G Ambroise Grandjean

P Berveiller

G Hossu

P Noble

M Chamagne

O Morel

Prospective assessment of reproducibility of threedimensional ultrasound for fetal biometry

Keywords: Gestational age, Reproducibility of results, Ultrasonography, prenatal, Ultrasonography, Three-dimensional ultrasound

Purpose: To compare fetal ultrasound measurements performed by two observers with different levels of experience and evaluate the potential contribution of the use of threedimensional (3D) ultrasound on repeatability, reproducibility and agreement of twodimensional (2D) and 3D-derived measurements.

Materials and methods: Two observers (one senior and one junior) measured head circumference (HC), abdominal circumference (AC) and femur length (FL) in 33 fetuses (20 to 40 weeks of gestation). Each observer performed two series of 2D measurements and two series of 3D measurements (i.e., measurements derived from triplane volume processing).

Measurements were converted into Z-scores according to gestational age. Variability between the different series of measurements was studied using Bland-Altmann plots and intra-class correlation coefficients (ICC).

Results: Agreement with the 2D measurements of the senior observer was higher in 3D than in 2D for the junior observer (systematic differences of -0.4, -0.2 and -0.8 Z-score vs. -0.1, -0.1 and -0.6 for HC, AC and FL on 2D and 3D datasets, respectively). The use of 3D ultrasound improved junior observer repeatability (ICC= 0.94, 0.88, 0.90 vs. 0.94, 0.94 and 0.96 for HC, AC and FL in 2D and 3D, respectively). The reproducibility was greater using the junior observer 3D datasets (ICC= 0.75, 0.60 and 0.45 vs. 0.79, 0.89 and 0.63 for HC, AC and FL, respectively).

Conclusion:

The use of 3D ultrasound improves the consistency of the measurements performed by a junior observer and increases the overall repeatability and reproducibility of measurements performed by observers with different levels of experience.

Introduction

Screening for fetal growth abnormalities relies primarily on accurate biometric measurements performed on two-dimensional (2D) ultrasound images, allowing the estimation of fetal weight [START_REF] Papageorghiou | International standards for fetal growth based on serial ultrasound measurements: the fetal growth longitudinal study of the INTERGROWTH-21st Project[END_REF]. As with all screening tests, it is essential to maintain strict control over the reproducibility of the measurements used [START_REF] Kha | Evaluation of a clinical test. I: assessment of reliability[END_REF][START_REF] Neto | True reproducibility of ultrasound techniques (TRUST): systematic review of reliability studies in obstetrics and gynecology[END_REF]. The operator-dependent nature of ultrasound compared with other imaging techniques is well understood [START_REF] Perni | Intraobserver and interobserver reproducibility of fetal biometry[END_REF] and impacts negatively on the quality of the measurements. The variability of ultrasound measurements is difficult to quantify probably due to several factors including the identification of anatomical landmarks, plane acquisition and caliper positioning [START_REF] Neto | True reproducibility of ultrasound techniques (TRUST): systematic review of reliability studies in obstetrics and gynecology[END_REF]. However, even if this variability is marginal and acceptable for experienced operators, it remains substantial for inadequately trained operators [START_REF] Perni | Intraobserver and interobserver reproducibility of fetal biometry[END_REF][START_REF] Benacerraf | Proceedings: beyond ultrasound first forum on improving the quality of ultrasound imaging in Obstetrics and Gynecology[END_REF][START_REF] Sarris | Standardization of fetal ultrasound biometry measurements: improving the quality and consistency of measurements[END_REF][START_REF] Weerasinghe | Cumulative sum (CUSUM) analysis in the assessment of trainee competence in fetal biometry measurement[END_REF].

The use of three-dimensional (3D) ultrasound allows a potential pathway to improve the accuracy of fetal measurements. Offline analysis of stored 3D volumes to select the plane, which corresponds to the conventional 2D ultrasound criteria, can be performed by identifying anatomical landmarks without specific skill at manipulating the probe [START_REF] Sarris | Fetal biometry: how well can offline measurements from three-dimensional volumes substitute real-time two-dimensional measurements?[END_REF]. It was previously demonstrated that repeatability and operator reproducibility could be improved by using 3D ultrasound volumes to perform biometric measurements [START_REF] Chan | Volumetric (3D) imaging reduces inter-and intraobserver variation of fetal biometry measurements[END_REF][START_REF] Lima | Biometry and fetal weight estimation by two-dimensional and three-dimensional ultrasonography: an intraobserver and interobserver reliability and agreement study[END_REF]. Improvement might be especially significant for inexperienced operators [START_REF] Yang | Fetal biometry by an inexperienced operator using two-and three-dimensional ultrasound[END_REF]. The use of 3D ultrasound was also shown to reduce the time needed to perform the ultrasound and to lead to an improved image quality.

However, in previously published reports, fetal biometric measurements were performed at different gestational ages, which did not allow for the direct comparison of data. Indeed, expressed in millimeters, the observed variability between repeated measurements of the same parameter increases with increasing gestational age. By using the Z-score, this difficulty can be overcome [START_REF] Sarris | Intra-and interobserver variability in fetal ultrasound measurements[END_REF].

Although 3D ultrasound has an a priori lower risk of variability than 2D ultrasound, it still presents a risk of intra-and inter-operator variability for two reasons: firstly, due to the quality of the acquisition of the volume [START_REF] Sarris | Fetal biometry: how well can offline measurements from three-dimensional volumes substitute real-time two-dimensional measurements?[END_REF] and secondly due to the quality of the subsequent analysis (selection of the correct plane and caliper placement). The specific impact of this second step (i.e., processing volumes) has not yet been assessed. The primary goal of this pilot study was to compare fetal ultrasound measurements performed by two observers of different levels of experience and evaluate the potential contribution of 3D ultrasound on repeatability, reproducibility and agreement of 2D and 3D-derived measurements. The additional study had the aim of assessing the impact of the observer's experience on the processing of the ultrasound volumes.

