused "by a disease not previously encountered."

Witt believes it is of more than just academic interest to investigate whether Chopin died of cystic fibrosis.

"It matters for those who are affected with cystic fibrosis," he said. "Can you believe what message you send saying that you might become a genius even if you have a disorder like that?"

Efficacy of Aerosol MP-376 (Levofloxacin Inhalation Solution) in Mouse Lung Infection Models due to Pseudomonas aeruginosa.

Efficacy of Aerosol MP-376 (Levofloxacin Inhalation Solution) in Mouse Lung Infection Models due to Pseudomonas aeruginosa.

Sabet M, Miller CE, Nolan TG, Senekeo-Effenberger K, Dudley MN, Griffith DC.

Mpex Pharmaceuticals, Inc., San Diego, CA.

Progressive respiratory failure due to Pseudomonas aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis patients. Pulmonary delivery of anti-microbial agents provides high concentrations of drug directly at the site of infection and attains PK-PD indices exceeding those which can be achieved with systemic dosing.

MP-376 is a new formulation of levofloxacin that enables the safe aerosol delivery of high concentrations of drug to pulmonary tissues. In vivo studies were conducted to demonstrate the efficacy of MP-376 in mouse models of pulmonary infection. The superiority of aerosol dosing over systemic dosing was demonstrated in both acute and chronic lung infection models. In an acute lung infection model, treatment with single or twice daily dosing of aerosolized MP-376 reduced the lung bacterial load to a greater extent than aerosolized tobramycin or aztreonam at similar or higher doses.

The bacterial killing observed in the lung with aerosolized MP-376 in the acute pulmonary infection model translated to improved survival (p<0.05). style="font-weight: bold; color: rgb(255, 0, 0);">In a chronic pulmonary infection model, aerosolized MP-376 had superior antimicrobial effects compared to aztreonam (p<0.05)>0.05).

In summary, these data show in vivo activity of aerosol MP-376 in the treatment of acute and chronic lung infections caused by P. aeruginosa.

Tuesday, June 16, 2009

Pharmaxis' Bronchitol inhaler has encouraging Phase 3 trial results for cystic fibrosis patients

Pharmaxis' Bronchitol inhaler has encouraging Phase 3 trial results for cystic fibrosis patients

company news image

Australian listed pharmaceutical company Pharmaxis (ASX:PXS, Nasdaq:PXSL) has received encourgaging results for its recently completed international Phase III trial of the company's Bronchitol portable inhaler in patients with cystic fibrosis.

Bronchitol has received Orphan Drug Designation and fast track status for cystic fibrosis from the U.S. Food and Drug Administration and Orphan Drug Designation from the European Medicines Agency.

The trial was a multi-centre, randomised, double blind, placebo controlled, 26 week study, with an optional further 6 month open label uncontrolled period. It was conducted in 40 centres in the United Kingdom, Ireland, Australia and New Zealand.

The primary endpoint of the trial was to assess whether Bronchitol improves lung function as measured by a change in FEV when administered twice per day for six months.

The key secondary endpoint of the trial was to assess whether Bronchitol further improves lung function in patients already being treated with the most commonly used CF therapeutic, rhDNase. Additional endpoints included changes in the Forced Vital Capacity of the lung, pulmonary exacerbations and antibiotic use.

Clinical Results showed there was a clinically meaningful change from baseline (119mL) and placebo (93mL) at week 26 with Bronchitol for FEV1 (p<0.001).>

For the subgroup of patients on concomitant rhDNase there was also a significant improvement in FEV1 from baseline (88mL) and from placebo (109mL) at week 26 with Bronchitol (p=0.001). Again, there was an immediate and sustained improvement in FEV1 over the 26 week period of the study (p=0.003).

While the study was not powered to show a reduction in the secondary endpoint of exacerbation, the rate of a protocol defined pulmonary exacerbation (PDPE) per subject for the 26 weeks was lower for Bronchitol versus control: overall reduction in rate of 25% (p=0.2).

