Jans Smpj

A ; De Jonge

Alm Lagro-Janssen

Maternal and perinatal outcomes among haemoglobinopathy carriers: A systematic review

Keywords: premature labour, low birth weight, intrauterine growth retardation, miscarriage, neonatal death, low apgar score, neural tube defects Haemoglobinopathy, sickle cell trait, thalassaemia minor, pregnancy

Background: With the introduction of screening programs for haemoglobinopathies, more women will be aware of their HbP status. Midwives and obstetricians need to know whether pregnant HbP carriers have any special needs besides the genetic risk.

The objective was to investigate the hypothesis that being a carrier of HbP has no consequences for the health of pregnant women and the outcome of their pregnancies.

Methods: A systematic search was carried out until august 2008 in the Cochrane Library; Medline; EMBASE and CINAHL databases. All references were inspected to identify further studies. The authors of key-publications were contacted for any unpublished research.

Selection criteria: Cohort and case control studies, pregnant women with a singleton pregnancy. Exposure: HbAS or thalassaemia minor. Outcomes: urinary tract infection, anaemia, (pre-)eclampsia, gestational diabetes,

Introduction

It is generally accepted that the pregnancy of a woman with homozygotic haemoglobinopathy (HbP) disease and some double heterozygous conditions, such as HbSC and HbS-β-thalassaemia, carries a higher risk. While the genetic implications of HbP carrier status are clear, the effect if any, on the outcome of pregnancy remains subject to debate.

Thalassaemia minor (carriers) constitutes of alpha and beta defects and some rare exceptions [START_REF] Harteveld | Novel 112 kb (epsilonGgammaAgamma) deltabeta-thalassaemia deletion in a Dutch family[END_REF]. The most well known of the structural defects is HbS, like beta thalassaemia this is a beta gene defect that is non-active in the fetus until birth. Over the years, several studies on the effect on pregnancy have been published but provide inconsistent conclusions. Some studies suggest an increased risk for adverse perinatal outcomes [START_REF] Abdulsalam | Pregnancy outcomes among Palestinian refugee women with sickle cell trait in Damascus, Syria[END_REF][START_REF] Roopnarinesingh | Decreased birth weight and femur length in fetuses of patients with the sickle-cell trait[END_REF][START_REF] Taylor | Pregnancy loss after first-trimester viability in women with sickle cell trait: time for a reappraisal?[END_REF] while others cannot confirm this risk [START_REF] Adeyemi | Outcome of pregnancy in a population of Nigerian women with sickle cell trait[END_REF][START_REF] Blank | Sickle cell trait and pregnancy[END_REF][START_REF] Stamilio | Pregnant women with the sickle cell trait are not at increased risk for developing preeclampsia[END_REF][START_REF] Tita | Perinatal and maternal outcomes in women with sickle or hemoglobin C trait[END_REF][START_REF] Whalley | Sickle cell trait and pregnancy[END_REF]. Pregnant women with homozygotic HbP disease need to be referred to a tertiary care centre. Pregnant women who are (known) HbP carriers are usually considered low risk as no risks for their health or the outcome of their pregnancies are assumed. However it is unclear whether these women have special needs during pregnancy. As far as we are aware, no systematic review has been conducted on this subject. with sickle cell disease or thalassaemia major or is a carrier, the parents will be offered genetic screening tests to confirm their carrier status which will then enable them to make appropriate reproductive choices concerning any subsequent pregnancies. Midwives and obstetricians will therefore increasingly encounter women who are aware of their HbP carrier status. The prevalence of an HbP carrier status is currently estimated to be 4-14 % amongst people with non-Northern European background in the Netherlands [START_REF] Giordano | Prevention of hereditary haemoglobinopathies in The Netherlands[END_REF] and according to the World Health Organisation approximately 5 % of the world population is a carrier of a significant variant of haemoglobin disorders such as sickle cell disorders or thalassaemias with as many as 40% carriers in some regions [START_REF] Modell | Global epidemiology of haemoglobin disorders and derived service indicators[END_REF]. It is therefore important that midwives and obstetricians are aware of any potential risks among HbP carriers during pregnancy.

In this systematic review we will address the possible risks for pregnant HbP carriers and investigate the hypothesis that being an HbP carrier has no consequences for the health of pregnant women and the outcome of their pregnancies.

Methods

With the aid of a librarian, we searched the Cochrane Library; Medline; EMBASE and CINAHL databases for publications in English, French, German, Spanish and Dutch, examining the effect of sickle cell trait and/or thalassaemia minor on maternal morbidity or perinatal morbidity and mortality.

The search was not restricted by publication date and was carried out until august 2008. We only searched for (prospective and retrospective) cohort studies and case control studies. We inspected the references of all studies found to identify any further studies. The authors of key-publications were contacted for any unpublished research.

The initial selection of studies by title and abstract was carried out by one reviewer (SJ) based on its relevance to the research question and, if possible, by the inclusion criteria (table 1). The studies found were assessed and scored by two independent reviewers (SJ and AdJ). Details of authors, affiliated institutions, journal name, and year of publication were removed for the second reviewer.

Selection for inclusion into the review was made on the basis of in-and exclusion criteria and quality assessment. The use of checklists is disputed by some [START_REF] Greenland | On the bias produced by quality scores in meta-analysis, and a hierarchical view of proposed solutions[END_REF]. Sanderson highlighted the lack of existence of an adequate tool, but he nevertheless advised to use a simple checklist rather than a scale and concluded that it is important to use a small number of key domains for quality assessment [START_REF] Sanderson | Tools for assessing quality and susceptibility to bias in observational studies in epidemiology: a systematic review and annotated bibliography[END_REF]. We decided to use the guidelines for assessing the quality of observational and case control studies as defined by the Dutch Institute for Healthcare Improvement to support our quality assessment [START_REF]Guidelines for assessing the quality of observational and case control studies[END_REF]. These include all the elements mentioned by Sanderson [START_REF] Sanderson | Tools for assessing quality and susceptibility to bias in observational studies in epidemiology: a systematic review and annotated bibliography[END_REF], and have been carefully developed by the institute in collaboration with the Dutch Cochrane Centre and are generally accepted as adequate in the Netherlands (table 2). We awarded a maximum of two points for each item on the list, meaning that each study could score a maximum of 16 points. We excluded any study which obtained a score of less than 12 points. This high cut-off point was chosen because almost all studies automatically scored high on "adequate follow-up" and "selective lossto-follow-up". Some authors were contacted for clarification of the study methods.

Data were extracted independently from the included studies by two investigators (SJ and AdJ) on study design, population and setting, screening method, exposition, outcome variables, confounders and point estimates. Differences were resolved by discussion.

(Statistical) analysis

We used population (ethnicity), exposition, screening method and (definition of) outcome variables to assess clinical heterogeneity. Study design, confounders and point estimates were used to assess methodological heterogeneity. Confounders were assessed for all separate outcomes. Both clinical and methodological homogeneity could not be established; therefore after consultation with a statistician, we decided not to pool the results.

Results

We initially identified 780 studies from which we selected 91 titles (fig. 1). After screening the abstracts, we read 36 papers. By inspecting the references of these papers, we identified a further 30 studies of which nine studies were read and assessed. Contact with the authors provided one additional study which was only available as a (PhD-) thesis (15). In total 46 studies were assessed for methodological quality. The two reviewers disagreed on six out of 46 studies (13 %). Consensus was reached by discussion.

Nine studies on HbAS met the inclusion criteria and scored 12 points or more and therefore were of sufficient methodological quality to be included in the review. Only one of these studies investigated the effect of thalassaemia minor on pregnancy (15). None of the studies into premature birth stated whether the expected date of delivery was confirmed by ultrasound scan (table 3). Bryant [START_REF] Bryant | Presence of the sickle cell trait and preterm delivery in African-American women[END_REF] showed a significant protective effect for delivery before 32 weeks in women with sickle cell trait (SCT), whereas Tita [START_REF] Tita | Perinatal and maternal outcomes in women with sickle or hemoglobin C trait[END_REF] showed a protective effect for deliveries at less than 37 weeks gestation. Stamilio [START_REF] Stamilio | Pregnant women with the sickle cell trait are not at increased risk for developing preeclampsia[END_REF] showed no difference between the two groups. The prospective cohort study of Landman (15) found that HbP carriers had a smaller but non-significant risk of a premature delivery.

The prospective cohort study by Larrabee [START_REF] Larrabee | Women with sickle cell trait are at increased risk for preeclampsia[END_REF] only looked at the mean gestational age and showed a significant reduction in the length of gestation by one week in women with SCT.

None of the four studies examining the risk of low birth weight in infants of women with SCT showed a significant effect (2;5;7;8) (table 3). Four studies (5;8;15;18) looked at intra uterine growth retardation (IUGR) (table 3). Two studies stated that dates were confirmed by ultrasound scan and only Tan [START_REF] Tan | Sickle-cell trait and small-for-gestational age babies: Is there a link?[END_REF] and Landman (15) gave a clear definition of IUGR. Tita [START_REF] Tita | Perinatal and maternal outcomes in women with sickle or hemoglobin C trait[END_REF] supplied the definition of IUGR and Landman (15) confirmed dating by ultrasound scan when contacted. None of the studies found any effect of HbP carrier status on the risk of IUGR.

Risk of urinary tract infection (UTI) and pyelonephritis:

None of the three studies into the risk of UTI in pregnant women with SCT showed an increased risk (2;5;15) (table 4). In only one study, a definition of UTI was given (15). Two studies showed a small but statistically significant increased risk for bacteriuria in pregnant women with SCT (8;19) and in one of them the risk of pyelonephritis was also increased [START_REF] Bail | Sickle trait and its association with birthweight and urinary tract infections in pregnancy[END_REF]. Only three included studies looked at anaemia in pregnant HbP carriers (2;5;15) (table 4).