Materials and methods

Patients

Data were collected during a prospective cross-sectional pilot study undertaken on women referred for ultrasound examination to an academic hospital. The standard management of pregnant women in our institution includes an ultrasound examination performed by an expert sonographer (with findings being reported in the patient's medical notes). An optional additional ultrasound examination may be performed by a student as part of training. Oral consent is obtained from the patient before the additional scan is undertaken. This additional scan results in a slightly longer ultrasound examination. Data acquired during this scan do not contain patient information or result in changes to the patient's care.

This study was performed from November 1 st , 2015 until the January 31 st , 2016.

Women referred for an ultrasound examination involving biometric measurements between 17 and 41 weeks' gestation (WG) were invited to participate. The non-inclusion criterion was the refusal to participate in the optional part of the examination. The exclusion criterion was the presence or suspicion of a fetal abnormality.

Ultrasound protocol and image analysis

The measurements were performed on a Voluson E8 ® (GE Healthcare) using a 4-8 MHz probe. During the ultrasound examinations, a mask was put on the screen to prevent the observer from seeing the measurements and gestational age-related percentiles. Each 2D and 3D measurement was performed twice by each observer to evaluate the reproducibility.

All 2D and 3D biometric measurements were performed according to the methodology published in the Intergrowth 21 study [START_REF] Papageorghiou | International standards for fetal growth based on serial ultrasound measurements: the fetal growth longitudinal study of the INTERGROWTH-21st Project[END_REF]. The procedures for analyzing the volumes were developed and implemented by adapting procedures from previous publications and using the co-author's expertise [START_REF] Sarris | Fetal biometry: how well can offline measurements from three-dimensional volumes substitute real-time two-dimensional measurements?[END_REF]. The 3D volume acquisition was obtained in triplane mode with the sweep angle set at 60°. This angle had been previously defined to encompass all the relevant structures in a fetus at 41 WG. In order to limit the distortion of the volumes, the acquisition planes were: a transverse transthalamic plane, a transverse abdominal plane and a plane allowing the visualization of the uppermost femur (with less than 40° angulation compared to the horizontal) for the volumes of the cephalic pole, the abdomen and the thigh respectively [START_REF] Sarris | Fetal biometry: how well can offline measurements from three-dimensional volumes substitute real-time two-dimensional measurements?[END_REF]. For very active fetuses, the speed of acquisition was increased. The pressure of the probe on the maternal abdomen was adjusted in order not to deform the contours of the fetal anatomy. All of these acquisition techniques, as well as the visual assessment of the absence of artifact caused by fetal movement during the volume acquisition, were covered in the teaching received by the junior observer before the study.

The time taken to obtain the 2D planes and 3D volumes, as well as the analysis of the volumes, were recorded. For 2D measurement procedures and 3D volumes acquisition, the observers kept an empty screenshot at the beginning and end of each process to calculate the duration. For volume processing procedures, the observers used an external timer.

Data collection and definitions

Data were collected during examinations performed successively by both a senior observer and a junior observer. The senior observer (G. A.) was an expert sonographer and a trainer for fetal biometry training programs. This observer has conducted over 2,000 ultrasound examinations, with both 2D and 3D technique, over the last five years. The junior observer (M. C.) was a trainee in obstetrics and gynecology and had performed less than 50 2D ultrasound examinations in the course of his training and had minimal experience in 3D ultrasound. Before the study commenced, the junior observer received two hours of theoretical training and four hours of hands-on training on how to perform 2D and 3D fetal biometry including measurement methodology, anatomical landmarks, volumes acquisition and 3D volume processing.

Conventional 2D measurements performed by the senior observer were chosen as the reference standard. The index tests assessed were 2D measurements performed by the junior observer and 3D measurements performed both by the senior and the junior observers. The STARD standards were used to report the results [START_REF] Bossuyt | an updated list of essential items for reporting diagnostic accuracy studies[END_REF].

For each fetus, datasets comprising of the head circumference (HC), the abdominal circumference (AC) and the femur length (FL) were recorded twice by the two observers and further referred to as datasets 2Dsenior and 2Djunior. Similarly, 3D volumes (cephalic pole, abdomen, and thigh) were acquired twice by both observers. At the end of the collection period, the volumes were reviewed off-line by their respective observers to obtain the corresponding measurements: HC, AC, and FL and further referred to as datasets 3Dsenior and 3Djunior.

As part of the additional study, the volumes acquired by the junior observer were reset and processed again by the senior observer (dataset 3Dreview) to evaluate the specific impact of off-line processing on inter-observer reproducibility and to assess the impact of 3D acquisitions quality on the processing step. The intra-observer reproducibility was not studied as part of this additional study.

The two observers processed all the measurements in a random order to limit the impact of memorization effect and training bias on variability. These procedures produced four 2Ddatasets for each fetus (two datasets 2Dsenior and two datasets 2Djunior) and five 3Ddatasets (two datasets 3Dsenior, two datasets 3Djunior and one dataset 3Dreview). Examples of measurements performed by processing 3D acquisition (triplane view and post-reviewing view) and a flowchart of the data acquisition procedure are provided in Figures 1 and2, respectively. For all women, indication for ultrasound examination, body mass index (BMI), maternal age, gestational age, complications or risk factors and fetal presentation were recorded.

