Federico Solla
email: fedesolla@hotmail.com

Jean Luc Clement

Vincent Cunin

Carlo M Bertoncelli

Vincent Fiere

Virginie Rampal

Jean-Luc Clément

Vincent Fière

Patient-specific rods for thoracic kyphosis correction in adolescent idiopathic scoliosis surgery: Preliminary results

Keywords: Adolescent idiopathic scoliosis, Posterior fusion, Patient-specific rods, Contouring, Thoracic kyphosis

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

Hypothesis:

The use of pre-bent patient-specific rods has a favourable effect on thoracic kyphosis at last follow-up.

Material and methods: 37 patients with AIS, including 17 with hypokyphosis, managed with patient-specific rods were included in a prospective study. The rod contouring angles were based on predefined pelvic incidence criteria (25° to 40° for the rod on the convex side and the same value plus 10° for the rod on the concave side). Thoracic kyphosis was assessed before surgery and at last follow-up, after 12-36 months (mean, 19 months). Student's t test was applied to compare means. Multivariate linear regression analysis was performed.

Results: At last follow-up, the mean increase in kyphosis was 14° and was comparable to the planned increase (mean difference=0, p=0.85). Factors associated with kyphosis at last follow-up were the concave rod contouring angle and the pre-operative kyphotic angle of the thoracic segment to be instrumented (p<0.05). Mean differences between kyphosis of the instrumented thoracic segment at last follow-up and target kyphosis were -5° in the subgroup with hypokyphosis (<20°) before surgery and +4° in the subgroup with normal kyphosis before surgery.

INTRODUCTION

The curves that characterise adolescent idiopathic scoliosis (AIS) develop in all three planes. In the sagittal plane, thoracic hypokyphosis and lumbar hypolordosis are common [START_REF] Clement | Relationship between thoracic hypokyphosis, lumbar lordosis and sagittal pelvic parameters in adolescent idiopathic scoliosis[END_REF][START_REF] Clement | Surgical Increase of Thoracic Kyphosis Increases Unfused Lumbar Lordosis in Selective Fusion for Thoracic Adolescent Idiopathic Scoliosis[END_REF][START_REF] Abelin-Genevois | Sagittal classification in adolescent idiopathic scoliosis: original description and therapeutic implications[END_REF]. Post-operative hypokyphosis is associated with trunk imbalance that results in osteoarthritis in adulthood [START_REF] Glassman | Correlation of radiographic parameters and clinical symptoms in adult scoliosis[END_REF][START_REF] Bernstein | Thoracal flat back is a risk factor for lumbar disc degeneration after scoliosis surgery[END_REF]. Whereas posterior vertebral instrumentation now ensures nearly complete curve correction in the coronal plane, outcomes in terms of sagittal alignment have varied widely across studies [START_REF] Clement | Relationship between thoracic hypokyphosis, lumbar lordosis and sagittal pelvic parameters in adolescent idiopathic scoliosis[END_REF][START_REF] Clement | Surgical Increase of Thoracic Kyphosis Increases Unfused Lumbar Lordosis in Selective Fusion for Thoracic Adolescent Idiopathic Scoliosis[END_REF][START_REF] Angelliaume | Titanium vs cobalt chromium: what is the best rod material to enhance adolescent idiopathic scoliosis correction with sublaminar bands?[END_REF][START_REF] Suk | Comparison of Cotrel-Dubousset pedicle screws and hooks in the treatment of idiopathic scoliosis[END_REF][START_REF] Kim | Proximal junctional kyphosis in adolescent idiopathic scoliosis after 3 different types of posterior segmental spinal instrumentation and fusions: incidence and risk factor analysis of 410 cases[END_REF][START_REF] Suk | Restoration of thoracic kyphosis in the hypokyphotic spine: a comparison between multiple-hook and segmental pedicle screw fixation in adolescent idiopathic scoliosis[END_REF][START_REF] Clement | Restoration of thoracic kyphosis by posterior instrumentation in adolescent idiopathic scoliosis: comparative radiographic analysis of two methods of reduction[END_REF].

The accepted range of normal overall thoracic kyphosis (TK) is 10° to 55° [START_REF] Clement | Relationship between thoracic hypokyphosis, lumbar lordosis and sagittal pelvic parameters in adolescent idiopathic scoliosis[END_REF][START_REF] Clement | Surgical Increase of Thoracic Kyphosis Increases Unfused Lumbar Lordosis in Selective Fusion for Thoracic Adolescent Idiopathic Scoliosis[END_REF][START_REF] Vialle | Radiographic analysis of the sagittal alignment and balance of the spine in asymptomatic subjects[END_REF][START_REF] Lenke | The Lenke classification of adolescent idiopathic scoliosis: how it organizes curve patterns as a template to perform selective fusions of the spine[END_REF][START_REF] Winter | Sagittal spinal alignment: the true measurement, norms, and description of correction for thoracic kyphosis[END_REF][START_REF] Mac-Thiong | Pediatric sagittal alignment[END_REF].

However, few data are available on the optimal amount of TK for each individual.

