Transmission and infection of H5N1
Transmission and infection of H5N1 from infected avian sources to humans has been a concern since the first documented case of human infection in 1997, due to the global spread of H5N1 that constitutes a pandemic threat.
Infected birds pass on H5N1 through their saliva, nasal secretions, and feces. Other birds may pick up the virus through direct contact with these excretions or when they have contact with surfaces contaminated with this material. Because migratory birds are among the carriers of the H5N1 virus it may spread to all parts of the world. Past outbreaks of avian flu have often originated in crowded conditions in southeast and east Asia, where humans, pigs, and poultry live in close quarters. In these conditions a virus is more likely to mutate into a form that more easily infects humans. A few isolated cases of suspected human to human transmission exist, with the latest such case in June 2006 (among members of a family in Sumatra). No pandemic strain of H5N1 has yet been found.
H5N1 vaccines for chickens exist and are sometimes used, although there are many difficulties, and it's difficult to decide whether it helps more or hurts more. H5N1 pre-pandemic vaccines exist in quantities sufficient to inoculate a few million people and might be useful for priming to "boost the immune response to a different H5N1 vaccine tailor-made years later to thwart an emerging pandemic". H5N1 pandemic vaccines and technologies to rapidly create them are in the H5N1 clinical trials stage but can not be verified as useful until after there exists a pandemic strain.
- 1 Environmental survival
- 2 Avian flu in birds
- 3 Avian flu in humans
- 4 Notes and references
- 5 See also
- 6 Further reading
Avian flu virus can last indefinitely at a temperature dozens of degrees below freezing, as is found in the northern most areas that migratory birds frequent.
Heat kills H5N1 (i.e. inactivates the virus).
Influenza A viruses can survive:
- Over 30 days at 0°C (32.0°F) (over one month at freezing temperature)
- 6 days at 37°C (98.6°F) (one week at human body temperature)
- decades in permanently frozen lakes
- on hard non-porous surface such as plastic or stainless steel for 24–48 hours
- on clothes, paper and tissues for 8–12 hours
While cooking poultry to 70°C (158°F) kills the H5N1 virus, it is recommended to cook meat to 74°C (165°F) to kill all foodborne pathogens.
Inactivation of the virus also occurs under the following conditions:
- 30 minutes 60°C (140.0°F) (half hour at a temperature that causes first and second degree burns in humans in ten seconds)
- Acidic pH conditions
- Presence of oxidizing agents such as sodium dodecyl sulfate, lipid solvents, and B-propiolactone
- Exposure to disinfectants: formalin, iodine compounds 
To kill avian flu viruses,
(the) World Health Organization recommends that environmental surfaces be cleaned by the following:
- Disinfectants such as sodium hypochlorite, 1% in-use dilution, 5% solution to be diluted 1:5 in clean water, for materials contaminated with blood and body fluids
- Bleaching powder seven grams per liter with 70% available chlorine for toilets and bathrooms
- 70% alcohol for smooth surfaces, tabletops, and other surfaces where bleach cannot be used
H5N1 "can remain infectious in municipal landfills for almost 2 years. [...] The two factors that most reduced influenza survival times were elevated temperature and acidic or alkaline pH."
Avian flu in birds
According to Avian Influenza by Timm C. Harder and Ortrud Werner:
- Following an incubation period of usually a few days (but rarely up to 21 days), depending upon the characteristics of the isolate, the dose of inoculum, the species, and age of the bird, the clinical presentation of avian influenza in birds is variable and symptoms are fairly unspecific. Therefore, a diagnosis solely based on the clinical presentation is impossible. The symptoms following infection with low pathogenic AIV may be as discrete as ruffled feathers, transient reductions in egg production or weight loss combined with a slight respiratory disease. Some LP strains such as certain Asian H9N2 lineages, adapted to efficient replication in poultry, may cause more prominent signs and also significant mortality. In its highly pathogenic form, the illness in chickens and turkeys is characterised by a sudden onset of severe symptoms and a mortality that can approach 100% within 48 hours.
The current method of prevention in animal populations is to destroy infected animals, as well as animals suspected of being infected. In southeast Asia, millions of domestic birds have been slaughtered to prevent the spread of the virus.