Statistical analysis

The term 'agreement' referred to the fact that two or more independent measurements of the same parameters were equal [START_REF] Bland | Applying the right statistics: analyses of measurement studies[END_REF]. The term 'repeatability' referred to the ability of a same observer to obtain similar results on iterative measurements performed on the same fetus [START_REF] Soyer | Agreement and observer variability[END_REF]. The term 'reproducibility' referred to the ability for different observers to obtain similar results measuring the same fetus. The terms 'random error' and 'systematic error' referred to the individual variability related to unpredictable changes due to changes in observer or technique and predictable changes related to a tendency to over-or under-estimate a magnitude.

Use of Z-scores to report fetal measurements resulted in variability remaining constant irrespective of fetal size [START_REF] Sarris | Intra-and interobserver variability in fetal ultrasound measurements[END_REF]. All of the measurements were, therefore, converted into Zscores using the references provided by the Intergrowth 21st study [START_REF] Papageorghiou | International standards for fetal growth based on serial ultrasound measurements: the fetal growth longitudinal study of the INTERGROWTH-21st Project[END_REF].The following cut-offs, Z-score < -1.3 and Z-score > 1.3 (corresponding to measurements < 10 th percentile and > 90 th percentile) were used to assess the distribution of measurements in each series.

To underline the clinical impact resulting from a difference in fetal biometric measurements, the estimated fetal weight (EFW) was calculated using the Hadlock formula [START_REF] Hadlock | Estimation of fetal weight with the use of head, body, and femur measurements: a prospective study[END_REF]. The intergrowth 50° centile for 32 WG was used as the reference.

A minimum of 30 observations (i.e., 30 fetuses) was required to provide a sample with a normal distribution as a part of a pilot study. A further 10% was added to mitigate the risk of missing data. Therefore, the protocol specified 33 fetuses needed to be scanned.

All quantitative data were expressed as mean ± standard deviation (SD) and ranges. Paired Welch two sample t-test was used for duration comparison. The differences between the different series of measurements were searched for with the Tukey's HSD test (honestly significant difference). Significance for difference was set at P < 0.05. The calculation of the intraclass correlation coefficients (ICC) with their 95% confidence interval (CI) allowed the assessment of the repeatability and reproducibility between the different series. Statistical analysis was performed using R software version 3.2.0 (R Core Team).

Agreement between the different series of measurements was evaluated using Bland-Altman plots [START_REF] Bland | Applying the right statistics: analyses of measurement studies[END_REF].

Results

Patients

Of the initially 40 consecutive eligible women, measurements and acquisitions were performed on 33 women because 7 refused the additional examination. The mean maternal age was 28 years ± 3.9 (SD) years (range: 20 -40 years) with a mean gestational age of 28 ± 5 (SD) WG (range: 20 -40 WG). The mean body mass index (BMI) of the women was 27± 5.18 (SD) kg/m 2 (range: 17 -36 kg/m 2 ) with 23 women having a BMI ≥ 25 kg/m 2 .

Fetal measurements and volume acquisitions were performed during routine 2 nd and 3 rd trimester ultrasound appointments for 20 of the 33 women. The other 13 women were scanned to monitor fetal growth. The pregnancies progressed normally in 28/33 women (85%), with the other five women having the following complications or risk factors: gestational diabetes (n = 3), arterial hypertension (n = 1) and antiphospholipid antibodies syndrome (n = 1). Fetal presentation was cephalic in 20/33 women (61%), breech in 7/33 women (21%), and transverse in 6/33 women (18%). The flowchart of the study population recruitment is presented in Figure 2.

Both observers acquired the necessary 2D images and 3D volumes for biometric measurements in all women. However, on processing his 3D volumes, the junior observer identified that on three occasions a volume of the shoulder and humerus had been mistakenly acquired instead of the pelvis and femur. The FL measurements corresponding to these three volumes were not calculated.

The mean time taken for fetal biometry in 2D & 3D is reported in Table 1. The senior observer obtained measurements significantly faster using both 2D images and 3D volumes than the junior observer. The use of 3D volume acquisition significantly reduced the "point of care" time for both observers. However, when the time for the complete procedure was analyzed (i.e., 3D acquisition and processing) 3D procedure time was significantly longer than 2D procedure time for junior and senior observers (P < 0.001).

Distribution of measurements

There were no significant differences between the different observers' measurements of the HC and AC or between the 2D and 3D measurements (Table 2). The mean of the biometric measurements obtained by the junior observer using 2D ultrasound (dataset 2Djunior) was lower with a higher standard deviation than using 3D. And significant differences in 2D measurements were found for FL between the junior and the senior observer (P = 0.038).

The number of measurements < -1.3 Z-score or > 1.3 Z-score (below the 10 th or above the 90 th centile) was reduced for the junior observer when using 3D ultrasound. The concordance between the two observers for the number of measurements between < -1.3 and > 1.3 Z-score increased when the junior observer used 3D volumes.

Senior observer

The senior observer demonstrated high degrees of agreement between all measurement techniques, with a 95% CI of less than 1 Z-score for the three parameters (Figure 3). This variability corresponded, in a clinically relevant manner, to 95% of EFWs being between 1534 g and 2039 g for a 1762 g fetus at 32 WG (Table 3). The repeatability of the measurements was high within all the series (ICC ≥ 0.90 for the datasets 2Dsenior and 3Dsenior) and an improvement was seen using 3D for the HC and FL parameters (Table 4).

Junior observer

The agreement between the reference measurements (dataset 2Dsenior) and the datasets 2Djunior and 3Djunior were weaker than those seen between the reference and the dataset 3Dsenior. For the HC and AC parameters, the 95% confidence interval was approaching 2 Z-scores in both 2D and 3D (Figure 3). A systematic difference of approximately -0.4 and -0.2 Z-score with the reference measurements was found for the dataset 2Djunior for these two parameters, respectively. This difference was consistent with the average differences seen in Table 2 (underestimation of measurements within the 2Djunior dataset compared to the reference dataset).

