- Email: [email protected]
Stem-cell therapy for myocardial diseases
CORRESPONDENCE
Sir—The article by Lars Klareskog and colleagues1 is impressive and encouraging, and offers hope for remission and avoidance of structural damage in a high proportion of patients with rheumatoid arthritis. However, some questions remain unresolved: why is improvement not more universal? and what is the optimum timing for combined treatment? I am not surprised that the combination of etanercept and methotrexate is not more universally effective since the range of autoimmune hyperactivity in rheumatoid arthritis is broad and suppression of tumour necrosis factor (TNF) activity by etanercept addresses only a fraction of that range. Methotrexate has a wider range of activity than does etanercept, but is not specific enough to counteract all aspects of immune hyperactivity found in rheumatoid arthritis. In view of the objectives of the research programme, that this article deals with the pharmacological aspects of rheumatoid arthritis is understandable; however, we should not lose sight of psychosocial factors. Restoration of a normal immune pattern might not be achievable by pharmacological means alone and could additionally require a psychosocial approach. Baker and Brewerton2 reported an association between stressful life circumstances and rheumatoid arthritis. Moreover, a link between long-term psychosocial stress, chronic fatigue, and rheumatic disorders has also been noted.3 Chronic fatigue syndrome can be triggered by infection, psychosocial stress, or both. It is accompanied by mild cortisol insufficiency4 and thus an overactive immune system, with, among other changes, overproduction of several cytokines, including TNF ␣ and TNF ; increased activity of CD4 (helper) T cells and B cells; and autoto antibody production5—similar changes found in rheumatoid arthritis. Overall treatment of patients with rheumatoid arthritis should include not only pharmacological means, such as the combined therapy used in the TEMPO trial,1 but also lifestyle changes, which help to counteract chronic fatigue—eg, increased rest, graded exercises, psychotherapy, and financial and social support where needed. Alex Poteliakhoff Sudbury, 16B Prince Arthur Road, London NW3 6AY, UK 1
Klareskog L, van der Heijde D, de Jager J, et al. Therapeutic effect of the combination of etanercept and methotrexate compared with each treatment alone in patients with rheumatoid arthritis: double-blind randomised controlled trial. Lancet 2004; 363: 675–81.
1734
2
3 4
5
Baker GHB, Brewerton DA. Rheumatoid arthritis: a psychiatric assessment. BMJ 1981; 282: 2014. Poteliakhoff A. Emotion and immunity. Lancet 1985; 326: 327. Poteliakhoff A. Adrenocortical activity and some clinical findings in acute and chronic fatigue. J Psychosom Res 1981; 25: 91–95. Poteliakhoff A. Fatigue syndromes and the aetiology of autoimmune disease. J Chronic Fatigue Syndr 1998; 4: 31–49.
African women, bride price, and AIDS Sir—The report by Charles Wendo (Feb 28, p 716)1 blamed bride price for the spread of AIDS in sub-Saharan Africa. It also cites bride price as an important factor in polygamy. However, the contribution of bride price to these problems is minuscule. Traditionally, payment of the bride price by the groom was a demonstration that he had come of age and had the means and capability to support himself and his wife. Acceptance of the bride price signifies the families’ support and blessing of the union. Rejection or nonacceptance of the bride price meant non-acceptance of the marriage proposal. Payment and acceptance of the bride price, whatever the currency used, seals the traditional marriage ceremony and authenticates the union between the man and woman. Once the woman is married, the poor treatment she might become subject to within the relationship has nothing to do with the bride price, but is a direct result of the fact that she has little say within the marriage as a result of her poor socioeconomic status. Therefore, even if the bride price were abolished, a woman’s lot within the marriage would not be different from what it currently is. The only thing that would change African women’s lot is the power to negotiate within the relationship. At the moment they have no powers, not because of the bride price, but because they have no education and are therefore totally dependent on their husbands.2 There is no doubt that like everything else the institution of bride price has been subject to abuse: prospective in-laws have used the bride price to make unreasonable and unrealistic demands of the groom. This situation can lead to tensions between the families and indeed couples, but so can many other sources of disagreement. The system therefore is in need of reform, but to blame it for the spread of HIV infection and polygamy is without
basis and a misdirection of the thrust for change. Polygamy will certainly spread HIV, but religion (Islam and African traditional religions allow polygamy) is responsible for that, not bride price. What is needed is a concerted effort in the education of African women. Parents, governments, and nongovernmental organisations need to promote this agenda. Education will empower African women to make better choices and negotiate within relationships. It would also give them independence and the means and ability to leave abusive relationships.3 Umo I Esen South Tyneside District Hospital, Harton Lance, South Shields, Tyne and Wear NE34 0PL, UK (e-mail: [email protected]) 1 2
3
Wendo C. African women denounce bride price. Lancet 2004; 363: 716. Esen UI. Violence against women in developing countries. East African Med J 2000; 77: 400. Harrison KA. The importance of the educated healthy woman in Africa. Lancet 1997; 349: 644–47.
