Gaelle Noel

Aina Harimanana

Akm Alfa

Lala Rafetrarivony

Ak Koba

Sl Razafimahatratra

Mohand Randriamoramanana Am

Ait Ahmed

Nicole Guiso

Betty Njanpop-Lafourcade

Alfa Akm

Koba Ak

Razafimahatratra Sl

Njanpop-Lafourcade Bm

J-M Collard

Transversal Pertussis Sero-Epidemiological Study in Fully Vaccinated Children and Adolescents in Antananarivo, Madagascar, and in Dapaong, Togo

Keywords: Pertussis, Togo, Madagascar, Whole-cell vaccine, Sero-prevalence List of abbreviations: PT: Pertussis toxin, IgG: Immunoglobulin G, LMIC: Low and middle-income countries, EPI: Expanded Program on Immunization, HIC: High-income countries, WHO: World Health Organization, wPV: Whole-cell vaccine, ELISA: Enzyme-linked immunosorbent assay

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Pertussis, also known as whooping cough or the 100-day cough, is a bacterial respiratory illness. It is often considered as a "forgotten disease" as specific vaccines are integrated into national infant immunization schedules since decades, including in low-and middle-income countries (LMIC) with the inception of the Expanded Program on Immunization (EPI) in 1974, which have led to the sharp reduction of cases worldwide. However, due to high contagiousness of its etiological bacteria Bordetella pertussis, vaccination coverage >90% is necessary in order to maintain a low incidence. In addition, as for many other vaccines, the vaccine-induced immunity declines over the years and booster doses are recommended in a lifetime in addition to the vaccination during his infancy [1]. Although severe to deadly illness mainly occurs in infants <3 months of age, pertussis may affect everyone contributing to the circulation of B. pertussis. In high-income countries (HIC), where surveillance system is largely implemented, pertussis is still considered as a public health concern due to the difficulty in hindering new cases of severe or deadly cases each year despite the use of effective infant and booster vaccines in children and adolescents. In LMIC, the pertussis epidemiological situation is poorly known due to absence of diagnosis platforms. World Health Organization (WHO) estimated in 2003 about 17.6 million cases of pertussis and about 279 000 fatal cases worldwide, 90% of which were in developing countries [START_REF]Managing pertussis outbreaks during humanitarian emergencies: WHO technical note[END_REF]. More recently, an updated model from Yeung and colleagues showed that there were 24.1 million pertussis cases and 160 700 deaths from pertussis in children younger than 5 years in 2014, with the African region contributing the largest proportions with 33% of cases and 58% of estimated deaths [START_REF] Yeung | An update of the global burden of pertussis in children younger than 5 years: a modelling study[END_REF].

It is critical to gather information to inform national public health authorities in African countries, especially as i. no booster vaccines are included in EPI, and ii. we do not know the effectiveness of, and the duration of protection induced by whole-cell pertussis vaccines (wPVs) currently produced and distributed with the EPI for vaccinating infants during their first year of life.

The wPVs are made of chemically or heat-inactivated B. pertussis and may suffer from disparities in production processes. Pooled efficacy of those produced in 1980-1990s was close to 80% but significantly varied among the different brands, with efficacy ranging from 46% to 92%. No data exists on currently available wPVs vaccines [1].

A few studies have evidenced the circulation of B. pertussis among infants, children, adolescents, and adults and reported outbreaks in several African countries [START_REF]Managing pertussis outbreaks during humanitarian emergencies: WHO technical note[END_REF][START_REF] Muloiwa | The burden of laboratory-confirmed pertussis in low-and middle-income countries since the inception of the Expanded Programme on Immunisation (EPI) in 1974: a systematic review and meta-analysis[END_REF][START_REF] Mitiku | Pertussis outbreak in Southern Ethiopia: challenges of detection, management and response[END_REF][START_REF] Razafimahatratra | Seroprevalence of pertussis in Madagascar and implications for vaccination[END_REF][START_REF] Semeeh | An epidemiological investigation of the 2019 suspected pertussis outbreak in northwestern Nigeria[END_REF][START_REF] Macina | Bordetella pertussis in School-Age Children, Adolescents, and Adults: A Systematic Review of Epidemiology, Burden, and Mortality in Africa[END_REF].

Materials and Methods

Study population and design

Children were recruited in schools in Antananarivo, Capital of Madagascar, and in Dapaong, northern Togo. Inclusion criteria were: being aged 3-15 years old and having completed the three injections of pertussis primary vaccination at least a year before sampling date. Presenting a proof of vaccination (i.e. vaccination booklet or diploma) was an additional inclusion criterion in Madagascar. In Togo, the following non-inclusion criteria were applied: having a known hemostasis disorder, or serious acute or chronic illness, or any illness that kept the child from attending the appointment planned for blood collection. Information related to pertussis vaccination history, sex, age, and blood sampling was collected on a standardized questionnaire and data recorded into a computer database. No information related to past or current respiratory illness was collected. A blood sample from the tip of a finger (approximately 200 µl) was collected using a lancet (Becton Dickinson, Sentry 23G) and a serum separating microtainer (Becton Dickinson, reference 365968).

Anti-pertussis toxin serology

Upon collection, blood samples were transferred within 6 hours to the Unité de Bactériologie Expérimentale -Institut Pasteur in Madagascar, and the Centre Hospitalier Regional de Dapaong in Togo, where serum was collected after a short spinning and then stored at -80°C. In Togo, frozen samples were then shipped to the Institut National d'Hygiène in Lomé.

As previously described [START_REF] Noel | Circulation of Bordetella pertussis in vaccinated Cambodian children: A transversal serological study[END_REF], anti-PT IgG titers were quantified using a commercial purified PT-containing Enzyme-linked immunosorbent assay (ELISA) kit (EUROIMMUN; reference EI 2050-G) [START_REF] Riffelmann | Performance of Commercial Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Bordetella pertussis[END_REF] and the WHO reference serum available from the National Institute for Biological Standards and Control (NIBSC). All tests had internal negative and positive controls and passed the validity criteria. Results were reported as International Units (IU)/ml. Lower limit of quantitation (LLOQ) was defined as 5 IU/ml. A titer of <5 IU/ ml corresponded to being seronegative and children with anti-PT IgG levels ≥5 IU/ml were considered as seropositive. Anti-PT titers were categorized using 40 IU/ml and 100 IU/ml cut-offs to evidence children who had contact with B. pertussis sometime during the past 12 months when [40-100[ IU/ml, and 6 months when ≥100 IU/ml [START_REF] Guiso | What to do and what not to do in serological diagnosis of pertussis: recommendations from EU reference laboratories[END_REF].

Pertussis immunization schedule

Pertussis vaccination schedule in Madagascar and in Togo include vaccine injections at ages 6, 10, and 14 weeks. Based on WHO recommendation, compliance with national schedule was defined as having received first injection at ≥38 days of life, and the second and third injections 24-70 days after the previous dose.

Statistical analysis

Continuous variables were described using the median, interquartile range (IQR), and comparisons were conducted using two-sided Kruskall-Wallis test. For categorical variables, percentages were estimated, and comparisons were conducted using two-sided chi² test. Logistic regression models were used to identify factors associated with compliance and with seropositivity.

Statistical analyses were undertaken using Stata software, version 15.1. A 5% alpha risk was considered for statistical tests.

Ethical aspects

After giving the information to the parents/guardians, informed consent of the parents/guardians and the oral assent for children aged >7 years were collected. Only children meeting the inclusion criteria and for whom written and oral consent/assent were obtained participated to the study. The study protocol was reviewed and approved by the Institutional Review Board of Institut

Results

Study population

A total of 1041 and 937 children were recruited in schools in Antananarivo, Madagascar (MDG) and in Dapaong, Togo (TGO), respectively (Figure 1). In Madagascar, children were recruited in 13 primary and 8 secondary schools, located in Antananarivo city and suburban areas, over a period of 4 months (February-May 2018). In Dapaong (TGO), recruitment took place in 7 primary and 3 secondary schools over a period of 3 months (January-March 2017). Several children who did not respond to inclusion criteria or with partial or missing information were subsequently excluded for analyses as detailed in Figure1. Pertussis serology and vaccination timeliness analyses were performed using information from 1033 and 996 children in Antananarivo (MDG; Figure 1A), and 908 and 772 children in Dapaong (TGO; Figure 1B), respectively. Age groups were defined as 3-5, 6-8, 9-11, and 12-15 years; each group included ≥170 children (Table 1). Information regarding pertussis vaccination was mainly obtained from the child vaccination booklet in Antananarivo (MDG; 97.6%; 1008/1033), and in Dapaong (TGO; 87.6%; 795/908), which provides details on vaccine injection dates. The official confirmation of a complete primary vaccination, but no injection date details, was obtained through the vaccination diploma for a few children in Antananarivo (MDG; 2.4%; 25/1033).