Concerning the femur measurements, the agreement between the 2D and 3D series of the junior operator and the reference measurements were even weaker with a 95% CI > 3.5 Zscores and an underestimation of measurements, compared to the reference dataset. This variability corresponds to 95% of EFWs being between 1176 g and 2281 g for a 1762 g fetus at 32 WG in 2D (Table 3). And, to 95% of EFWs being between 1270 g and 2215 g in 3D (Table 3). The repeatability of the measurements was raised in 3D with ICCs close to those seen within the 2Dsenior dataset (Table 4).

Reproducibility

The inter-operator intra-technique reproducibility was higher when both operators measured from 3D volumes rather than in 2D. A particular benefit was seen for AC and FL (ICC = 0.60 and 0.45 vs. 0.89 & 0.63 for 2D and 3D, respectively) (Table 4).

Impact of acquisition quality on 3D volumes processing

Analysis based on Bland Altmann plots showed that when the senior observer processed the 3D volumes acquired by the junior observer (dataset3Dreview) the agreement with the reference range (dataset 2Dsenior) was lower than the agreement of the 3Djunior dataset with the reference range. Similarly, reproducibility between 3Dreview & 2Dsenior was lower than 3Djunior & 2Dsenior for the measurements of HC and AC. Those results demonstrated a reduction in reproducibility in connection with the processing by the senior operator (Table 4). However, this same comparison for the FL shows a raised reproducibility within the 3Dreview dataset with a beneficial impact of the review by the expert operator (Table 4). The variability between 3Dreview & dataset 2Dsenior corresponds to 95% of EFWs being between 1301g and 2341g for a 1762g fetus at 32 WG (Table 3).

Discussion

This pilot study demonstrated that, when taking fetal biometric measurements, agreement with the senior observer was higher when the junior observer used 3D ultrasound volumes.

The distribution of measurements was closer to the reference range, and repeatability and reproducibility were increased. Taking fetal biometric measurements using 3D volumes was significantly slower for the junior observer when taking into account the entire procedure but allowed a significant shortening of the duration of the "point of care" examination.

On the opposite, using 2D ultrasound, the junior observer under-estimated measurements. This was most notably seen for the FL. A possible explanation for this could be a confusion between identifying the humerus instead of the femur. This confusion was detected in three cases when reviewing the 3D volumes and these were excluded. As this potential confusion was not detected at the time of the 2D scan no measurements were excluded from the 2D dataset. In clinical practice this could lead to errors in obstetric management which could be mitigated by the use of 3D volumes.

In our study, there was a limited impact of the imaging technique (2D or 3D) on the biometric measurements obtained by the senior observer. The slight improvement of repeatability obtained using 3D for the measurements of HC and FL was balanced out by the duration of the procedure which was significantly longer than the time taken to obtain the measurements in 2D.

The patients included in this study were done so in a consecutive manner without exclusion for growth abnormality. This explains the distribution seen within the cohort, with a large proportion of measurements < -1.3 Z-score and > 1.3 Z-score. This choice reflected a desire to study the impact of measurement techniques on a population representative of a standard clinic setting (physiological and pathological growth, variable fetal presentations, maternal BMI representative of the local population). Within this series, there were several overweight patients (23 patients out of 33 patients included), which is a factor that limits the quality of ultrasound measurements. However, it appears that the use of 3D ultrasound is feasible as volumes were obtained in all patients and indeed may have a beneficial impact when performing biometric measurements.

Regarding the differences in the range of measurements performed on 2D images and from 3D volumes, this has been already reported in a previous study and is potentially explained by the difficulties encountered in 2D measuring with the associated risk of random error and of a more significant under-or over-estimation of measurements (systematic errors) [START_REF] Chan | Volumetric (3D) imaging reduces inter-and intraobserver variation of fetal biometry measurements[END_REF]. This bias seems to be limited by using the 3D technique especially for the junior observer [START_REF] Yang | Fetal biometry by an inexperienced operator using two-and three-dimensional ultrasound[END_REF], however, the specific composition of our cohort (a large proportion of measurements < -1.3 Z-score and > 1.3 Z-score) may have accentuated this effect. This finding calls for caution over the clinical interpretation of 3D measurements. Current reference ranges referring to 2D data are probably not superimposable on a 3D series.

The starting hypothesis of the additional 3D review study was to confirm that the senior observer's review of the junior observer's 3D volumes would result in increased reproducibility and agreement of the measurements. However, this point was not confirmed (inter-operator ICC was weaker for the HC and AC parameters for the 3Dreview dataset). The only advantage observed concerned the agreement of FL measurements with the reference measurements. These results underline that the experience of the observer appears to have a more significant impact when using 2D ultrasound than 3D ultrasound. This difference could be partly explained by the standardization of analyzing 3D volumes, limiting the observerdependent nature of performing measurements. This would need to be confirmed on a more extensive series involving different observers from different institutions to reduce the interpretation bias related to each observer. However, this finding echoes the concerns recently put forward in the Beyond Ultrasound First Forum [START_REF] Benacerraf | Proceedings: beyond ultrasound first forum on improving the quality of ultrasound imaging in Obstetrics and Gynecology[END_REF] regarding quality control of data and the evolution of ultrasound within different imaging techniques. It encourages research aimed at automating the measurement process, especially regarding the analysis of 3D volumes [START_REF] Hur | Feasibility of three-dimensional reconstruction and automated measurement of fetal long bones using 5D Long Bone[END_REF][START_REF] Ambroise Grandjean | Artificial intelligence assistance for fetal head biometry: assessment of automated measurement software[END_REF][START_REF] Ambroise Grandjean | Optimization of fetal biometry with 3D ultrasound and image recognition (EPICEA): protocol for a prospective cross-sectional study[END_REF].

