# Single- versus multiple-sampling strategy for blood cultures in the diagnosis of infective endocarditis: the prospective multicenter UniEndo study

François Goehringer, Marc Soudant, Corentine Alauzet, Christine Selton-Suty, Nelly Agrinier, Jean-Marc Virion, Benjamin Lefevre, Nejla Aissa, François Alla, Yvon Ruch, et al.

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François Goehringer, Marc Soudant, Corentine Alauzet, Christine Selton-Suty, Nelly Agrinier, et al. Single- versus multiple-sampling strategy for blood cultures in the diagnosis of infective endocarditis: the prospective multicenter UniEndo study. Clinical Infectious Diseases, 2025, pp.ciaf163. 10.1093/cid/ciaf163. hal-05033711

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1 Single- versus multiple-sampling strategy for blood cultures in the diagnosis of infective

2 endocarditis: the prospective multicenter UniEndo study

3

4 François Goehringer¹, Marc Soudant², Corentine Alauzet³, Christine Selton-Suty⁴, Nelly

5 Agrinier², Jean-Marc Virion², Benjamin Lefevre¹, Nejla Aissa⁵, François Alla⁶, Yvon Ruch⁷,

6 Yohan N'Guyen⁸, Lionel Piroth⁹, Kevin Bouiller¹⁰, Pierre-Yves Royer¹¹, Vincent Le Moing¹²,

7 Bruno Hoen * 13, 14, Xavier Duval * 15, 16 and the UniEndo-AEPEI Study group.

8

9 ¹ Service de maladies infectieuses et tropicales, CHRU Nancy, F-54000, Nancy, France.

10 ² CHRU Nancy, Université de Lorraine, Inserm, CIC, Epidémiologie clinique, F-54000,

11 Nancy, France.

12 ³ Service de microbiologie, CHRU de Nancy, UR SIMPA, Université de Lorraine, F-54000,

13 Nancy, France.

14 ⁴ Département de cardiologie médicale, CHRU-Nancy, CIC-EC, F-54000 Nancy, France.

15 ⁵ Laboratoire de microbiologie, CHU de Guadeloupe, Pointe-à-Pitre, Guadeloupe, France.

16 ⁶ Service de prévention, CHU de Bordeaux, F-33000, Bordeaux, France.

17 ⁷ Service de maladies infectieuses et tropicales, Nouvel Hôpital Civil, CHRU Strasbourg, F-

18 67000, Strasbourg, France.

19 ⁸ Consultations médecine interne, maladies infectieuses et immunologie clinique, Hôpital

20 Robert Debré CHU Reims, Avenue Général Koenig, 51100 Reims, France.
21 ⁹ CHU Dijon-Bourgogne, INSERM, Université de Bourgogne, CIC 1432, Module
22 Epidémiologie Clinique, F-21000, Dijon, France.

23 ¹⁰ Université de Franche-Comté, CHU Besançon, UMR-CNRS 6249 Chrono-environnement,
24 Department of infectious and tropical diseases, F-25000, Besançon, France.

25 ¹¹ Service de maladies infectieuses et tropicales, Hôpital Nord Franche-Comté, 90400,
26 Trévenans, France.

27 ¹² Service des maladies infectieuses et tropicales, CHU de Montpellier, F-34000, Montpellier,
28 France.

29 ¹³ Université de Lorraine, Inserm, INSPIIRE, F-54000, Nancy, France.

30 ¹⁴ CHU de Besançon, Service de Maladies Infectieuses et Tropicales, F-25000 Besançon,
31 France.

32 ¹⁵ Université Paris Cité and Université Sorbonne Paris Nord, Inserm 1137, IAME, F-75018,
33 Paris, France.

34 ¹⁶ Inserm CIC 1425, Centre d'investigation clinique, AP-HP, Hôpital Bichat, F-75018, Paris,
35 France.

36 * these two authors contributed equally to the study

37
38 **Short Title:** Single sampling for blood cultures in IE

39
40
41

**Keywords:** Infective endocarditis, blood culture, diagnostic strategy, blood sampling
42 **Summary:** A single sampling strategy for performing blood cultures is at least as effective as

43 the conventional multiple sampling strategy in diagnosing and classifying infective

44 endocarditis

45

46

47 *Corresponding author:*

48 François Goehringer, MD,

49 Centre Hospitalier Régional Universitaire de Nancy,

50 Service de Maladies Infectieuses et Tropicales,

51 Rue du Morvan

52 54511 Vandœuvre-Lès-Nancy Cedex, France

53 f.goehringer@chru-nancy.fr: +33 6 6229 5723

54

55 Alternate corresponding author

56 Pr Xavier Duval

57 xavier.duval@aphp.fr
58 **Abstract**

59 **Background:** Single-sampling strategy (SSS) for blood cultures (BC) has not been evaluated
60 in infective endocarditis (IE). We assessed the diagnostic performance of SSS vs conventional
61 multi-sampling strategy (MSS) in IE diagnosis.

62 **Methods:** Patients suspected of IE were prospectively enrolled in 8 tertiary-care centers. Five
63 BC bottle pairs were sampled from each patient. Pairs 1, 2 and 3 were sampled
64 simultaneously, followed by 2 additional separate pairs (4 and 5) sampled more than one hour
65 later. Pairs 1, 2, and 3 emulated SSS and pairs 1, 4, and 5 emulated MSS. The sensitivity and
66 specificity of the major microbiologic criterion of the 2015-ESC IE diagnostic criteria, based
67 on the SSS and MSS BC results, were calculated using the endocarditis team's diagnosis as
68 the gold standard.

69 **Results:** An IE was diagnosed in 101 (39.4%) of the 256 patients enrolled. Sensitivity rates of
70 SSS and MSS were 50.5% (95%CI [40.7–60.2]) and 45.5% (95%CI [35.8–55.3]),
71 respectively (p=0.063), while specificity rates were 94.8% (95%CI [91.4–98.3]) and 95.5%
72 (95%CI [92.2–98.8]), respectively (p=1). In IE patients, SSS as compared to MSS accurately
73 upgraded the diagnosis from possible to definite IE in one patient and downgraded it in none.

74 **Conclusion:** Using SSS to define the major microbiologic criterion was as sensitive and
75 specific as using MSS for diagnosing IE. Using SSS instead of MSS BC results did not lead to
76 erroneous changes in diagnostic class according to the 2015 ESC criteria. Consequently, SSS
77 may be regarded as standard practice for IE diagnosis.
78 **Introduction**

Along with echocardiography and other cardiac imaging techniques, blood cultures (BC) are
key to the diagnosis of infective endocarditis (IE). Unlike other bloodstream infections, where
bacteremia is usually intermittent, bacteremia is deemed to be permanent in IE, because of the
intravascular location of the infection site. This is accounted for in IE diagnostic criteria,
where a sustained bacteremia, confirmed by the positivity of several BCs drawn apart, is
required to qualify as a major microbiological criterion [1-5].

85

As the diagnostic yield of BC is strongly correlated with the volume of blood collected and
cultured [6,7], it is key to collect a sufficient volume of blood to optimize the chance of
identifying the causative microorganism. It is also crucial to minimize the risk of sample
contamination. For these reasons, it has been advocated that a single sampling strategy (SSS),
in which the entire desired volume of blood is obtained from a single venipuncture should
replace the conventional multi-sampling strategy (MSS) [8-10]. Apart from IE, several
studies showed that SSS is non-inferior or even superior to MSS for the diagnosis of
bacteremia in various clinical situations, including patients admitted to an emergency
department and patients with sepsis [11-13].