Poultry farming practices
There have been a number of farming practices that have changed in response to outbreaks of the H5N1 virus, including:
- vaccinating poultry against bird flu
- vaccinating poultry workers against human flu
- limiting travel in areas where H5N1 is found
- increasing farm hygiene
- reducing contact between livestock and wild birds
- reducing open-air wet markets
- limiting workers contact with cock fighting
- reducing purchases of live fowl
- improving veterinary vaccine availability and cost.
For example, after nearly two years of using mainly culling to control the virus, the Vietnamese government in 2005 adopted a combination of mass poultry vaccination, disinfecting, culling, information campaigns and bans on live poultry in cities.
Dealing with outbreaks
The majority of H5N1 flu cases have been reported in southeast and east Asia. Once an outbreak is detected, local authorities often order a mass slaughter of birds or animals
Use of vaccines
Dr. Robert G. Webster et al. write
- Transmission of highly pathogenic H5N1 from domestic poultry back to migratory waterfowl in western China has increased the geographic spread. The spread of H5N1 and its likely reintroduction to domestic poultry increase the need for good agricultural vaccines. In fact, the root cause of the continuing H5N1 pandemic threat may be the way the pathogenicity of H5N1 viruses is masked by cocirculating influenza viruses or bad agricultural vaccines."
Webster speculates that substandard vaccines may be preventing the expression of the disease in the birds but not stopping them from carrying or transmitting the virus through feces, or the virus from mutating.
In order to protect their poultry from death from H5N1, China reportedly made a vaccine based on reverse genetics produced with H5N1 antigens, that Dr Wendy Barclay, a virologist at the University of Reading believes have generated up to six variations of H5N1.
According to the United Nations FAO, wild water fowl likely plays a role in the avian influenza cycle and could be the initial source for AI viruses, which may be passed on through contact with resident water fowl or domestic poultry, particularly domestic ducks. A newly mutated virus could circulate within the domestic and possibly resident bird populations until highly pathogenic avian influenza (HPAI) arises. This new virus is pathogenic to poultry and possibly to the wild birds that it arose from.
Wild birds found to have been infected with HPAI were either sick or dead. This could possibly affect the ability of these birds to carry HPAI for long distances. However, the findings in Qinghai Lake-China, suggest that H5N1 viruses could possibly be transmitted between migratory birds. Additionally, the new outbreaks of HPAI in poultry and wild birds in Russia, Kazakhstan, Western China and Mongolia may indicate that migratory birds probably act as carriers for the transport of HPAI over longer distances. Short distance transmission between farms, villages or contaminated local water bodies is likewise a distinct possibility.
The AI virus has adapted to the environment in ways such as using water for survival and to spread, and creating a reservoir (ducks) strictly tied to water. The water in turn influences movement, social behavior and migration patterns of water bird species. It is therefore of great importance to know the ecological strategy of influenza virus as well, in order to fully understand this disease and to control outbreaks when they occur. Most research is needed concerning HPAI viruses in wild birds. For example, small birds like sparrows and starlings can be infected with deadly H5N1 strains and they can carry the virus from chicken house to chicken house causing massive epidemics among the chickens. However, pigeons do not present a risk as they neither catch nor carry the virus.
Avian flu in humans
Human to human transmission
The WHO believes that another influenza pandemic is as likely to occur at any time since 1968, when the last century's third of three pandemics took place. The WHO describes a series of six phases, starting with the inter-pandemic period, where there are no new influenza virus subtypes detected in humans, and progressing numerically to the pandemic period, where there is efficient and sustained human-to-human transmission of the virus in the general population. At the present moment, we are at phase 3 on the scale, meaning a new influenza virus subtype is causing disease in humans, but is not yet spreading efficiently and sustainably among humans.
So far, H5N1 infections in humans are attributed to bird-to-human transmission of the virus in most cases. Until May 2006, the WHO estimate of the number of human to human transmission had been "two or three cases". On May 24, 2006, Dr. Julie L. Gerberding, director of the United States Centers for Disease Control and Prevention in Atlanta, estimated that there had been "at least three." On May 30, Maria Cheng, a WHO spokeswoman, said there were "probably about half a dozen," but that no one "has got a solid number." A few isolated cases of suspected human to human transmission exist. with the latest such case in June 2006 (among members of a family in Sumatra). No pandemic strain of H5N1 has yet been found.