The use of Z-scores to compare ultrasound measurements prevents an over-estimation of the agreement and reproducibility for the measurements in early gestations (where a low variability is seen of measurements when presented in millimeters) and similarly an underestimation of the same parameters at later gestations [START_REF] Sarris | Intra-and interobserver variability in fetal ultrasound measurements[END_REF]. By using Z-scores in this study it allowed comparison of these data with previously published data. Variability seen between repeated measurements of the same parameter in 2D and 3D by the senior observer is equivalent to that seen in conventional 2D ultrasound of large cohorts. The use of Z-scores also allows a comparison of the 3D ultrasound measurements by the junior observer with data published in the same format, opening the possibility to subsequent comparisons to verify these preliminary results.

There are two main limitations to this study. The first relates to the small population size and the number of observers performing the scans. This limits the possibility of extrapolating the results to standard care. The second relates to the use of 2D measurements obtained by the senior observer as the gold standard for the reference range.

In conclusion, the results of this pilot study confirm prior results regarding the use of 3D ultrasound in reducing differences in fetal measurements due to the level of observer experience and also in reducing the duration of "point of care" examination for non-expert observers. These preliminary results support the possibility to standardize measurement procedures by using 3D ultrasound volumes, allowing us to reflect on the use of this technique in the training of physicians and sonographers, in the quality control of data and in the development of new measurement techniques.

Legends for figures

Figure 1 .

 1 Figure 1. Examples of measurements performed by three-dimensional processing acquisition (triplane view and post-reviewing view). a = head measurements; b = abdominal circumference; c = femur length. Arrows indicate caliper locations.

Figure 2 .

 2 Figure 2. Flowchart of the selection of patients and data acquisition procedures by the senior and junior observers.

Figure 3 .

 3 Figure 3. Graphs show agreement with reference measurements (2Ddataset) for 3Dsenior, 2Djunior, 3Djunior & 3Dreview datasets. Head circumference (a), abdominal circumference (b) and femur length (c).

Table 1 .

 1 Duration times for fetal biometry using two-dimensional and three-dimensional ultrasound.

Table 2 .

 2 Distribution of measurements for 2Dsenior, 3Dsenior, 2Djunior and 3Djunior datasets.

Table 3 .

 3 Examples of fetal weight estimation (EFW) confidence interval at 32 weeks when comparing each technique to the reference set (Dataset 2Dsenior).

Table 4 .

 4 Repeatability & reproducibility of fetal measurements by to different observers using intraclass correlation coefficient.Data are given in minutes:seconds and expressed as means ± standard deviations; numbers in brackets are ranges. Paired Welch two sample t-test was used for comparison.Intraclass correlation coefficient were calculated from data given in Z-score and given along with their confidence intervals in parentheses. HC = Head circumference, AC = Abdominal circumference, FL = femur length.

		2D		3D		P value*
		Mean time 2D	Mean time volume	Mean time volume	Total mean time
			acquisition	processing	3D procedure
	Junior	6:02 ± 2:20	2:49 ± 1:09	8:50 ± 2:45	11:40 ± 3:12	< 0.001
	(MC)	[1:55 -11:50]	[1:30 -7:21]	[4:47 -15:55]	[6:54-19:51]
	Senior	2:54 ± 0:44	1:56 ± 0:32	7:24 ± 1:28	9:20 ± 1:40	< 0.001
	(GA)	[1:23 -4:45]	[1:06 -3:36]	[5:26 -11:43]	[7:13 -13:42]
	P value*	< 0.001	< 0.001	0.008	< 0.001	-

** Comparison between repeated measurements of the same parameter by the same observer ǂ Comparison between the first measurement of each dataset for each technique & observer

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.

Authors' contributions

GA, MC, and OM designed the study; GA, PB, and PN wrote the manuscript; the English version was corrected by PN; GA and MC collected and entered and the data; GH did the statistical analysis and all authors reviewed and substantially contributed to the manuscript.

Ethics approval and consent to participate

The study was conducted in accordance with Good Clinical Practice guidelines. As a noninterventional monocentric study, this research was approved by the ethics committee of CHRU Nancy.

Competing interests

The authors declare that they have no conflicts of interest to disclose in relation with this article.


Philippe Gautret

J.-C Lagier

Philippe Parola

Thuan Van

Line Hoang

Morgane Meddeb

Barbara Mailhe

Johan Doudier

Valérie Courjon

Vera Esteves Giordanengo

Vieira

Jean-Christophe Lagier

Van Thuan Hoang

Line Meddeb

Morgane Mailhe

Barbara Doudier

Johan Courjon

Valérie Giordanengo

Vera Esteves Vieira

Tissot Dupont

Stéphane Honoré

Philippe Colson

Eric Chabrière

Bernard La Scola

Jean-Marc Rolain

Philippe Brouqui

Didier Raoult
email: didier.raoult@gmail.com

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

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

Despite its small sample size our survey shows that hydroxychloroquine treatment is significantly associated with viral load reduction/disappearance in COVID-19 patients and its effect is reinforced by azithromycin.