85
86
87
88
89
90
91
92
93

Based on these considerations, according to the 2023 Duke-ISCVID criteria, while best
practice recommendations endorse separate venipuncture for each blood culture whenever
possible, it is no longer required by the Duke Criteria to qualify positive BCs as a major
microbiological criterion [14]. Of note, the result of several BCs drawn apart is still required
in the 2023 ESC classification [15]. To our knowledge, the effectiveness of SSS in the
microbiologic diagnosis of IE has never been evaluated or compared with that of MSS.
94
95
96
97
98
99

The aim of this study was to compare SSS and MSS in the clinical diagnosis and diagnostic
classification of IE. The primary objective was to assess the sensitivity and specificity of the
100
101
major microbiologic criterion of IE diagnosis classifications depending on whether the results
of either SSS or MSS BCs were considered. We also wanted to evaluate the potential impact
of SSS on case categorization of IE as definite, possible, or rejected according to the 2015
ESC criteria.

106

## 107 Patients and methods

Patients were prospectively and consecutively enrolled in this multicenter, cross-sectional
single-gate study. They were recruited in 8 tertiary-care hospitals from November 2017 to
May 2022 and underwent both strategies, SSS being assessed against MSS as the reference
standard method.

112

## 113 Participants

Eligible patients were adults hospitalized and evaluated for a clinical suspicion of IE. To
maximize the proportion of patients with an IE, patients had to have either evidence of
endocardial involvement (presence of the major imaging criterion), or at least two minor non-
microbiologic criteria, based on the 2015 ESC classification [5]. Patients with prior positive
BC or who already received antibiotic treatment for more than 24h over the prior 7 days by
the time of screening were not eligible.

120

## 121 Procedures

Each BC consisted of a pair of aerobic and anaerobic bottles sampled consecutively. For each
patient, five pairs of BC bottles were sampled: pairs 1, 2, and 3 were sampled at the same time
from the same venipuncture site, followed by 2 additional pairs (4 and 5) sampled more than
one hour later. As a result, the two sampling strategies were performed and could be
compared in all patients, each patient serving as their own control. BC bottles were labelled 1
through 5. Pairs 1, 2, and 3 emulated SSS and pairs 1, 4, and 5 emulated MSS (Figure 1).

The exact timing of sampling was recorded for each bottle pair, as was the duration of each
blood sampling procedure. Each bottle was to be filled with 8-10 mL of blood and the volume
of blood was recorded as well. Blood volume was estimated either by visual comparison with
an abacus or automatically when the BC incubator made it possible. BC bottles were
transported at room temperature to the local hospital microbiology laboratory where they
were incubated according to each laboratory procedures. Times from sampling to incubation
and from incubation to bacterial detection were both recorded.

BCs were incubated at 36°C until they signaled positive or for up to 10 days. For positive
BCs, Gram staining, subcultures, and species identification as well as antibiotic susceptibility
testing were performed in each center as usual (see Table S1 in appendix for the analytical
methods of the 8 centers).

139

## Patients' data collection

For each patient, an e-CRF with all clinical, laboratory, microbiological, and imaging data
required for applying the 2015 ESC criteria was filled out.

143

## Interpretation of blood culture results

All BC results, whether they were clinically relevant or contaminants, were included in the
analysis with application of the 2015 ESC criteria.

146
147 For MSS, according to 2015 ESC criteria (Table S2), the BC result met the conditions for a
148 major microbiological criterion if either at least two distinct pairs among those labeled 1, 4,
149 and 5 had at least one positive bottle for a microorganism typical of IE, or all three pairs
150 labeled 1, 4, and 5 had at least one positive bottle for a microorganism consistent with IE.

151 For SSS, the same algorithm as above was used, based on the results of the bottle pairs 1, 2,
152 and 3.

153 Detected isolates were defined as either clinically relevant growth or contaminant growth,
154 according to the guidelines of the French Society of Microbiology [16]. Relevant bacterial
155 growth was defined as growth of pathogenic microorganisms in at least one BC bottle. For
156 MSS, contaminant growth was defined as one or both BC bottles of the same pair positive for
157 coagulase-negative staphylococci, *Cutibacterium acnes*, α-hemolytic streptococci or *Bacillus*
158 spp, with no other microbiological sample (other protocol and off-protocol BCs, valve sample
159 etc...) positive for the same microorganism. For SSS, contaminant growth was defined as only
160 one BC bottle of the 6 bottles positive for one of the same above-mentioned microorganisms,
161 in the absence of isolation of this microorganism in any other microbiological sample.

162 The BC results were considered discordant between SSS and MSS if either the number of
163 positive bottles was different and led to a difference in interpretation of the microbiological
164 criterion (no, minor, or major criterion), or if the isolated microorganisms were different.

165

166 Gold standard

167 The gold standard was the discharge diagnosis, established binarily (IE vs no IE) by each
168 endocarditis team, consisting of at least an infectious disease specialist, a cardiologist and a
169 clinical microbiologist. The endocarditis team considered all the microbiological results,
170 including those obtained after the completion of the BCs required by the protocol.
171

172 Statistical analysis

173 Patient characteristics were summarized using descriptive statistics. Continuous variables
174 were presented with their median and interquartile range (IQR); categorical variables were
175 presented using numbers and percentages. Exploratory comparisons between SSS and MSS
176 were performed using the McNemar test for binary variables and the paired t-test for
177 continuous variables.

178 For the primary analysis, the sensitivity was computed as the number of patients who fulfilled
179 the definition of a major microbiological criteria for SSS and MSS as defined above, divided
180 by the number patients with IE by gold standard. The specificity was computed as the number
181 of patients who did not fulfill the definition of a major microbiological criteria for SSS and
182 MSS as defined above, divided by the number patients with no IE by gold standard. 95%
183 confidence intervals were calculated using either normal approximation when applicability
184 was verified or Wilson score method otherwise [17]. McNemar and prevalence-adjusted bias-
185 adjusted kappa (PABAK) tests were used to compare sensitivities and specificities and to
186 estimate SSS MSS reliability [18].

187 We also determined the number of patients for whom the 2015 ESC criteria diagnostic
188 assignations (definite /possible /rejected) were discordant depending on whether the results of
189 SSS or MSS were used.

190 All statistical analyses were performed using SAS/STAT 9.04.01 and AdClin Table
191 Production Framework version 3.5.

192

193 Sample size and power
Persistently positive BCs are known to be 62% sensitive and 96% specific for diagnosing definite IE [2]. With an expected 20% rate of IE in our study sample [19], we calculated that enrolling 100 cases of IE would be associated with a precision of the 95% confidence interval of the sensitivity and the specificity of +/-9.4% and +/-2.0% respectively. As a result, we planned to enroll 500 patients and stop enrollment once 100 patients were diagnosed with IE.