Iron Deficiency Anaemia (IDA) and folate deficiency

None of them took physiological haemodilution into account. Adeyemi [START_REF] Adeyemi | Outcome of pregnancy in a population of Nigerian women with sickle cell trait[END_REF] did not give a definition of IDA and found no statistically significant difference between groups. Abdulsalaam (2) found significantly more anaemia in the HbAS group. Landman (15) found a difference between groups when IDA was found in combination with folate deficiency. A significantly higher percentage of anaemia with a Hb ≤ 90 g/l was found in the HbAS group (5.5% vs 3.6%, p=<0.05). Looking at the specific causes of anaemia, IDA occurred more often in the control group but this difference was not statistically significant.

A combined iron and folate deficiency occurred significantly more frequently in the HbAS group. The group with thalassaemia trait had less IDA but this was not statistically significant. Folate deficiency and combined iron and folate deficiency, occurred more in the thalassaemia group but this was not statistically significant.

High blood pressure: Pregnancy Induced Hypertension (PIH) and Preeclampsia (PE)

Three studies (5;8;15) investigated the relationship between being a HbP carrier and PIH of which two (8;15) gave a definition of PIH (table 5). None of the three studies was able to demonstrate a significant effect. Four studies (2;7;8;17) looked at pre-eclampsia of which only one [START_REF] Larrabee | Women with sickle cell trait are at increased risk for preeclampsia[END_REF] found a significantly increased risk for PE in women with SCT.

Miscarriage, fetal and neonatal death

Three studies into the risk of fetal and neonatal death found opposite effects but none of these were significant (2;5;8) (table 5). Another study (15) found only one case in the control group (results not in table ). Two studies investigated the effect of SCT on stillbirths alone (5;8). These studies showed a protective effect but in one this was not significant [START_REF] Adeyemi | Outcome of pregnancy in a population of Nigerian women with sickle cell trait[END_REF]. Tita (8) found a very small non-significant reduction in fetal death. No studies examined the risk of miscarriage.

Low Apgar score

Two studies looked at the Apgar score (table 5) (5;15). One study showed a decreased incidence of low Apgar scores among women with HbAS (5) and another study showed an insignificantly increased risk (15). None of the included studies examined the effect of HbP carrier status on gestational diabetes or neural tube defects.

Discussion

As far as we know, this is the first systematic review that examines possible risks of pregnant women who are HbP carriers. Besides the obvious need for genetic counselling, it is important for midwives and obstetricians to be aware of any risk factors if these do exist. Some studies showed a protective effect of sickle cell trait for premature birth, low Apgar score and stillbirth: there was some evidence of an increased risk of anaemia and bacteriuria. No significant effect of HbP carrier status was found on low birth weight, growth retardation, urinary tract infection or high blood pressure. These results should be interpreted with caution because of several methodological problems. All studies gave adequate definitions of the exposure factor and of the population under investigation; however, many authors failed to give adequate definitions of the outcomes assessed in their studies and they often did not control for confounders In some studies (7;8;16;20) it was not clear how the expected date of delivery was calculated and if this was confirmed by ultrasound scan. Nevertheless, considering the time frame and the setting in which the studies took place (academic hospitals) it could be assumed that women had an ultrasound scan to assess the expected date of delivery.

However, in view of the fact that only one author [START_REF] Tita | Perinatal and maternal outcomes in women with sickle or hemoglobin C trait[END_REF] was able to confirm this when contacted, we need to be cautious with the overall conclusion.

In some studies (7;17) ethnicity was poorly defined and SCT may therefore have been underreported resulting in considerable bias.

The testing method to confirm carrier status was a problem in most studies. Only two studies used adequate methods that identified the presence of thalassaemia (15;18). The other studies used a sickle cell test and / or electrophoresis to establish HbP status of pregnant women. Two studies tested all women with a sickle test and confirmed only the positive women with electrophoresis (7;17). According to The British Committee for Standards in Haematology (BCHS) a sickle test may not detect low levels of HbS and should only be used in an emergency (www.bcshguidelines.com). Carriers may have been missed in these studies giving rise to further bias. According to the BCHS, electrophoresis is not recommended for the quantification of HbA2 necessary to establish carrier status of thalassaemia: HPLC or micro column chromatography should be used [START_REF]Working Party of the General Haematology Task Force of the British Committee for Standards in Haematology. Guidelines on the laboratory diagnosis of haemoglobinopathies[END_REF]. Most studies did not specifically state whether other fractions such as HbAC, HbAE or any of the thalassaemias were found and whether or not they were excluded. With the exception of two studies (18;15) none of the studies gave any information on the co-existence of HbS and thalassaemia. Many people of Afro-Caribbean descent with HbAS have a combination of HbAS and α-thalassaemia. High HbA2 β-thalassaemia or any other trait found in early pregnancy and suspected to be associated with thalassaemia major, sickle cell disease or with other severe pathologies, should be confirmed at DNA level in particular when a couple at risk is suspected [START_REF] Mosca | The role of haemoglobin A(2) testing in the diagnosis of thalassaemias and related haemoglobinopathies[END_REF].

Protective effect

A protective effect was found for preterm labour in carriers of HbP. However in view of the many methodological pitfalls, the conclusion of a protective effect is only tentative.

Besides, the patho-physiological pathways underlying these results are unclear.

In order to find a significant effect on stillbirth or perinatal mortality, a large population needs to be studied. Only Tita investigated a large group of women and found a small reduction in perinatal mortality and stillbirth which was borderline significant [START_REF] Tita | Perinatal and maternal outcomes in women with sickle or hemoglobin C trait[END_REF]. Apart from chance playing a role in this study, the antenatal policy of serial urine culture and the reduction in premature labour may also have had an effect on the rate of perinatal mortality.

Two studies found opposite effects on stillbirth (2;5). Consanguinity may have played a role in the study which found an increased risk (2). Bittles [START_REF] Bittles | Consanguineaous marriage and childhoodhealth[END_REF] showed that consanguinity is responsible for 4,4% of deaths between 6 months of pregnancy and 10 years of age. The other studies did not take consanguinity into account. The effect may be particularly important in studies carried out in the Middle East where the incidence of consanguine marriages is relatively high [START_REF] Bittles | The global prevalence of consanguinity[END_REF].

HbP carrier status appears to offer a small protective effect on perinatal mortality. However the patho-physiological mechanism behind this remains unclear and in view of the methodological flaws, the results remain questionable. No statistically significant difference was found for the risk of low birth weight (due to prematurity or growth retardation). Two of the studies were carried out in developing countries where both the health care standards can differ considerably and the population may have different needs than those in developed countries [START_REF]Worldwide prevalence of anaemia 1993-2005. WHO Global database on anaemia[END_REF]. As both a poor nutritional status and anaemia increase the risk of low birth weight (26), a difference based on HbP status may not have been detected. On the basis of these studies, it cannot be concluded that HbAS has any effect on the risk of low birth weight.

No effect

We found no increased risk of intra uterine growth retardation among women with a positive carrier status for HbP.

Only Larrabee (17) found a significantly higher risk of both (pre-) eclampsia and pregnancy induced hypertension in women who are a carrier of SCT. Other studies found no significant differences (2;5;7;8;15). This could be due to methodological problems as discussed earlier. None of the authors corrected for maternal weight in their study. We conclude that the relation between HbP carrier status and the risk of (pre-) eclampsia or pregnancy induced hypertension is inconclusive. Further studies are urgently needed to shed light on this issue. Especially since hypertensive disorders in pregnancy are the leading cause of maternal death in the Netherlands [START_REF] Schuitemaker | Confidential enquiry into maternal deaths in The Netherlands 1983-1992[END_REF], rank fourth place in the United Kingdom [START_REF]Why mothers die. The confidential enquiries into Maternal Deaths in the United Kingdom[END_REF] and are the second direct cause of maternal death in the United States of America (www.mchb.hrsa.gov). Moreover, a recent study carried out in the Netherlands showed that non-Western immigrant women had a 1.3-fold increased risk of severe maternal morbidity, including PIH, when compared with Western women [START_REF] Zwart | Severe maternal morbidity during pregnancy, delivery and puerperium in the Netherlands: a nationwide population-based study of 371,000 pregnancies[END_REF]. The generally accepted claim that pregnant sickle cell carriers have a higher risk of UTI in pregnancy (30;31) could not be substantiated in this review. Two of the three included studies (2;5;15) failed to give a definition of UTI which may have led to classification bias.

Although we could not confirm an increased risk for UTI in the pregnancies of HbP carriers, we did find an association between sickle cell trait and an increased risk of bacteriuria in two studies (8;19). Treatment of asymptomatic bacteriuria reduces the risk of pyelonephritis in pregnancy (32). Surprisingly, only three studies were included which investigated the risk of anaemia amongst pregnant women with an HbP-trait. It is generally accepted that a positive carrier status for HbP can give rise to anaemia in pregnancy (32); however this has not been very well investigated. Although the overall consensus seems to be that sickle cell trait is asymptomatic and thalassaemia minor may cause mild microcytic or hypochromic anaemia in the general population at risk (33) (www.thalassaemia.org.cy), this systematic review shows that few investigators have adequately studied the effect of HbP carrier status on anaemia in pregnancy. Small numbers in all three studies may be the reason that little or no effect was found.

Despite the limited power, Landman (15) found that women with HbAS or ß-thalassaemia trait have a higher chance of having a combined iron and folate deficiency anaemia. This study did not take confounders for anaemia into account, such as twin-pregnancy, poor dietary habits or a low socio-economic status (SES) and the study was carried out amongst a high risk population which may have included women that were already referred because of an increased risk of anaemia. Abdulsalaam (2) did not use anaemia as an outcome measure but instead used it as a risk factor for the outcomes to be studied and subsequently found a significantly increased percentage of women with HbAS that were anaemic. Whether this is due to iron and/or folate deficiency was not clarified in this study. It is possible that women who were sickle cell carriers had in fact a combination of sickle cell trait and α-thalassaemia which was not investigated. It is very likely that a higher risk of anaemia amongst HbP carriers is caused by the (combined) effect of thalassaemias as these are known for ineffective erythropoiesis which in pregnancy because of the increase in blood volume, could result in more pronounced anaemia especially in the second trimester. Because of the disturbed Hb synthesis in HbP carriers, it is very likely that extra folic acid is needed. This may very well be the reason that most textbooks (30;31;34) recommend supplementing these pregnant women with folic acid. However we found no study that investigated and confirmed this theory.

