Download Pre-hospital thrombolysis is associated with mortality reduction and

Pre-hospital thrombolysis when compared with In-hospital thrombolysis is associated with
mortality reduction and prognosis improvement in patients with suspected ST-elevation
myocardial infarction
Ana Luísa Frutuoso, Ana Rita Magalhães, Bárbara Almeida, Carolina Fonseca, Catarina Machado,
Diana Monteiro, Irina Rosa, João Pedro Silva, João Magalhães, Lília Monteiro, Mariana Antunes,
Sofia Lopes, [email protected]
Filipa Almeida, class 7
ABSTRACT
Background: Time to thrombolysis remains a key modifiable determinant of mortality in ST-elevation
myocardial infarction (STEMI)
1,2,3.
Despite numerous medical advances, the time from symptom
onset to thrombolysis has remained at large unchanged, with a median of 2.5–3 h1. A pre-hospital
treatment strategy when compared with in-hospital thrombolysis may reduce time to thrombolysis with
a subsequent decrease of in-hospital mortality.
Problem: There are sparse and scattered data on the impact of pre-hospital thrombolysis (PHT) in
real-life patients with ST-elevation myocardial infarction (STEMI).
Aim: Evaluate treatment delays, outcome and complications in clinical trials and prospective cohort
studies with real-life patients with STEMI myocardial infarction according to thrombolysis delivered on
site (PHT) or in the hospital (IHT).
Methods and Results: Systematic review of clinical trials and prospective cohort studies with
subsequent metanalysis. Median time from symptom onset to treatment was 113 min for PHT and
185 min for in-hospital thrombolysis. PHT group in-hospital mortality, 1-month mortality and 1-year
mortality were associated with a risk ratio (IV, Random, 95% CI) of 0.70, 0.73 and 0.64, respectively,
when compared with the IHT group. PHT is also associated with fewer complications; PHT
reinfarction rate, ventricular fibrillation and Killip class > 1 risk ratio (IV, Random, 95% CI) were 0.88,
0.76 and 0.75, respectively, when compared with the IHT group.
KEY-WORDS: Acute Myocardial Infarction (MeSH); Pre-hospital thrombolysis (MeSH); Alteplase;
Reteplase; Tecneplase; Treatment delay; Mortality (MeSH).
INTRODUCTION
Diseases of the cardiovascular system, of which acute myocardial infarction is the most serious
manifestation, constitute the main cause of mortality in developed countries5.
Acute myocardial infarction (AMI) is the destruction of myocardial tissue resulting from insufficient
supply of oxygen to the heart muscle
5,6.
The leading cause of AMI is atherosclerosis6 which is
formation of plaques in blood vessels7. Theses plaques are made of fatty substances, cholesterol,
cellular waste products, calcium and fibrin that accumulate in the inner lining of the arteries 7,8,9.
Plaques decrease the lumen of blood vessels which consequently reduces blood flow and tissue
oxygenation. The fissure or bleeding of the plaque activates the plaquetar aggregation and the
coagulation cascade and thrombin, generating a thrombus that occludes the coronary artery 8,10,11.
The resultant death of the myocardial tissue due to lack of oxygenation constitutes an acute coronary
syndromes (ACS) which could imply a cardiogenic shock (state in which the heart has been damaged
so much that it is unable to supply enough blood to the organs of the body) 19. The ACS encompasses
three disorders of related etiology: ST-elevation myocardial infarction (STEMI), non-ST elevation
myocardial infarction (NSTEMI) and unstable angina (UA)
5,6.
The management of STEMI differs from
that for UA and NSTEMI which may be considered as one clinical entity 12.
The diagnosis of acute coronary syndrome is based on three fundamental aspects: signs and
symptoms, the electrocardiogram and biochemical data
12,13,14.
The ST-segment variation allows
distinguishing between ST-elevation myocardial infarction and non-ST elevation myocardial
infarction. For this reason the initial electrocardiogram remains a pillar in the diagnosis of STEMI and
one of the most important aspects in the decision of reperfusion therapy. A way to measure the
severity of heart failure with myocardial infarction is a scale named Killip Classification through which
patients are separated by symptoms in four categories15.