Anti-pertussis toxin serology

At the time of the studies, the shortest timespan since last pertussis vaccine injection was 2.7 years in Antananarivo (MDG), and 2.3 years in Dapaong (TGO). The global seropositivity rate was 56.9% (588/1033) in Antananarivo (MDG), and 62.2% (565/908) in Dapaong, (TGO; Table 2 and Figure 2). In Antananarivo (MDG), seropositivity rate varied from 45.6% (83/182) to 63.0% (174/276) among the age groups, and significant increase in antibody titers was observed in children 6 years old or older, with respect to 3-5 years old children. The 3). Vaccine timeliness was found to be significantly associated to the age groups, with children aged 3-5 years showing a better compliance with the recommendations in both countries (Table 3). While girls were significantly more compliant as compared to boys in Antananarivo (MDG), pertussis seropositivity was not associated to vaccine timeliness in both study populations. possible to determine whether the study was run during a period of low or high circulation of B. pertussis. WHO monitoring system reports that annual number of cases ranged from 0 to 350 in Madagascar, and from 10 to 180 in Togo in the past 15 years, including several years for which no data is available for both countries [START_REF]Incidence time series for Madagascar (MDG). WHO vaccine-preventable diseases: monitoring system 2020 global summary[END_REF][START_REF]Incidence time series for Togo (TGO). WHO vaccine-preventable diseases: monitoring system 2020 global summary[END_REF]. Serological evidence of recent cases occurring in both Antananarivo (MDG) and Dapaong (TGO) in all age groups was found in these studies. Loss of vaccine-induced immunity was well described with wPVs in historical and more recent studies, including in African settings [START_REF] Jenkinson | Duration of effectiveness of pertussis vaccine: evidence from a 10 year community study[END_REF][START_REF] Broutin | Loss of immunity to pertussis in a rural community in Senegal[END_REF][START_REF] Bassey | Bordetella Pertussis Antibody Levels in DPT/Pentavalent Immunized Preschool Nigerian Children: A Cross-Sectional Study[END_REF]. This observation, also true when acellular pertussis vaccines are used, have lead the health authorities to recommend several booster doses including in toddlers and preschool children [1]. Study results are not surprising given the insufficient coverage recorded in these two countries, and vaccination schedule restricted the primary vaccination in newborns. Indeed, despite the improvement in the beginning of the 2000s, vaccine coverage remains below the 90% goal of WHO Global Vaccine Action Plan in both countries. Estimates given by the United Nations of International Children's Emergency Fund (UNICEF) and the WHO show that coverage for third dose of wPV was less than 60% in 2000 in Madagascar; it slightly increased thereafter ranging from 70% to 80% [START_REF]WHO UNICEF estimates time series for Madagascar (MDG). WHO vaccine-preventable diseases: monitoring system. 2020 global summary[END_REF]. Similarly, coverage was 64% in Togo in 2000, and then has stayed to approximately 80% since 2005 [START_REF]WHO UNICEF estimates time series for Togo (TGO). WHO vaccine-preventable diseases: monitoring system. 2020 global summary[END_REF].

Antananarivo (MDG) -N=996 Dapaong (TGO) -N=772

Age at injection (wks) median (IQR) median (IQR)

The success of the immunization program and population-level immunity is determined by the national coverage, but also by the ability of delivering the three doses within a narrow time window to provide early and adequate protection, and the quality of the wPVs [START_REF] Heininger | The concept of vaccination failure[END_REF]. In Antananarivo (MDG), timeliness of vaccination, in particular intervals in between two doses, was as high as 89.0%. In absence of booster doses, vaccinees become more susceptible for infection and disease as time goes by, and contribute to maintain circulation that may be critical for the very young and unvaccinated children. This delay is also highly dependent on the quality of wPVs that are currently available, which is unknown. Studies on wPVs used in the past have evidenced high variability in immunogenicity, efficacy, effectiveness among the products [START_REF]Immunological basis for immunization: Pertussis[END_REF]. This lack of data of contemporary vaccines is a concern that is added to the fact that wPVs distributed in LMICs are often acquired from different vaccine producer's year-to-year.

In this research, the same study protocol was applied in two very different regions of the African continent. On one hand, recruitment took place in Antananarivo (city and suburban areas), which is Madagascar's main city with as very high population density that reached 1.9 million inhabitants in 2018. On the other hand, the study was implemented in Dapaong, the main city of the Savanes region in Northern Togo. Its population was estimated to be 69,900 in 2020. In addition, recruitment of children took place over two different periods, at approximately one-year interval. While the sero-epidemiological results cannot be compared, they spotlight pertussis as a public health concerned that demands a better surveillance in Madagascar and Togo, in order to determine the most adapted health strategy to adopt.

Conclusions

The present results showed that B. pertussis substantially circulates among Malagasy and Togolese children vaccinated during infancy, with increasing contact from 6 years old in Antananarivo but at a steady rate in Dapaong. Further efforts are necessary to initiate vaccination schedule as early as 6 weeks of age, and timeliness of vaccination should be improved in Dapaong. These data are local evidence of the need for improving pertussis surveillance in Madagascar and Togo. The presence of a platform for laboratory diagnosis is critical to estimate the burden of the disease and to rapidly confirm the diagnosis to clinicians to eventually adapt the antibiotic therapy. The results also support the extension of the vaccination program with boosters.

  Pasteur, France (decision number 2016-04/IRB), by the Ethics Committee of Biomedical Research of the Ministry of Public Health of Madagascar (decision number 065-MSANP/CE), and by the Comité de Bioéthique pour la Recherche en Santé in Togo (decision number 268/2016/ MSPS/CAB/SGIDPML/CBRS). Authorization for data processing has been obtained from French legal authority (Commission Nationale Informatique et Liberté), and pseudonymization was performed assigning a unique code specific to each participant. The ClinicalTrials.gov identifier of the studies is: NCT02983487.

Figure 1 :

 1 Figure 1: Flow chart of participants in Antananarivo (MDG; A), and Dapaong (TGO; B)

Figure 2 :

 2 Figure 2: Distribution of anti-PT IgG titers by age groups. Proportions of children exhibiting <5, [5-40, [40-100] and ≥100 IU/ml in Antananarivo (MDG; A), and in Dapaong (TGO; B) are represented by age groups

  6-8 years old age group included the highest frequency of children exhibiting serological sign of a recent infection with 10.2% (21/206) and 8.7% (18/206) of children having anti-PT titers [40-100] and ≥100 IU/ml, respectively. In Dapaong (TGO), seropositivity rate was the lowest (56.3%; 180/320) in the 9-11 years old age group, and reached 70.1% (162/231) in the 12-15 years old age group. Rate of children exhibiting serological sign of recent infection was similar across the age groups and totalized 11.7% (106/908) and 2.1% (19/908) of children having anti-PT titers [40-100] and ≥100 IU/ml, respectively. Neither sex nor availability of the vaccination booklet was associated to seropositivity.

			Antananarivo (MDG)			Dapaong (TGO)
	Age groups	3-5	6-8	9-11	12-15 Total p value 3-5	6-8	9-11	12-15 Total	p value
	N	182 (100)	206 (100)	369 (100)	276 (100)	1033 (100)	-	170 (100)	187 (100)	320 (100)	231 (100)	908 (100)	-
	Gender						0.196						0.010
	Boys	97 (53.3)	112 (54.4)	172 (46.6)	131 (47.5)	512 (49.6)		94 (55.3)	84 (44.9)	157 (49.1)	90 (39.1)	425 (46.9)*
	Girls	85 (46.7)	94 (45.6)	197 (53.4)	145 (52.5)	521 (50.4)
	Origin of
	pertussis vaccination						<0.001						0.539
	history
	Declarative	-	-	-	-	-		16 (9.4)	23 (12.3)	40 (12.5)	33 (14.3)	112 (12.3)*
	Vaccination diploma	2 (1.1)	15 (7.3)	7 (1.9)	1 (0.4)	25 (2.4)		-	-	-	-	-
	Vaccination	180	191	362	275	1008		154	163	280	198	795
	booklet	(98.9)	(92.7)	(98.1)	(99.6)	(97.6)		(90.6)	(87.2)	(87.5)	(85.7)	(87.6)**

* 1 child with no information regarding sex ** 1 child with no information on origin of pertussis vaccination history

Table 1: Distribution of the study population recruited in Antananarivo, MDG (left), and in Dapaong, TGO (right) shown by age groups. Results are shown as n (%)

Table 2 :

 2 AnalysisDue to the lack of vaccination booklet, or incomplete dates of birth or of vaccine injections, assessment of vaccination timeliness was performed on the data from 996 and 772 children, in Antananarivo (MDG) and Dapaong (TGO), respectively. Median age at each vaccine dose is presented in Table3. Overall, median age for each dose was older than recommended in both countries. The rate of children who initiated the vaccination at age >8 weeks was 33.4% in Antananarivo (MDG) and