Besides this, a recent study found that women of Non-Northern European descent had a significantly increased risk of becoming anaemic in pregnancy [START_REF] Jans | Are pregnant women of non-Northern European descent more anaemic than women of Northern European descent? A study into the prevalence of anaemia in pregnant women in Amsterdam[END_REF]. The researchers were not able to identify the cause of this increased risk. In view of the evidence found in this systematic review the increased risk could partly be explained by women who are a carrier of HbP.

Conclusion

In conclusion, the findings of this study show that pregnant women who are HbP carriers have a low risk of adverse outcomes of pregnancy. In expectation of further research on this subject, midwives and obstetricians should be aware of the possibility of anaemia, asymptomatic bacteriuria and the subsequent risk of pyelonephritis and investigate pregnant women who are known HbP carriers more frequently for these conditions.

If carrier status of these women is known and anaemia is present, the cause of it should be investigated further before any treatment is started. Each woman should be correctly diagnosed and be offered partner testing if necessary so that she can make a well informed women with SCT or thalassaemia minor are given correct advice on how this will affect their pregnancy and be reassured that it will not increase their risk of developing complications during pregnancy or shortly thereafter. We found no reason at present to refer pregnant women who are a carrier of HbP to a high risk setting.

This study highlights the lack of evidence of the effect of a positive HbP status on the outcome of pregnancy and the need for large, well controlled prospective studies. With routine investigations into HbP becoming more standard in many countries, it will become easier to conduct further research to thoroughly investigate any possible effect that this may have on pregnancy.

F

Inclusion criteria

Types of studies

All observational (prospective and retrospective) cohort studies and case control studies examining the effect of sickle cell trait and/or thalassaemia minor on maternal morbidity and neonatal morbidity and mortality.

Types of participants

Pregnant women with singleton pregnancy.

Types of exposure

Sickle cell trait or heterozygous α-/β-thalassaemia or homozygous α+ thalassaemia versus no trait or no thalassaemia.

Sickle cell trait is defined as the presence of HbAS. Carrier ship of thalassaemia is defined as α+-thalassaemia heterozygote; α0-thalassaemia heterozygote; α+-thalassaemia homozygote or β-thalassaemia heterozygote. -Were the groups that were compared clearly identified?

Types of outcome measures

-Could selection bias be sufficiently excluded?

-Was the exposure factor clearly identified and was the method for assessing the exposure factor adequate?

-Was the outcome clearly identified and was the method for assessing the outcome adequate?

-Was the outcome assessed blinded for the exposure factor?

-Was the follow-up adequate?

-Could selective loss-to-follow-up be excluded?

  cell disease has been added to the existing neonatal screening program in several countries, such as the Netherlands and England. If a baby has been diagnosed

  were: Pregnant women, sickle cell, sickle cell trait, sickle cell anaemia, sickle cell disease, thalassaemia, heterozygote thalassaemia, haemoglobinopathy, abnormal haemoglobines, pregnancy outcome, complications of pregnancy, urinary tract infections, iron deficiency anaemia, folic acid deficiency anaemia, (pre-)eclampsia, gestational diabetes, premature labour, low birth weight, miscarriage, fetal / neonatal death, low Apgar score and neural tube defects.

6

 6 Premature birth/ (Low)Birth weight (LBW)/ Intra Uterine Growth retardation(IUGR)

Deleted:

  Confirmation of traits other than those associated with sickle cell disease should be done at DNA level

  order to avoid unexpected outcomes. It also remains important that

Table 1 :

 1 Inclusion criteria

	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 2 :

 2 Quality assessment criteria

Table 3 :

 3 Premature birth, Low birth weight, Small for gestational age / Intra uterine growth retardation

Table 4 :

 4 Urinary tract infection, Anaemia in pregnancy

Table 5 :

 5 Pregnancy induced hypertension / pre-eclampsia, Post natal mortality rate / still

	birth, Low apgar score
	Fig.1: Flowchart of search

Table 1 .

 1 Inclusion criteria

Table 2 .

 2 Quality assessment criteria

	Urinary tract infections
	Iron deficiency anaemia
	Folic acid deficiency anaemia
	(Pre-) eclampsia
	Gestational diabetes
	Premature labour
	Low birth weight
	Intra uterine growth retardation
	Spontaneous abortion
	Fetal / neonatal death
	Low Apgar score
	Neural tube defects

Page 27 of 27 International Journal of Clinical Practice International Journal of Clinical Practice

  Taylor 2006 (4), Switzer[START_REF] Switzer | The sickle cell trait; incidence and influence in pregnant colored women[END_REF],Whalley 1963 (9), Roopnarinesingh 1986 (3), Hoff 1983 (37), Lam 1999(38), Landman 1985 (39), Miller 1983 (40), Okunofua 1990 (41), Thurman 2006 (42), White 1985 (43), Lao 2001 (44), Abrahams 1959 (45), Balgir 1997 (46), Tuck 1983 (47), Whalley 1964 (48), Ssebabi 1977 (49), Roopnarinesingh 1986 (50), Rimer 1975 (51), Ibba 2003 (52), Hamdi 2002 (53), Cooley 1984 (54), Brown 1972 (55), Blattner 1977(56), Pearson 1969 (57), Pritchard 1973 (58), McCurdy 1964 (59), Morrison 1979(60), Pakes 1970 (61), Pastore 1999 (62), Whiteside 1973 (63), Pastore 1999(64), Hoff 2001 (65), Sheiner 2004 (66), Lewis 1982 (67), White 1986(68).

	study 2003	Health clinics (Primary care	controls (77) Fig. 1 Flowchart of search PNMR: no definition given
		setting)			Homozygote α-		25.0% vs 2.4%
	Stamilio (7)	1996	PE:		thal.2 (8) vs SCT (87) vs	AOR = 0.5	(ns)
	USA Retrospective cohort study 780 titles identified Ethnicity mixed University hospital Initial selection by first author	Blood pressure of >140/90 mm Hg on 2 separate	Black controls (83) none-SCT (1909)	(CI: 0.2 -1.6)
	2003	*Subanalysis on	occasions, at least
		afro-american	6 hrs apart and		689 titles excluded for following reasons:
		women: author contacted but no reply	proteinuria >300mg/24 hrs or 1+ to 2+ on serial	Study not related to pregnancy or research design did not match inclusion criteria in research protocol
			quali exam.
	91 abstracts were assessed			55 abstracts excluded for following reasons:
	For all tables: SCT: Sickle cell trait			Not related to pregnancy or not according to inclusion criteria as per protocol.
	EDD: Expected date of delivery
	-Were the most important confounders identified and was correction for the F o r F o r F o r F o r US: Ultra sound OR: Odds ratio AOR: Adjusted odds ratio RR: Relative risk F o author contact yielded another 31 titles r Snowball method of references and 21 abstracts excluded
	confounders carried out? CI: Confidence interval P: P-value significant if < 0.05	P P P P P
	NS: not significant		e e e e e
	PNM: Post natal mortality rate PE: Pre-eclampsia In Total 36 + 9+1 full studies assessed by two researchers	e r e r e r e r 37 studies excluded: did not match inclusion criteria as per protocol or e r failed quality assessment
	PIH: Pregnany Induced Hypertension
	MSU: Mid stream specimen of urine 9 studies included into the review		R R R R R
					e e e e e
						v i e v i e v i e v i e v i e
						w w w w w
							O n l O n l O n l O n l O n l
							y y y y y
							25 26 27

International Journal of Clinical PracticeInternational Journal of Clinical Practice

Acknowledgements:

We thank Elmie Peters for her help in searching for the literature, Hans Bor for his advice on statistics and Steve A. Abbott for his editing. Special thanks to Hans Landman MD, PhD who gave clinical advice and commented on the final draft of the study.

Disclosure of interests: none


Edmund Leung
email: ed.leung@doctors.org.uk

Jennie Grainger
email: jenniegrainger@doctors.org.uk

Nageswar Bandla
email: nrbandla@gmail.com

Ling Wong
email: lingswong@doctors.org.uk

Mr Edmund Leung

THE EFFECTIVENESS OF THE 'TWO-WEEK WAIT' REFERRAL SERVICE FOR COLORECTAL CANCER

whether they are published or not. The documents may come from teaching and research institutions in France or

Colorectal cancer (CRC) is the third commonest cause of cancer related death (after breast and lung) in the United Kingdom. Around 100 new cases of colorectal cancer are diagnosed in the UK each day [START_REF]ACPGBI colorectal cancer guidelines. ACPGBI referral guidelines for colorectal cancer[END_REF] . Despite recent advances in medical treatment and diagnostic technology, a high proportion of patients with CRC present with advanced disease with 5-year survival rates of 49.6% in men and 50.8% in women [START_REF] Dua | Meleagros L The two-week rule in colorectal cancer. Can it deliver its promise?[END_REF] . Furthermore, mortality from CRC in the UK is above that of our European counterparts [START_REF]Editorial: Is the two week rule for cancer referrals working[END_REF] . Some cancer patients were experiencing significant delays waiting for an out-patient appointment in secondary care, which may lead to delay in diagnosis and advancing stage of disease [START_REF] Dua | Meleagros L The two-week rule in colorectal cancer. Can it deliver its promise?[END_REF] . In order to facilitate access to a colorectal specialist, the Department of Health compiled the NHS Cancer Plan in 2000. Part of this NHS Cancer Plan consisted of the Two-Week Rule (TWR), which states that all units were required to see 95% of potential cancer patients.

The aim of the TWR was in part designed so patients with symptoms highly suspicious of CRC were seen in secondary care within 14 days, leading to prompt assessment and investigation. This in turn may lead to prompt treatment and therefore reduce morbidity and mortality. A set of referral criteria for the TWR was devised for each type of cancer.

These TWR referral criteria for CRC, introduced by the UK Government in 2000, were specifically designed to guide general practitioners (GPs) decide which patients merited referral on this basis, selecting symptomatology that was associated with a high risk of CRC [START_REF] Dua | Meleagros L The two-week rule in colorectal cancer. Can it deliver its promise?[END_REF][START_REF] Chohan | How has the 'two-week wait' rule affected the presentation of colorectal cancer?[END_REF] (Table 1). Therefore any patient suspected of having CRC by their GPs will be seen within two weeks of the referral date [START_REF] Nhs Executive | The New NHS -Modern, Dependable[END_REF] .