Key words: 2019-nCoV; SARS-CoV-2; COVID-19; hydroxychloroquine; azithomycin; clinical trial

Introduction

In late December 2019, an outbreak of an emerging disease (COVID-19) due to a novel coronavirus (named SARS-CoV-2 latter) started in Wuhan, China and rapidly spread in China and outside [START_REF] Lai | Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges[END_REF][START_REF] Wang | A review of the 2019 Novel Coronavirus (COVID-19) based on current evidence[END_REF]. The WHO declared the epidemic of COVID-19 as a pandemic on March 12 th 2020 [START_REF]General's opening remarks at the media briefing on COVID-19 -11[END_REF]. According to a recent Chinese stud, about 80% of patients present with mild disease and the overall case-fatality rate is about 2.3% but reaches 8.0% in patients aged 70 to 79 years and 14.8% in those aged >80 years [START_REF] Wu | Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72 314 cases from the Chinese Center for Disease Control and Prevention[END_REF]. However, there is probably an important number of asymptomatic carriers in the population, and thus the mortality rate is probably overestimated. France is now facing the COVID-19 wave with more than 4500 cases, as of March 14 th 2020 [START_REF]Infection au nouveau Coronavirus (SARS-CoV-2), COVID-19[END_REF]. Thus, there is an urgent need for an effective treatment to treat symptomatic patients but also to decrease the duration of virus carriage in order to limit the transmission in the community. Among candidate drugs to treat COVID-19, repositioning of old drugs for use as antiviral treatment is an interesting strategy because knowledge on safety profile, side effects, posology and drug interactions are well known [START_REF] Colson | Chloroquine for the 2019 novel coronavirus SARS-CoV-2[END_REF]7].

A recent paper reported an inhibitor effect of remdesivir (a new antiviral drug) and chloroquine (an old antimalarial drug) on the growth of SARS-CoV-2 in vitro, [8] and an early clinical trial conducted in COVID-19 Chinese patients, showed that chloroquine had a significant effect, both in terms of clinical outcome and viral clearance, when comparing to controls groups [9,10]. Chinese experts recommend that patients diagnosed as mild, moderate and severe cases of COVID-19 pneumonia and without contraindications to chloroquine, be treated with 500 mg chloroquine twice a day for ten days [11].

Hydroxychloroquine (an analogue of chloroquine) has been demonstrated to have an anti-SARS-CoV activity in vitro [START_REF] Biot | Design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities[END_REF]. Hydroxychloroquine clinical safety profile is better than that of chloroquine (during long-term use) and allows higher daily dose [START_REF] Marmor | Recommendations on Screening for Chloroquine and Hydroxychloroquine Retinopathy (2016 Revision)[END_REF] and has fewer concerns about drug-drug interactions [START_REF] Yao | In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)[END_REF]. Our team has a very comprehensive experience in successfully treating patients with chronic diseases due to intracellular bacteria (Q fever due to Coxiella burnetii and Whipple's disease due to Tropheryma whipplei) with long-term hydroxychloroquine treatment (600 mg/day for 12 to 18 months) since more than 20 years. [START_REF] Raoult | Treatment of Q fever endocarditis: comparison of 2 regimens containing doxycycline and ofloxacin or hydroxychloroquine[END_REF]16] We therefore started to conduct a clinical trial aiming at assessing the effect of hydroxychloroquine on SARS-CoV-2-infected patients after approval by the French Ministry of Health. In this report we describe our early results, focusing on virological data in patients receiving hydroxychloroquine as compared to a control group.

Study population and Methods

Setting

This ongoing study is coordinated by The Méditerranée Infection University Hospital Institute in Marseille. Patients who were proposed a treatment with hydroxychloroquine were recruited and managed in Marseille centre. Controls without hydroxychloroquine treatment were recruited in Marseille, Nice, Avignon and Briançon centers, all located in South France.

Patients

Hospitalized patients with confirmed COVID-19 were included in this study if they fulfilled two primary criteria: i) age >12 years; ii) PCR documented SARS-CoV-2 carriage in nasopharyngeal sample at admission whatever their clinical status.

Patients were excluded if they had a known allergy to hydroxychloroquine or chloroquine or had another known contraindication to treatment with the study drug, including retinopathy, G6PD deficiency and QT prolongation. Breastfeeding and pregnant patients were excluded based on their declaration and pregnancy test results when required.

Informed consent

Before being included in the study, patients meeting inclusion criteria had to give their consent to participate to the study. Written informed signed consent was obtained from adult participants (> 18 years) or from parents or legal guardians for minors (<18 years). An information document that clearly indicates the risks and the benefits associated with the participation to the study was given to each patient. Patients received information about their clinical status during care regardless of whether they participate in the study or not. Regarding patient identification, a study number was assigned sequentially to included participants, according to the range of patient numbers allocated to each study centre. The study was conducted in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines of good clinical practice, the Helsinki Declaration, and applicable standard operating procedures.

The protocol, appendices and any other relevant documentation were submitted to the French National Agency for Drug Safety (ANSM) (2020-000890-25) and to the French Ethic Committee (CPP Ile de France) (20.02.28.99113) for reviewing and approved on 5 th and 6 th March, 2020, respectively. This trial is registered with EU Clinical Trials Register, number 2020-000890-25.

Procedure

Patients were seen at baseline for enrolment, initial data collection and treatment at day-0, and again for daily follow-up during 14 days. Each day, patients received a standardized clinical examination and when possible, a nasopharyngeal sample was collected. All clinical data were collected using standardized questionnaires. All patients in Marseille center were proposed oral hydroxychloroquine sulfate 200 mg, three times per day during ten days (in this preliminary phase ,we did not enrolled children in the treatment group based in data indicating that children develop mild symptoms of COVID-19 [START_REF] Wu | Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72 314 cases from the Chinese Center for Disease Control and Prevention[END_REF]). Patients who refused the treatment or had an exclusion criteria, served as controls in Marseille centre. Patients in other centers did not receive hydroxychloroquine and served as controls. Symptomatic treatment and antibiotics as a measure to prevent bacterial super-infection was provided by investigators based on clinical judgment. Hydroxychloroquine was provided by the National Pharmacy of France on nominative demand.