Once the STEMI consists in a total occlusion of a coronary artery with subsequent ischemia and
necrosis of myocardial tissue, the treatment goal is reperfusion as soon as possible, ideally in the first
2 hours after the onset of symptoms. It can be done by mechanical methods (angioplasty) or
pharmacological therapy16. The former can take place only in the hospital 16. The latter consists in
administering thrombolytic therapy intravenously to promote dissolution of the blood clot and
thrombus and consequent opening of the coronary artery17. An earlier reperfusion reduces the short
and long term mortality rate and the incidence of left ventricle dysfunction and heart failure1,2,3.
The thrombolysis consists in dissolving the fibrin clot of the thrombus that is blocking the coronary
artery. This lysis of the clot is due to the action of a specific enzyme, the plasmin, derived from the
proenzyme plasminogen. Since these drugs operate on plasminogen they are known as the
“plasminogen activators." However, the imbalance triggered between the plasminogen-plasmin
system increases possible hemorrhages, and this treatment is contra-indicated in patients with a
history of cerebrovascular accident, intracranial neoplasm, suspected aortic dissection, severe
uncontrolled hypertension, current use of anticoagulants, recent trauma, internal bleeding or peptic
ulcer in activity12,17.
As the key factor in treating AMI is speed, the early treatment is essential in reducing the percentage
of affected myocardium and thus complications and cardiovascular death. The pre-hospital
thrombolytic therapy is possible and can reduce time between symptoms and reperfusion therapy.
Recent studies have shown that pre-hospital thrombolytic therapy (PHT) can reduce the mortality
caused by AMI between 25 and 50%
1,2,3,
and other reports have raised concerns about the relative
safety of PHT18.
The main objective of this study is to define if PHT is beneficial and safe in terms of morbidity and
mortality for patients with ST-elevation myocardial infarction when compared with intra-hospital
reperfusion therapy, either thrombolysis or mechanical reperfusion.
PARTICIPANTS AND METHODS
Study objectives and design
We performed a systematic review and meta-analysis of randomized controlled trials and cohort
studies reporting outcomes of PHT versus intra-hospital reperfusion in patients with STEMI. The
primary endpoints were determine if pre-hospital thrombolysis reduces early and late (1 year)
cardiovascular mortality in patients with ST elevation myocardial infarction and evaluate if prehospital thrombolysis improves prognosis in terms of: reinfarction rates, left ventricular ejection
fraction (an estimation is made of the volume of blood in the ventricle at the end of diastole and the
volume of blood remaining in the ventricle at the end of systole), ventricular fibrillation rates, and killip
class on admission values. The secondary endpoint evaluated was if pre-hospital thrombolysis
performed by non medical teams increases complication rates. The outcomes of interest were
mortality, myocardial infarction, killip class on admission values, left ventricle dysfunction and
hemorrhagic complications.
Population and Sampling Methods
We performed a systematic review of randomized controlled trials and cohort studies that compared
PHT with in-hospital thrombolytic therapy in adults presenting with suspected myocardial infarction
with ST- elevation. The defined query was used in Medline, SCOPUS and ISI Web of Knowledge,
using various combinations of the MESH terms Acute Myocardial Infarction; Pre-hospital
thrombolysis; Mortality, Treatment delay, Alteplase; Reteplase and Tecneplase. Two reviewers
identified articles eligible for further review by performing a screen of abstracts and titles. If a study
was deemed relevant, the manuscript was obtained and reviewed according to the inclusion and
exclusion criteria. The inclusion criteria consisted of: 1) only clinical trials and cohort studies, 2)
studies that compare pre-hospital thrombolysis with in-hospital thrombolysis, 3) studies that use new
generation thrombolytics, 4) follow-ups till 1 year, 5) cases of patients with suspect of ST segment
elevation myocardial infarction, 6) comparison between pre-hospital and in-hospital treatment. The
exclusion criteria are: 1) non-english studies, 2) articles with no abstract, 3) articles that don’t have
full text, 4) studies that used first generation thrombolytics, 5) non related studies, 6) studies that do
not compare pre-hospital thrombolysis with aspects connected to our study aim, 7) comparison
between AMI and thrombolytic treatment inexistent, 8) articles that do not fulfill any of the inclusion
criteria.