## *Good clinical practice and ethics*

This study complies with the principles outlined in the Declaration of Helsinki, was registered with Clinicaltrials.gov (NCT03153384), and was authorized by the French Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé (CCTIRS, acceptance number N° 16-813) and by the ethics committee of the Nancy University Hospital (acceptance number N° 370). All patients were informed of their participation in the study, and their written consent was not required under current French law.

## **Results**

### *Patients' characteristics*

Between November 2017 and May 2022, 269 patients were enrolled. Among these, 13 were secondarily excluded (Figure 2).

Characteristics of the remaining 256 patients are displayed in Table 1. Median age was 70 years, 71.5% were males, 125 (48.8%) had a prosthetic valve, 72 (28.1%) had a known native valve disease, 49 (19.1%) a prior history of IE and 46 (18.0%) a cardiac implantable electronic device (CIED). At the time of enrollment, 149 (58.2%) patients presented with
217 fever, 69 (27.0%) with cardiac failure, 41 (16.0%) with impaired general condition, 28

218 (10.9%) with an embolic event, and 58 (22.7%) had antibiotics started for less than 24 hours

219 prior to enrolment in the study. All patients underwent TTE and/or TEE, 117 (45.7%) a

220 cardiac CT, and 110 (42.9%) a [18F]-FDG-PET/CT. When combining the results of all

221 cardiac imaging modalities, 124 (48.4%) patients had a major imaging criterion.

222 By gold standard, IE was diagnosed in 101 (39.5%) patients. The discharge diagnoses of the

223 155 non-IE patients are displayed in Table 1.

224

225 Blood culture sampling

226 The sampled blood volume was adequate for all bottles (above 8 mL per bottle) in 195

227 (76.2%) and 182 (71.1%) of patients by SSS and MSS strategies, respectively (p= 0.003)

228 (Table 2). For MSS, the required minimum time interval of 1 hour between the first and the

229 last sampling was met in all cases. In terms of nurse workload per patient, the median time

230 spent on BC sampling was shorter for SSS (10.0 min IQR [5.0 - 15.0]) than for MSS (16.5

231 min, IQR [7.5 - 30.0], p<0.001). The cumulative time spent performing BCs for the 184

232 patients for whom information was available was 35.8 hours for SSS and 66.3 hours for MSS.

233

234 Blood culture results

235 Of the total 1536 bottles obtained by either strategy, 341 (22.2%) and 307 (20.0%) were

236 positive by SSS and MSS, respectively. When excluding the first pair, which was common to

237 both strategies, the number of patients with at least one positive BC was 67 (26.2%) for SSS

238 and 63 (24.6%) for MSS (p=0.28) (Table 2). Among the 101 IE patients, these figures were

239 56 (55.4%) and 51 (50.5%) (p=0.03) (Table 3); the microorganisms most frequently isolated
240 from BCs were distributed as follows: 22 (21.8%) streptococci, 19 (18.8%) staphylococci, and
241 13 (12.9%) enterococci (Table 3).

242

243 *Sensitivity and specificity of each sampling strategy*

244 The number of patients with a major microbiological criterion was 59 (23.0%) and 53
245 (20.7%) in SSS and MSS respectively.

246 The major microbiological criterion had a sensitivity of 50.5% (95%CI [40.7–60.2]) and
247 45.5% (95%CI [35.8–55.3]) using SSS and MSS respectively (p=0.063; PABAK=0.90
248 (95%CI [0.82–0.99))); the corresponding specificities were 94.8% (95%CI [91.4–98.3]) and
249 95.5% (95%CI [92.2–98.8]) (p=1; PABAK=0.99 (95%CI [0.96–1.0]), Figure 3).

250

251 *Patients with discordant BC results between SSS and MSS*

252 Fourteen (5.5%) patients had discordant BC results between SSS and MSS, 7 of whom had an
253 IE and 7 had not (Table 4).

254 In the 7 patients with IE, all had positive BC bottles in SSS (5 interpreted as major criterion
255 and 2 as minor), while 5 had positive BC bottles in MSS, all of them interpreted as minor
256 criteria. For one patient, the microorganism detected in BCs was discordant: *Streptococcus*
257 *gallolyticus* in SSS and *Escherichia coli* in MSS.

258 In the 7 patients without IE, 2 had positive BC bottles with SSS, one interpreted as a major
259 microbiological criterion and one as a minor criterion. Conversely, with MSS, 6 had positive
260 BC bottles, all interpreted as a minor microbiological criterion.
261

Finally, among these 14 patients, these discrepancies led to a difference in classification

262

category according to the 2015 ESC classification for only one patient, who was adequately

263

upgraded from possible IE using MSS BC results to definite IE using SSS BC results (Table

264

4). The classification was downgraded for no patient.

265

266

Discussion

267

To our knowledge, this study is the first to address the diagnostic yield of SSS BCs in patients

268

with a suspicion of IE. First, we demonstrated that the sensitivity and the specificity of the

269

major microbiologic criterion of IE diagnosis classification were maintained when the results

270

of BCs were those of SSS compared to conventional MSS. Second, the use of SSS BC results

271

instead of MSS BC results did not lead to erroneous changes in diagnostic class according to

272

the 2015 ESC criteria.

273

There is no unique clinical pattern for a patient with suspicion of IE. We defined our inclusion

274

criteria with the aim of obtaining a sufficient number of patients with definite IE to properly

275

assess the diagnostic performance of the two sampling strategies. As a result, 101 (39.5%) of

276

the enrolled patients did have an IE. This reflects the high likelihood of IE within the study

277

population, which is greater than that observed in all-comers undergoing BCs [12,19]. This

278

also explains the high rate of IE on intracardiac devices. Moreover, for methodological

279

reasons, patients who were already known to have positive BCs by the time of screening were

280

excluded from the study, despite this being a reasonable indication for suspecting IE. This

281

exclusion criterion may have resulted in an under-representation of patients with bloodstream

282

infections caused by a rapidly growing microorganism, such as *Staphylococcus aureus*. This

283

may partly explain why the proportion of patients with *S. aureus* IE (18.8%) among those

284

with IE was lower in our study than in recent large longitudinal series of IE [20,21].
The proportion of positive bottles was greater with SSS than with MSS. This may be explained by a higher rate of optimal bottle filling when BCs were taken in SSS versus MSS, resulting in a higher bacterial inoculum, which has been previously reported [8]. Of note, suboptimal bottle filling may have resulted in undercapture of some IE cases. In some patients, this resulted in discrepancies between SSS and MSS results, both in patients with and without IE. Improved rates of optimal bottle filling for samples obtained with the SSS strategy likely improved both the microbiologic yield and the ability to diagnose IE.

Moreover, despite the increased sensitivity of SSS, the number of false-positive BCs was lower when SSS was used instead of MSS. This was possibly due to the contamination of a small number of samples.

A further advantage of SSS BCs could be the reduced risk of contamination of BCs by skin microorganisms [11,12]. This was not observed in our study, with a similar number of contaminated bottles with each sampling strategy (n = 3), a figure that is considerably lower than that typically reported in the literature [11,12,22]. This might have resulted from the greater attention paid to aseptic procedures in this experimental study [23]. Furthermore, as anticipated, the median nursing time allocated to BC collection was reduced by 39% with SSS in comparison to MSS. This equates to a total of 30.4 hours saved for the 184/256 patients for whom the data on sampling times were available.