Clinical classification

Patients were grouped into three categories: asymptomatic, upper respiratory tract infection (URTI) when presenting with rhinitis, pharyngitis, or isolated low-grade fever and myalgia, and lower respiratory tract infections (LRTI) when presenting with symptoms of pneumonia or bronchitis.

PCR assay

SARS-CoV-2 RNA was assessed by real-time reverse transcription-PCR [START_REF] Amrane | Rapid viral diagnosis and ambulatory management of suspected COVID-19 cases presenting at the infectious diseases referral hospital in Marseille, France, -January 31st to March 1st, 2020: A respiratory virus snapshot[END_REF].

Hydroxychloroquine dosage

Native hydroxychloroquine has been dosed from patients' serum samples by UHPLC-UV using a previously described protocol [START_REF] Armstrong | Simultaneous UHPLC-UV analysis of hydroxychloroquine, minocycline and doxycycline from serum samples for the therapeutic drug monitoring of Q fever and Whipple's disease[END_REF]. The peak of the chromatogram at 1.05 min of retention corresponds to hydroxychloroquine metabolite. The serum concentration of this metabolite is deduced from UV absorption, as for hydroxychloroquine concentration. Considering both concentrations provides an estimation of initial serum hydroxychloroquine concentration.

Culture

For all patients, 500 µL of the liquid collected from the nasopharyngeal swab were passed through 0.22-µm pore sized centrifugal filter (Merck millipore, Darmstadt, Germany), then were inoculated in wells of 96-well culture microplates, of which 4 wells contained Vero E6 cells (ATCC CRL-1586) in Minimum Essential Medium culture medium with 4% fetal calf serum and 1% glutamine. After centrifigation at 4,000 g, microplates were incubated at 37°C.

Plates were observed daily for evidence of cytopathogenic effect. Presumptive detection of virus in supernatant was done using SU5000 SEM (Hitachi) then confirmed by specific RT-PCR.

Outcome

The primary endpoint was virological clearance at day-6 post-inclusion. Secondary outcomes were virological clearance overtime during the study period, clinical follow-up (body temperature, respiratory rate, long of stay at hospital and mortality), and occurrence of sideeffects.

Statistics

Assuming a 50% efficacy of hydroxychloroquine in reducing the viral load at day 7, a 85% power, a type I error rate of 5% and 10% loss to follow-up, we calculated that a total of 48 COVID-19 patients (ie, 24 cases in the hydroxychloroquine group and 24 in the control group) would be required for the analysis (Fleiss with CC). Statistical differences were evaluated by Pearson's chi-square or Fisher's exact tests as categorical variables, as appropriate. Means of quantitative data were compared using Student's t-test. Analyses were performed in Stata version 14.2.

Results (detailed results are available in supplementary Table 1)

Demographics and clinical presentation

We enrolled 36 out of 42 patients meeting the inclusion criteria in this study that had at least six days of follow-up at the time of the present analysis. A total of 26 patients received hydroxychloroquine and 16 were control patients. Six hydroxychloroquine-treated patients were lost in follow-up during the survey because of early cessation of treatment. Reasons are as follows: three patients were transferred to intensive care unit, including one transferred on day2 post-inclusion who was PCR-positive on day1, one transferred on day3 post-inclusion who was PCR-positive on days1-2 and one transferred on day4 post-inclusion who was PCRpositive on day1 and day3; one patient died on day3 post inclusion and was PCR-negative on day2; one patient decided to leave the hospital on day3 post-inclusion and was PCR-negative on days1-2; finally, one patient stopped the treatment on day3 post-inclusion because of nausea and was PCR-positive on days1-2-3. The results presented here are therefore those of 36 patients (20 hydroxychloroquine-treated patients and 16 control patients). None of the control patients was lost in follow-up. Basic demographics and clinical status are presented in Table 1.

Overall, 15 patients were male (41.7%), with a mean age of 45.1 years. The proportion of asymptomatic patients was 16.7%, that of patients with URTI symptoms was 61.1% and that of patients with LRTI symptoms was 22.2%). All patients with LRTI symptoms, had confirmed pneumonia by CTScan. Hydroxychloroquine-treated patients were older than control patients (51.2 years vs. 37.3 years). No significant difference was observed between hydroxychloroquine-treated patients and control patients with regard to gender, clinical status and duration of symptoms prior to inclusion (Table 1). Among hydroxychloroquine-treated patients six patients received azithromycin (500mg on day1 followed by 250mg per day, the next four days) to prevent bacterial super-infection under daily electrocardiogram control.

Clinical follow-up and occurrence of side-effects will be described in a further paper at the end of the trial.

Discussion

For ethical reasons and because our first results are so significant and evident we decide to share our findings with the medical community, given the urgent need for an effective drug against SARS-CoV-2 in the current pandemic context.

We show here that hydroxychloroquine is efficient in clearing viral nasopharyngeal carriage of SARS-CoV-2 in COVID-19 patients in only three to six days, in most patients. A significant difference was observed between hydroxychloroquine-treated patients and controls starting even on day3 post-inclusion. These results are of great importance because a recent paper has shown that the mean duration of viral shedding in patients suffering from COVID-19 in China was 20 days (even 37 days for the longest duration) [START_REF] Zhou | Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study[END_REF] Very recently, a Chinese team published results of a study demonstrating that chloroquine and hydroxychloroquine inhibit SARS-CoV-2 in vitro with hydroxychloroquine (EC50=0.72%µM) found to be more potent than chloroquine (EC50=5.47%µM) [START_REF] Yao | In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)[END_REF]. These in vitro results corroborate our clinical results. The target values indicated in this paper [START_REF] Yao | In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)[END_REF] were reached in our experiments. The safer dose-dependent toxicity profile of hydroxychloroquine in humans, compared to that of chloroquine [START_REF] Marmor | Recommendations on Screening for Chloroquine and Hydroxychloroquine Retinopathy (2016 Revision)[END_REF] allows using clinical doses of hydroxychloroquine that will be over its EC50 observed in vitro [START_REF] Yao | In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)[END_REF].