Stem-cell therapy for myocardial diseases Sir—We wish to raise several issues regarding the publication by Hyun-Jae Kang and colleagues (Mar 6, p 751)1 on the role of intracoronary infusion of peripheral-blood stem cells mobilised with granulocyte colony stimulating factor (G-CSF) in patients who had had a myocardial infarction. First, data on the effects of G-CSF in the 11 enrolled patients are incomplete. Although the baseline total white-cell count is provided for all patients, there is no documentation of the change in the white-cell count and CD34 count during the 4 days of treatment with GCSF in these patients, and how these changes correlated with changes in cardiac function. This information is important because a potential reason for the differences seen between the two groups is that the group who received only G-CSF did not mobilise their bone marrow as well as the group who received G-CSF plus cell infusion—a fact not acknowledged in the Discussion. Kang and colleagues’ acceptance that there is little experience in the use of G-CSF in patients after myocardial infarction, the previously documented association between GCSF and three deaths,2 and the fact that the CD34 count was used as the stemcell marker by Kang and colleagues makes it vital that all such data is presented to allow a more complete analysis of their results.
THE LANCET • Vol 363 • May 22, 2004 • www.thelancet.com
For personal use. Only reproduce with permission from The Lancet publishing Group.
CORRESPONDENCE
Second, in the cell infusion group, any remaining white blood cells and peripheral-blood stem cells that were not infused via the intracoronary route were reinfused via the peripheral intravenous route. Apheresis produced a mean of 9·0⫻109 leucocytes (SD 3·0⫻109), with a mean of 1·5⫻109 (0·5⫻109) being reinfused via the intracoronary route. Therefore most of the white blood cells and peripheral-blood stem cells were returned via the intravenous route. This fact is not controlled for in the G-CSF only group and no comment is made about its possible confounding effect on the results. In light of this possibility and the lack of information about haematological variables, we find it difficult to support the authors’ conclusion that the intracoronary infusion of peripheral-blood stem cells was responsible for the improvement in cardiac function seen in the G-CSF plus cell infusion group. Future trials involving G-CSF in this setting should document the changes in white-cell count and CD34+ cells, as well as the number and phenotype of the cells reinfused with these parameters being correlated with outcome. Such data would provide insight into the efficacy of G-CSF in this group of acutely unwell patients, and will allow for proper control between treatment groups. Changes in total white-cell count and CD34+ cells could potentially be surrogate markers for restoration of cardiac function, and a potential marker of adverse events and complications such as restenosis. Finally, since the true stem cell for cardiac regeneration is not known, phenotyping is vital if a systematic approach is to be taken to attempt its identification, especially because future researchers might choose to infuse different stem-cell preparations.3 *Carlo Palmieri, David Vigushin, R Charles Coombes Department of Cancer Medicine, Cancer Research UK Laboratories, 5th Floor MRC Cyclotron Building, Faculty of Medicine, Imperial College, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK (e-mail: [email protected]) 1
2
3
Kang H-J, Kim H-S, Zhang S-Y, et al. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet 2004; 363: 751–56. Hill JM, Paul JD, Powell TH, et al. Efficacy and risk of granulocyte colony stimulating factor administration in patients with severe, coronary artery disease. Circulation 2003; 108 (suppl IV): 478. Korbling M, Estrov Z. Adult stem cells for tissue repair-new therapeutic concept? N Engl J Med 2003; 349: 570–82.