Study identification
We searched through for possible studies from January 1, 1990 to February 28, 2009. These dates
were thought to be appropriate as the new generation thrombolytics were not used until 1990. From
the search we obtained 464 articles in Medline, 692 in Scopus and 415 in ISIWeb. After this selection
we restrained our articles down to 9 from Medline, 11 from Scopus and 3 from ISIWeb, using the
inclusion and exclusion criteria. We read the 23 articles and included 8 (4 from Medline and 4 from
Scopus) in our review (3 randomized trials, and 5 cohort studies), which directly compared the two
treatment forms. From the 15 excluded, 5 articles compared pre-hospital thrombolysis with aspects
not connected to our study aim (3 from ISIWeb and 2 from Scopus), 6 used first generation
thrombolytics (3 from Scopus and 3 from Medline) and 4 didn’t have full text (3 from Scopus and 1
from Medline) (Flowchart 1).
Flowchart 1: Included studies.
Data collection methods
Two reviewers extracted independently pre-specified data elements from each study, and data was
organized in four different categories: Study Sample, Study Sample Characteristics, Time to
Treatment, Mortality and In-Hospital Complications. Charts were used to compile the data of each
category. After creating the charts, we inserted the data in SPSS and proceeded to an initial statistic
analysis. We calculated the mean values of each variable with weighted cases by number of patients
on first and then with weighted cases off. The purpose of this first analysis was to give us an idea of
the impact of PHT trough variable tendencies.
Variable description and study design
The following variables we created and inserted in SPSS according to the four categories: 1) Study
Sample variables - PHT given by paramedics [Yes/No], Thrombolytic Type, Patients given PHT (%),
Patients given IHT (%) and Total Patients; 2) Study Sample Characteristics - PHT group age, PHT
female sex (%), IHT female sex (%), PHT Diabetes Mellitus (%), IHT Diabetes Mellitus (%), PHT
Current Smoker (%), IHT Current Smoker (%), PHT Hypertension (%) and IHT Hypertension (%); 3)
Time to Treatment and Mortality parameters - PHT Symptom onset to Treatment Time, IHT Symptom
onset to Treatment Time, PHT In-Hospital Mortality (%), IHT In-Hospital Mortality, PHT 1-Month
mortality (%), IHT 1-Month mortality (%), PHT 1-Year mortality (%) and IHT 1-Year mortality (%); 4)
In-Hospital Complications - PHT Reinfarction rate, IHT Reinfarction rate, PHT Ventricular Fibrillation,
IHT Ventricular Fibrillation, PHT Killip Class>1 on admission and IHT Killip Class>1 on admission.
Statistical analysis
Data were analyzed on an intention to treat basis. Where appropriate, data from all trials were
combined using the, metanalysis software in Review Manager. All the outcome measures of this
review were dichotomous. Data were combined using random effects modeling to determine a
summary estimate of the relative risk and the 95% confidence interval. Heterogeneity was statistically
assessed using the chi-square test (p<0.10) for all end points and the I2 statistic (Higgins 2003) for
selected end points. The I2 statistic is displayed on the forest plots for all analyses.
RESULTS
Patients’ baseline characteristics
Patient’s baseline characteristics are summarized in the table below (Table 1). Patients
characteristics are similar both in the pre-hospital and in-hospital groups. Still, the number of female
patients in the in-hospital group is larger; the same occur for patients with diabetes mellitus. This can
be associated with the larger risk of complications in these patients. In all ambulances without
physicians diagnosis was established by a physician at the hospital using telemedicine (ECGs always
available for the physician in the hospital).
Table 1: Summary of patient’s baseline characteristics for each study.
Time until Thrombolysis
Median time from symptom onset to
treatment was lower for the pre-
Study ID
Median Time to
Thrombolysis in the
PHT Group (mins)
Median Time to
Thrombolysis in the
IHT Group (mins)
Median Time
Difference (mins)
hospital group in all studies (Table 2).
European Heart J. 2006
113
165
52
In 4 of the 6 studies in which data
European Heart J. 2003
138
240
102
GREAT Study BMJ 1992
regarding median time from symptom
101
240
139
JAMA 1993
77
110
33
onset to treatment was available, the
JAMA 2006
120
167
47
statistical analyses indicated that
New Eng. J. of Med. 1993
130
190
60
Mean of Medians
113
185
72
there
was
statistical
significance
between pre-hospital group and in-
Table 2: Median time from symptom onset to thrombolysis in both pre-hospital and in-hospital
groups and respective median time difference.
hospital group. In the remaining 2 studies the statistical analyses comparing the two groups was not
performed (or at least not published in the full article).