Our preliminary results also suggest a synergistic effect of the combination of hydroxychloroquine and azithromycin. Azithromycin has been shown to be active in vitro against Zika and Ebola viruses [START_REF] Retallack | Zika virus cell tropism in the developing human brain and inhibition by azithromycin[END_REF][START_REF] Madrid | Evaluation of Ebola Virus Inhibitors for Drug Repurposing[END_REF][START_REF] Bosseboeuf | Azithromycin inhibits the replication of Zika virus[END_REF] and to prevent severe respiratory tract infections when administrated to patients suffering viral infection [START_REF] Bacharier | Early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: A randomized clinical trial[END_REF]. This finding should be further explored to know whether a combination is more effective especially in severe cases. Speculated potential risk of severe QT prolongation induced by the association of the two drugs has not been established yet but should be considered. As for each treatment, the cost benefits of the risk should be evaluated individually. Further studies on this combination are needed, since such combination may both act as an antiviral therapy against SARS-CoV-2 and prevent bacterial super-infections.

The cause of failure for hydroxychloroquine treatment should be investigated by testing the isolated SARS-CoV-2 strains of the non-respondents and analyzing their genome, and by analyzing the host factors that may be associated with the metabolism of hydroxychloroquine.

The existence of hydroxychloroquine failure in two patients (mother and son) is more suggestive of the last mechanism of resistance.

Such results are promising and open the possibility of an international strategy to decisionmakers to fight this emerging viral infection in real-time even if other strategies and research

including vaccine development could be also effective, but only in the future. We therefore recommend that COVID-19 patients be treated with hydroxychloroquine and azithromycin to cure their infection and to limit the transmission of the virus to other people in order to curb the spread of COVID-19 in the world. Further works are also warranted to determine if these compounds could be useful as chemoprophylaxis to prevent the transmission of the virus, especially for healthcare workers. Our study has some limitations including a small sample size, limited long-term outcome follow-up, and dropout of six patients from the study, however in the current context, we believe that our results should be shared with the scientific community.

[ 7 ]Figure 1 .

 71 Figure 1. Percentage of patients with PCR-positive nasopharyngeal samples from inclusion to

Figure 2 .

 2 Figure 2. Percentage of patients with PCR-positive nasopharyngeal samples from inclusion to

Table 1

 1 Characteristics of the study population. tract respiratory infection, LRTI: lower tract respiratory infection, POS: positive PCR, NEG: negative PCR (CT value >35), ND: PCR not done

		Age (years)		Male gender	Clinical status	Time between onset of symptoms and inclusion (days)
		Mean ± SD	t	p-value	n (%)	p-value Asymptomatic	URTI	LRTI	p-value Mean ± SD	t	p-value
	Hydroxychloroquine
	treated patients	51.2 ± 18.7			9 (45.0)	2 (10.0)	12 (60.0) 6 (30.0)	4.1 ± 2.6
	(N=20)		-1.95	0.06		0.65			0.30	-0.15	0.88
	Control patients (N=16)	37.3 ± 24.0			6 (37.5)	4 (25.0)	10 (62.5) 2 (12.5)	3.9 ± 2.8
	All patients (36)	45.1 ± 22.0			15 (41.7)	6 (16.7)	22 (61.1) 8 (22.2)	4.0 ± 2.6
	URTI: upper tract respiratory infection, LRTI: lower tract respiratory infection

Acknowledgements:

We thank Céline Boschi, Stéphanie Branger, Véronique Filosa, Géraldine Gonfier, Nadège Palmero, Magali Richez and all the clinical, technical and paramedical staffs of the hospitalization units and laboratories for their support in this difficult context.

Hydroxychloroquine dosage

Mean hydroxychloroquine serum concentration was 0.46 µg/ml+0.2 (N=20).

Effect of hydroxychloroquine on viral load

The proportion of patients that had negative PCR results in nasopharyngeal samples significantly differed between treated patients and controls at days 3-4-5 and 6 post-inclusion (Table 2). At day6 post-inclusion, 70% of hydroxychloroquine-treated patients were virologicaly cured comparing with 12.5% in the control group (p= 0.001).

When comparing the effect of hydroxychloroquine treatment as a single drug and the effect of hydroxychloroquine and azithromyc in combination, the proportion of patients that had negative PCR results in nasopharyngeal samples was significantly different between the two groups at days 3-4-5 and 6 post-inclusion (Table 3). At day6 post-inclusion, 100% of patients treated with hydroxychloroquine and azithromycin combination were virologicaly cured comparing with 57.1% in patients treated with hydroxychloroquine only, and 12.5% in the control group (p<0.001). These results are summarized in Figures 1 and2. Drug effect was significantly higher in patients with symptoms of URTI and LRTI, as compared to asymptomatic patients with p<0.05 (data not show).

Of note, one patient who was still PCR-positive at day6-post inclusion under hydroxychloroquine treatment only, received azithromycin in addition to hydroxychloroquine at day8-post inclusion and cured her infection at day-9 post infection. In contrast, one of the patients under hydroxychloroquine and azithromycin combination who tested negative at day6 post-inclusion was tested positive at low titer at day8 post-inclusion.

Cultures

We could isolate SARS-CoV-2 in 19 out of 25 clinical samples from patients.