In September 2008 Pharmaxis submitted a marketing application in Australia for Bronchitol as a treatment for bronchiectasis. This follows a long-term Phase 3 safety study of more than 360 subjects finding that Bronchitol is safe, with no serious adverse events attributable to the medicine. This supports results from an earlier stage of the trial showing that Bronchitol delivered a highly significant improvement in users' quality of life.

The medicine is expected to be approved for bronchiectasis first in Australia, and later in the U.S. and Europe following the completion of a further Phase 3 trial. Bronchitol will be the first targeted medication for sufferers in over 20 years.

Small cap fund manager Acorn Capital is one of the largest shareholders in Pharmaxis owning 7.8% of the company.


Saturday, June 13, 2009

Symptomatic pancreatitis in patients with cystic fibrosis and association with R117H

Symptomatic pancreatitis in patients with cystic fibrosis.

Gooding I, Bradley E, Puleston J, Gyi KM, Hodson M, Westaby D.

Department of Gastroenterology, Hammersmith Hospital, Du Cane Road, London, UK. ian_gooding@yahoo.co.uk

OBJECTIVES: A small group of cystic fibrosis (CF) patients develop symptomatic pancreatitis. The clinical characteristics of these cases are not well documented in the literature. Most cases are in pancreatic sufficient (PS) patients, but it is not clear whether pancreatitis does occur in pancreatic insufficient (PI) patients. There is no information on how the group with PS and pancreatitis differs from the group with PS that does not develop pancreatitis.

METHODS: The Royal Brompton Hospital database of adult CF patients was searched to identify all patients with symptomatic pancreatitis. Clinical details were taken from the case notes. PS pancreatitis patients were then compared with an age- and sex-matched PS control group drawn from the database.

RESULTS: Sixteen patients (9 males) had suffered symptomatic pancreatitis, representing 1.6% of the total database. The mean age at CF diagnosis was 18.7 years, and at presentation with pancreatitis it was 28.8 years. Twelve were PS at diagnosis of CF. At presentation with pancreatitis, seven patients were PS and at the most recent follow-up or death, two remained PS. There was a median of three hospital admissions with pancreatitis. Eight cases developed pancreatic or hepatobiliary complications. In the comparison of pancreatitis patients with controls, there was no difference in survival but pancreatitis patients were significantly more likely to develop PI status. Mild CF transmembrane conductance regulator mutations in general, and R117H in particular, were found more often in pancreatitis patients.

CONCLUSIONS: Symptomatic pancreatitis is a significant problem in 1-2% of patients with CF. These patients are PS at birth but are more likely to develop late PI status than PS patients without pancreatitis. R117H may be associated with this phenotype.

PMID: 19491865 [PubMed - in process]

Azithromycin Treatment Alters Gene Expression in Inflammatory, Lipid Metabolism, and Cell Cycle Pathways in Well-Differentiated Human Airway Epithelia

Azithromycin Treatment Alters Gene Expression in Inflammatory, Lipid Metabolism, and Cell Cycle Pathways in Well-Differentiated Human Airway Epithelia

Carla Maria P. Ribeiro,1,2* Harry Hurd,3 Yichao Wu,3¤ Mary E. B. Martino,1 Lisa Jones,1 Brian Brighton,1 Richard C. Boucher,1,2 and Wanda K. O'Neal1,2

Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex.

To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes.

Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment.

AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected.

On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression.

These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help explain the diverse immunomodulatory roles of macrolides. (edited to add by me: and why zithro used to work so well with me and now it doesn't really do all that much, or so it seems, for me)

Full article: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=19503797

Respiratory Microbiology of Patients With CF in the US, 1995-2005.

Respiratory Microbiology of Patients With Cystic Fibrosis in the United States, 1995-2005.

Razvi S, Quittell L, Sewall A, Quinton H, Marshall B, Saiman L.