There is also concern, although no definitive proof, that other animals — particularly cats — may be able to act as a bridge between birds and humans. So far several cats have been confirmed to have died from H5N1 and the fact that cats have regular close contact with both birds and humans means monitoring of H5N1 in cats, and dogs will need to continue.
Notwithstanding possible mutation of the virus, the probability of a "humanized" form of H5N1 emerging through genetic recombination in the body of a human co-infected with H5N1 and another influenza virus type (a process called reassortment) could be reduced by widespread seasonal influenza vaccination in the general population. It is not clear at this point whether vaccine production and immunization could be stepped up sufficiently to meet this demand.
If an outbreak of pandemic flu does occur, its spread might be slowed by increasing hygiene in aircraft, and by examining airline cabin air filters for presence of H5N1 virus.
The American Centers for Disease Control and Prevention advises travelers to areas of Asia where outbreaks of H5N1 have occurred to avoid poultry farms and animals in live food markets. Travelers should also avoid surfaces that appear to be contaminated by feces from any kind of animal, especially poultry.
There are several H5N1 vaccines for several of the avian H5N1 varieties. H5N1 continually mutates rendering them, so far for humans, of little use. While there can be some cross-protection against related flu strains, the best protection would be from a vaccine specifically produced for any future pandemic flu virus strain. Dr. Daniel Lucey, co-director of the Biohazardous Threats and Emerging Diseases graduate program at Georgetown University has made this point, "There is no H5N1 pandemic so there can be no pandemic vaccine."  However, "pre-pandemic vaccines" have been created; are being refined and tested; and do have some promise both in furthering research and preparedness for the next pandemic. Vaccine manufacturing companies are being encouraged to increase capacity so that if a pandemic vaccine is needed, facilities will be available for rapid production of large amounts of a vaccine specific to a new pandemic strain.
The human incubation period of avian influenza A (H5N1) is 2 to 17 days. Once infected, the virus can spread by cell-to-cell contact, bypassing receptors. So even if a strain is very hard to initially catch, once infected, it spreads rapidly within a body. For highly pathogenic H5N1 avian influenza in a human, "the time from the onset to presentation (median, 4 days) or to death (median, 9 to 10 days) has remained unchanged from 2003 through 2006."
- See also Pneumonia.
Avian influenza HA bind alpha 2-3 sialic acid receptors while human influenza HA bind alpha 2-6 sialic acid receptors. Usually other differences also exist. There is as yet no human form of H5N1, so all humans who have caught it so far have caught avian H5N1.
Human flu symptoms usually include fever, cough, sore throat, muscle aches, conjunctivitis and, in severe cases, severe breathing problems and pneumonia that may be fatal. The severity of the infection will depend to a large part on the state of the infected person's immune system and if the victim has been exposed to the strain before, and is therefore partially immune. No one knows if these or other symptoms will be the symptoms of a humanized H5N1 flu.
Highly pathogenic H5N1 avian influenza in a human appears to be far worse, killing over 50% of humans reported infected with the virus, although it is unknown how many cases (with milder symptoms) go unreported. In one case, a boy with H5N1 experienced diarrhea followed rapidly by a coma without developing respiratory or flu-like symptoms.
As of February 2008, the "median age of patients with influenza A (H5N1) virus infection is approximately 18 years [...] The overall case fatality proportion is 61% [...] Handling of sick or dead poultry during the week before the onset of illness is the most commonly recognized risk factor [...] The primary pathologic process that causes death is fulminant viral pneumonia."
There have been studies of the levels of cytokines in humans infected by the H5N1 flu virus. Of particular concern is elevated levels of tumor necrosis factor-alpha (TNFα), a protein that is associated with tissue destruction at sites of infection and increased production of other cytokines. Flu virus-induced increases in the level of cytokines is also associated with flu symptoms including fever, chills, vomiting and headache. Tissue damage associated with pathogenic flu virus infection can ultimately result in death. The inflammatory cascade triggered by H5N1 has been called a 'cytokine storm' by some, because of what seems to be a positive feedback process of damage to the body resulting from immune system stimulation. H5N1 type flu virus induces higher levels of cytokines than the more common flu virus types such as H1N1  Other important mechanisms also exist "in the acquisition of virulence in avian influenza viruses" according to the CDC.