In the group of patients with IE, 56/101 (55.4%) exhibited positive BC when the results of the five pairs of bottles drawn in accordance with the study protocol were pooled (Table 3). This figure may appear relatively low in comparison to the 80% rate of microbiological documentation reported in contemporary observational studies on IE [20,21]. However, it should be noted that our data set analysis disregarded all other microbiological results, including any BCs taken prior to or subsequent to the five protocol-required BC pairs, as well as any serological or molecular biology results, such as those obtained through PCR. A
microbiological documentation was obtained in 72.3% of cases when all microbiological
results collected throughout the patients' hospital stay were considered (Table S3). Moreover,
it is noteworthy that 22.7% of the patients enrolled had received antibiotics for less than 24
hours in the preceding seven days, as permitted by the study protocol. This may have lowered
the yield of BCs.

We acknowledge some limitations to our study. Firstly, it should be remembered that our
study sample may not be representative of the full range of patients in whom blood cultures
are ordered for suspected IE. Secondly, in addition to BCs, any further diagnostic
investigations, whether laboratory-based or imaging-based, were conducted at the discretion
of the attending physicians, based on their diagnostic assumptions. Consequently, not all
patients underwent all the investigations that can be performed in the workup of patients
suspected of having IE, including TEE, cardiac CT or PET scans. This could have resulted in
missing the diagnosis of IE in some patients. Finally, using the 2023 ESC criteria instead of
the 2015 ESC criteria for the diagnosis of IE as a reference would not have affected our
results, as the two classifications were almost identical in terms of microbiological criteria.

In conclusion, although our results should be replicated through additional studies, the use of
SSS BC results for the diagnosis of IE may be a reliable and secure approach, as proposed by
the authors of the 2023 Duke-ISCVID criteria. Moreover, the reduced nursing time and the
expected improved patients' comfort provided by SSS could lead to the recommendation of
this strategy as the new standard for routine clinical use in the diagnosis of IE, especially in
patients with high likelihood of having an IE.
331 Acknowledgments

332

333 Author contributions

334 FG, XD, BH and FA contributed to the design of the study. FG, XD, BH, CSS, CA and BL
335 contributed to the analysis of the results and to the writing of the manuscript. MS and JMV
336 developed the database and performed the statistical analyses. NA and FA supervised the
337 methodological process. All authors contributed to the acquisition of the data and provided
338 critical feedback on the manuscript.

339

340 UniEndo-AEPEI study group:

341 **Scientific Committee:** Nelly Agrinier, Corentine Alauzet, Nejla Aissa, François Alla, Xavier
342 Duval, François Goehringer, Bruno Hoen, Benjamin Lefevre, Christine Selton-Suty

343 **Methodology and statistics group:** Nelly Agrinier, Marie-Line Erpelding, Nadine Juge,
344 Marc Soudant, Jean-Marc Virion.

345 **Investigators:** Nejla Aissa, Corentine Alauzet, Antoine Fraix, Marine Frayssinoux, Francois
346 Goehringer, Olivier Huttin, Benjamin Lefevre, Nathalie Pace, Firouzé Bani-Sadr, Maxime
347 Hentzien, Anne Limelette, Pierre Nazeyrollas, Yohan N'guyen, Véronique Vernet-Garnier,
348 Louis Bohard, Kévin Bouiller, Catherine Chirouze, Damien Fournier, Basile Mouhat, Noémie
349 Tissot, Xavier Argemi, Pierre Boyer, Angéline Chabaud, François Danion, Nawal Douiri,
350 Antoine Grillon, Nicolas Lefebvre, Aurélie Martin, Yvon Ruch, Emilie Talagrand-Reboul,
351 Axel Ursenbach, Julien Bador, Lionel Piroth, Pauline Garnier, Vincent Gendrin, Timothée
352 Klopfenstein, Julie Plantin, Pierre-Yves Royer, Lynda Toko, Jean-Baptiste Vuillemenot,
353 Souheil Zayet, François Bénézit, Loren Dejoies, Pierre Tattevin, Laurene Deconinck, Xavier
354 Duval, Nathalie Grall, Emilia Ilic-Habensus, Bernard Iung
355 Centers' Study nurses and technicians: Catherine Campagnac, Sandrine Gerset, Isabelle

356 Petitgenet, Tubanur Unal, Morgane Cangero, Benjamin Cuinet, Magali Rebucci-Peixoto,

357 Céline Thiébaut, Carole Charles, Sandrine Gohier, Charlotte Bourgoin, Lynda Chalal, Sylvie

358 Le Gac

359

360 *Conflict of interest:*

361 VLM disclosed consulting fees, payment for lecture and support for attending meeting from

362 Pfizer laboratory, and payment for lectures from Advanz Pharma laboratory. All the other

363 co-authors had none to declare.

364

365 *Funding/supports:*

366 This work was supported by the French Ministry of Health [GIRCI Est APJ 2015/N15], the

367 Association pour l'Etude et la Prévention de l'Endocardite Infectieuse, the Association des

368 Chefs de Service du Centre Hospitalier Universitaire de Nancy, and BioMérieux.

369

370 Availability of data and material: the datasets, scripts and additional medical data are

371 available on demand.

372
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443
444 **Figure legends**

445

446 **Figure 1: Schematic representation of the experimental protocol**

447

448 Note: MSS, multiple-sampling strategy; SSS, single sampling strategy.

449

450 **Figure 2: Flow-chart of the study**

451

452 Note: BC, blood culture.

453

454 **Figure 3: Compared sensitivities and specificities of the major microbiologic criterion of the**

455 2015 ESC classification for the diagnosis of infective endocarditis, using single- versus

456 multiple-sampling strategy for blood cultures

457

458 Note: MSS, multiple-sampling strategy; SSS, single sampling strategy; TP, true positives;

459 TN, true negatives; PABAK, prevalence-adjusted bias-adjusted kappa test; ESC, European

460 society of Cardiology.