Authors’ reply Sir—In response to Carlo Palmieri and colleagues’ questions, we present updated results from nine patients in the cell infusion group and seven patients in the G-CSF group who completed a 6-month follow-up assessment. We did not find any significant differences between the cell infusion and the G-CSF group in terms of changes in white-cell counts. Baseline counts were 7864 per L (SD 2210) in the cell infusion group and 6723 per L (2119) in the G-CSF group (p=0·254). After 4 days of G-CSF injection, white-cell counts increased to 46 983 per L (8798) and 45 522 per L (14 243), respectively (p=0·789). Also there were no significant differences in the proportion of peripheral blood mononuclear cells between the two groups after G-CSF injection (15·4% [4·7] in the cell infusion group vs 14·2% [5·1] in the G-CSF group; p=0·594). White-cell counts after G-CSF did not show any significant correlation with change in left ventricular systolic function (r=–0·252, p=0·346) nor myocardial perfusion (r=–0·276, p=0·301) in either group. We did not measure peripheral blood CD34+ cell counts in this study, only CD34 counts in the apheresis product. There were no significant correlations between improvements in systolic function and such counts (r=0·075, p=0·861) nor the proportion of collected and infused CD34+ cells (r=–0·018, p=0·966). We did not think that intravenous infusion of collected cells could significantly change the outcomes in the cell infusion group. Although remaining collected cells were infused through a vein in the forearm, we might not have excluded a possible “first pass effect” of highly concentrated peripheral blood stem cells and mononuclear cells on the coronary circulation. However, only small numbers of intravenously infused cells can reach the coronary circulation.1,2 If we adopt Strauer’s calculation,1 only 3% of cardiac output can reach the coronary circulation. Therefore, less than 3% of intravenously infused cells could reach the infarcted myocardium. When compared with the numbers of cells infused directly into the target coronary artery, only small numbers of intravenously infused cells could have contributed to improvement of cardiac function and myocardial perfusion. We also saw no significant
THE LANCET • Vol 363 • May 22, 2004 • www.thelancet.com
correlation between intravenously infused cell numbers and change in left ventricular systolic function (r=–0·092, p=0·813). *Hyo-Soo Kim, Hyun-Jae Kang, Young-Bae Park Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea (e-mail: [email protected]) 1
2
Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002; 106: 1913–18. Gregg DE, Fisher LC. Blood supply to the heart. In: Handbook of physiology, volume 2. Washington, DC: American Physiological Society, 1963: 1517–84.
Sir—Hyun-Jae Kang and colleagues1 describe the effects of G-CSF alone or intracoronary infusion of G-CSFmobilised, unselected peripheral blood leucocytes at the time of angioplasty and stenting, 2–6 days after a myocardial infarction. There were no acute negative effects, and patients who received peripheral blood leucocytes showed improvements in several endpoints. The trial was stopped early because of excessive instent restenosis at late follow-up—a finding noted also in patients who received only G-CSF. In-stent restenosis, due to neointimal proliferation (probably as a result of vessel injury by balloon or stent struts), is rapidly progressive initially, but after the acute insult (3–6 months), typically does not progress.2 Moreover, as discussed in an accompanying Commentary,3 two other coronary stem-cell infusion studies have been done (that did not use G-CSF). No increase in in-stent restenosis was noted in these trials. It seems reasonable to conclude, therefore, that G-CSF given at the time of stenting can cause excessive instent restenosis. In his Commentary, Hiroaki Matsubara3 argues against the use of G-CSF in all patients with coronary disease, largely on the basis of the findings of Kang and colleagues and a preliminary report of one death in 12 patients with severe, refractory coronary disease who received G-CSF.4 However, given the severity of disease in the high-risk patients in this latter study, it is difficult to attribute this single death, more than a week after cessation of treatment, to G-CSF. In animal models of ischaemia, G-CSF has impressive beneficial effects. In the same issue, Richard Vulliet and colleagues5 describe features
1735
For personal use. Only reproduce with permission from The Lancet publishing Group.