The NS1 protein of the highly pathogenic avian H5N1 viruses circulating in poultry and waterfowl in Southeast Asia is currently believed to be responsible for the enhanced proinflammatory cytokine response. H5N1 NS1 is characterized by a single amino acid change at position 92. By changing the amino acid from glutamic acid to aspartic acid, researchers were able to abrogate the effect of the H5N1 NS1. This single amino acid change in the NS1 gene greatly increased the pathogenicity of the H5N1 influenza virus.
In short, this one amino acid difference in the NS1 protein produced by the NS RNA molecule of the H5N1 virus is believed to be largely responsible for an increased pathogenicity (on top of the already increased pathogenicity of its hemagglutinin type which allows it to grow in organs other than lungs) that can manifest itself by causing a cytokine storm in a patient's body, often causing pneumonia and death.
Neuraminidase inhibitors are a class of drugs that includes zanamivir and oseltamivir, the latter being licensed for prophylaxis treatment in the United Kingdom. Oseltamivir inhibits the influenza virus from spreading inside the user's body. It is marketed by Roche as Tamiflu. This drug has become a focus for some governments and organizations trying to be seen as making preparations for a possible H5N1 pandemic. In August 2005, Roche agreed to donate three million courses of Tamiflu to be deployed by the WHO to contain a pandemic in its region of origin. Although Tamiflu is patented, international law gives governments wide freedom to issue compulsory licenses for life-saving drugs.
A second class of drugs, which include amantadine and rimantadine, target the M2 protein, but have become ineffective against most strains of H5N1, due to their use in poultry in China in the 1990s, which created resistant strains. However, recent data suggest that some strains of H5N1 are susceptible to the older drugs, which are inexpensive and widely available.
Research indicates that therapy to block one cytokine to lessen a cytokine storm in a patient may not be clinically beneficial.
From the first laboratory-confirmed case through January 24, 2012, the number confirmed human cases of H5N1 reported to WHO stands at 583, with 344 fatalities, reflecting a 59% fatality rate.
The global case fatality ratio looks only to the official tally of cases confirmed by the WHO. It takes no account of other cases, such as those appearing in press reports. Nor does it reflect any estimate of the global extent of mild, asymptomatic, or other cases which are undiagnosed, unreported by national governments to the WHO, or for any reason cannot be confirmed by the WHO. While the WHO's case count is clearly the most authoritative, these unavoidable limitations result in an unknown number of cases being omitted from it.
Notes and references
- Brown, David (2012), "Flu scientists agree to 60-day ‘pause’ in bird-flu research", The Washington Post (20 Jan 2012), washingtonpost.com, retrieved 21 Jan 2012
- Lauerman, John (May 23, 2006). Robert Simison, ed. "Seven Indonesian Bird Flu Cases Linked to Patients". Bloomberg L.P. bloomberg.com.
- Jones, Kathy (23 Jun 2006), WHO confirms human transmission in Indonesian bird flu cluster, Sherwood, Oregon: Foodconsumer.org, archived from the original on 29 Jun 2006, retrieved 23 Jun 2006
- "HHS has enough H5N1 vaccine for 4 million people". CIDRAP. July 5, 2006.
- "Study supports concept of 2-stage H5N1 vaccination". CIDRAP. October 13, 2006.
- "The prevention and treatment of viral respiratory disorders". Retrieved September 2007.
Restricted access; only summary available without login.
- CIDRAP article Germany finds H5N1 in frozen duck meat published September 10, 2007
- "Hot Water Burn & Scalding Graph". Retrieved 2006-09-15.
- "Avian flu biofacts". CIDRAP.
- "Study: Chlorination inactivates avian flu strain", Water Technology (Grand View Media, published 10 Sep 2007), 2007, WaterTech Online, archived from the original on 17 Oct 2007,
Researchers from the US Environmental Protection Agency (EPA), the University of Georgia (Athens, GA) and US Department of Agriculture (USDA) found that the maintenance of a free chlorine residual of 0.52 to 1.08 milligrams per liter (mg/L) was sufficient to inactivate the virus by greater than three orders of magnitude within an exposure time of one minute, according to the study. They noted that EPA specifications for public water supplies that the free chlorine residual values be 6 to 8 mg/L per minute would be “more than sufficient” to inactivate H5N1 in the water environment.