The means of the median times from symptom onset to treatment available in the 6 studies were 113
minutes for the pre-hospital group and 185 minutes for the in-hospital group (median time difference
approximately 72 minutes). This interval difference supports the finding that pre-hospital thrombolysis
significantly improved time to needle for thrombolysis.
Outcomes
Data collected from the 8 studies
ID
included supports that pre-hospital
thrombolysis is associated with lower
mortality until hospital discharge, 1-
Great Study 2
New Eng. J. M. 1993
21
InHospital
Mortality
(%) PHT
In-Hospital
Mortality
(%)IHT
1-Month
Mortality
(%) PHT
1-Month
Mortality
(%) IHT
1-Year
Mortality
(%) PHT
1-Year
Mortality
(%) IHT
6,7
11,5
-
-
-
-
9,1
10,5
9,7
11,1
-
-
5,7
8,1
-
-
month mortality and 1-year mortality
-
-
BMJ 2003 22
11
12
-
-
-
-
(Table 3), although that difference was
European Heart J. 2003 23
7
13
-
-
-
-
8
-
-
6
11
8,8
7,6
11,4
10,3
15,9
8,3
7,2
11,8
JAMA 1993
20
Circulation 2004 24
not always statistically significant. Our
JAMA 2006 25
3,3
5,9
outcome measures also support those
European Heart J. 2006 1
-
-
5,4
findings; in-hospital mortality, 1-month
Mean
7
10,3
7,6
10,3
7,8
13
Weighted Mean
6,3
9,2
8
11
9,5
15
mortality and 1-year mortality in the
Table 3: In-Hospital, 1-Month and 1-Year Mortality in both pre-hospital and in-hospital groups.
PHT group is associated with risk ratio
(IV, Random, 95% CI) of 0.70, 0.73 and 0.64, respectively, when compared with the IHT group
(Figures 1, 2 and 3 respectively).
.
Figure 1: Forest Plot comparing In-Hospital Mortality for PHT and IHT patients.
Figure 2: Forest Plot comparing 1-Month Mortality for PHT and IHT patients.
.
Figure 3: Forest Plot comparing 1-Year Mortality for PHT and IHT patients.
Complications
Data collected regarding reinfarction rates, ventricular fibrillation rates and killip class >1 on
admission when comparing the PHT group with IHT group suggests that these complications are less
frequent in pre-hospital patients; reinfarction rates, ventricular fibrillation and Killip class > 1 risk ratio
(IV, Random, 95% CI) in the prehospital treated group were 0.88,
0.76 and 0.75, respectively, when
compared
with
the
IHT
group
.
(Figures 4, 5 and 6, respectively).
Occurrence of cerebral bleedings in
PHT groups and IHT groups was
studied only by one of the included
studies
(Erik
European
According
Bjo¨rklund
Heart
to
et
Journal
this
study
al.
2006).
the
occurrence of these complications is
similar in both groups.
Killip Class > 1
Killip Class > 1
on Admission
on Admission
PHT (%)
IHT (%)
-
-
-
-
-
-
-
-
-
-
-
-
-
25
23
4
5
11
14
-
-
22,8
30,9
2,3
4
14,9
21,2
3,2
4,2
5,3
18,4
22,3
2,3
4,3
6
20,9
28,4
Reinfarction
Reinfarction
Ventricular
Ventricular
PHT (%)
IHT (%)
Fib. PHT (%)
Fib. IHT (%)
-
-
-
-
1993
-
-
6,2
7
JAMA 1993
-
-
-
BMJ 2003
-
-
-
-
2004
3,4
4
JAMA 2006
3
2
Heart J. 2006
3,1
3,4
Mean
3,2
3
ID
Great Study
New Eng. J. M.
European
Heart J. 2003
Circulation
European
Weighted
Mean
Table 4: In-Hospital complications in both pre-hospital and in-hospital groups.
Figure 4: Forest Plot comparing reinfarction rates (%) in pre-hospital and in-hospital groups.
Figure 5: Forest Plot comparing ventricular fibrillation rates (%) in pre-hospital and in-hospital groups.
Figure 6: Forest Plot comparing killip class >1 on admission (%) in pre-hospital and in-hospital groups.
DISCUSSION
.