	39.4% in Dapaong (TGO). On the other hand, 1.8% and 3.4% of children received their third vaccine dose at age >12 months
	old in Antananarivo (MDG) and Dapaong (TGO), respectively. Compliance with national vaccination recommendation was
	89.0% (886/960) and 77.2 % (596/772) in Antananarivo (MDG) and Dapaong (TGO), respectively (Figure

of pertussis seropositivity according to age groups and other factors in Antananarivo (MDG; left), and in Dapaong (TGO; right) Journal of Immunology and Infectious Diseases Volume 8 | Issue 1 Vaccination timeliness

Timeliness for entire primary vaccination n (%) OR (95% CI) p value n (%) OR (95% CI) p value

	1		7.3 (6.6-8.9)			7.4 (6.6-9.7)
	Dose 2		12.4 (11.4-14.4)		13.0 (11.6-16.0)
	Dose 3		17.6 (16.3-20.0)		18.7 (16.4-23.4)
	Initiation of primary	n (%)	OR (95% CI) p value n (%)	OR (95% CI) p value
	vaccination at age >8 wks
	Total		337 (33.4) -	-	304 (39.4) -	-
	Age group	3-5	49 (27.4)	1	ref	52 (34.2)	1	ref
		6-8	62 (33.0)	1.3 (0.8-2.0)	0.243	49 (30.3)	0.8 (0.5-1.3) 0.453
		9-11	128 (35.9) 1.5 (1.0-2.2)	0.050	106 (39.7) 1.3 (0.8-1.9) 0.265
		12-15	98 (36.0)	1.5 (1.0-2.3)	0.056	97 (50.8)	2.0 (1.3-3.1) 0.002
	Sex	Boys	162 (32.3) 1	ref	147 (41.2) 1	ref
		Girls	175 (35.4) 1.1 (0.9-1.5)	0.314	157 (37.8) 0.9 (0.7-1.2) 0.343
	Anti-PT IgG	<5 IU/ml 155 (36.4) 1	ref	110 (36.8) 1	ref
	titers
		≥5 IU/ml 182 (31.9) 0.8 (0.6-1.1)	0.142	194 (41.0) 1.2 (0.9-1.6) 0.242
	Total		886 (89.0) -	-	596 (77.2) -	-
	Age group	3-5	169 (94.4) 1	ref	134 (88.2) 1	ref
		6-8	166 (88.3) 0.4 (0.2-1.0)	0.042	134 (82.7) 0.6 (0.3-1.2) 0.175
		9-11	310 (86.8) 0.4 (0.2-0.8)	0.009	204 (76.4) 0.4 (0.2-0.8) 0.004
		12-15	241 (88.6) 0.5 (0.2-1.0)	0.040	124 (64.9) 0.3 (0.1-0.4) <0.001
	Sex	Boys	432 (86.2) 1	ref	275 (77.0) 1	ref
		Girls	454 (91.7) 1.8 (1.2-2.7)	0.006	321 (77.4) 1.0 (0.7-1.4) 0.916
	Anti-PT IgG	<5 IU/ml 383 (89.9) 1	ref	230 (76.9) 1	ref
	titers
		≥5 IU/ml 503 (88.3) 0.8 (0.6-1.3)	0.409	366 (77.4) 1.0 (0.7-1.4) 0.883

Table 3 :

 3 Age at pertussis vaccination and factors associated to vaccination timeliness in Antananarivo (MDG; left), and in Dapaong (TGO; right). Wks: weeks Journal of Immunology and Infectious Diseases

	Volume 8 | Issue 1

  However, >30% of Malagasy children failed to initiate their pertussis vaccination program at ≤8 weeks of age, which is a WHO recommendation [1]. Similar observation was found in a recent large analysis studying coverage in six districts in Madagascar[START_REF] Jones | The Challenge of Achieving Immunity Through Multiple-Dose Vaccines in Madagascar[END_REF]. Of note, this delay in initiating the vaccination program was significantly lower among the youngest children, which suggest

an improvement of the national vaccination campaign. In Dapaong (TGO), compliance was not as high, with 77.2% of children who were vaccinated following the national recommendations; delay in administering doses 2 and 3 concerned 7 and 8% of the children, respectively. In addition, 39.4% of the children started the vaccination program at age >8 weeks of age. Similarly to what was observed in Antananarivo (MDG), the youngest children (3-8 years old) represented those who most frequently received their first dose at ≤ 8 weeks of age.

Acknowledgements

We thank all the children and their parents or legal guardians for participating to these studies, and the Directors of the schools in Antananarivo and Dapaong, and Father Pedro Opeka, founder of the Akamasoa community in Antananarivo, for giving us access to the schools. We also thank Sandra Corre for her participation in laboratory staff training, David Tavel for his support in data cleaning, Yoann Madec for his advices on data analysis, Tsinjo Rasoanaivo for her help in observing good clinical practices, Harimanana Ramanantsoa and Avosaina Rasamimanana for their participation in the field coordination, Mializay Ralalanombana and Hortensia M. Heriniaina in Madagascar, and Essofa Oubote and Didier Mounkoro in Togo, for their contribution in serum collection and logistic organization, Haoua Tall for her help in transferring to the study site, and Benoit Garin and Jennifer Moisi for their support in the early discussions for the study implementation. We thank the members of the PERILIC Programme's Scientific Committee for their recommendations and advice in interpreting the data. We thank the Clara Belliveau Foundation for its donation, and the Total Foundation for financing this research.

s Emergency Fund.

Discussions

The present sero-epidemiological studies are novel in addressing the circulation of B. pertussis in fully vaccinated children in Madagascar and in Togo. The approach using indirect diagnosis applied in these studies is particularly interesting in LMIC settings as i) it is easy to implement on a large scale, and ii) there is no booster vaccine that may interfere with infection-induced anti-PT IgG detected by ELISA. It allows, not only to diagnose recent infection and address immune responses to vaccines, but also evaluate immunity in populations. These studies, focusing on vaccinated 3-15 years old children and adolescents, evidenced the circulation of B. pertussis in Antananarivo, Madagascar, and in Dapaong, Togo, with recent cases occurring, thus suggesting insufficient immune protection at least 3 years from the primary vaccination. Details on vaccination history also highlighted delay in implementing early vaccination, although it appeared to have improved in recent years.

The trend in seropositivity rates observed across the age groups in Antananarivo (MDG) was similar to what we observed in Cambodia, where the same study was run and where the same pertussis immunization schedule is applied [START_REF] Noel | Circulation of Bordetella pertussis in vaccinated Cambodian children: A transversal serological study[END_REF]. There was a significant increase in frequency of seropositive children among those aged 6 years and older with respect to the 3-5 years age group, anti-PT titers being the highest among the 6-8 years old age group. However, in Antananarivo (MDG), rates were higher with roughly 10% increase for each age category with respect to what was observed in Cambodia, and there were more recent infection cases detected, reaching 18.9% of the 6-8 years old children. By contrast, seropositivity rate in Dapaong (TGO) was as high as 65.3% in the 3-5 age group, with no significant variation among older children. Global seropositivity rates were 56.9% and 62.2% in Antananarivo (MDG) and in Dapaong (TGO), respectively, while it was 47.0% in Cambodia. However, comparison of global seropositivity is difficult since incidence rate varies across the years in a given country. Indeed, pertussis is an endemic disease with 3-5 years cyclic increase of incidence, depending on rate of susceptible individuals. This is linked to demographic differences and different vaccine coverage [START_REF] Bouchez | Bordetella pertussis, B. parapertussis, vaccines and cycles of whooping cough[END_REF]. With the scarce surveillance in Madagascar and Togo, it is not


M.D. Valentin Coirier
email: valentin.coirier@chu-rennes.fr

Mathieu Lesouhaitier

Florian Reizine

Benoit Painvin

Quentin Quelven

Adel Maamar

Arnaud Gacouin

Jean-Marc Tadie

Yves Le Tulzo

Christophe Camus

Tolerance and complications of therapeutic plasma exchange by centrifugation: A single center experience

Keywords: NCT03888417) therapeutic plasma exchange, fresh frozen plasma, albumin, hypotension, hypocalcemia, allergic reaction albumin, A+FFP: albumin + fresh frozen plasma, ACD-A: acid citrate dextrose, solution A, EPV: estimated plasma volume, FFP: fresh frozen plasma, HES: hydroxyethyl starch, ICU: Intensive Care Unit, TPE: therapeutic plasma exchange, UERTU: Urgent Extracorporeal Replacement Therapy Unit

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Tolerance and complications of therapeutic plasma exchange by centrifugation: a single center experience

Valentin Coirier1,2 *, Mathieu Lesouhaitier 1,2 , Florian Reizine 1,2 , Benoît Painvin 1,2 , Quentin Quelven 1,2 , Adel Maamar 1,2 , Arnaud Gacouin 1,2 , Jean-Marc Tadié 1,2 , Yves Le Tulzo 1,2 , and Christophe Camus 1,2 .