Sir—The article by Lars Klareskog and colleagues1 is impressive and encouraging, and offers hope for remission and avoidance of structural damage in a high proportion of patients with rheumatoid arthritis. However, some questions remain unresolved: why is improvement not more universal? and what is the optimum timing for combined treatment? I am not surprised that the combination of etanercept and methotrexate is not more universally effective since the range of autoimmune hyperactivity in rheumatoid arthritis is broad and suppression of tumour necrosis factor (TNF) activity by etanercept addresses only a fraction of that range. Methotrexate has a wider range of activity than does etanercept, but is not specific enough to counteract all aspects of immune hyperactivity found in rheumatoid arthritis. In view of the objectives of the research programme, that this article deals with the pharmacological aspects of rheumatoid arthritis is understandable; however, we should not lose sight of psychosocial factors. Restoration of a normal immune pattern might not be achievable by pharmacological means alone and could additionally require a psychosocial approach. Baker and Brewerton2 reported an association between stressful life circumstances and rheumatoid arthritis. Moreover, a link between long-term psychosocial stress, chronic fatigue, and rheumatic disorders has also been noted.3 Chronic fatigue syndrome can be triggered by infection, psychosocial stress, or both. It is accompanied by mild cortisol insufficiency4 and thus an overactive immune system, with, among other changes, overproduction of several cytokines, including TNF ␣ and TNF ; increased activity of CD4 (helper) T cells and B cells; and autoto antibody production5—similar changes found in rheumatoid arthritis. Overall treatment of patients with rheumatoid arthritis should include not only pharmacological means, such as the combined therapy used in the TEMPO trial,1 but also lifestyle changes, which help to counteract chronic fatigue—eg, increased rest, graded exercises, psychotherapy, and financial and social support where needed. Alex Poteliakhoff Sudbury, 16B Prince Arthur Road, London NW3 6AY, UK 1
Klareskog L, van der Heijde D, de Jager J, et al. Therapeutic effect of the combination of etanercept and methotrexate compared with each treatment alone in patients with rheumatoid arthritis: double-blind randomised controlled trial. Lancet 2004; 363: 675–81.
1734
2
3 4
5
Baker GHB, Brewerton DA. Rheumatoid arthritis: a psychiatric assessment. BMJ 1981; 282: 2014. Poteliakhoff A. Emotion and immunity. Lancet 1985; 326: 327. Poteliakhoff A. Adrenocortical activity and some clinical findings in acute and chronic fatigue. J Psychosom Res 1981; 25: 91–95. Poteliakhoff A. Fatigue syndromes and the aetiology of autoimmune disease. J Chronic Fatigue Syndr 1998; 4: 31–49.
African women, bride price, and AIDS Sir—The report by Charles Wendo (Feb 28, p 716)1 blamed bride price for the spread of AIDS in sub-Saharan Africa. It also cites bride price as an important factor in polygamy. However, the contribution of bride price to these problems is minuscule. Traditionally, payment of the bride price by the groom was a demonstration that he had come of age and had the means and capability to support himself and his wife. Acceptance of the bride price signifies the families’ support and blessing of the union. Rejection or nonacceptance of the bride price meant non-acceptance of the marriage proposal. Payment and acceptance of the bride price, whatever the currency used, seals the traditional marriage ceremony and authenticates the union between the man and woman. Once the woman is married, the poor treatment she might become subject to within the relationship has nothing to do with the bride price, but is a direct result of the fact that she has little say within the marriage as a result of her poor socioeconomic status. Therefore, even if the bride price were abolished, a woman’s lot within the marriage would not be different from what it currently is. The only thing that would change African women’s lot is the power to negotiate within the relationship. At the moment they have no powers, not because of the bride price, but because they have no education and are therefore totally dependent on their husbands.2 There is no doubt that like everything else the institution of bride price has been subject to abuse: prospective in-laws have used the bride price to make unreasonable and unrealistic demands of the groom. This situation can lead to tensions between the families and indeed couples, but so can many other sources of disagreement. The system therefore is in need of reform, but to blame it for the spread of HIV infection and polygamy is without
basis and a misdirection of the thrust for change. Polygamy will certainly spread HIV, but religion (Islam and African traditional religions allow polygamy) is responsible for that, not bride price. What is needed is a concerted effort in the education of African women. Parents, governments, and nongovernmental organisations need to promote this agenda. Education will empower African women to make better choices and negotiate within relationships. It would also give them independence and the means and ability to leave abusive relationships.3 Umo I Esen South Tyneside District Hospital, Harton Lance, South Shields, Tyne and Wear NE34 0PL, UK (e-mail: [email protected]) 1 2
3
Wendo C. African women denounce bride price. Lancet 2004; 363: 716. Esen UI. Violence against women in developing countries. East African Med J 2000; 77: 400. Harrison KA. The importance of the educated healthy woman in Africa. Lancet 1997; 349: 644–47.