461

462

463
<table><caption>464 Table 1: Patients' characteristics</caption><thead><tr><th></th><th>No IE (N=155)</th><th>IE (N=101)</th><th>Total (N=256)</th></tr><tr><th></th><th>n (%) or<br>Median [IQR]</th><th>n (%) or<br>Median [IQR]</th><th>n (%) or<br>Median [IQR]</th></tr></thead><tbody><tr><td colspan="4"><strong>Patients' characteristics</strong></td></tr><tr><td>Age (years)</td><td>69 [57-78]</td><td>70 [59-78.4]</td><td>70 [58-78.1]</td></tr><tr><td>Male gender</td><td>103 (66.5)</td><td>80 (79.2)</td><td>183 (71.5)</td></tr><tr><td colspan="4"><strong>Cardiac history</strong></td></tr><tr><td>Underlying heart disease</td><td></td><td></td><td></td></tr><tr><td>Native valve heart disease</td><td>40 (25.8)</td><td>32 (31.7)</td><td>72 (28.1)</td></tr><tr><td>Prosthetic valvular material</td><td>69 (44.5)</td><td>56 (55.4)</td><td>125 (48.8)</td></tr><tr><td>Congenital heart disease</td><td>7 (4.5)</td><td>9 (8.9)</td><td>16 (6.3)</td></tr><tr><td>CIED</td><td>17 (11.0)</td><td>29 (28.7)</td><td>46 (18.0)</td></tr><tr><td>Previous IE</td><td>27 (17.4)</td><td>22 (21.8)</td><td>49 (19.1)</td></tr><tr><td colspan="4"><strong>Origin of infection</strong></td></tr><tr><td>Community acquired</td><td>99 (63.9)</td><td>70 (69.3)</td><td>169 (66.0)</td></tr><tr><td>Health care associated IE, hospital-acquired</td><td>13 (8.4)</td><td>11 (10.9)</td><td>24 (9.4)</td></tr><tr><td>Health care associated IE, non-hospital-acquired</td><td>9 (5.8)</td><td>11 (10.9)</td><td>20 (7.8)</td></tr><tr><td>Injection drug use</td><td>14 (9.0)</td><td>7 (6.9)</td><td>21 (8.2)</td></tr><tr><td>Unknown</td><td>20 (12.9)</td><td>2 (2.0)</td><td>22 (8.6)</td></tr><tr><td colspan="4"><strong>Clinical features at time of inclusion</strong></td></tr><tr><td>Impaired general condition*</td><td>18 (11.6)</td><td>23 (22.8)</td><td>41 (16.0)</td></tr><tr><td>Fever</td><td>81 (52.3)</td><td>68 (67.3)</td><td>149 (58.2)</td></tr><tr><td>Heart failure</td><td>38 (24.5)</td><td>31 (30.7)</td><td>69 (27.0)</td></tr><tr><td>Embolic event</td><td>17 (11.0)</td><td>11 (10.9)</td><td>28 (10.9)</td></tr><tr><td>Cerebral embolism</td><td>12 (7.7)</td><td>7 (6.9)</td><td>19 (7.4)</td></tr><tr><td>Visceral embolism</td><td>3 (1.9)</td><td>3 (3.0)</td><td>6 (2.3)</td></tr><tr><td>Unspecified</td><td>2 (1.3)</td><td>1 (1.0)</td><td>3 (1.2)</td></tr><tr><td>Arthralgia/Rachialgia</td><td>20 (12.9)</td><td>8 (7.9)</td><td>28 (10.9)</td></tr><tr><td colspan="4"><strong>Clinical features including those detected during hospitalization</strong></td></tr><tr><td>Cerebral embolism</td><td>16 (10.3)</td><td>23 (22.8)</td><td>39 (15.2)</td></tr><tr><td>Symptomatic</td><td>15 (9.7)</td><td>13 (12.9)</td><td>28 (10.9)</td></tr><tr><td>Cerebral hemorrhage</td><td>2 (1.3)</td><td>3 (3.0)</td><td>5 (2.0)</td></tr><tr><td>Transient ischemic attack</td><td>1 (0.6)</td><td>1 (1.0)</td><td>2 (0.8)</td></tr><tr><td>Visceral embolism</td><td>16 (10.3)</td><td>29 (28.7)</td><td>45 (17.8)</td></tr><tr><td>Limb embolism</td><td>7 (4.5)</td><td>6 (5.9)</td><td>13 (5.1)</td></tr><tr><td>Spondylodiscitis</td><td>10 (6.5)</td><td>6 (5.9)</td><td>16 (6.3)</td></tr><tr><td>Janeway lesions</td><td>0 (0.0)</td><td>3 (3.0)</td><td>3 (3.0)</td></tr><tr><td>Conjunctival hemorrhage</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (1.0)</td></tr><tr><td>Subungual hemorrhage</td><td>0 (0.0)</td><td>2 (2.0)</td><td>2 (2.0)</td></tr><tr><td>Osler's nodes</td><td>1 (0.6)</td><td>1 (1.0)</td><td>2 (0.8)</td></tr><tr><td>Glomerulonephritis</td><td>12 (7.7)</td><td>10 (9.9)</td><td>22 (8.6)</td></tr><tr><td>Splenomegaly</td><td>4 (2.6)</td><td>7 (6.9)</td><td>11 (4.3)</td></tr></tbody></table>
<table><thead><tr><th></th><th>No IE (N=155)</th><th>IE (N=101)</th><th>Total (N=256)</th></tr><tr><th></th><th>n (%) or<br>Median [IQR]</th><th>n (%) or<br>Median [IQR]</th><th>n (%) or<br>Median [IQR]</th></tr></thead><tbody><tr><td colspan="4"><strong>Biological features at inclusion</strong></td></tr><tr><td>White-blood cell count (G/L)</td><td>9.1 [6.6 - 13]</td><td>9.1 [7.3 - 12.9]</td><td>9.1 [7.0 - 12.9]</td></tr><tr><td>Platelets (G/L)</td><td>233 [188 - 327]</td><td>199 [155 - 272]</td><td>221 [172 - 290]</td></tr><tr><td>Positive rheumatoid factors</td><td>18 (11.6)</td><td>20 (19.8)</td><td>38 (14.8)</td></tr><tr><td colspan="4"><strong>Imaging features</strong></td></tr><tr><td>Transthoracic and/or transesophageal echo (n=256)</td><td></td><td></td><td></td></tr><tr><td>At least one lesion suggestive of vegetation</td><td>26 (16.8)</td><td>66 (65.3)</td><td>92 (35.9)</td></tr><tr><td>Intracardiac abscess /aneurysm</td><td>1 (0.6)</td><td>16 (15.8)</td><td>17 (6.6)</td></tr><tr><td>Prosthetic dehiscence</td><td>2 (1.3)</td><td>9 (8.9)</td><td>11 (4.3)</td></tr><tr><td>18-FDG PET CT or WBC SPECT/CT(n=110)</td><td></td><td></td><td></td></tr><tr><td>Prosthetic uptake</td><td>13 (8.4)</td><td>28 (27.7)</td><td>41 (16.0)</td></tr><tr><td>Cardiac CT (n=117)</td><td></td><td></td><td></td></tr><tr><td>Abscess, Pseudo aneurysm</td><td>0 (0.0)</td><td>17 (16.8)</td><td>17 (6.6)</td></tr><tr><td colspan="4"><strong>Management and outcome</strong></td></tr><tr><td>Antibiotics started for less than 24 h within 7 days preceding the protocol</td><td>34 (21.9)</td><td>24 (23.8)</td><td>58 (22.7)</td></tr><tr><td>Antibiotic treatment started as for IE</td><td>42 (27.1)</td><td>98 (97.0)</td><td>140 (54.7)</td></tr><tr><td>Valvular surgery performed</td><td>9 (5.8)</td><td>35 (34.7)</td><td>44 (17.2)</td></tr><tr><td>All cause in-hospital mortality</td><td>6 (3.9)</td><td>21 (20.8)</td><td>27 (10.5)</td></tr><tr><td colspan="4"><strong>Final diagnosis</strong></td></tr><tr><td>IE</td><td>-</td><td>101 (100)</td><td>101 (39.5)</td></tr><tr><td>Pneumonia</td><td>20 (12.9)</td><td>-</td><td>20 (7.8)</td></tr><tr><td>Septic arthritis or spondylodiscitis</td><td>15 (9.7)</td><td>-</td><td>15 (5.9)</td></tr><tr><td>Uncomplicated bacteremia</td><td>13 (8.4)</td><td>-</td><td>13 (5.1)</td></tr><tr><td>Aseptic valvular disease</td><td>12 (7.7)</td><td>-</td><td>12 (4.7)</td></tr><tr><td>Thrombotic disease</td><td>9 (5.8)</td><td>-</td><td>9 (3.5)</td></tr><tr><td>Fibrino-cruoric thrombus</td><td>7 (4.5)</td><td>-</td><td>7 (2.7)</td></tr><tr><td>Septic thrombophlebitis</td><td>2 (1.3)</td><td>-</td><td>2 (0.8)</td></tr><tr><td>Heart failure</td><td>8 (5.2)</td><td>-</td><td>8 (3.1)</td></tr><tr><td>Systemic disease</td><td>8 (5.2)</td><td>-</td><td>8 (3.1)</td></tr><tr><td>Stroke</td><td>7 (4.5)</td><td>-</td><td>7 (2.7)</td></tr><tr><td>Marastic endocarditis</td><td>6 (3.9)</td><td>-</td><td>6 (2.3)</td></tr><tr><td>Prosthetic valve dysfunction</td><td>6 (3.9)</td><td>-</td><td>6 (2.3)</td></tr><tr><td>Pyelonephritis</td><td>4 (2.6)</td><td>-</td><td>4 (1.6)</td></tr><tr><td>Clostridioides difficile colitis</td><td>2 (1.3)</td><td>-</td><td>2 (0.8)</td></tr><tr><td>Other</td><td>21 (13.5)</td><td>-</td><td>21 (8.2)</td></tr><tr><td>None</td><td>24 (15.5)</td><td>-</td><td>24 (9.4)</td></tr></tbody></table>
467 **Note:** IE, infective endocarditis; CIED, cardiac implantable electronic device; 18-FDG PET