METHODS:

All patients referred to colorectal clinic via the TWR referral system in a national CRC screening centre was assessed over a 12 month period. Data relating to final diagnosis following secondary care investigations and cancer staging was collected retrospectively. Moreover, comparison was made with CRC patients presenting via other routes (i.e. not via the TWR system) during the same time period. Chi square test with Yates' correction was used to assess statistical significance.

Non-TWR referrals were categorised into urgent (consultation occurred within 4 weeks) or routine out-patients referrals (consultation occurred within 18 weeks), patients presented with rectal bleeding referred from other secondary care specialities, pilot CRC screening patients and patients referred by primary care as an emergency. For the purpose of our study, even patients with cancer metastasis were grouped into Dukes C.

RESULTS:

Over the 12 month period of the study a total of 1100 patients were referred to colorectal clinics via the TWR referral system. All of these patients (100%) were seen by the target time of two weeks.

Of these, 85.3% (n=938) of patients fulfilled the criteria for referral via the TWR. Of those referred, 7.3% (n=81) of patients were diagnosed with CRC.

MODE OF PRESENTATION:

Over the study period there were 217 patients diagnosed with CRC for the first time. Of these 63% (n=136) were referred via alternative routes to the TWR (Table 2). The two most common referral methods were via the TWR (37%) and urgent out-patient clinic appointments (40%). Dukes C accounted for 51.6% of all CRC diagnosed in the study year. This cohort of patients, who were referred via the TWR system, had a significantly higher incidence of Dukes' C cancers (33.0%) than those patients referred by pilot screening (4.5%), routine appointments (6.3%), rectal bleeding (3.6%) and emergency referrals (7.1%) (p=0.0001). There was a similar incidence of Dukes' C cancers compared with groups referred via urgent clinic appointments (45.5%) (p=0.11). The TWR referral system was initially introduced so that patients with symptoms suspicious of a CRC were seen within 14 days by a colorectal specialist in order to aid speedy investigations and diagnosis. By achieving faster diagnosis, treatment could be initiated earlier leading to a reduction in morbidity and mortality.

The results of our study have shown that very few patients (7.3%) who were referred via the TWR were actually diagnosed with CRC. This means that more than 90% of patients who fulfilled the set criteria for a TWR referral (see Table 1) actually had benign disease requiring no urgent treatment. Despite all the research that went into formulating the TWR guidelines 1 , it would appear that they do not exhibit much specificity and therefore the detection of CRC has been generally low [START_REF] Raj | Prioritization of colorectal referrals: a review of the 2-week wait referral system[END_REF] . Such guidelines imposing on these patients being consulted within two weeks, this puts excessive pressure on specialist surgeons despite the fact the majority actually had benign disease. Just as many cancers presented via urgent referrals rather than TWR, but with clinic time emphasis being put on TWR, patients being referred via other means were potentially not seen as quickly.

One study 7 has shown that there is a wide variability in the detection of CRC depending on which primary care trust has referred them. In that study, GP practices that were found to have a low correlation between the number of TWR referrals and CRC diagnosis were also found to be under utilizing the pathway, meaning they were referring actual patients who had cancer via alternative routes. Furthermore, it found that 22% of GPs were unaware of the TWR guidelines and only 8% knew the number of referral criteria. This would account for the large numbers of CRC patients referred via alternative routes and also those referred via the TWR pathway gave a low yield. Our study has shown that of all patients that were diagnosed with CRC during our study period, 63% patients were referred via a method other than the TWR system. There was no difference in cancer staging between TWR system and urgent referral, suggesting that selective patients could be seen within 4 weeks as opposed to 2 weeks. Obviously, the cancer patients detected by pilot screening would have been asymptomatic and never fulfilled the TWR set criteria.

The TWR system defends solely on the assessment of GPs and appointments given to patients are then dependent on the referral letters. However, Aljarabah et al [START_REF] Aljarabah | Referral letters for 2-week wait suspected colorectal cancer do not allow a 'straight-to-test' pathway[END_REF] concluded in their study that GP referral letters for patients with suspected CRC were largely inaccurate and appropriate investigations and suspected diagnosis could only be adequately carried out once a colorectal specialist had consulted and examined the patient.

It is ostensible that the TWR criteria may require revision in order to increase its specificity and positive predictive value. With many hospital trusts introducing one-stop clinics with history, examination and flexible sigmoidoscopy, resources are overwhelmed. One example that is seen frequently in secondary care is that, despite patients fulfilling the set criteria for TWR referral, these patients had already been fully investigated within 12 months but were still referred. GPs need to consider the guidelines set out by the British Society of Gastroenterology (figure 1) for those previously investigated prior to sending these patients on the TWR system again. The TWR referral system has a moderate yield for detecting patients with CRC, but did not appear to improve prognosis even if patients were consulted within 14 days. The set criteria for TWR may warrant revision. Our preliminary results suggest that as long as patients were seen reasonably urgently, there would be no significant difference in prognosis.

Figure 1 .

 1 Figure 1. British Society of Gastroenterology guidelines for surveillance by colonoscopy

Table 1 :

 1 Specific Referral Guidelines for Two Week Rule

	1	Patient ≥ 40 with rectal bleeding AND change in bowel
		habit +/-increased stool frequency for >6 weeks
	2	Patients ≥ 60 with change in bowel habit for >6 weeks
		WITHOUT rectal bleeding
	3	Patients ≥60 with rectal bleeding for >6 weeks
		WITHOUT anal symptoms and WITHOUT change in
		bowel habit
	4	Any patient with lower abdominal mass
	5	Any patient with palpable rectal mass
	6	Men of any age with unexplained iron-deficiency
		anaemia and Hb <11g/100ml
	7	Post menopausal women with unexplained iron-
		deficiency anaemia and Hb < 10g/100ml
	The provision of consulting 95% of referrals by colorectal specialists within 14 days has a
	significant impact on the workload of any colorectal surgeon. Since all TWR patients are high risk
	for CRC, therefore, more time for each consultation is required. It increases pressures on clinic
	times and has a knock-on effect on routine referrals. Choghan et al 4 in 2001 reported that the TWR
	did accelerate access to specialist colorectal clinic, however, pick-up for CRC in this population of
	patients was low at 27%.

The aim of this study was to assess the effectiveness of the TWR referral system in terms of CRC detection and the impact it has on cancer staging.

Table 2 :

 2 Different modes of colorectal cancer referrals.Dukes A accounted for 15.7% of all CRC diagnosed in the study year. 14.7% and 8.8% of Dukes' A CRC patients were referred via pilot screening and routine clinic appointments respectively. 41.2% of patients with Dukes A CRC patients were referred via the TWR system (Table3). There were no

	Two-week referral	N=81	37%
	Pilot Screening	N=18	8%
	Routine out-patient referral	N=12	6%
	Emergency presentation	N=13	6%
	Rectal bleeding	N= 7	3%
	Urgent out-patient referral	N=86	40%
	CANCER STAGING:
	Dukes A:

Dukes' A those patients, who presented as an emergency. Statistically, TWR referrals identified significantly more Dukes A patients compared with referrals from pilot screening, routine appointments, rectal bleeding and emergency presentation (p=0.0001). Urgent referrals had similar share of Dukes A patients (p=0.24).

Table 3 :

 3 Dukes' Staging According to Referral Method:

	REFERRAL	DUKES' A	DUKES' B	DUKES' C
	Two-week referral	N=14,	N=30, 42.3% N=37, 33.0%
		41.2%
	Pilot Screening	N= 5,	N= 8, 11.3% N= 5, 4.5%
		14.7%
	Routine Out-patient	N= 3, 8.8% N= 2, 2.8%	N= 7, 6.3%
	Referral
	Emergency Presentation	N= 0, 0%	N= 5, 7.0%	N= 8, 7.1%
	Rectal Bleeding	N= 1, 2.9% N= 2, 2.8%	N= 4, 3.6%
	Urgent Out-Patient	N=11,	N=24, 33.8% N=51, 45.5%
	Referral	32.4%

DISCUSSION:


BSc(Hons Stephen Thomas Ward

Bruce Jones
email: bruce.jones@heartofengland.nhs.uk

Alan J Jewkes
email: alan.jewkes@heartofengland.nhs.uk

Ward St

Jones Bg

Corresponding

A two millimetre free margin from invasive cancer minimises residual disease in breast-conserving surgery

Keywords: Surgical margins, Re-excision, Breast-conserving surgery, Breast neoplasm

Aims

In breast-conserving surgery, the width of free margin around a tumour to ensure adequate excision is controversial. The aim of this study was firstly to evaluate the frequency of residual disease in wider excision specimens in patients who undergo further surgery due to close margins of less than 5 millimetres.

Secondly, the ability of demographic and tumour-related factors to predict the close margins was appraised.

Patients and methods

Three-hundred-and-three patients were included in the study. Patients undergoing wider excision were assessed for the presence of residual disease and this was tested for association with the width of the initial free margin. Various factors were studied for association with close or involved margins by univariate analysis.

Results

Fifty-three percent of patients were eligible for re-excision based on the need for a 5 millimetre clearance.

With a free margin of 2 mm or more from invasive cancer, the probability of finding residual disease was 2.3%. The probability of residual disease was higher for DCIS and did not decline with increasing free margin width.

Tumour size, lobular cancer type, vascular invasion and nodal involvement, were associated with close margins.

Conclusions

We suggest that a free margin of 2 mm from invasive cancer is adequate to minimise residual disease, whereas the equivalent free margin for DCIS is unclear. Patients with large tumours and lobular cancer type should be counselled at the time of first surgery concerning the higher risk of further excision and mastectomy.

What is already known about this topic?

Following breast-conserving surgery, a margin involved with tumour is associated with residual disease and an increased risk of local recurrence. The necessary width of free margin to minimise both residual disease and local recurrence is controversial. The relationship between residual disease and local recurrence is also unclear.

What does this article add?

Our department requested re-excision of margins if the free margin width was less than 5 millimetres.