INTRODUCTION

Therapeutic plasma exchange (TPE) was first utilized in human medicine at the end of the 1950s [START_REF] Schwab | Treatment of Waldenström's Macroglobulinemia by Plasmapheresis[END_REF]. Since then, the efficacy of TPE has been well evaluated [START_REF] Van Der Meché | A Randomized Trial Comparing Intravenous Immune Globulin and Plasma Exchange in Guillain-Barré Syndrome. Dutch Guillain-Barré Study Group[END_REF][START_REF] Jayne | Randomized Trial of Plasma Exchange or High-Dosage Methylprednisolone as Adjunctive Therapy for Severe Renal Vasculitis[END_REF][START_REF] Rock | Comparison of Plasma Exchange With Plasma Infusion in the Treatment of Thrombotic Thrombocytopenic Purpura. Canadian Apheresis Study Group[END_REF][START_REF] Larsen | High-volume Plasma Exchange in Patients With Acute Liver Failure: An Open Randomised Controlled Trial[END_REF] and international recommendations and the levels of evidence are frequently updated [START_REF] Padmanabhan | Guidelines on the Use of Therapeutic Apheresis in Clinical Practice-Evidence-Based Approach from the Writing Committee of the American Society for Apheresis: The Eight Special Issue[END_REF]. TPE is currently recommended as first-or second-line therapy for approximately 20 disorders, with highquality evidence for only four of them.

The mechanism of action of TPE is not completely understood [START_REF] Reeves | The mechanisms of action of plasma exchange[END_REF]. It is largely accepted that two main elements are the removal of pathological substances from the blood [START_REF] Kaplan | Moderator's view: High-volume plasma exchange: pro, con and consensus[END_REF] and/or the replacement of deficient plasma components [START_REF] George | Syndromes of Thrombotic Microangiopathy[END_REF]. TPE is currently performed using either a high-permeability filter with standard multifunctional renal replacement equipment or a centrifugation device [START_REF] Madore | Plasmapheresis. Technical aspects and indications[END_REF].

Most authors have used national registries to report complications associated with TPE procedures [START_REF] Norda | Therapeutic apheresis in Sweden: update of epidemiology and adverse events[END_REF][START_REF] Korach | Plasma exchange in France: Epidemiology 2001[END_REF][START_REF] Rock | The Canadian apheresis registry[END_REF] and described complications associated with various types of apheresis (TPE, cytapheresis, LDL-adsorption). Registries have the advantage of gathering multicenter observational data. However, such studies do not guarantee completeness because data collection is heterogeneous between centers. Moreover, the various plasmapheresis techniques used were generally not described. We assessed the type and frequency of adverse effects during TPE performed by a dedicated staff using a standardized protocol.

MATERIALS AND METHODS

Study design and patients

We conducted a single center retrospective observational study at the Rennes University Hospital between January 2011 and December 2017. Eligibility criteria were being an adult and having had at least one TPE session during the study period. Patients under legal protection, minors, those who declined to participate in the study, and those for whom monitoring forms were not found were not included. The study protocol was approved by the Rennes Hospital Institutional Review Board (agreement number 18.59) before the beginning of the study.

TPE sessions

The TPE sessions were performed by nurses specifically trained in the TPE technique in the for critically ill patients in other ICUs, for whom TPE was performed on site. The indications and number of sessions were decided according to international recommendations (6), in agreement with the patients' specialist physician and the TPE referent intensivist. The nature of the replacement fluid depended on the indication, the bleeding risk, and the interval between TPE sessions. The type and number of fresh frozen plasma (FFP) units were provided by the French Blood Establishment (Établissement Français du Sang), depending on available stocks. When 4% albumin (A) was used as the replacement fluid, a small proportion of hydroxyethylstarch (HES) was included at the beginning of the TPE session, as recommended [START_REF] Korach | Adult and paediatric therapeutic hemapheresis[END_REF][START_REF] Agreda-Vásquez | Starch and albumin mixture as replacement fluid in therapeutic plasma exchange is safe and effective[END_REF].

The plasmapheresis technique used in our center was exclusively centrifugation, regardless of the indication. It was chosen because of the higher efficiency of plasma extraction relative to filtration [START_REF] Hafer | Membrane versus centrifuge-based therapeutic plasma exchange: a randomized prospective crossover study[END_REF] and the possibility of using peripheral venous access. The device used was the Comtec Cell Separator (Fresenius Kabi -61352 Bad Homburg, Germany). Anticoagulation of the extracorporeal circuit was achieved using acid citrate dextrose solution A (ACD-A). The total volume of ACD-A used during the procedure and the volume reinfused to the patient were calculated by the centrifuge. Hypocalcemia was prevented by continuously infusing calcium during the TPE procedure, the amount based on the measurement of prior ionized calcium levels and the type of replacement fluid used. Generally, 2 g calcium gluconate per hour was infused with A and 2 g calcium chloride per hour with FFP.

The choice of venous access depended on the indication for TPE, the number of sessions planned, the patient's status, and the condition of the peripheral veins of the upper limbs.

Ultrasound-guided insertion of a dialysis catheter at the jugular or subclavian site was systematically checked by chest x-ray to ensure proper positioning and the absence of any complication (e.g. pneumothorax).

Standardized monitoring forms were designed for the surveillance of TPE sessions and were contemporaneously completed by the nurses. (Supplementary material 1).

Data collection

Patient characteristics were collected, including age, height, weight, and TPE indication.

Cardiorespiratory monitoring parameters during sessions were recorded (blood pressure, heart rate, pulse oximetry, and respiratory rate), as well as data related to the TPE procedures (volumes processed and treated, nature and site of venous access, hypocalcemia or anaphylaxis symptoms, ACD-A volume used, dose of calcium infused, device-related problems, discontinuation of the session, and any other event considered to be an adverse effect). Biological data collected included ionized calcium levels, pH, and hematocrit.

Ionized calcium and pH were measured in whole blood. Bacteriological results of the catheter culture were collected, as well as all blood cultures performed during the treatment period.

Adverse effects: definitions and classifications (Supplementary Material 2)

Hypotension was defined as the occurrence of a mean arterial pressure <65 mmHg and/or a decrease in systolic arterial pressure >30 mmHg during the session relative to baseline and was classified into three severity grades. Hypocalcemia was diagnosed if the ionized calcium level was <1.10 mmol/L. The severity of hypocalcemia was determined according to the Lee classification [START_REF] Lee | Anticoagulation techniques in apheresis: from heparin to citrate and beyond[END_REF]. Allergic reactions were classified from grade I to IV according to the Ring and Messmer classification [START_REF] Ring | Incidence and classification of adverse reactions to plasma substitutes[END_REF].

Among complications associated with venous access (pain, bleeding at the catheter insertion site and dysfunction of the catheter, the single-use kits, or the centrifuge) were identified from monitoring forms. Chest x-rays after insertion of the dialysis catheter at the superior vena cava site were examined to assure proper positioning and the absence of pneumothorax.

Venous Doppler examination was performed if catheter-related thrombosis was suspected.

The catheter tip was submitted for bacteriological culture if there was local inflammation or systemic signs. The diagnosis of infection related to venous access was performed in accordance with international recommendations [START_REF] Mermel | Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America[END_REF]. Cases with catheter-tip positive cultures were classified as catheter-associated bloodstream infection if the blood cultures were positive for the same microorganism or, otherwise, as catheter colonization.

Outcomes and statistical analysis

The main outcome was the rate of all-type adverse effects, expressed as the number (percent) of sessions and the number (percent) of patients. The rates of each type of adverse effect and overall severe adverse effects were secondary outcomes. Proportions were estimated with the 95%confidence interval (95% CI) by the method of Clopper and Pearson. Statistical analysis was performed using StatView 5.0 version, SAS. Quantitative variables were expressed as medians (25th percentile-75th percentile) and comparisons between groups were performed using the Mann Whitney U test or Kruskall-Wallis test. Categorical variables were compared using the chi-square test or Fisher's exact test, as appropriate. All tests were two-sided and a p-value <0.05 was considered statistically significant. Post-hoc multiple comparisons between groups were performed according to the Bonferroni correction (a p-value <0.0167 was considered statistically significant for three comparisons).

RESULTS

Description of the population and TPE sessions

Session characteristics

In total, 185 patients were included, allowing the analysis of 1,895 TPE sessions (Figure 1). 2). The exchanged volume varied depending on the TPE indication. A particularly large volume was prescribed for the treatment of acute liver failure (2.5 x EPV). Other characteristics of the sessions are reported in Table 2.