468 CT, 18F-fluorodeoxyglucose positron emission tomography-computed tomography; WBC-

469 SPECT/CT, radio-labelled white blood cell single photon emission computed tomography;

470 Cardiac CT, Cardiac computed tomography.
471 **Table 2:** Blood culture characteristics according to multiple or simple sampling strategy in the

472 256 patients

<table><thead><tr><th></th><th>MSS<br>N=256<br>n (%)<br>or Median [IQR]</th><th>SSS<br>N=256<br>n (%)<br>or Median [IQR]</th></tr></thead><tbody><tr><td colspan="3"><strong>Blood culture Sampling informations</strong></td></tr><tr><td>Blood volume above 8 mL blood volume in all bottles*</td><td>182 (71.1)</td><td>195 (76.2)</td></tr><tr><td>Bottle 1</td><td>205</td><td>(80.1)</td></tr><tr><td>Bottle 2</td><td>209</td><td>(81.6)</td></tr><tr><td>Bottle 3</td><td>199 (77.7)</td><td>203 (79.3)</td></tr><tr><td>Bottle 4</td><td>201 (78.5)</td><td>206 (80.5)</td></tr><tr><td>Bottle 5</td><td>206 (80.5)</td><td>206 (80.5)</td></tr><tr><td>Bottle 6</td><td>208 (81.3)</td><td>205 (80.1)</td></tr><tr><td>Median time between sampling and bottle incubation (h)</td><td>2.5 [1.3 - 4.4]</td><td>2.5 [1.2 - 4.4]</td></tr><tr><td>Median time between first and last sampling (h)</td><td>2.3 [1.3 - 4.4]</td><td>NA</td></tr><tr><td>Median duration of sampling nursing care (min)<sup>π</sup></td><td>16.5 [7.5 - 30.0]</td><td>10.0 [5.0 - 15.0]</td></tr><tr><td colspan="3"><strong>Blood culture results</strong></td></tr><tr><td>Median time to positivity (h)</td><td>15.1 [10.5 - 24.1]</td><td>15.1 [10.9 - 24.1]</td></tr><tr><td>Number of patients with at least 1 positive bottle</td><td>71 (27.7)</td><td>69 (27.0)</td></tr><tr><td>Number of positive bottle per patient</td><td></td><td></td></tr><tr><td>1 bottle</td><td>13 (5.1)</td><td>7 (2.7)</td></tr><tr><td>2 bottles</td><td>5 (2.0)</td><td>4 (1.6)</td></tr><tr><td>3 bottles</td><td>5 (2.0)</td><td>6 (2.3)</td></tr><tr><td>4 bottles</td><td>8 (3.1)</td><td>0 (0.0)</td></tr><tr><td>5 bottles</td><td>3 (1.2)</td><td>4 (1.6)</td></tr><tr><td>6 bottles</td><td>37 (14.5)</td><td>48 (18.8)</td></tr><tr><td>At least 1 positive among bottles common to both strategies (#1,2)</td><td>62</td><td>(24.2)</td></tr><tr><td>At least 1 positive among strategy-specific bottles (#3, 4, 5, 6)</td><td>63 (24.6)</td><td>67 (26.2)</td></tr><tr><td>Number of contaminated bottles</td><td>3 (1.2)</td><td>3 (1.2)</td></tr></tbody></table>

473

474 *p=0.003 (MacNemar test); π p <0.001 (paired Wilcoxon test).