Most other units aim for less than a 5 millimetre free margin and we were thus well-positioned to study the relationship between free margin width and residual disease.

INTRODUCTION

Breast-conserving surgery (BCT) followed by adjuvant radiotherapy has been validated as an alternative to mastectomy over 20 years follow-up in selected patients [START_REF] Fisher | Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer[END_REF][START_REF] Veronesi | Twenty-year follow-up of a randomized study comparing breast-conserving surgery with radical mastectomy for early breast cancer[END_REF]. These studies have demonstrated similar survival in both groups although there is an increased rate of local recurrence in patients undergoing BCT.

Local recurrence has a detrimental effect on survival with nearly 50% patients with local recurrence having coincident distant disease [START_REF] Veronesi | Local recurrence and distant metastases after conservative breast cancer treatments: partly independent events[END_REF][START_REF] Clarke | Effects of radiotherapy and the differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: An overview of the randomized trials[END_REF].

There are no defined anatomical compartments in the breast and surgeons therefore rely on a rim of normal tissue around the tumour known as the free margin as evidence of complete removal. The most important factor determining local recurrence is the status of the free margin of the excised specimen [START_REF] Luini | The surgical margin status after breast-conserving surgery: discussion of an open issue[END_REF]. Local recurrence occurs more frequently when there is no margin of normal tissue around the tumour despite adjuvant radiotherapy [START_REF] Singletary | Surgical margins in patients with early-stage breast cancer treated with breast conservation therapy[END_REF]. The width of free margin necessary to conclude that the tumour has been adequately excised is controversial and may differ for invasive cancer and ductal carcinoma in-situ (DCIS) [START_REF] Luini | The surgical margin status after breast-conserving surgery: discussion of an open issue[END_REF][START_REF] Von Smitten | Margin status after breast-conserving treatment of breast cancer: How much free margin is enough?[END_REF].

A survey of two hundred breast surgeons from the United Kingdom revealed a wide variation in what is considered to be an adequate margin with 24% wanting a clear margin of 1 mm and 65% wanting a margin of 2 mm or more [START_REF] Young | A review of current practices in breast conservation surgery in the UK[END_REF]. This study highlights a disparity in practice across different units and the need for evidence-based guidelines.

Marginal involvement is an indication for further surgery in the form of either wider excision or mastectomy and is frequently associated with residual disease in the further surgical specimen [START_REF] Singletary | Surgical margins in patients with early-stage breast cancer treated with breast conservation therapy[END_REF]. Further surgery when margins are not involved has been undertaken in order to gain a free margin clearance of 1, 2 or 5 mm [START_REF] Young | A review of current practices in breast conservation surgery in the UK[END_REF]. Further surgery is obviously desirable if it leads to a risk reduction of local recurrence but negatively impacts on cosmesis as well as patient psychology and hospital resources. The relationship between residual disease and local recurrence is unclear [START_REF] Singletary | Surgical margins in patients with early-stage breast cancer treated with breast conservation therapy[END_REF]. With a policy in place to undertake further surgery when required to gain a free margin of certain width, pre-operative factors that could predict the likelihood of inadequate margins in BCT specimens could be useful in both patient counselling and the selection of patients for more extensive primary surgery or routine cavity shaving. Previous studies have found inconsistent associations of a wide range of factors available pre-operatively with the presence of involved margins and residual disease [START_REF] Singletary | Surgical margins in patients with early-stage breast cancer treated with breast conservation therapy[END_REF][START_REF] Dillon | A pathologic assessment of adequate margin status in breastconserving therapy[END_REF].

During the study period, our unit aimed for a free margin of 5 mm in patients undergoing breast-conserving surgery for both invasive cancer and DCIS. The association between the width of free margin and residual disease in wider excision specimens was evaluated to guide local policy. Various demographic and tumour-related factors were also studied for an association with inadequate margins in order to establish how these factors could influence the nature or extent of surgery.

International

PATIENTS AND METHODS

The Good Hope Hospital Breast Cancer Database (GHHBCD) identified 310 patients who underwent breast-conserving surgery for breast cancer between 1 st October 2002 and 22 nd October 2008. These patients all had a pre-operative histological diagnosis of probable invasive cancer or DCIS, but seven patients were excluded due to absent or benign disease following excision, leaving a total of 303 patients in the study population.

The BCT specimens were oriented by the surgeon using sutures and in some instances, accompanied by one or more cavity shaves before submission for histopathological analysis. For ease of discussion, cavity shave specimens taken at the initial operation will be referred to as primary cavity shaves.

Surgical specimens were immediately placed in 10% neutral buffered formalin and transported to the pathology department. The specimens were left in formalin for a minimum period of 24 hours. BCT specimens were sliced at 5 mm intervals usually in the direction of the shortest axis. Every radial and deep margin of BCT specimens were analysed, taking at least three blocks of tumour from the closest macroscopic margins. Most or all of the tissue from cavity shave specimens were embedded for microscopic examination.

The distance between the tumour and the margin on microscopic assessment was noted for the following margins: medial, lateral, superior and inferior by analysis of pathology reports. A free margin width of less than 5 mm for either invasive cancer or DCIS was deemed to be inadequate in our unit, prompting either a wider excision or mastectomy. Anterior (superficial) and deep margins were not assessed in this study because generally wider excision was not performed if the anterior or deep free margin was less than 5 mm.

The ability to perform a wider excision of the anterior and deep margins is frequently limited by the location of the tumour and there is evidence that most recurrences in relation to breast tissue occur along radial margins [START_REF] Dixon | Breast incisions for conservation surgery [Comment, Letter][END_REF].

For those patients who underwent wider excision following BCT, the presence of residual disease was determined. Residual disease was defined as the presence of either invasive cancer or DCIS. Those patients who underwent completion mastectomy or who were treated with chemoradiotherapy between the primary and further operations were excluded from the analysis. Some patients underwent wider excision of two or more margins sent as separate specimens, enabling the histopathological assessment of each margin and its wider excision to contribute separately to the analysis.

In the second part to the study, patients were classified into two groups: Close margins (one or more free margins of less than 2 mm at BCT) and clear margins (all free margins 2 mm or more). The two groups were compared for differences in demographic factors (age at diagnosis, tumour laterality), pathological factors (tumour grade, tumour type, presence of vascular invasion, use of a primary cavity shave) and radiological factors (ultrasound-determined tumour size). Ultrasound-determined measurements were chosen because they were thought to be more objective than clinical measurements and were more frequently performed than mammographic measurements.

The experimental protocol was approved by the Good Hope Hospital Research and Ethics Committee. All breast cancer database data, pathology report data and radiology report data were tabulated using Microsoft Excel 2007 and statistical analysis was performed using StatView (v5.0, SAS Inst. Inc.). The Mann-Whitney U-test was used to test for statistical significant differences in age, and tumour size while Chisquared analysis was used to test for statistical significant differences in tumour grade and type and the presence of vascular invasion. χ 2 and P-values were calculated to three significant figures.

RESULTS

Of the 303 patients in the study population, 93 (31%) patients underwent a wider excision and 39 (13%) a completion mastectomy. This left 171 (56%) patients who did not proceed to further surgery. Eleven patients in the wider excision group progressed to a third operation, all mastectomies.

Based on the criteria for free radial margins of 5 mm, 161 (53%) patients were eligible for further surgery although only 132 (44%) actually proceeded to this.

Of the 93 patients undergoing a wider excision, 140 wider excision specimens (88 for invasive cancer and 52 for DCIS) were analysed, a mean of 1.5 wider margins per patient.

Width of free margin and residual disease in wider excision specimens

The percentage of wider excision specimens found to contain residual disease declined with increasing width of the free margin from invasive cancer at first surgery (see table 1). With a free margin of 2 mm or more, the probability of finding residual disease was 2.3%.

Regarding the relationship between the width of the free margin from DCIS and the percentage of wider excision specimens with residual disease, the overall probability of residual disease was higher for DCIS than for invasive cancer and this probability did not appear to decline as rapidly with an increasing width of free margin.

Demographic and tumour-related factors

Comparison between the close and clear patient groups revealed a statistically significant association between tumour size, the presence of vascular invasion and nodal involvement with close margins (see table 2). Despite no overall association between the various tumour types and close margins, there was a statistically significant association when comparing the frequency of only ductal and lobular invasive carcinomas. Of the above factors, tumour size (as determined by ultrasound) and tumour type would be available to the surgeon pre-operatively.

DISCUSSION

Our results clearly show a relationship between the width of the free margin and the likelihood of finding residual disease at further surgery. In order to minimise the risk of residual disease, we would recommend aiming for a free margin from invasive cancer of 2 mm. A free margin of 2 mm from DCIS was associated with a higher probability of residual disease but this analysis was based on fewer numbers.

From a logical standpoint, adequate excision should only demand a margin that is not directly involved with disease. However, the methodology employed to pathologically assess margin status is imprecise [START_REF] Luini | The surgical margin status after breast-conserving surgery: discussion of an open issue[END_REF][START_REF] Von Smitten | Margin status after breast-conserving treatment of breast cancer: How much free margin is enough?[END_REF]. A single haematoxylin and eosin slide of a 2-3 mm block from a BCT specimen visualizes less than a thousandth of its surface area and therefore evaluation of margins is made by random and not complete examination of the surface [START_REF] Mccready | Keeping abreast of marginal controversies[END_REF]. A width of free margin is thus simply a marker of increased probability of adequate local excision of the tumour. A study of mastectomy specimens found that if hypothetical BCT surgery had been performed with free margins as great as 20 mm, foci of invasive tumour would remain in the breast in 14% cases [START_REF] Holland | Histologic multifocality of Tis, T 1-2 breast carcinomas: Implications for clinical trials of breast conserving surgery[END_REF]. This is because breast cancer is a disease of multifocality, multicentricity and discontinuous spread.