Replacement fluids

The replacement fluids used were FFP alone, 4% A alone, or a combination of 4% A and FFP (A+FFP) for 596 (31.5%), 933 (49.2%) and 366 (19.3%) TPE sessions, respectively (Tables 1 and2). Among the total of 13,472 FFP units, four different types of FFP were delivered during the study period: either untreated quarantined units (38.9%) or pathogen-inactivated units (amotosalen-inactivated: 24.6%, solvent/detergent-treated: 21%, methylene bluetreated: 15.5%). HES was given in 80.4% of sessions using A alone and 26.2% of sessions using A+FFP.

ACD-A and calcium use

The total volume of ACD-A used during the sessions (

Allergic reactions

An allergic reaction was observed in 56 of 1,890 sessions (3.0% [2.3%-3.8%]), exclusively with FFP as the replacement fluid (6.9% of FFP sessions [5.0%-9.3%], Table 3). Of the 56 reactions, 52 were grade I (93%), three were grade II, and only one was grade III (anaphylactic shock). There was an increasing risk of allergic reaction per liter of FFP infused (OR: 1.34 [1.11-1.63], p=0.003).

The incidence of allergic reactions was significantly lower with the use of pathogeninactivated FFP units (4.2%) than exclusively untreated quarantined FFP units (9.3%, p=0.002), with a similar number of FFP units used in both settings (14 units [START_REF] Kaplan | Moderator's view: High-volume plasma exchange: pro, con and consensus[END_REF][START_REF] George | Syndromes of Thrombotic Microangiopathy[END_REF][START_REF] Madore | Plasmapheresis. Technical aspects and indications[END_REF][START_REF] Norda | Therapeutic apheresis in Sweden: update of epidemiology and adverse events[END_REF][START_REF] Korach | Plasma exchange in France: Epidemiology 2001[END_REF][START_REF] Rock | The Canadian apheresis registry[END_REF][START_REF] Korach | Adult and paediatric therapeutic hemapheresis[END_REF][START_REF] Agreda-Vásquez | Starch and albumin mixture as replacement fluid in therapeutic plasma exchange is safe and effective[END_REF][START_REF] Hafer | Membrane versus centrifuge-based therapeutic plasma exchange: a randomized prospective crossover study[END_REF][START_REF] Lee | Anticoagulation techniques in apheresis: from heparin to citrate and beyond[END_REF][START_REF] Ring | Incidence and classification of adverse reactions to plasma substitutes[END_REF][START_REF] Mermel | Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America[END_REF] and 15 units [START_REF] Madore | Plasmapheresis. Technical aspects and indications[END_REF][START_REF] Norda | Therapeutic apheresis in Sweden: update of epidemiology and adverse events[END_REF][START_REF] Korach | Plasma exchange in France: Epidemiology 2001[END_REF][START_REF] Rock | The Canadian apheresis registry[END_REF][START_REF] Korach | Adult and paediatric therapeutic hemapheresis[END_REF][START_REF] Agreda-Vásquez | Starch and albumin mixture as replacement fluid in therapeutic plasma exchange is safe and effective[END_REF][START_REF] Hafer | Membrane versus centrifuge-based therapeutic plasma exchange: a randomized prospective crossover study[END_REF][START_REF] Lee | Anticoagulation techniques in apheresis: from heparin to citrate and beyond[END_REF][START_REF] Ring | Incidence and classification of adverse reactions to plasma substitutes[END_REF] respectively, p=0.71).

Other adverse effects

Digestive were stopped prematurely (Table 3).

Non-infectious venous access-related complications

Among the 1,891 sessions analyzed, 202 adverse effects were associated with complications related to venous access (10.7% [9.3%-12.2]) ( respectively; all p<0.0001).

Infectious venous access-related complications

Among 163 dialysis catheters used, 74 catheter tips (45.4%) were submitted for culture due to a suspected infection. There were five cases of catheter-related bacteremia (Supplementary Material 3), resulting in an incidence rate of 3.5‰ catheter-days (1.1‰-8.1‰). The incidence rate of catheter colonization was 11.5‰ catheter-days. Staphylococcus epidermidis was found in 13 of 17 cases of colonization (76%). There were no documented infections associated with the use of other types of venous access.

Severe adverse effects

Severe complications were those that were potentially life-threatening (grade III hypotension, grade III allergic reactions, and grade IV hypocalcaemia). Bacteremia was recorded as a patient-related rather than session-related complication. Severe complications were reported for 5 of 1895 sessions (0.3% [0.1-0.6%]) and 10 of 185 patients (5.4% [2.6-9.7%], including bacteremia).

DISCUSSION

This is the first recent study to reassess the adverse effects of TPE. Previous studies examined periods prior to 2010 [START_REF] Bramlage | Predictors of complications in therapeutic plasma exchange[END_REF], most often the late 1990s and early 2000s [START_REF] Basic-Jukic | Complications of therapeutic plasma exchange: experience with 4857 treatments[END_REF][START_REF] Couriel | Complications of therapeutic plasma exchange: a recent assessment[END_REF]. The most important result concerns the safety of TPE, which showed excellent clinical and biological tolerance (Table 3) and only rare severe adverse effects. The replacement fluid was the main determinant for the occurrence of adverse effects.

Hypotension was frequent, but largely asymptomatic or mild. A higher rate of hypotension with the use of A than that of FFP has already been reported by Shemin et al. [START_REF] Shemin | Complications of therapeutic plasma exchange: a prospective study of 1,727 procedures[END_REF]. The mechanism is poorly understood, because the osmolarity of the two solutions is similar [START_REF] Reutter | Incidence of allergic reactions with fresh frozen plasma or cryo-supernatant plasma in the treatment of thrombotic thrombocytopenic purpura[END_REF].

Severe hypotension during TPE has been reported in patients with acute inflammatory neuropathy, CIDP and MG and was twice as more frequent in those with inflammatory neuropathy than in other patients [START_REF] Yeh | Plasmapheresis-related hypotension[END_REF]. Hypovolemia due to insufficient oral intake and semirecumbent position may contribute to hypotension. Because it has been associated with intradialytic instability in hemodialysis patients [START_REF] Shafi | Autonomic dysfunction as a mechanism of intradialytic blood pressure instability[END_REF] and frequently reported in CIDP [START_REF] Stamboulis | Clinical and subclinical autonomic dysfunction in chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Figueroa | Autonomic dysfunction in chronic inflammatory demyelinating polyradiculoneuropathy[END_REF], autonomic dysfunction may also play a role in TPE-associated hypotension in CIDP patients.

Our observation of an association between hypotension and HES use was unexpected. The use of HES was recommended in France to improve haemodynamic tolerance of TPE [START_REF] Korach | Adult and paediatric therapeutic hemapheresis[END_REF][START_REF] Guidet | Efficacy and tolerance of Elohes in plasma exchanges[END_REF][START_REF] Varrier | Fluid composition and clinical effects[END_REF]. An allergic reaction due to HES without a cutaneous manifestation was unlikely [START_REF] Laxenaire | Anaphylactoid reactions to colloid plasma substitutes: incidence, risk factors, mechanisms. A French multicenter prospective study[END_REF].

Because HES was infused at the beginning of the procedure, it is possible that rapid elimination of the HES volume (>75%) during TPE resulted in hypotension [START_REF] Brecher | Washout kinetics of colloidal starch as a partial or full replacement for plasma exchange[END_REF]. However, other confounding factors could not be excluded. This finding led us to abandon the use of HES during TPE.

Our study estimated the incidence of hypocalcemia to be approximately 20%, despite calcium infusion during the TPE sessions, and it was more frequent with the use of FFP. This side effect was however largely asymptomatic (73% of cases). Our calcium replacement protocol contributed to the reduction of both the degree of hypocalcemia and associated symptoms.

However, systematic calcium replacement is not a uniform practice among TPE centers.

FFP was strongly associated with allergic reactions, whereas A and HES were not. It is already known that allergic reactions are not rare during TPE, in particular in the treatment of thrombotic thrombocytopenic purpura [START_REF] Reutter | Incidence of allergic reactions with fresh frozen plasma or cryo-supernatant plasma in the treatment of thrombotic thrombocytopenic purpura[END_REF][START_REF] Garraud | Types of fresh plasma with focus on therapeutic plasma exchange[END_REF]. The association between the volume of FFP infused and the risk of developing an allergic reaction was shown. This intuitive result underscores the need to curtail the volume of FFP required. Moreover, we observed a higher risk of allergic reaction when using quarantined-FFP than pathogen-inactivated FFP. This finding is in accordance with those already published by the International Hemovigilance Network [START_REF] Saadah | Comparing transfusion reaction risks for various plasma products -an analysis of 7 years of ISTARE haemovigilance data[END_REF]. Whether pooled FFP should be used for large volume FFP TPE in order to reduce allergic reactions deserves further investigation.