- Source of quotation: Chotani, Rashid A. (2006), "Part 5 of 6: Interventions", The Impact of Pandemic Influenza on Public Health (PDF; slide pack), Johns Hopkins Center for Public Health Preparedness, p. 28
- physorg.com Reprint from: American Chemical Society ; article "Bird flu virus remains infectious up to 600 days in municipal landfills" published May 27th, 2009
- A.R. Elbers, G. Kock and A. Bouma (2005). "Performance of clinical signs in poultry for the detection of outbreaks during the avian influenza A (H7N7) epidemic in The Netherlands in 2003". Avian Pathol 34 (3): 181–7. doi:10.1080/03079450500096497. PMID 16191700.
- I. Capua and F. Mutinelli (2001). "Low pathogenicity (LPAI) and highly pathogenic (HPAI) avian influenza in turkeys and chicken". A Colour Atlas and Text on Avian Influenza: 13–20.
- S. Bano S, K. Naeem K, S.A. Malik (2003). "Evaluation of pathogenic potential of avian influenza virus serotype H9N2 in chicken". Avian Dis 47 (Suppl): 17–22. doi:10.1637/0005-2086-47.s3.817. PMID 14575070.
- C Li, K Yu, G TiaG, D Yu, L Liu, B Jing, J Ping, H. Chen (2005). "Evolution of H9N2 influenza viruses from domestic poultry in Mainland China". Virology 340 (1): 70–83. doi:10.1016/j.virol.2005.06.025. PMID 16026813.
- D.E. Swayne, D.L. Suarez (2000). "Highly pathogenic avian influenza". Rev Sci Tech 19: 463–8.
- Timm C. Harder and Ortrud Werner. "Avian Influenza". Influenza Report.
- "The Threat of Global Pandemics". Council on Foreign Relations. June 16, 2005. Retrieved 2006-09-15.
- "Vietnam to unveil advanced plan to fight bird flu". Reuters. April 28, 2006.
- Webster RG, Peiris M, Chen H, Guan Y (January 2006). "H5N1 outbreaks and enzootic influenza". Emerging Infect. Dis. 12 (1): 3–8. doi:10.3201/eid1201.051024. PMC 3291402. PMID 16494709.
- "Expert: Bad vaccines may trigger China bird flu". MSNBC. December 30, 2005. Retrieved 2006-09-15.
- Morelle, Rebecca (February 22, 2006). "Bird flu vaccine no silver bullet". BBC. Retrieved 2006-09-15.
- "Wild birds and Avian Influenza". FAO. Retrieved 2006-09-15.
- "Small birds must be kept out of poultry farms". World Poultry. December 12, 2006.
- Rudi Hendrikx, DVM. "Avian Influenza in Pigeons". Retrieved 2014-02-06.
- Panigrahy B, Senne DA, Pedersen JC, Shafer AL, Pearson JE (1996). "Susceptibility of pigeons to avian influenza". Avian Diseases 40 (3): 600–4. doi:10.2307/1592270. PMID 8883790.
- Perkins LE, Swayne DE (2002). "Pathogenicity of a Hong Kong-origin H5N1 highly pathogenic avian influenza virus for emus, geese, ducks, and pigeons". Avian Diseases 46 (1): 53–63. doi:10.1637/0005-2086(2002)046[0053:poahko]2.0.co;2. PMID 11924603.
- Current WHO phase of pandemic alert
- WHO Global Influenza Preparedness Plan
- Donald G. McNeil Jr. (June 4, 2006). "Human Flu Transfers May Exceed Reports". New York Times.
- "Avian influenza – situation in Indonesia – update 17". WHO. June 6, 2006.
- National Center for Infectious Diseases, Division of Global Migration and Quarantine (March 24, 2005). "Interim Guidance about Avian Influenza A (H5N1) for U.S. Citizens Living Abroad". Travel Notices. U.S. Centers for Disease Control and Prevention. Retrieved 2006-10-27.
- Jennifer Schultz (November 28, 2005). "Bird flu vaccine won't precede pandemic". United Press International. Retrieved 2006-10-27.
- Promising research into vaccines includes:
- Martin Enserink (August 12, 2005). "Avian Influenza: 'Pandemic Vaccine' Appears to Protect Only at High Doses". Science 309 (5737): 996. doi:10.1126/science.309.5737.996b. PMID 16099954.