Studies have demonstrated an association between the width of the free margin and the probability of residual disease following wider excision [START_REF] Von Smitten | Margin status after breast-conserving treatment of breast cancer: How much free margin is enough?[END_REF][START_REF] Dillon | A pathologic assessment of adequate margin status in breastconserving therapy[END_REF][START_REF] Pittinger | Importance of margin status in outcome of breastconserving surgery for carcinoma[END_REF][START_REF] Papa | Positive margins of breast biopsy: Is reexcision always necessary?[END_REF]. A study of 211 patients treated by BCT showed residual disease in 44% cases with involved margins, 24% cases with free margins of less than 3 mm and in no cases where the free margin was greater than 3 mm [START_REF] Pittinger | Importance of margin status in outcome of breastconserving surgery for carcinoma[END_REF]. In contrast to our findings, one study demonstrated residual disease in 45% patients with a free margin between 2 and 5 mm [START_REF] Dillon | A pathologic assessment of adequate margin status in breastconserving therapy[END_REF]. This may be due to the free margin of 2-5 mm surrounding in-situ as opposed to invasive disease.

Rationally, residual disease ought to be avoided but its importance in relation to local recurrence is unclear.

The importance of the free margin width in terms of local recurrence has been corroborated by studies showing that re-excision to achieve clear margin status results in a low rate of local recurrence, equivalent to if the margins were clear initially [START_REF] Freedman | Patients with early stage invasive cancer with close or positive margins treated with conservative surgery and radiation have an increased risk of breast recurrence that is delayed by adjuvant systemic therapy[END_REF]. There is also evidence that local recurrence rates are generally higher with free margins of less than 2 mm [START_REF] Luini | The surgical margin status after breast-conserving surgery: discussion of an open issue[END_REF]. The use of routine cavity shaving in patients all treated by the same surgeon, has demonstrated a higher rate of local recurrence and lower overall survival in those with positive primary cavity shaves [START_REF] Besana-Ciani | The importance of margins status after breast conservative surgery and radiotherapy in node positive patients: a follow-up of 10-15 years[END_REF] after 10 years of follow-up. This difference was however only statistically significant in the patient group with node negative disease.

With regard to DCIS, the full extent of disease is often non-palpable and is underestimated by mammography [START_REF] Von Smitten | Margin status after breast-conserving treatment of breast cancer: How much free margin is enough?[END_REF] making adequate surgical margins more difficult to obtain. Our data was not able to determine a free margin width associated with minimal residual DCIS due to our policy of not re-excising free margins of more than 5 mm. The difficulty in obtaining a free margin around DCIS has led some investigators to suggest a free margin of 10 mm to minimise local recurrence [START_REF] Silverstein | The influence of margin width on local control of ductal carcinoma in situ of the breast[END_REF], although a recent metaanalysis has reported no benefit from a free margin of more than 2 mm when combined with radiotherapy [START_REF] Dunne | Effect of Margin Status on Local Recurrence After Breast Conservation and Radiation Therapy for Ductal Carcinoma In Situ[END_REF].

It is accepted that the most important factor leading to local recurrence after BCT and adjuvant radiotherapy, and the only factor under surgical control, is the presence of involved margins, and that these require re-excision [START_REF] Singletary | Surgical margins in patients with early-stage breast cancer treated with breast conservation therapy[END_REF]. The controversy over the width of free margin required to avoid re-excision has been caused by conflicting results from the literature. This has probably arisen from a wide variation in patient characteristics, selection criteria leading to BCT over mastectomy, inclusion of patients undergoing diagnostic excisions, surgical technique, the use of radiotherapy including the use of radiotherapy boosts and differences in radiological and pathological evaluation. For this reason, it is wise for every breast unit to evaluate their individual rates of residual disease from wider excisions and local recurrence rates.

We have shown a statistical association between tumour size and close margins. One study found associations between positive margins and a variety of factors, but interestingly, only tumour size was found to uphold a significant association after multivariate analysis [START_REF] Tartter | Lumpectomy margins, reexcision, and local recurrence of breast cancer[END_REF]. A proportion of operations in this study were diagnostic excisions as opposed to wide local clearances. However, tumour size appears to consistently be associated with close or involved margins after adjusting for confounding factors [START_REF] Singletary | Surgical margins in patients with early-stage breast cancer treated with breast conservation therapy[END_REF][START_REF] Tartter | Lumpectomy margins, reexcision, and local recurrence of breast cancer[END_REF][START_REF] Park | Outcome at 8 years after breast-conserving surgery and radiation therapy for invasive breast cancer: Influence of margin status and systemic therapy on local recurrence[END_REF].

There is also published support for the association between involved lymph nodes and vascular invasion with involved margins [START_REF] Park | Outcome at 8 years after breast-conserving surgery and radiation therapy for invasive breast cancer: Influence of margin status and systemic therapy on local recurrence[END_REF], consistent with our results. A similarly-conducted study to ours [START_REF] Dillon | A pathologic assessment of adequate margin status in breastconserving therapy[END_REF] showed an association between close margins and pathological tumour size, an extensive intraductal component to the tumour, referral from a symptomatic rather than population-based screening program and the absence of pre-operative diagnosis. We did not assess all of these factors in our study and there is some evidence that an extensive intraductal tumour component is associated with residual disease after adjusting for confounding factors [START_REF] Wazer | The influence of age and extensive intraductal component histology upon breast lumpectomy margin assessment as a predictor of residual tumor[END_REF]. Other factors which may be important are the operating surgeon, tumour multcentricity and palpability but these were not analysed in our study. In agreement with our results regarding tumour type, there is evidence that lobular cancers are associated with higher rates of margin involvement [START_REF] Moore | Association of infiltrating lobular carcinoma with positive surgical margins after breast-conservation therapy[END_REF][START_REF] Dillon | Identifying patients at risk of compromised margins following breast conservation for lobular carcinoma[END_REF].

Pre-operative factors such as tumour size and type could be used to counsel patients about the increased risk following BCT of wider excision or mastectomy to obtain clear margins. It is possible that routine cavity shaving, used selectively in patients with large tumours, may prove a useful adjunct to increase the proportion of patients with clear margins.

CONCLUSION

This study has demonstrated that in patients undergoing BCT, a free margin of 2 mm from invasive cancer is associated with a low risk of residual disease. A free margin of up to 5 mm from DCIS is associated with residual disease in one third of patients. Large tumour size, as determined pre-operatively by ultrasound, and lobular cancer type are associated with close margins and these patients should be counselled at the time of first surgery concerning the higher risk of further excision and mastectomy.

TABLE 1

 1 The percentage of wider excision specimens containing residual disease by width of free margin from the tumour at first surgery

					Page
	INVASIVE CANCER
	Free margin (mm)	Residual disease	No residual	Total	% Residual disease
			disease (clear)
	Involved	6	11	17	35.3
	0.1 to 0.9	2	4	6	33.3
	1.0 to 1.9	2	17	19	10.5
	2.0 to 4.9	1	40	41	2.4
	> 5.0	0	4	4	0.0
	Total	11	76	87
	DCIS
	Free margin (mm)	Residual disease	No residual	Total	% Residual disease
			disease (clear)
	Involved	8	10	18	44.4
	0.1 to 0.9	4	3	7	57.1
	1.0 to 1.9	3	6	9	33.3
	2.0 to 4.9	6	11	17	35.3
	> 5.0	0	1	1	0.0
	Total	21	31	52

14 of 15 International Journal of Clinical Practice International Journal of Clinical Practice

	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y

Acknowledgements:

The authors would like to acknowledge Dr T Salim for informing us of the processes involved in histopathological assessment of the specimens.

Univariate analysis of various factors between patients with clear margins of 2 mm or more against those with at least one close margin less than 2 mm. * P-values considered to be statistically significant.

Factor


Audrey Tupin

Maxime Gualtieri

Françoise Roquet-Banères

Zakia Morichaud

Konstantin Brodolin

Jean-Paul Leonetti

Resistance to rifampicin: at the crossroads between ecological, genomic and medical concerns

Keywords:

A c c e p t e d M a n u s c r i p t 2 E-mail address: audrey.tupin@univ-montp1.fr (A. Tupin).

A c c e p t e d M a n u s c r i p t

ABSTRACT

The first antibiotic of the ansamycin family, rifampicin (RIF), was isolated in 1959 and was introduced into therapy in 1962; it is still a first-line agent in the treatment of diseases such as tuberculosis, leprosy and various biofilm-related infections. The antimicrobial activity of RIF is due to its inhibition of bacterial RNA polymerase (RNAP).

Most frequently, bacteria become resistant to RIF through mutation of the target; however, this mechanism is not unique. Other mechanisms of resistance have been reported, such as duplication of the target, action of RNAP-binding proteins, modification of RIF and modification of cell permeability. We suggest that several of these alternative resistance strategies could reflect the ecological function of RIF, such as autoregulation and/or signalling to surrounding microorganisms. Very often, resistance mechanisms found in the clinic have an environmental origin. One may ask whether the introduction of the RIF analogues rifaximin, rifalazil, rifapentine and rifabutin in the therapeutic arsenal, together with the diversification of the pathologies treated by these molecules, will diversify the resistance mechanisms of human pathogens against ansamycins.

Introduction

Ansamycin derivatives such as rifampicin (RIF) are first-line antibiotics in the treatment of tuberculosis (TB), leprosy and a growing number of Gram-positive bacteria such as multidrug-resistant Staphylococcus aureus. Ansamycin was isolated in 1959 by Sensi et al. [START_REF] Sensi | Rifomycin. I. Isolation and properties of rifomycin B and rifomycin complex[END_REF][START_REF] Sensi | Rifomycin O, a new antibiotic of the rifomycin family[END_REF] from a strain of Amycolatopsis mediterranei (previously known as Streptomyces mediterranei and Nocardia mediterranei) and was introduced into therapy in 1962.