From the Department of Pediatrics (Drs. Razvi, Quittell, and Saiman), Columbia University, New York, NY; Morgan Stanley Children's Hospital of NewYork-Presbyterian (Drs. Razvi, Quittell, and Saiman), New York, NY; Sewall, Inc (Dr. Sewall), Bethesda, MD; the Department of Medicine (Dr. Quinton), Dartmouth Medical School, Hanover, NH; and the Cystic Fibrosis Foundation (Dr. Marshall), Bethesda, MD.

Numerous improvements in diagnostic and therapeutic strategies for patients with cystic fibrosis (CF) have occurred during the past two decades. We hypothesized that these changes could impact trends in respiratory microbiology.

Data from the CF Foundation Patient Registry were used to examine trends in the incidence and prevalence of bacterial pathogens isolated from CF patients in the United States from 1995 to 2005.

The number of CF patients in the Patient Registry increased from 19,735 in 1995 to 23,347 in 2005. During the study period, the reported annual prevalence of Pseudomonas aeruginosa significantly declined from 60.4% in 1995 to 56.1% in 2005 (p < 0.001). The decline was most marked in children 6-10 years old (48.2% to 36.1%) and adolescents 11-17 years old (68.9% to 55.5%). Both the incidence (21.7% in 1995 and 33.2% in 2005) and prevalence (37.0% in 1995 and 52.4% in 2005) of methicillin-susceptible Staphylococcus aureus significantly increased and the age-specific prevalence was highest in patients 6-17 years old. The prevalence of methicillin-resistant S. aureus increased from 0.1% in 1995 to 17.2% in 2005 and from 2002 to 2005 was highest in adolescents 11-17 years old. Both the prevalence and incidence of Burkholderia cepacia complex declined, while the prevalence of Haemophilus influenzae, Stenotrophomonas maltophilia and Alcaligenes xylosoxidans increased.

Data from the Patient Registry suggest that the epidemiology of bacterial pathogens in CF patients changed during the study period. Future studies should continue to monitor changing trends and define the association between these trends and care practices in CF.

PMID: 19505987 [PubMed - as supplied by publisher]

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

Geraint B Rogers,1 Thomas WV Daniels,2 Andrew Tuck,3 Mary P Carroll,4 Gary J Connett,5 Gondi JP David,1 and Kenneth D Brucecorresponding author1

Drawing from previous studies, the traditional routine diagnostic microbiology evaluation of samples from chronic respiratory conditions may provide an incomplete picture of the bacteria present in airways disease. Here, the aim was to determine the extent to which routine diagnostic microbiology gave a different assessment of the species present in sputa when analysed by using culture-independent assessment.

Six different media used in routine diagnostic microbiology were inoculated with sputum from twelve patients. Bacterial growth on these plates was harvested and both RNA and DNA extracted. DNA and RNA were also extracted directly from the same sample of sputum. All nucleic acids served as templates for PCR and reverse transcriptase-PCR amplification of "broad range" bacterial 16S rRNA gene regions. The regions amplified were separated by Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling and compared to assess the degree of overlap between approaches.

A mean of 16.3 (SD 10.0) separate T-RF band lengths in the profiles from each sputum sample by Direct Molecular Analysis, with a mean of 8.8 (SD 5.8) resolved by DNA profiling and 13.3 (SD 8.0) resolved by RNA profiling. In comparison, 8.8 (SD 4.4) T-RF bands were resolved in profiles generated by Culture-derived Molecular Analysis. There were a total of 184 instances of T-RF bands detected in the direct sputum profiles but not in the corresponding culture-derived profiles, representing 83 different T-RF band lengths. Amongst these were fifteen instances where the T-RF band represented more than 10% of the total band volume (with a mean value of 23.6%). Eight different T-RF band lengths were resolved as the dominant band in profiles generated directly from sputum. Of these, only three were detected in profiles generated from the corresponding set of cultures.

Due to their focus on isolation of a small group of recognised pathogens, the use of culture-dependent methods to analyse samples from chronic respiratory infections can provide a restricted understanding of the bacterial species present. The use of a culture-independent molecular approach here identifies that there are many bacterial species in samples from CF and COPD patients that may be clinically relevant.

Full text of the publication available here: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=19368727