Other manifestations, not associated with hypotension, hypocalcemia, or an allergic reaction, were frequently reported during the TPE sessions (malaise, digestive symptoms). They may have been due to ACD-A induced hypomagnesemia (unmonitored) or ACD-A toxicity alone.

Catheter-related complications were frequent, generally not serious and mostly associated with the use of a peripheral venous catheter. Our results suggest that the use of a dialysis catheter is reasonable when TPE sessions are frequent and large volumes are to be exchanged.

Our study had several strengths. First, the same standardized protocol was used for all TPE sessions, leading to the collection of data that was more homogenous than that of previously published studies [START_REF] Korach | Plasma exchange in France: Epidemiology 2001[END_REF][START_REF] Rock | The Canadian apheresis registry[END_REF][START_REF] Stegmayr | World apheresis registry report[END_REF]. Second, the prospective recording of adverse effects during the procedures by experienced nurses permitted the precise classification of adverse effects and not only a description of the symptoms [START_REF] Shemin | Complications of therapeutic plasma exchange: a prospective study of 1,727 procedures[END_REF]. The reporting of adverse effects was therefore more exhaustive than that based on data from registries. It is likely that the 3 to 5% complication rates reported in these studies were considerable underestimates [START_REF] Korach | Plasma exchange in France: Epidemiology 2001[END_REF][START_REF] Basic-Jukic | Complications of therapeutic plasma exchange: experience with 4857 treatments[END_REF][START_REF] Cid | Efficacy and safety of plasma exchange: ann 11-year single-center experience of 2730 procedures in 317 patients[END_REF].

Our study also had a number of limitations, in particular its being a single center study and the retrospective nature of the work. Moreover, our monitoring form did not include surveillance of the corporeal temperature, whereas Shemin et al. [START_REF] Mermel | Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America[END_REF] reported that fever occurred during 7.7% of TPE sessions. This could be helpful for the diagnosis of transfusionrelated adverse events or for the better detection of infections. No other published study has reported fever during TPE sessions.

In conclusion, we report a high rate of adverse effects, mostly asymptomatic or mild. Serious adverse effects were rare. The main determinant of the complication rate was the replacement fluid. FFP was associated with the occurrence of allergic reactions and hypocalcemia.

Hypotension was more frequent with the use of 4% A and was associated with the use of HES. Most catheter-related complications occurred with the use of a peripheral venous catheter.

Figure 1 .

 1 Figure 1. Flow chart

  Urgent Extracorporeal Replacement Therapy Unit (UERTU) of the Infectious Diseases andMedical Intensive Care Department. Procedures were generally performed in the Intensive

Care Unit (ICU) TPE room within the Medical Intensive Care Department. Most patients hospitalized in other wards were transferred to the TPE room for the TPE treatment except

Table 2

 2 Hypocalcemia occurred during 370 of the 1,891 sessions (19.6% [17.8%-21.4%]) (Table3).

	) was 559 mL [476-663] and

Description of adverse effects

At least one adverse effect was reported for 805 of the 1,895 sessions (42.5% [29.9%-70.1%]) and 171 of the 185 patients (92.4% [87.6%-95.8%]). regression analysis, CIDP (OR: 2.70

[1.73-4.22]

, p<0.0001) and the use of HES (OR: 2.17

[1.42-3.31]

, p<0.001) were associated with the occurrence of hypotension (Table

4

).

Table 5

 5

	). Most (88.1%) consisted of

TABLE 1 .

 1 Indications for therapeutic plasma exchange sessions Flow chart. TPE, therapeutic plasma exchange

Supplementary material 1. Prescription and monitoring forms. Supplementary material 2. Adverse event classification.

	Supplementary material 3
	Hypotension
	Once the diagnosis of hypotension was made, it was classified according to severity:
	-grade I: asymptomatic hypotension if there were no associated symptoms and no specific
	therapy was given
	-grade II: non-severe symptomatic hypotension if there was a need for volume expansion or
	increasing the dose of a vasoactive agent
	-grade III: severe symptomatic hypotension if TPE session had to be stopped because of low
	blood pressure
	Hypocalcemia
	The severity of hypocalcemia was determined according to the Lee classification (17)
	-grade I: asymptomatic (symptomless hypocalcemia)
	-grade II: mild (perioral and/or distal paresthesia, sneezing, dizziness, flushes, chills,
	headache)
	-grade III: moderate (nausea/vomiting, irritability, abdominal cramps, spasms, hypotension,
	tremor)
	-grade IV: severe (heart rhythm disorders, attack)
	Allergic reactions
	The severity of allergic reactions was determined according to the Ring and Messmer classification
	(18):
	-grade I: general skin signs (erythema, urticaria, edema)
	-grade II: moderate multi-visceral disorders (skin/mucosal signs, hypotension or unusual
	tachycardia, bronchial hyperreactivity)
	-grade III: life threatening multi-visceral disorders that required specific therapy (collapse,
	tachycardia or bradycardia, heart rhythm disorders, bronchospasm)
	-grade IV: allergic reactions complicated by cardiac arrest

. Bacteriological documentation of catheter-related infections.

	Catheter-related infection	Bacteriological documentation	Catheter site
	Catheter-related bacteremia
	-catheter n°1	Methicillin-susceptible Staphylococcus aureus	jugular vein
	-catheter n°2	Escherichia coli (Extended spectrum beta-lactamase-	femoral vein
	-catheter n°3	producing)	jugular vein
		Methicillin-resistant Staphylococcus epidermidis
	-catheter n°4	Pseudomonas aeruginosa, wild-type phenotype	jugular vein
	-catheter n°5	Methicillin-resistant Staphylococcus aureus	jugular vein
		Methicillin-susceptible Staphylococcus aureus
	Colonization
	-catheter n°6	Methicillin-susceptible Staphylococcus epidermidis	jugular vein
	-catheter n°7	Methicillin-resistant Coagulase-negative Staphylococcus	jugular vein
	-catheter n°8	Methicillin-resistant Staphylococcus epidermidis	subclavian vein
	-catheter n°9	Methicillin-resistant Staphylococcus epidermidis	jugular vein
	-catheter n°10	Methicillin-resistant Staphylococcus epidermidis	jugular vein
	-catheter n°11	Staphylococcus epidermidis	jugular vein
	-catheter n°12	Methicillin-resistant Staphylococcus epidermidis	femoral vein
	-catheter n°13	Staphylococcus epidermidis	jugular vein
	-catheter n°14	Methicillin-resistant Coagulase-negative Staphylococcus	jugular vein
	-catheter n°15	Methicillin-resistant Staphylococcus epidermidis	jugular vein
	-catheter n°16	Staphylococcus epidermidis,	jugular vein
		Pseudomonas aeruginosa, wild-type phenotype
	-catheter n°17	Methicillin-susceptible Staphylococcus epidermidis	jugular vein
	-catheter n°18	Staphylococcus capitis	jugular vein
	-catheter n°19	Methicillin-resistant Staphylococcus epidermidis	jugular vein
	-catheter n°20	Staphylococcus epidermidis	jugular vein
	-catheter n°21	Escherichia coli (penicillinase-producing)	jugular vein
	-catheter n°22	Methicillin-resistant Staphylococcus epidermidis	jugular vein

Maladies Infectieuses et Réanimation Médicale, CHU de Rennes, Rennes, France.

Université de Rennes 1, Faculté de Médecine, Rennes, France.

Acknowledgments: We would like to thank the nurses in the UERTU team (Magalie Anne, Sonia Antin, Kelly Bajoc, Frédérique Charpentier, Fabienne Chamblay, Mara Gérard, Valérie Lallonder, Guénaëlle Le Scouarnec, Jean-Marc Morin) and the Department of Haemovigilance (Dr Hervé Gouezec, Dr Isabelle Grulois and Isabelle Lerenard) for their help in conducting this study.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Funding sources

None.


Clément Bellini

Clément Orione

Jean-François Carpentier

Yann Sarazin

Tailored Cyclic and Linear Polycarbosilazanes via Bariumcatalysed N-H/H-Si Dehydrocoupling Reactions

2]2(THF)3 catalyses the fast and controlled dehydrogenative polymerisation of Ph2SiH2 and p-xylylenediamine to afford polycarbosilazanes. The structure (cyclic vs. linear; endgroups) and molecular weight of the macromolecules can be tuned by adjusting the Ph2SiH2/diamine feed ratio. A detailed analysis of the resulting materials (mol. wt. up to ca. 10,000 g mol -1 ) is provided.

[14] Barium compounds in particular catalyse the coupling of diamines and di(hydrosilanes). [14a,b] We present here an extension of this work to the Ba-mediated production of polycarbosilazanes. The rapid and controlled syntheses of either linear or cyclic polymers by dehydropolymerisation of p-xylylenediamine ( H2N^^NH2) and diphenylsilane, together with detailed characterisations, are reported (Scheme 1).