Other complications were considered in the included studies. Unfortunately, those complications
were not studied in all the included studies or they were evaluated using different scales or
parameters in each study and, consequently, the metanalysis was not possible for those
complications. However is important to make a reference to the results regarding each of those
complications in the included studies.
Regarding left ventricular ejection fraction, according with [JAMA 1993] and [Circulation 2004] LVEF
differences between PHT and IHT patients were not statistically significant, however [European Heart
Journal 2006] study shows that LVEF is higher in PHT patients with statistical significance.
.
Cerebral bleedings were also considered in the [European Heart Journal 2006] study. 1,4% of PHT
and 0.9% of IHT patients developed cerebral bleedings; statistical analysis demonstrated that the
difference
between
groups
is
nonsignificant.
Regarding
non-cerebral
bleedings,
[New Eng. J. of Medicine 1993] and [JAMA 1993] did not found any differences in the incidence of
non-cerebral bleedings; [JAMA 2006] study also found no differences in the number of deaths caused
by non-cerebral bleedings.
Hospital stay time was only considered by the [Circulation 2004] study. Median hospital stay time was
shown to lesser by 1 day in PHT [ 5d (4-7) ] patients than in IHT patients [ 6d (4-8) ].
Diagnosis accuracy was excellent in both ambulances staffed by medics, nurses or paramedics. ECG
was performed in site and sent to a specialist in the hospital that received the patient in all studies.
This step helps establishing the right diagnosis with almost perfect accuracy (around 95% of success
in the [JAMA 2003] study.
All studies show that initiating the thrombolytic treatment in site greatly reduces symptom onset to
"needle time". Nevertheless, more important that initiated treatment inside or outside the hospital, is
to initiate the reperfusion as soon as possible. Mortality reduction is a result from the time saved from
PHT.
As mentioned before, many times was impossible to proceed to the metanalysis because data,
related to the same issue, is presented in many different ways. In future studies, study groups should
be defined and same for the variables and parameters used. Only so, data collected could be used in
systematic reviews and metanalysis in order to find results even more significant.
CONCLUSIONS
Studies have demonstrated that pre-hospital thrombolysis when compared with in-hospital
thrombolysis can reduce time from symptom onset to treatment and improve short-term, 1-month and
1-year outcomes without the risk of increased complications. Further efforts are needed to expand
the pre-hospital-treated population in real-life.
REFERENCES
1 Erik Björklund, Ulf Stenestrand, Johan Lindbäck, et al. Pre-hospital thrombolysis delivered by
paramedics is associated with reduced time delay and mortality in ambulance-transported real-life
patients with ST-elevation myocardial infarction. European Heart Journal 2006 May; 27(10):114652.
2. GREAT Group. Feasibility, safety, and efficacy of domiciliary thrombolysis by general
practitioners: Grampian region early anistreplase trial. British Medical Journal September 5 1992;
305(6853): 548–553.
3 Danchin N, Durand E, Blanchard D. Pre-hospital thrombolysis in perspective. Overview of
current guidelines and data from trials and registries on the best perfusion approach for acute MI
with ST-elevation. European Heart Journal 2008 October 23. [Epub ahead of print]
4 J Wilkinson, K Foo, N Sekhri et al. Interaction between arrival time and thrombolytic treatment
in determining early outcome of acute myocardial infarction. Heart 2002 Dec; 88(6):583-6.
5 World Health Organization. What are Cardiovascular Diseases? Fact sheet N°317; February
2007. Available from: http://www.who.int/mediacentre/factsheets/fs317/en/
6 Drew Evan Fenton, MD. Myocardial Infarction [Internet] New York: eMedicine Clinical
Knowledge Base; [updated Oct 10, 2008]. Available from: http://emedicine.medscape.com
/article/759321-overview
7 American Heart Association. Atherosclerosis. [updated March 11, 2008] Available from:
http://www.americanheart.org/presenter.jhtml?identifier=228
8 Ursula Rauch, MD; Julio I. Osende, MD; Valentin Fuster, MD, PhD; James H. Chesebro, MD et
al. Thrombus Formation on Atherosclerotic Plaques: Pathogenesis and Clinical Consequences.
Ann Intern Med. 2001;134:224-238.