Stem-cell therapy for myocardial diseases Sir—We wish to raise several issues regarding the publication by Hyun-Jae Kang and colleagues (Mar 6, p 751)1 on the role of intracoronary infusion of peripheral-blood stem cells mobilised with granulocyte colony stimulating factor (G-CSF) in patients who had had a myocardial infarction. First, data on the effects of G-CSF in the 11 enrolled patients are incomplete. Although the baseline total white-cell count is provided for all patients, there is no documentation of the change in the white-cell count and CD34 count during the 4 days of treatment with GCSF in these patients, and how these changes correlated with changes in cardiac function. This information is important because a potential reason for the differences seen between the two groups is that the group who received only G-CSF did not mobilise their bone marrow as well as the group who received G-CSF plus cell infusion—a fact not acknowledged in the Discussion. Kang and colleagues’ acceptance that there is little experience in the use of G-CSF in patients after myocardial infarction, the previously documented association between GCSF and three deaths,2 and the fact that the CD34 count was used as the stemcell marker by Kang and colleagues makes it vital that all such data is presented to allow a more complete analysis of their results.
THE LANCET • Vol 363 • May 22, 2004 • www.thelancet.com
For personal use. Only reproduce with permission from The Lancet publishing Group.
CORRESPONDENCE
Second, in the cell infusion group, any remaining white blood cells and peripheral-blood stem cells that were not infused via the intracoronary route were reinfused via the peripheral intravenous route. Apheresis produced a mean of 9·0⫻109 leucocytes (SD 3·0⫻109), with a mean of 1·5⫻109 (0·5⫻109) being reinfused via the intracoronary route. Therefore most of the white blood cells and peripheral-blood stem cells were returned via the intravenous route. This fact is not controlled for in the G-CSF only group and no comment is made about its possible confounding effect on the results. In light of this possibility and the lack of information about haematological variables, we find it difficult to support the authors’ conclusion that the intracoronary infusion of peripheral-blood stem cells was responsible for the improvement in cardiac function seen in the G-CSF plus cell infusion group. Future trials involving G-CSF in this setting should document the changes in white-cell count and CD34+ cells, as well as the number and phenotype of the cells reinfused with these parameters being correlated with outcome. Such data would provide insight into the efficacy of G-CSF in this group of acutely unwell patients, and will allow for proper control between treatment groups. Changes in total white-cell count and CD34+ cells could potentially be surrogate markers for restoration of cardiac function, and a potential marker of adverse events and complications such as restenosis. Finally, since the true stem cell for cardiac regeneration is not known, phenotyping is vital if a systematic approach is to be taken to attempt its identification, especially because future researchers might choose to infuse different stem-cell preparations.3 *Carlo Palmieri, David Vigushin, R Charles Coombes Department of Cancer Medicine, Cancer Research UK Laboratories, 5th Floor MRC Cyclotron Building, Faculty of Medicine, Imperial College, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK (e-mail: [email protected]) 1
2
3
Kang H-J, Kim H-S, Zhang S-Y, et al. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet 2004; 363: 751–56. Hill JM, Paul JD, Powell TH, et al. Efficacy and risk of granulocyte colony stimulating factor administration in patients with severe, coronary artery disease. Circulation 2003; 108 (suppl IV): 478. Korbling M, Estrov Z. Adult stem cells for tissue repair-new therapeutic concept? N Engl J Med 2003; 349: 570–82.