475 **Note:** MSS, multiple sampling strategy; SSS, simple sampling strategy; NA, not applicable.
<table><caption>476 **Table 3: Blood culture results**</caption><thead><tr><th></th><th>No IE (N=155)</th><th>IE (N=101)</th><th>Total (N=256)</th></tr><tr><th></th><th>n (%)</th><th>n (%)</th><th>n (%)</th></tr></thead><tbody><tr><td><strong>Per protocol blood cultures</strong></td><td></td><td></td><td></td></tr><tr><td>At least one positive bottle</td><td>18 (11.6)</td><td>56 (55.4)</td><td>74 (28.9)</td></tr><tr><td>At least one positive bottle SSS</td><td>11 (7.1)</td><td>56 (55.4)</td><td>67 (26.2)</td></tr><tr><td>At least one positive bottle MSS</td><td>12 (7.7)</td><td>51 (50.5)</td><td>63 (24.6)</td></tr><tr><td>Identified microorganisms</td><td></td><td></td><td></td></tr><tr><td>Staphylococcaceae</td><td>6 (3.9)</td><td>19 (18.8)</td><td>25 (9.8)</td></tr><tr><td>Staphylococcus aureus</td><td>3 (1.9)</td><td>12 (11.9)</td><td>15 (5.9)</td></tr><tr><td>Staphylococcus epidermidis</td><td>1 (0.6)</td><td>6 (5.9)</td><td>7 (2.7)</td></tr><tr><td>Staphylococcus capitis</td><td>2 (1.3)</td><td>0 (0.0)</td><td>2 (0.8)</td></tr><tr><td>Staphylococcus haemolyticus</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (0.4)</td></tr><tr><td>Streptococcaceae</td><td>8 (5.2)</td><td>35 (34.7)</td><td>43 (16.8)</td></tr><tr><td>Streptococci</td><td>3 (1.9)</td><td>22 (21.8)</td><td>25 (9.8)</td></tr><tr><td>Oral streptococci</td><td>2 (1.3)</td><td>11 (10.9)</td><td>13 (5.1)</td></tr><tr><td>Streptococcus sanguinis</td><td>0 (0.0)</td><td>4 (4.0)</td><td>4 (1.6)</td></tr><tr><td>Streptococcus mitis/oralis</td><td>1 (0.6)</td><td>3 (3.0)</td><td>4 (1.6)</td></tr><tr><td>Streptococcus mutans</td><td>0 (0.0)</td><td>2 (2.0)</td><td>2 (0.8)</td></tr><tr><td>Streptococcus gordonii</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (0.4)</td></tr><tr><td>Streptococcus salivarius</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (0.4)</td></tr><tr><td>Streptococcus constellatus</td><td>1 (0.6)</td><td>0 (0.0)</td><td>1 (0.4)</td></tr><tr><td>Group D streptococci</td><td>0 (0.0)</td><td>10 (9.9)</td><td>10 (3.9)</td></tr><tr><td>Streptococcus gallolyticus</td><td>0 (0.0)</td><td>8 (7.9)</td><td>8 (3.1)</td></tr><tr><td>Streptococcus infantarius</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (0.4)</td></tr><tr><td>Streptococcus lutetiensis</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (0.4)</td></tr><tr><td>Other streptococci</td><td>1 (0.6)</td><td>1 (1.0)</td><td>2 (0.8)</td></tr><tr><td>Streptococcus agalactiae</td><td>1 (0.6)</td><td>0 (0.0)</td><td>1 (0.4)</td></tr><tr><td>Streptococcus dysgalactiae</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (0.4)</td></tr><tr><td>Enterococci</td><td>3 (1.9)</td><td>13 (12.9)</td><td>16 (6.3)</td></tr><tr><td>Enterococcus faecalis</td><td>3 (1.9)</td><td>11 (10.9)</td><td>14 (5.5)</td></tr><tr><td>Community-acquired</td><td>2 (1.3)</td><td>9 (8.9)</td><td>11 (4.3)</td></tr><tr><td>Enterococcus faecium</td><td>0 (0.0)</td><td>2 (2.0)</td><td>2 (0.8)</td></tr><tr><td>Community-acquired</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (0.4)</td></tr><tr><td>Others</td><td></td><td></td><td></td></tr><tr><td>Cutibacterium acnes</td><td>0 (0.0)</td><td>3 (3.0)</td><td>3 (1.2)</td></tr><tr><td>Escherichia coli</td><td>1 (0.6)</td><td>1 (1.0)</td><td>2 (0.8)</td></tr><tr><td>Pseudomonas aeruginosa</td><td>0 (0.0)</td><td>2 (2.0)</td><td>2 (0.8)</td></tr><tr><td>Anaerococcus</td><td>1 (0.6)</td><td>0 (0.0)</td><td>1 (0.4)</td></tr><tr><td>Escherichia hermanii</td><td>1 (0.6)</td><td>0 (0.0)</td><td>1 (0.4)</td></tr><tr><td>Haemophilus aphrophilus</td><td>0 (0.0)</td><td>1 (1.0)</td><td>1 (0.4)</td></tr><tr><td>Micrococcus spp.</td><td>1 (0.6)</td><td>0 (0.0)</td><td>1 (0.4)</td></tr><tr><td>Streptobacillus moniliformis</td><td>1 (0.6)</td><td>0 (0.0)</td><td>1 (0.4)</td></tr><tr><td>≥2 microorganisms</td><td>0 (0.0)</td><td>5 (5.0)</td><td>5 (2.0)</td></tr></tbody></table>

477

478 **Note:** IE, infective endocarditis; SSS, simple sampling strategy; MSS, multiple sampling

479 strategy.
480 **Table 4:** Microbiological results and final diagnosis of the 14 patients with discrepancies of the microbiological criterion among the 256 patients

481 according to 2015 ESC classification depending on SSS or MSS

<table><thead><tr><th rowspan="3">Final diagnosis (Gold standard)</th><th colspan="3">SSS</th><th colspan="3">MSS</th><th colspan="2">2015 ESC Major microbiological criteria</th><th colspan="2">2015 ESC Minor microbiological criteria</th><th colspan="2">2015 ESC classification</th></tr><tr><th>Microorganism detected on blood culture</th><th>Number of positive bottles</th><th>Number of pairs with at least one positive bottle</th><th>Microorganism detected on blood culture</th><th>Number of positive bottles</th><th>Number of pairs with at least one positive bottle</th><th>SSS</th><th>MSS</th><th>SSS</th><th>MSS</th><th>SSS</th><th>MSS</th></tr></thead><tbody><tr><td>no IE</td><td>Streptobacillus moniliformis</td><td>6</td><td>3</td><td>Streptobacillus moniliformis</td><td>2</td><td>1</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>E</td><td>E</td></tr><tr><td>no IE</td><td>Enterococcus faecalis</td><td>1</td><td>1</td><td>-</td><td>-</td><td>-</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>P</td><td>P</td></tr><tr><td>no IE</td><td>-</td><td>-</td><td>-</td><td>Micrococcus spp.</td><td>1</td><td>1</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>P</td><td>P</td></tr><tr><td>no IE</td><td>-</td><td>-</td><td>-</td><td>Streptococcus mitis/oralis</td><td>1</td><td>1</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>E</td><td>E</td></tr><tr><td>no IE</td><td>-</td><td>-</td><td>-</td><td>Escherichia coli</td><td>1</td><td>1</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>E</td><td>E</td></tr><tr><td>no IE</td><td>-</td><td>-</td><td>-</td><td>Staphylococcus capitis</td><td>1</td><td>1</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>E</td><td>E</td></tr><tr><td>no IE</td><td>-</td><td>-</td><td>-</td><td>Streptococcus constellatus</td><td>1</td><td>1</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>E</td><td>E</td></tr><tr><td>IE</td><td>Streptococcus gordonii</td><td>6</td><td>3</td><td>Streptococcus gordonii</td><td>2</td><td>1</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>D</td><td>D</td></tr><tr><td>IE</td><td>Streptococcus salivarius</td><td>5</td><td>3</td><td>Streptococcus salivarius</td><td>1</td><td>1</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>D</td><td>D</td></tr><tr><td>IE</td><td>Staphylococcus epidermidis</td><td>6</td><td>3</td><td>Staphylococcus epidermidis</td><td>4</td><td>2</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>D</td><td>D</td></tr><tr><td>IE</td><td>Streptococcus dysgalactiae</td><td>2</td><td>2</td><td>Streptococcus dysgalactiae</td><td>1</td><td>1</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>D</td><td>D</td></tr><tr><td>IE</td><td>Streptococcus gallolyticus</td><td>2</td><td>2</td><td>Escherichia coli</td><td>1</td><td>1</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>D</td><td>D</td></tr><tr><td>IE</td><td>Staphylococcus epidermidis</td><td>1</td><td>1</td><td>-</td><td>-</td><td>-</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>D</td><td>D</td></tr><tr><td>IE</td><td>+ Pseudomonas aeruginosa</td><td>1</td><td>1</td><td>-</td><td>-</td><td>-</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>D</td><td>P</td></tr><tr><td>IE</td><td>Cutibacterium acnes</td><td>1</td><td>1</td><td>-</td><td>-</td><td>-</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>D</td><td>P</td></tr></tbody></table>

482

483

484 **Note:** SSS, single sampling strategy; MSS, multiple sampling strategy; IE, infective endocarditis; E, excluded infective endocarditis; P, possible

485 infective endocarditis; D, definite infective endocarditis; ESC, European society of Cardiology.
486