- "GlaxoSmithKline Initiates Human Trial Programme With Two H5N1 Pandemic Flu Vaccines" (PDF). March 30, 2006. Archived from the original on 2006-10-10. Retrieved 2006-10-27.
- "Murtha eager to speed vaccine". Tribune-Review. January 28, 2006. Retrieved 2006-10-27. A promising new bird flu vaccine developed by University of Pittsburgh researchers could provide better protection and be made more quickly than other experimental vaccines.
- "Oseltamivir (Tamiflu)". National Institutes of Health. January 13, 2000. Revised on January 10, 2001.
- The Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A/H5 (September 29, 2005). "Avian Influenza A (H5N1) Infection in Humans". N Engl J Med 353 (13): 1374–85. doi:10.1056/NEJMra052211. PMID 16192482.
- T Jacob John (November 12, 2005). "Bird Flu: Public Health Implications for India". Economic and Political Weekly.
- Abdel-Ghafar AN, Chotpitayasunondh T, Gao Z et al. (January 2008). "Update on avian influenza A (H5N1) virus infection in humans". N. Engl. J. Med. 358 (3): 261–73. doi:10.1056/NEJMra0707279. PMID 18199865.
- de Jong MD, Bach VC, Phan TQ et al. (February 17, 2005). "Fatal Avian Influenza A (H5N1) in a Child Presenting with Diarrhea Followed by Coma". N Engl J Med 352 (7): 686–691. doi:10.1056/NEJMoa044307. PMID 15716562.
- Robert G. Webster and Elizabeth Jane Walker (2003). "Influenza: The world is teetering on the edge of a pandemic that could kill a large fraction of the human population". American Scientist 91 (2): 122. doi:10.1511/2003.2.122.
- Chan MC, Cheung CY, Chui WH et al. (2005). "Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells". Respiratory Research 6 (1): 135. doi:10.1186/1465-9921-6-135. PMC 1318487. PMID 16283933.
- Martin Hirst, Caroline R. Astell, Malachi Griffith, Shaun M. Coughlin, Michelle Moksa, Thomas Zeng et al. (December 2004). "Novel Avian Influenza H7N3 Strain Outbreak, British Columbia". Emerg Infect Dis 10 (12): 2192–5. doi:10.3201/eid1012.040743. PMC 3323367. PMID 15663859.
- Alan Sipress (June 18, 2005). "Bird Flu Drug Rendered Useless: Chinese Chickens Given Medication Made for Humans". Washington Post.
- "WHO sees role for older antivirals in some H5N1 cases". CIDRAP. May 22, 2006.
- CIDRAP article Study: Inhibiting cytokine response might not reverse H5N1 infections published July 16, 2007
- World Health Organization (24 January 2012). "Cumulative number of confirmed human cases of avian influenza A(H5N1) reported to WHO" (PDF).
- http://www.medpagetoday.com/InfectiousDisease/URItheFlu/tb/5964 Options For Influenza Control VI (Conference, Toronto Canada, June 18, 2007) Even those who were in close contact with both infected birds and infected people showed no sign of ever having been infected, Dr. Dejpichai and colleagues found. The study is consistent with findings in Hong Kong, China, and Cambodia, which showed viral seroprevalence of no more than 10% among poultry workers and people living in villages where H5N1 outbreaks occurred, she said. But it contradicts a population-based study in Vietnam, published last year, that concluded that mild cases of the virus were likely to be common. (see Mild Avian Flu Transmission May Be Common) http://www.medpagetoday.com/InfectiousDisease/URItheFlu/tb/2450
|Wikinews has related news: Category:Avian Flu|
- "Epidemiology of WHO-confirmed human cases of avian influenza A(H5N1) infection" (pdf).
- "WHO Avian influenza resource (updated)".
- CDC "Facts About Avian Influenza (Bird Flu) and Avian Influenza A (H5N1) Virus".
- "FAO information on Avian Influenza - Latest news, Disease Card, Maps, Animations".
- "Avian Influenza (Bird Flu): Agricultural and Wildlife Considerations". CIDRAP.
- Juckett G (September 2006). "Avian influenza: preparing for a pandemic". Am Fam Physician 74 (5): 783–90. PMID 16970022.