Authors’ reply Sir—In response to Carlo Palmieri and colleagues’ questions, we present updated results from nine patients in the cell infusion group and seven patients in the G-CSF group who completed a 6-month follow-up assessment. We did not find any significant differences between the cell infusion and the G-CSF group in terms of changes in white-cell counts. Baseline counts were 7864 per L (SD 2210) in the cell infusion group and 6723 per L (2119) in the G-CSF group (p=0·254). After 4 days of G-CSF injection, white-cell counts increased to 46 983 per L (8798) and 45 522 per L (14 243), respectively (p=0·789). Also there were no significant differences in the proportion of peripheral blood mononuclear cells between the two groups after G-CSF injection (15·4% [4·7] in the cell infusion group vs 14·2% [5·1] in the G-CSF group; p=0·594). White-cell counts after G-CSF did not show any significant correlation with change in left ventricular systolic function (r=–0·252, p=0·346) nor myocardial perfusion (r=–0·276, p=0·301) in either group. We did not measure peripheral blood CD34+ cell counts in this study, only CD34 counts in the apheresis product. There were no significant correlations between improvements in systolic function and such counts (r=0·075, p=0·861) nor the proportion of collected and infused CD34+ cells (r=–0·018, p=0·966). We did not think that intravenous infusion of collected cells could significantly change the outcomes in the cell infusion group. Although remaining collected cells were infused through a vein in the forearm, we might not have excluded a possible “first pass effect” of highly concentrated peripheral blood stem cells and mononuclear cells on the coronary circulation. However, only small numbers of intravenously infused cells can reach the coronary circulation.1,2 If we adopt Strauer’s calculation,1 only 3% of cardiac output can reach the coronary circulation. Therefore, less than 3% of intravenously infused cells could reach the infarcted myocardium. When compared with the numbers of cells infused directly into the target coronary artery, only small numbers of intravenously infused cells could have contributed to improvement of cardiac function and myocardial perfusion. We also saw no significant
THE LANCET • Vol 363 • May 22, 2004 • www.thelancet.com
correlation between intravenously infused cell numbers and change in left ventricular systolic function (r=–0·092, p=0·813). *Hyo-Soo Kim, Hyun-Jae Kang, Young-Bae Park Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea (e-mail: [email protected]) 1
2
Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002; 106: 1913–18. Gregg DE, Fisher LC. Blood supply to the heart. In: Handbook of physiology, volume 2. Washington, DC: American Physiological Society, 1963: 1517–84.
Sir—Hyun-Jae Kang and colleagues1 describe the effects of G-CSF alone or intracoronary infusion of G-CSFmobilised, unselected peripheral blood leucocytes at the time of angioplasty and stenting, 2–6 days after a myocardial infarction. There were no acute negative effects, and patients who received peripheral blood leucocytes showed improvements in several endpoints. The trial was stopped early because of excessive instent restenosis at late follow-up—a finding noted also in patients who received only G-CSF. In-stent restenosis, due to neointimal proliferation (probably as a result of vessel injury by balloon or stent struts), is rapidly progressive initially, but after the acute insult (3–6 months), typically does not progress.2 Moreover, as discussed in an accompanying Commentary,3 two other coronary stem-cell infusion studies have been done (that did not use G-CSF). No increase in in-stent restenosis was noted in these trials. It seems reasonable to conclude, therefore, that G-CSF given at the time of stenting can cause excessive instent restenosis. In his Commentary, Hiroaki Matsubara3 argues against the use of G-CSF in all patients with coronary disease, largely on the basis of the findings of Kang and colleagues and a preliminary report of one death in 12 patients with severe, refractory coronary disease who received G-CSF.4 However, given the severity of disease in the high-risk patients in this latter study, it is difficult to attribute this single death, more than a week after cessation of treatment, to G-CSF. In animal models of ischaemia, G-CSF has impressive beneficial effects. In the same issue, Richard Vulliet and colleagues5 describe features
1735
For personal use. Only reproduce with permission from The Lancet publishing Group.