Ansamycins are active against a large variety of organisms, including bacteria and eukaryotes. They have a broad activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative bacteria. Interestingly, ansamycins are also active against the transcription machinery of the eukaryotic parasite Plasmodium falciparum [START_REF] Gardner | A circular DNA in malaria parasites encodes an RNA polymerase like that of prokaryotes and chloroplasts[END_REF][START_REF] Pukrittayakamee | Antimalarial effects of rifampin in Plasmodium vivax malaria[END_REF]. Resistance to RIF by mutation of the target is widespread but not unique. Other mechanisms of resistance have been reported, such as duplication of the target, action of RNAP-binding proteins, modification of RIF and modification of cell permeability (Table 1). Bacteria and fungi are not isolated in nature, but exist in communities and need to communicate to be competitive. A majority of the low molecular weight compounds synthesised by microbes at low concentrations apparently act as cellsignalling molecules and directly or indirectly modulate transcription to favour symbiosis between these microorganisms [START_REF] Yim | Antibiotics as signalling molecules[END_REF]. As expected from a key player in the bacterial signalling network, RNAP is targeted by a variety of antibiotics that contain similar binding sites to RIF [START_REF] Villain-Guillot | Progress in targeting bacterial transcription[END_REF], and bacteria have developed highly diverse strategies to grow in the presence of these antibiotics. Some of these mechanisms confer resistance to very high levels of RIF, whilst others only protect the bacteria against lower concentrations. These natural resistances cast light on the potential evolution of resistance to RIF and on other new transcription inhibitors in clinical development in the future. [START_REF] Campbell | Structural mechanism for rifampicin inhibition of bacterial RNA polymerase[END_REF] and its binding site is highly conserved among bacteria (Fig. 1). Binding of the molecule to RNAP involves 12 amino acid residues (Fig. 2). Mutagenesis of each of these residues, except one, generates a resistant phenotype [START_REF] Campbell | Structural mechanism for rifampicin inhibition of bacterial RNA polymerase[END_REF]. Spontaneous resistance to RIF is mainly associated with single point mutations resulting in amino acid substitutions and is less frequently associated with a few insertions or deletions. Ninetyfive percent of these mutations map to four regions in the N-terminal half of the -subunit polypeptide [START_REF] Floss | Rifamycin-mode of action, resistance, and biosynthesis[END_REF] involved in the binding of RIF. In E. coli, RIF resistance mutations are usually located in the central region of the polypeptide, within cluster I (amino acids 507-533), cluster II (amino acids 563-572) and cluster III [START_REF] Jin | RpoB8, a rifampicin-resistant termination-proficient RNA polymerase, has an increased K m for purine nucleotides during transcription elongation[END_REF][START_REF] Severinov | Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the  subunit of Escherichia coli RNA polymerase[END_REF], and they can also occur near the N-terminus of the -subunit [START_REF] Severinov | Rif R mutations in the beginning of the Escherichia coli rpoB gene[END_REF]. In M. tuberculosis, resistance to RIF is mainly associated with mutations in an 81-bp hot-spot region (codons 432-458 in cluster I) of rpoB [START_REF] Telenti | Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis[END_REF], with 41% of resistant clinical isolates carrying a mutation of S455, 36% with a mutation of H440 and 9% with a mutation of D430 [START_REF] Ramaswamy | Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update[END_REF]. A mutation at the beginning of the rpoB gene, the amino terminal of cluster I, has also been reported (V176F) [START_REF] Heep | Mutations at four distinct regions of the rpoB gene can reduce the susceptibility of Helicobacter pylori to rifamycins[END_REF][START_REF] Heep | Mutations in the beginning of the rpoB gene can induce resistance to rifamycins in both Helicobacter pylori and Mycobacterium tuberculosis[END_REF]. This mutation confers high-level resistance to RIF and may account for more than 1% of all RIF R strains [START_REF] Heep | Frequency of rpoB mutations inside and outside the cluster I region in rifampinresistant clinical Mycobacterium tuberculosis isolates[END_REF]. RIF resistance mutations have also been found in the N-terminus (V135F) and in cluster I (Q469K, Q469L, Q469R, H482D, H482P, H482R, H482Y, S487F, S487L and S487Y) of B. subtilis rpoB [START_REF] Perkins | Uncovering new metabolic capabilities of Bacillus subtilis using phenotype profiling of rifampin-resistant rpoB mutants[END_REF] as well as in Helicobacter pylori (V149F) [START_REF] Heep | Frequency of rpoB mutations inside and outside the cluster I region in rifampinresistant clinical Mycobacterium tuberculosis isolates[END_REF].

Resistance by mutation of the target

In most cases, a point mutation in rpoB only confers RIF resistance, but recently a point mutation was shown to confer resistance to and dependence on RIF [START_REF] Maguire | Identification of a mutation conferring antibiotic dependence in Escherichia coli[END_REF]. The molecular A c c e p t e d M a n u s c r i p t 7 basis of this effect is not yet understood, but the finding itself casts doubt on the historical view that RIF acts by plugging the path of the elongating RNA as mentioned by Campbell et al. [START_REF] Campbell | Structural mechanism for rifampicin inhibition of bacterial RNA polymerase[END_REF].

Resistance to transcription inhibitors that arise through point mutations of the target is not neutral for bacteria. In the case of RIF, these alterations in the target were repeatedly involved in deregulation of gene expression [START_REF] Sonenshein | Isolation and characterization of rifampin-resistant and streptolydigin-resistant mutants of Bacillus subtilis with altered sporulation properties[END_REF][START_REF] Kawamura | Participation of Rho-dependent transcription termination in oxidative stress sensitivity caused by an rpoB mutation[END_REF][START_REF] Hu | Activation of antibiotic biosynthesis by specified mutations in the rpoB gene (encoding the RNA polymerase  subunit) of Streptomyces lividans[END_REF][START_REF] Bergval | Specific mutations in the Mycobacterium tuberculosis rpoB gene are associated with increased dnaE2 expression[END_REF][START_REF] Jin | Characterization of the termination phenotypes of rifampicin-resistant mutants[END_REF], possibly due to altered rho-dependent transcription termination [START_REF] Jin | Characterization of the termination phenotypes of rifampicin-resistant mutants[END_REF]. A striking example is the recent report that many alterations in substrate utilisation patterns were observed in B. subtilis RIF R mutants [START_REF] Perkins | Uncovering new metabolic capabilities of Bacillus subtilis using phenotype profiling of rifampin-resistant rpoB mutants[END_REF]. The consequences of this deregulation are, in most cases, reduced virulence [START_REF] Adone | Protective properties of rifampin-resistant rough mutants of Brucella melitensis[END_REF][START_REF] Björkman | Virulence of antibiotic-resistant Salmonella typhimurium[END_REF] of the RIF R mutants and a reduced fitness [START_REF] Gagneux | The competitive cost of antibiotic resistance in Mycobacterium tuberculosis[END_REF][START_REF] Enne | Rifampicin resistance and its fitness cost in Enterococcus faecium[END_REF]. More prolonged treatment with RIF can result in strains with no fitness defect owing to compensatory mutations, and these strains with low-or no-cost resistance mutations are also the most frequent among clinical isolates [START_REF] Gagneux | The competitive cost of antibiotic resistance in Mycobacterium tuberculosis[END_REF].

Resistance by duplication of the target

Several bacterial species are naturally resistant to RIF owing to mutations in rpoB. Nocardia sp. are Gram-positive saprophytes present in the environment, with several species causing severe infections. Nocardia farcinica is an opportunistic pathogen that is considered a major multidrug resistance problem that is particularly frequent in Japan [START_REF] Kageyama | Nocardial infections in Japan from 1992 to 2001, including the first report of infection by Nocardia transvalensis[END_REF] and Europe [START_REF] Schaal | Actinomycete infections in humans-a review[END_REF][START_REF] Wauters | Distribution of Nocardia species in clinical samples and their routine rapid identification in the laboratory[END_REF]. The genome of N. farcinica IFM 10152 was recently sequenced [START_REF] Ishikawa | The complete genomic sequence of Nocardia farcinica IFM 10152[END_REF] and the presence of two different RNAP -subunit genes, rpoB and rpoB2, was detected. These two genes share a high degree of homology (88.8%) and careful resistance [START_REF] Ishikawa | Contribution of rpoB2 RNA polymerase  subunit gene to rifampin resistance in Nocardia species[END_REF]. The role of the altered gene in conferring resistance was confirmed by knockout of rpoB2, shedding light on a novel resistance mechanism. Such a mechanism may not be a rare event, as rpoB duplication is apparently widespread in Nocardia sp. and has recently been found in another closely related actinomycete, Actinomadura sp. strain ATCC 39727 [START_REF] Vigliotta | Natural merodiploidy involving duplicated rpoB alleles affects secondary metabolism in a producer actinomycete[END_REF]. It is not yet known whether the actinomycete A. mediterranei, which produces RIF, uses this strategy of duplication to resist this antibiotic. RIF mutations are known to affect gene expression and may result in an increase in antibiotic production in Streptomyces [START_REF] Hu | Activation of antibiotic biosynthesis by specified mutations in the rpoB gene (encoding the RNA polymerase  subunit) of Streptomyces lividans[END_REF] and a change in carbon source assimilation in B. subtilis [START_REF] Inaoka | RNA polymerase mutation activates the production of a dormant antibiotic 3,3'-neotrehalosadiamine via an autoinduction mechanism in Bacillus subtilis[END_REF]. In light of these findings, the abovementioned duplications of mutated rpoB could either reflect signalling mechanisms between N. farcinica or Actinomadura sp. and other bacteria producing molecules sharing the same binding site as RIF, or the presence of dormant genes coding for molecules targeting transcription in their genomes.