Scheme 1. Dehydropolymerisation of Ph2SiH2 and H2N^^NH2 catalysed by A. Ba[CH(SiMe3)2]2•(THF)3 (A), identified as the most efficient of our alkalino-earth precatalysts for N-H/H-Si coupling reactions thanks in particular to its combination of highly reactive metal and alkyl groups, [14] catalyses the polycondensation of Ph2SiH2 and H2N^^NH2 upon release of H2 as sole by-product (Table 1). 1 H NMR indicated complete conversion of 100 equiv of the default monomer per metal within 10 min at 60 °C.

Table 1. Dehydropolymerisation of Ph2SiH2 and H2N^^NH2 catalysed by A. [a] [H2N^^NH2]0/ [Ph2SiH2]0/[A]0 Conv [%] [b] Xn,theo [c] Mn,end-group [g mol -1 ] [d] Mn,DOSY [g mol -1 ] [e] Xn,exp [f] P1 20:20:1 >99 n/a [g] n/a [g] 5,600 n/a P2 100:100:1 >99 n/a [g] n/a [g] [a] Reactions in C6H6 at 60 °C for 10 min. [b] Conversion of the default monomer determined by 1 H NMR. [c] Theoretical number-average degree of polymerisation calculated for 99% conversion using Carothers' equation: Xn,theo = (1 + r) / (1 + r -2.r.p) where r is the stoichiometric ratio and p is the extent of the reaction. [d] Number-average molecular weight established by 1 H NMR end-group analysis. [e] Number-average molecular weight estimated by 1 H DOSY NMR. [f] Experimental degree of polymerisation, calculated using the average values of Mn,endgroup and Mn,DOSY.

[g] Not applicable for the cyclic polymers recovered under these conditions.

[a] C. Rennes Cedex France E-mail: yann.sarazin@univ-rennes1.fr; jeanfrancois.carpentier@univ-rennes1.fr [b] C. Orione CRMPO, Université de Rennes 1, Campus de Beaulieu Supporting information for this article is given via a link at the end of the document.

The resulting polycarbosilazanes featured estimated molecular weights in the range 1,400-9,800 g mol -1 , [START_REF]For linear polycarbosilazanes, the values of estimated molecular weights quoted in the text are the average of the values determined by[END_REF] depending on the N-H/H-Si feed ratio. They were isolated in 75-80% yield, taking care to use dry, aprotic solvents due to their hydrolytic sensitivity. Reactions at 25 °C for 60 min gave similar outcomes. Although the polycarbosilazanes were soluble in organic solvents, their molecular weights could not be determined by size exclusion chromatography, owing to their sensitivity under analytical conditions. They were evaluated instead by 1 H DOSY NMR spectroscopy in C6D6, [START_REF]The calibration curve for the diffusion coefficientmol. wt. analysis was established using commercial calibrants, two Jeffamines™ (Mn = 800 and 2,000 g mol -1 ) and four monodisperse[END_REF] following the diffusion coefficientmolecular weight (Dfm.w.) method described by Grubbs and co-workers. [START_REF] Li | [END_REF] This analysis confirmed the existence of a single main population of polymer chains. Exclusive formation of cyclic polymers (vide infra) was seen for polymerisations performed with equimolar amounts of Ph2SiH2 and H2N^^NH2 (entries 1-2), whereas linear materials were selectively obtained when one of the comonomers was used in excess (entries 3-7). For linear polymers, the Dfm.w. analyses were corroborated by 1 H NMR end-group analysis. In a reasonably good first approximation, the experimental number-average degree of polymerisation (Xn,exp, calculated as the ratio of the experimental molecular weight [START_REF]For linear polycarbosilazanes, the values of estimated molecular weights quoted in the text are the average of the values determined by[END_REF] divided by Flory's mean molecular weight of the Ph2SiH2 and H2N^^NH2 segments [18] ) was commensurate with its theoretical value (Xn,theo) given by Carothers' equation. [18][START_REF]Principles of Polymerization[END_REF] The question of the controlled formation of cyclic or linear polycarbosilazanes with these systems is both fascinating and central. The macromolecules produced by dehydropolymerising equimolar amounts of the two comonomers are exclusively cyclic, as revealed by spectroscopic and mass spectrometric analyses. The 1 H NMR spectrum of P2 (Table 1, entry 2) exhibits diagnostic resonances: a doublet centred on 1H = 4.09 ppm with a relative integration for 4H and belonging to the methylene hydrogens ( 3 JH-H = 8.0 Hz), a triplet at 1H 1.41 ppm ( 3 JH-H = 8.0 Hz) integrating for 2H and corresponding to the NH groups, and a broad resonance at 1H 7.73 ppm for the four o-H of the C6H5 groups (Fig. 1). This assignment was confirmed by 1 H-1 H COSY NMR. No resonance for end-group moieties was detected, consistently with a cyclic structure. [START_REF]With a molecular weight of ca. 7,000 g mol -1 , the end-groups of a linear structure would[END_REF] Full assignment of all resonances in the 1 H and 13 C{ 1 H} NMR spectra was straightforward on the basis of the 1 H-13 C HMBC and HMQC spectra. The resonance for the CH2 methylene carbon atom appears at 13C = 45.80 ppm in the 13 C{ 1 H} NMR spectrum.

The INEPT 29 Si spectrum of the polymer features a unique resonance at 29Si = -24.32 ppm (Fig. 1), confirming the existence of a single type of silicon atoms as expected for a cyclic structure. The assignment of the key 1 H resonances and the absence of Si-H end-group was further corroborated by 29 Si-1 H HMQC NMR data. The presence of -(Ph2SiNH^^NH)-repetitive units (exact mass 316.1395 g mol -1 ) was substantiated by ASAP(+) mass spectrometric analysis of the low molecular weight component, where the main peak was found at 632.2757 g mol -1 (Xn = 4), and another peak was located at 949.4177 g mol -1 (Xn = 6). In the ATR-FTIR spectrum of the polymer, a single, characteristic absorption band of medium intensity was found for the N-H stretching vibration at (N-H) = 3,383 cm -1 ; no band for SiH or NH2 moieties could be detected. The diffusion coefficient (1.456×10 -10 m 2 s -1 ) vs. molecular weight (Mn,DOSY = 7,100 g mol -1 ) analysis for the polymer is displayed in Fig. 2. The spectroscopic features for polymer P1, also a cyclic polymer, were identical to those of P2; only the diffusion coefficient and hence the value of Mn,DOSY for P1 (1.776×10 -10 m 2 s -1 and 5,600 g mol -1 , respectively), differed from those of P2. Details are available in the Supporting Information. ) analysis for P2 (). Calibration curve established by 1 H DOSY NMR (C6D6, 298 K, 400.1 MHz), using four monodisperse PMMA and two Jeffamines™ () as commercial calibrants. [START_REF]The calibration curve for the diffusion coefficientmol. wt. analysis was established using commercial calibrants, two Jeffamines™ (Mn = 800 and 2,000 g mol -1 ) and four monodisperse[END_REF] The 1 H DOSY analysis for P2 itself (insert) shows the existence of a single main population.