9 Geoffrey H. Tofler, MB, MD; James E. Muller, MD. Triggering of Acute Cardiovascular Disease
and Potential Preventive Strategies. Circulation. 2006;114:1863-1872.
10 Shyam Prabhakaran, Rajinder Singh et al. Presence of calcified carotid plaque predicts
vascular events: The Northern Manhattan Study. Atherosclerosis. 2007 Nov;195(1):e197-201.
Epub 2007 May 4.
11 Fillipo Crea, M.D; Maria Luisa Finocchiaro, M.D. Thrombogenesis in Acute Coronary
Syndromes. J Interv Cardiol. 1995 Dec;8(6 Suppl):724-9.
12 American College of Cardiology (ACC) and American Heart Association (AHA) (2004).
ACC/AHA Guidelines for the management of patients with ST-elevation myocardial infarction.
Report of the ACC/AHA Task Force on Practice Guidelines. Circulation. 2004;110:588-636.
13 Kaya Z, Göser S, Buss SJ, Leuschner F et al. Identification of cardiac troponin I sequence
motifs leading to heart failure by induction of myocardial inflammation and fibrosis. Circulation.
2008 Nov 11;118(20):2063-72. Epub 2008 Oct 27.
14 Drew Evan Fenton, MD. Myocardial Infarction: Differential Diagnoses & Workup [Internet] New
York: eMedicine Clinical Knowledge Base; [updated Oct 10, 2008]. Available from:
http://emedicine.medscape.com/article/155919-diagnosis
15 Killip T, Kimball JT (Oct 1967). "Treatment of myocardial infarction in a coronary care unit. A
two year experience with 250 patients". Am J Cardiol. 20 (4): 457–64.
16 Aversano T, et al. Thrombolytic therapy vs. primary percutaneous coronary intervention for
myocardial infarction in patients presenting to hospitals without on-site cardiac surgery. JAMA
2002 April 7; 287(15): 1943–1951.
17 Wanda L Rivera-Bou, MD; Jose G Cabanas, MD. Thrombolytic Therapy [Internet] New York:
eMedicine
Clinical
Knowledge
Base;
[updated
November
20,
2008]
Available
from:
http://emedicine.medscape.com/article/811234-overview
18 M Woollard, K Pitt, A J Hayward, N C Taylor. Limited benefits of ambulance telemetry in
delivering early thrombolysis: a randomised controlled trial. Emerg Med J 2005;22:209–215. doi:
10.1136/emj.2003.013482
19 Topalian S, Ginsberg F, Parrillo JE. Cardiogenic shock. Crit Care Med. 2008 Jan;36(1
Suppl):S66-74.
20 W. D. Weaver, M. Cerqueira, A. P. Hallstrom, P. E. Litwin, J. S. Martin, P. J. Kudenchuk and
M. Eisenberg. Prehospital-initiated vs hospital-initiated thrombolytic therapy. The Myocardial
Infarction Triage and Intervention Trial. JAMA. September 8, 1993-Vol 270, No.10
21 European Myocardial Infarction Project Group. Prehospital Thrombolytic Therapy in Patients
with Suspected Acute Myocardial Infarction. New Eng. J. Med. August 5, 1993;329:383-389.
22 David K Pedley, Kim Bissett, Elizabeth M Connolly, Carol G Goodman, Ian Golding, T H
Pringle, G P McNeill, S D Pringle and M C Jones. Prospective observational cohort study of time
saved by prehospital thrombolysis for ST elevation myocardial infarction delivered by paramedics.
5 July, BMJ 2003;327;22-26.
23 T.P. Mathew, I.B.A. Menown, D. McCarty, H. Gracey, L. Hill, A.A.J. Adgey. Impact of prehospital care in patients with acute myocardial infarction compared with those first managed inhospital. European Heart Journal 2003;24, 161–171
24 Nicolas Danchin, Didier Blanchard et al. Impact of Prehospital Thrombolysis for Acute
Myocardial Infarction on 1-Year Outcome: Results from the French Nationwide USIC 2000
Registry. Circulation Sept 2004;110;1909-1915
25 Ulf Stenestrand; Johan Lindbäck; Lars Wallentin; et al. Long-term Outcome of Primary
Percutaneous Coronary Intervention vs Prehospital and In-Hospital Thrombolysis for Patients with
ST-Elevation Myocardial Infarction JAMA. October 11, 2006;296(14):1749-1756