Resistance induced by RNA polymerase-binding proteins

Although most cases of RIF resistance are due to mutations in the rpoB gene, some bacteria use other strategies. The actinomycete Streptomyces coelicolor is intrinsically resistant to low concentrations of RIF, and recently Newell et al. [START_REF] Newell | The RNA polymerase-binding protein RbpA confers basal levels of rifampicin resistance on Streptomyces coelicolor[END_REF] showed that an RNAP-binding protein, RbpA, confers basal levels of RIF resistance to this microorganism. Experiments have demonstrated that rbpA expression is induced by low concentrations of RIF and that this molecule inhibited a promoter directing the DnaA is another RNAP-binding protein that partially protects RNAP from the effect of RIF. Transcription and replication are intricate mechanisms [START_REF] Pomerantz | The replisome uses mRNA as a primer after colliding with RNA polymerase[END_REF], since RNA is used as a primer for replication. The RNAP located at a replication origin provides the transcription activity necessary for initiation of replication and is protected from the action of RIF by origin-bound DnaA. In vitro experiments showed that the DnaA protein interacts directly with RNAP, and both the ATP-form and the ADP-form of the DnaA protein were found to alleviate the inhibition of RNAP by RIF at the dnaA promoter, although a higher concentration was necessary to achieve the effect when the ADP-form of DnaA was used [START_REF] Flåtten | DnaA protein interacts with RNA polymerase and partially protects it from the effect of rifampicin[END_REF].

 factors are accessory proteins that bind to the core of RNAP to form the RNAP holoenzyme. Binding of the  factor allows transcription initiation by directing specific recognition of the promoter and isomerisation of the RNAP-DNA complex. Wegrzyn et al. [START_REF] Wegrzyn | Differential inhibition of transcription from sigma70-and sigma32-dependent promoters by rifampicin[END_REF] have reported a differential effect on the inhibition of transcription by RIF A c c e p t e d M a n u s c r i p t 10 depending on the  factor. RIF was shown to inhibit transcription from the  70 -dependent P L promoter at a significantly lower concentration than transcription from the  32dependent P groE promoter. These results may only reflect a selectivity of RIF according to the nature of the  factor bound to the core of RNAP and are probably more related to a response to stress rather than a true resistance.

Resistance by modification of rifampicin

Another strategy of resistance is to neutralise the antibiotic by covalent modification.

Bacteria can inactivate RIF in a variety of ways, such as glucosylation, ribosylation, phosphorylation and decolorisation (Fig. 3). Mycobacterium smegmatis DSM 43756, a pathogen intrinsically resistant to this drug, inactivates RIF by ribosylation [START_REF] Imai | Identification and characterization of a new intermediate in the ribosylative inactivation pathway of rifampin by Mycobacterium smegmatis[END_REF].

Resistance by modification of permeability

To be effective, an antibiotic has to reach its specific target in an efficient time frame and at sufficient concentration. One strategy to impair the action of an antibiotic is to prevent its entry to the cell. Some bacterial species that do not allow multiple antibiotics to accumulate in the cytoplasm are called 'intrinsically' resistant, whilst other antibioticsusceptible bacteria can become resistant to multiple antibiotics. This occurs through two mechanisms: (i) the bacteria can be resistant or acquire resistance by decreasing the permeability of the membrane barriers; and (ii) they can also overexpress membrane-associated energy-driven efflux pumps reminiscent of the eukaryotic multidrug resistance pumps.

Mycobacteria are intrinsically resistant to many antibiotics and this property is often attributed to a low permeability of the mycobacterial cell wall and its specific lipid-rich character. Decades ago, a strain of Mycobacterium intracellulare exhibiting natural resistance to RIF was isolated. Its RNAP was found to be susceptible to RIF and experiments suggested that a permeability barrier against RIF was responsible for this resistance [START_REF] Hui | Permeability barrier to rifampin in mycobacteria[END_REF]. Several efflux pumps are also suspected to contribute to mycobacteria multidrug resistance. Siddiqi et al. [START_REF] Siddiqi | Mycobacterium tuberculosis isolate with a distinct genomic identity overexpresses a tap-like efflux pump[END_REF] have pointed out a correlation between drug resistance and Rv1258c gene transcription levels of a clinical isolate of M. tuberculosis resistant to multiple antibiotics (RIF, ofloxacin, isoniazid and minomycin). Reverse of Rv1258c increased by a factor of 10 when the bacteria are grown in the presence of RIF or ofloxacin. These results suggest that efflux pumps can be induced in response to antibiotic treatment in Mycobacteria, consistent with the hypothesis of the involvement of efflux pumps in detoxification [START_REF] Fernandes | Solvent tolerance in bacteria: role of efflux pumps and cross-resistance with antibiotics[END_REF]. The mtr operon of Neisseria gonorrhoeae encodes an efflux pump system composed of the proteins MtrCDE [START_REF] Maness | Multiple antibiotic resistance due to a single mutation in Neisseria gonorrhoeae[END_REF]49]. This pump transports several antibiotics, and mutations in the mtrR gene, a transcriptional repressor of mtrCDE, induce overexpression of this efflux pump. This confers resistance to several antimicrobials, including rifamycins.

Conclusions

Bacteria use a variety of mechanisms to counter the actions of antibiotics, and RIF resistance or tolerance is a perfect example of this diversity. One may wonder what these genes are doing in these bacteria and whether they have additional functions. The RIF-binding domain is located in the catalytic site of the bacterial RNAP and is highly conserved among bacteria, but it has a high propensity to mutate, as observed, when RIF selection pressure is applied. These discrepancies remain to be understood.

The existence of multiple antibiotics sharing, at least partially, the same binding site as RIF, the presence of duplicated genes resistant and sensitive to RIF in the same bacteria, and the involvement of several RNAP-binding proteins in adaptation to low RIF concentrations are probably more revealing of the true function of this antibiotic.

Bacteria live in a community and they must be competitive to survive in their A c c e p t e d M a n u s c r i p t 13 environment. Apart from their killer function, RIF and these various other molecules at low concentrations could also find their usefulness in regulating the genes of surrounding bacteria to the benefit of the producer or the genes of the producer itself.

We now have strong genomic evidence that these resistance mechanisms found in the clinic have an environmental origin. The most common soil bacteria are a reservoir of a previously underappreciated diversity of resistance genes that can be mobilised under selection pressure such as antibiotic therapy. This raises concerns about the future of our antibacterial arsenal. One may ask whether the introduction of the RIF analogues rifaximin, rifapentine, rifabutin and rifalazil to the therapeutic arsenal together with the diversification of the pathologies treated by these molecules will diversify the resistance mechanisms of human pathogens to ansamycins. was built from the Protein Data Bank co-ordinates of the Thermus aquaticus core complex (1I6V [START_REF] Campbell | Structural mechanism for rifampicin inhibition of bacterial RNA polymerase[END_REF]) and from a Thermus thermophilus core scaffold complex (2O5I [START_REF] Vassylyev | Structural basis for transcription elongation by bacterial RNA polymerase[END_REF]).

The -subunit is represented in yellow, the -subunit in blue, the '-subunit in magenta, the DNA double helix in red and green, RNA in orange and RIF in bright yellow.

  Ansamycins are structurally characterised by a planar naphthoquinone ring in which positions 3 and 4 have been extensively modified by hemisynthesis to yield commercial antibiotics such as RIF. Interest in these molecules has recently increased with the US Food and Drug Administration (FDA) approval of several new ansamycin derivatives for treatment of a broader range of infectious diseases. For now, the ansamycins currently in therapeutic use are RIF, rifaximin, rifapentine, rifalazil and rifabutin. The antimicrobial activity of ansamycins is due to their inhibition of bacterial RNA polymerase (RNAP). RIF binds to conserved amino acids in the active centre of the enzyme and blocks transcription initiation. A large proportion of the encountered resistance to RIF is due to mutations of these amino acids. These mutations often occur A c c e p t e d M a n u s c r i p t 5 with high frequency, which compels the use of RIF almost exclusively in drug combinations, most commonly with isoniazid, as well as the restriction of its use to the treatment of TB and to emergencies.

  Many RIF-resistant (RIF R ) strains of Escherichia coli, S. aureus, Bacillus subtilis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, etc. have been characterised, showing that resistance is most frequently conferred by mutations in

A c c e p t e d M a n u s c r i p t 6 the

 6 RNAP active centre. RIF interacts only with the -subunit of RNAP encoded by rpoB

  amino acid sequences revealed that the Nocardia rpoB2 protein contains amino acid substitutions in RIF clusters I and II that are likely to confer RIF

  ribosomal RNA operon. In vitro experiments confirmed that RIF induces  R -dependent transcription of the rbpA gene and that RbpA stimulates  HrdBdependent transcription from rrnDp3. The data presented also suggest that RbpA confers basal levels of resistance to RIF and that it is a novel regulator of rRNA synthesis in S. coelicolor. The authors point out that genes homologous to rbpA are present in M. tuberculosis, M. leprae and Corynebacterium diphtheriae. These genes could account for the transcriptional activity detected in M. tuberculosis following treatment with RIF[START_REF] Hu | Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide[END_REF].

  Structural studies have shown that the final product is obtained by formation of 23-(O-ADP-ribosyl)-RIF followed by removal of AMP and dephosphorylation, leading to 23ribosyl-RIF. The strain expresses Arr enzymes that act as mono (ADP-ribosyl) transferases and catalyse the reaction of ADP-ribosylation of rifamycins. Arr homologues are widely distributed in environmental bacteria and in Stenotrophomonas maltophilia, Burkholderia cenocepacia, S. coelicolor, Corynebacterium glutamicum, Desulfitobacterium hafniense and Rhodopseudomonas palustris. It was shown that RIF analogues modified at position C25 generate derivatives that are resistant to inactivation by ADP-ribosyl transferases from M. smegmatis [42]. Other species use a different mechanism of modification. For example, Nocardia sp. modify the antibiotic by glucosylation (23-OH group) and phosphorylation, but ribosylation is not observed. Bacillus strains and actinomycete-related species were also found to inactivate RIF A c c e p t e d M a n u s c r i p t 11 through phosphorylation of the 21-OH group, RIF glucosylation (23-OH group) or decolorisation [43,44].

  reaction (RT-PCR) showed that the level of transcription

22 Fig. 1 .

 221 Fig. 1. Location of rifampicin (RIF) resistance mutations in the RNA polymerase (RNAP)

Fig. 2 .

 2 Fig. 2. Rifampicin (RIF)-binding site in the active site of RNA polymerase. The model

Fig. 3 .

 3 Fig. 3. ADP-ribosylation of rifampicin (RIF) by Mycobacterium smegmatis. RIF is

Fig 1 :

 1 Fig 1 : The rifampicin resistant regions of the RNAP β-subunit

Fig 2 :

 2 Fig 2 : Rifampicin-binding site in the active site of RNA polymerase

Acknowledgments

KB is supported by a fellowship from the RTRS Infectiopole Sud (CPBS, Montpellier, France). AT is supported by the CNRS (Montpellier, France) and the Region Languedoc-Roussillon.

Funding

This work was funded by the CNRS (Montpellier, France).

Competing interests

None declared.

Ethical approval