**Figure 1:** Schematic representation of the experimental protocol

<figure><img src="image_2.png" /></figure>

487

488

**Note:** MSS, multiple-sampling strategy; SSS, single sampling strategy.

489
<figure><img src="image_3.png" /><figcaption>490 **Figure 2:** Flow-chart of the study</figcaption></figure>

491

492 **Note:** BC, blood culture.
493 **Figure 3:** Compared sensitivities and specificities of the major microbiologic criterion of the 2015 ESC classification for the diagnosis of

494 infective endocarditis, using single- versus multiple-sampling strategy for blood cultures

495

<figure><img src="image_4.png" /></figure>

496

497 **Note:** MSS, multiple-sampling strategy; SSS, single sampling strategy; TP, true positives; TN, true negatives; PABAK, prevalence-adjusted

498 bias-adjusted kappa test, ESC, European society of Cardiology.
UNIENDO, SUPPLEMENTARY MATERIALS

<table><caption>Table S1: Microbiological analytic methods of the centers</caption><thead><tr><td>Center number</td><td>Blood culture detection system</td><td>Incubation time</td><td>Microbial identification system</td></tr></thead><tbody><tr><td>1 Nancy</td><td>Bactec® FX</td><td>10 days</td><td>MALDI-TOF: Bruker®</td></tr><tr><td>2 Reims</td><td>November 2017 to January 2020: Bact/Alert® 3D<br/>February 2020 to May 2022: Bact/Alert® 3D VIRTUO</td><td>10 days</td><td>MALDI-TOF: Bruker®</td></tr><tr><td>4 Besançon</td><td>Bactec® FX</td><td>10 days</td><td>MALDI-TOF: Bruker®</td></tr><tr><td>5 Strasbourg</td><td>Bactec® FX</td><td>10 days</td><td>MALDI-TOF: Bruker®</td></tr><tr><td>6 Dijon</td><td>Bactec® FX400</td><td>10 days</td><td>MALDI-TOF: Bruker®</td></tr><tr><td>9 HNFC</td><td>Bact/Alert® 3D</td><td>14 days</td><td>MALDI-TOF: Bruker®</td></tr><tr><td>10 Bichat</td><td>Bactec® FX</td><td>10 days</td><td>MALDI-TOF: Bruker®</td></tr><tr><td>11 Rennes</td><td>Bactec® FX</td><td>10 days</td><td>MALDI-TOF: Bruker®</td></tr></tbody></table>
<table><caption>Table S2: Qualification of blood cultures according to classifications</caption><thead><tr><th></th><th>2015 ESC</th><th>2023 Duke-ISCVID</th></tr></thead><tbody><tr><td><strong>Major microbiologic criteria</strong></td><td></td><td></td></tr><tr><td>Typical microorganisms</td><td></td><td></td></tr><tr><td>2 positive and separate blood cultures</td><td>X</td><td>X</td></tr><tr><td>2 blood cultures without timing or separate venipunctures restriction*</td><td></td><td>X</td></tr><tr><td>Microorganisms consistent with IE</td><td></td><td></td></tr><tr><td>2 positive cultures of blood samples drawn 12 hours apart, or 3 or a majority of ≥4 separate cultures of blood (first and last sample drawn at least 1 h apart)</td><td>X</td><td>X</td></tr><tr><td>3 blood cultures without timing or separate venipunctures restriction*</td><td></td><td>X</td></tr></tbody></table>

<footnote>*Separate venipunctures are recommended but no longer mandatory.</footnote>
<table><caption>Table S3: IE-causing microorganisms, based on all microbiological investigations</caption><thead><tr><th>Number of IE cases (N=101)</th><th>n</th><th>(%)</th></tr></thead><tbody><tr><td><strong>Staphylococcaceae</strong></td><td>22</td><td>(21.8)</td></tr><tr><td>Staphylococcus aureus</td><td>14</td><td>(13.9)</td></tr><tr><td>Staphylococcus epidermidis</td><td>4</td><td>(4.0)</td></tr><tr><td>Staphylococcus haemolyticus</td><td>1</td><td>(1.0)</td></tr><tr><td>Staphylococcus lugdunensis</td><td>1</td><td>(1.0)</td></tr><tr><td>Gemella morbillorum</td><td>1</td><td>(1.0)</td></tr><tr><td>Gemella spp.</td><td>1</td><td>(1.0)</td></tr><tr><td><strong>Streptococcaceae</strong></td><td>38</td><td>(37.6)</td></tr><tr><td>Streptococci</td><td>25</td><td>(24.8)</td></tr><tr><td>Streptococcus gallolyticus</td><td>8</td><td>(7.9)</td></tr><tr><td>Streptococcus sanguinis</td><td>4</td><td>(4.0)</td></tr><tr><td>Streptococcus mitis/oralis</td><td>3</td><td>(3.0)</td></tr><tr><td>Streptococcus dysgalactiae subsp. equisimilis</td><td>2</td><td>(2.0)</td></tr><tr><td>Streptococcus mutans</td><td>2</td><td>(2.0)</td></tr><tr><td>Streptococcus gordonii</td><td>1</td><td>(1.0)</td></tr><tr><td>Streptococcus pneumoniae</td><td>1</td><td>(1.0)</td></tr><tr><td>Streptococcus salivarius</td><td>1</td><td>(1.0)</td></tr><tr><td>Streptococcus bovis</td><td>1</td><td>(1.0)</td></tr><tr><td>Streptococcus infantarius</td><td>1</td><td>(1.0)</td></tr><tr><td>Streptococcus lutetiensis</td><td>1</td><td>(1.0)</td></tr><tr><td>Enterococci</td><td>12</td><td>(11.9)</td></tr><tr><td>Enterococcus faecalis</td><td>11</td><td>(10.9)</td></tr><tr><td>Enterococcus faecium</td><td>1</td><td>(1.0)</td></tr><tr><td>Other</td><td>1</td><td>(1.0)</td></tr><tr><td>Granulicatella adiacens</td><td>1</td><td>(1.0)</td></tr><tr><td><strong>Others</strong></td><td>10</td><td>(9.9)</td></tr><tr><td>Haemophilus aphrophilus</td><td>1</td><td>(1.0)</td></tr><tr><td>Cutibacterium acnes</td><td>3</td><td>(3.0)</td></tr><tr><td>Bartonella henselae</td><td>2</td><td>(2.0)</td></tr><tr><td>Pseudomonas aeruginosa</td><td>2</td><td>(2.0)</td></tr><tr><td>Bacteroides caccae</td><td>1</td><td>(1.0)</td></tr><tr><td>Chlamydia pneumoniae</td><td>1</td><td>(1.0)</td></tr><tr><td>Serratia marcescens</td><td>1</td><td>(1.0)</td></tr><tr><td><strong>Polymicrobial</strong></td><td>2</td><td>(2.0)</td></tr><tr><td>Enterococcus faecalis + Enterococcus faecium</td><td>1</td><td>(1.0)</td></tr><tr><td>Cutibacterium acnes + Staphylococcus capitis</td><td>1</td><td>(1.0)</td></tr><tr><td><strong>Unknown</strong></td><td>28</td><td>(27.7)</td></tr></tbody></table>

Note: IE, infective endocarditis.