Crucially, with excess H2N^^NH2, linear macromolecules having identifiable chain-ends are returned. Polycarbosilazane P4 of estimated molecular weight ca. 6,000 g mol -1 was obtained with 10 mol-% excess of the diamine (entry 4), while a 25 mol-% initial excess of the diamine yielded the shorter polymer P3 (entry 3). In agreement with Carothers' theory for linear polycondensation, Xn,exp decreases as the deviation from equimolar contents of the comonomers increases. The two products only differ by their molecular weights, but their main spectroscopic features (NMR, FTIR) are mostly identical, and only those of the shorter P3 are discussed here. Its 1 H NMR spectrum is displayed in Figure 3. In addition to the same resonances as those observed for P2, it also contains a quadruplet centred on 1H = 3.59 ppm ( 3 JH-H = 8.0 Hz) and a broad resonance at 1H = 0.76 ppm, which are scalarly coupled to each other according to 1 H-1 H COSY NMR data. Since the INEPT 29 Si spectrum of P3 exhibits the same, sole sharp singlet as that seen for P2 (29Si = -24.32 ppm), these resonances were respectively attributed to terminal H2NCH2-methylene and H2NCH2-amine residues. This assignment is further corroborated by examination of the 13 C{ 1 H} (Fig. 3) and 1 H- 13 C HMQC NMR spectra, the aliphatic region of which contains only two resonances, a main one at 13C = 45.79 ppm (the same as for P2) and a less intense one at 13C 46.58 ppm, assigned to internal (backbone) -HNCH2-and terminal H2NCH2-groups, respectively. The 29 Si-1 H HMQC NMR spectrum indicated that the resonances at 1H = 3.59 and 0.76 ppm are not coupled to any silicon atom. In the FTIR spectrum of P3 (in Nujol), the two absorption bands at  = 3,400 (m) and 3,308 (m) cm -1 were assigned to the stretching modes of N-H bonds in internal -NH-and terminal -NH2 moieties, respectively. These spectroscopic data are congruent with a linear structure such as that depicted in Fig. 3, capped by -NHCH2C6H4CH2NH2 at each chain-end. When Ph2SiH2 is used in excess compared to H2N^^NH2, the dehydropolymerisations catalysed by A also produce linear polycarbosilazanes (Table 1, entries 5-7). With 10% excess, polymer P6 of molecular weight ca. 4,500 g mol -1 , corresponding to Xn,exp = 28, was obtained (entry 6). With a larger excess (25%), the shorter P5 was synthesised, with Xn,exp = 9 (entry 5). Polycarbosilazane P7 of comparatively high molecular weight (ca. 9,800 g mol -1 ) was produced when the excess in Ph2SiH2 was reduced to 5% (entry 7). Hence, here again, the observed molecular weight decreases rapidly as the deviation from the 1:1 stoichiometry becomes greater. The spectroscopic features of these three polymers are almost identical. The 1 H and 29 Si NMR spectra are complicated, and show several new resonances compared to the cyclic P2. This is likely due to coupling between reactive NH groups within the polymer chain and the excess Ph2SiH2, leading to the formation of dangling N-SiPh2H silazanes and perhaps even N2Si2 cyclodisilazane (the formation of which can be catalysed by A in coupling reactions between dihydrosilanes, e.g. Ph2SiH2, and primary amines). [14] Besides, two types of end-groups are also to be expected: -NHSiPh2H and -N(SiPh2H)2. These different silicon environments to be found in P5-P7 are depicted in Figure 4. [START_REF]dangling groups undistinguishable from the main chain end-groups[END_REF] The main in the 1 H ( 4.09 and 1.42 ppm), 13 C{ 1 H} ( 45.79 ppm) and 29 Si{ 1 H} ( -24.32 ppm) NMR spectra of P5-P7 match those for P2 and P3, confirming the preponderance of regular repetitive units in the polymer backbone (Fig. 4). The data for P6 are discussed in the followings. In the 1 H NMR spectrum there are other identifiable minor resonances: singlets at 1H = 5.75 and 5.74 (SiH atoms of disilazane endgroups), 5.65 (for the SiH atom of monosilazane end-groups) and 4.38 (CH2N atoms of disilazane end-groups) ppm, a doublet at 3.91 ppm (CH2N atoms of monosilazane end-groups), and a multiplet at 1.04 ppm (NH atom of monosilazane end-groups). The ratio of monosilazane to disilazane end-groups is ca. 3/2. In the 29 Si{ 1 H} spectrum, minor resonances at 29Si = -12.92 and -17.10, -24.51 and -29.47 ppm are also visible. [START_REF]A minor resonance at 29Si = -7.43 ppm[END_REF] They were assigned to the different end-groups and irregular backbone units on the basis of the 1 H, 29 Si and HMQC 1 H-29 Si NMR data (Fig. 4). The INEPT 29 Si spectrum enhanced the intensities of specific resonances and showed the presence of an otherwise hardly undetectable singlet at 29Si = -11.82 ppm. Previous knowledge [14] allowed us to assign it to minute amounts of cyclodisilazane units; the pertaining resonances for Hg and Cg in the 1 H and 13 C{ 1 H} spectra were too small to be detected. In the aliphatic region, the 13 C{ 1 H} spectra contain the expected main resonance at 13C = 45.79 ppm, and minor ones at 13C = 50. 45, 46.97, 45.71 and 45.37 ppm; the assignments in Fig. 4 were achieved by combining 13 C{ 1 H} and 13 C-1 H HMBC and HMQC NMR data. This analysis suggests a complicated microstructure, where the backbone consists mostly of regular motives with minor inclusions of core/tethered motives and hardly detectable cyclosilazane units, and where the end-groups are distributed between mono-and disilazanes moieties. The identification of the chain-ends is eased by FTIR analyses. The ATR-FTIR spectrum of P6 features two key absorption bands of medium intensities at 3,393 and 2,112 cm -1 , the latter being diagnostic of hydrosilanes. They were assigned to the stretching vibration modes of backbone N-H and chain end Si-H bonds, respectively. No band was detected around 3,300 cm -1 , testifying to the absence of NH2 end-groups as expected from the use of excess dihydrosilane.

Such dehydropolymerisations catalysed by A without doubt follow the mechanistic pathway detailed previously for the bariumcatalysed cross-dehydrocoupling of amines and hydrosilanes. [14] Starting from the barium bis(alkyl) A, the key successive steps involve: (i) the formation of the competent barium amide species by protonolysis with H2N^^NH2, (ii) nucleophilic attack of the Namide atom onto the incoming hydrosilane, thus generating a hypervalent silicate, and (iii) a rate-limiting -H transfer to barium with concomitant release of the silazane, producing a Ba hydride species which will then react with another amine to regenerate the catalytically active Ba amide compound.

The Ba-mediated dehydropolymerisation of diamines and dihydrosilanes introduced here is a clean, fast and versatile route to bespoke polycarbosilazanes. Fundamentally, the cyclic or linear microstructures (and, for linear polymers, end-group identity) can be tuned by adjusting the initial comonomer ratio. We are endeavouring to improve productivity figures, and so far we are able to obtain cyclic polymers by fully converting 500 equiv of each comonomers (500:500:1) within 2 h at 60 °C (Mn,DOSY = 6,000 g mol -1 ). This methodology can a priori be extended to other (co)monomers, for instance aryl-functionalised dihydrosilanes, di(hydrosilane)s, or other diamines (e.g. 1,2-ethylenediamine, piperazine) including functionalised ones. We are for now probing the use of phenylsilane to obtain higher molecular weight and/or reticulated polymers (early attempts seem successful, as insoluble materials have been obtained; this methodology now requires optimisation), N,N'-dimethyl-p-xylylenediamine (to prevent the potential formation of backbone cyclodisilazane), and H2NCH2C6H4CH2NHSiPh2H, a self-sufficient ,-bifunctional monomer, to synthesise polycarbosilazanes with new properties.

Keywords: cross-dehydrogenative coupling • polycarbosilazane • barium catalyst • diamine • dihydrosilane

Figure 1 .

 1 Figure 1. 1 H (C6D6, 298 K, 400.1 MHz) and 29 Si (C6D6, 298 K, 79.5 MHz) NMR spectra of the cyclic polycarbosilazanes P2.

Figure 2 .

 2 Figure2. Molecular weight determination by coefficient) vs. log (mol. wt.) analysis for P2 (). Calibration curve established by 1 H DOSY NMR (C6D6, 298 K, 400.1 MHz), using four monodisperse PMMA and two Jeffamines™ () as commercial calibrants.[START_REF]The calibration curve for the diffusion coefficientmol. wt. analysis was established using commercial calibrants, two Jeffamines™ (Mn = 800 and 2,000 g mol -1 ) and four monodisperse[END_REF] The 1 H DOSY analysis for P2 itself (insert) shows the existence of a single main population.

Figure 3 .

 3 Figure 3. 1 H (C6D6, 298 K, 400.1 MHz) and aliphatic region of the 13 C{ 1 H} (C6D6, 298 K, 100.1 MHz) NMR spectra of the linear polycarbosilazane P3.

Figure 4 .

 4 Figure 4. Possible silicon environments, in the polymer backbone and as endgroups, in P5-P7 prepared in barium-promoted dehydropolymerisations with excess Ph2SiH2, with the assignment of the 1 H and 29 Si NMR chemical shifts.

COMMUNICATION

Entry for the Table of Contents

COMMUNICATION

The rapid and controlled bariumcatalysed dehydrogenative polymerisation of a diamine with diphenylsilane affords thoroughly characterised polycarbosilazanes with microstructures (cyclic vs. linear), end-groups and molecular weights that can be tailored by selection of the comonomers feed ratio.


Célia Fraga Malfatti

Jane Zoppas Ferreira

Claudia Trindade Oliveira

Esther S Rieder

Jean-Pierre Bonino

Electrochemical behavior of Ni-P-SiC composite coatings: effect of heat treatment and SiC particle incorporation

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  : Fraga Malfatti, Célia and Ferreira, J. Z. and Oliveira, C. T. and Rieder, E. S. and Bonino, Jean-Pierre. Electrochemical behavior of Ni-P-SiC composite coatings: effect of heat treatment and SiC particle incorporation. (2010). Materials and Corrosion, vol. 63 (n° 1). pp. 36-43. ISSN 0947-5117 Open Archive TOULOUSE Archive Ouverte (OATAO) OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible. This is an author-deposited version published in : http://oatao.univ-toulouse.fr/ Eprints ID : 8702 To link to this article : DOI:10.1002/maco.200905611 URL : http://dx.doi.org/10.1002/maco.200905611