Nonetheless, geometric associations exist between pelvic incidence and lumbar lordosis, as well as between TK and pelvic parameters [START_REF] Clement | Relationship between thoracic hypokyphosis, lumbar lordosis and sagittal pelvic parameters in adolescent idiopathic scoliosis[END_REF][START_REF] Clement | Surgical Increase of Thoracic Kyphosis Increases Unfused Lumbar Lordosis in Selective Fusion for Thoracic Adolescent Idiopathic Scoliosis[END_REF][START_REF] Abelin-Genevois | Sagittal classification in adolescent idiopathic scoliosis: original description and therapeutic implications[END_REF][START_REF] Vialle | Radiographic analysis of the sagittal alignment and balance of the spine in asymptomatic subjects[END_REF]. When performing surgery to correct AIS, an important goal is to restore a degree of TK consistent with optimal sagittal alignment.

Most surgeons use their personal experience to contour the rods during surgery, without first determining the desired amount of kyphosis or measuring the angle created in the rod.

The use of rods that are industrially pre-bent according to patient-specific data has been found effective in correcting spinal deformities in adults [START_REF] Solla | Patient specific rods for surgical correction of sagittal imbalance in adults: technical aspects and preliminary results[END_REF]. We suggest using patient-specific rods for AIS surgery, with the goal of obtaining a kyphotic angle similar to the planned value, thereby optimising post-operative sagittal alignment. Other expected advantages are a decrease in operative time and better mechanical properties of machine-contoured rods [START_REF] Solla | Patient specific rods for surgical correction of sagittal imbalance in adults: technical aspects and preliminary results[END_REF].

The objective of this study was to assess the preliminary outcomes of posterior fusion for AIS using patient-specific rods that were pre-contoured based on pelvic incidence. The primary evaluation criterion was the kyphotic angle of the instrumented thoracic segment at last follow-up (postopKA-ITS). Associations linking TK at last follow-up to other angle values and quality of life were also evaluated. The working hypothesis was that the use of patient-specific rods has a favourable effect on postopKA-ITS and, therefore, on overall TK at last follow-up.

2) MATERIAL AND METHODS

2.1) Ethics

All procedures performed for the study complied with the ethical requirements set forth in the 1964 Declaration of Helsinki and its subsequent amendments. The study was approved by the appropriate ethics committee (#14.009). Written informed consent was obtained from all patients and parents.

2.2) Patients

Between 2015 and 2017, two surgeons (JLC and FS) prospectively included patients scheduled for AIS surgery who met the following criteria: instrumentation including at least the T4-T11 segment, use of machine-bent patient-specific rods, availability of a comprehensive radiographic workup including standing full-spine antero-posterior and lateral views and bending views, reduction by simultaneous translation on two rods with a high anchor density [START_REF] Clement | Restoration of thoracic kyphosis by posterior instrumentation in adolescent idiopathic scoliosis: comparative radiographic analysis of two methods of reduction[END_REF][START_REF] Solla | Adolescent Idiopathic Scoliosis exceeding 70°: a single center surgical experience[END_REF][START_REF] Allia | Influence of Derotation Connectors on 3D Surgical Correction of Adolescent Idiopathic Scoliosis[END_REF] and hybrid constructs (pedicle screws, claw hooks and, in some cases, sublaminar bands), and follow-up of at least 1 year. Patients with anterior release or halo traction before posterior fusion were excluded. We also excluded patients with Lenke 5 AIS, who were managed by selective fusion excluding mid-thoracic spine [START_REF] Solla | Prognostic role of rib hump in overlying thoracic curve correction above selective fusion for Lenke 5 idiopathic adolescent scoliosis[END_REF].

2.3) Patient subgroups

The pre-operative kyphotic angle of the instrumented thoracic segment (preopKA-ITS), between the proximal and distal thoracic vertebras included in the instrumentation, was measured (Figure 1). Thus, the patients were divided into two groups based on whether preopKA-ITS was normal or decreased. A value ≤20° was taken to define hypokyphosis and a value >20° normal kyphosis [START_REF] Clement | Relationship between thoracic hypokyphosis, lumbar lordosis and sagittal pelvic parameters in adolescent idiopathic scoliosis[END_REF] (Table 1).

2.4) Surgical planning

When determining the limits of the instrumentation, Lenke's criteria were applied to determine which curves required instrumentation and Groupe d'Étude de la Scoliose (French Scoliosis Study Group) criteria to select the end vertebrae [START_REF] Lenke | The Lenke classification of adolescent idiopathic scoliosis: how it organizes curve patterns as a template to perform selective fusions of the spine[END_REF][START_REF] Clément | Five-year outcomes of the First Distal Uninstrumented Vertebra after posterior fusion for Adolescent Idiopathic Scoliosis Lenke 1 or 2[END_REF].

Overall TK was defined as the greatest value of the kyphotic curve between the cranial transition vertebra (at the junction of the TK and cervical lordosis) and the caudal transition vertebra (between the TK and the lumbar lordosis) (Figure 1).

Overall target TK was determined during planning for each patient using pre-operative calibrated radiographs (Figures 2 and3), based on pelvic incidence [START_REF] Legaye | Sagittal plane alignment of the spine and gravity: a radiological and clinical evaluation[END_REF]. Target TK increased with pelvic incidence and ranged from 27° to 44° (Table 2). The pre-operative value of the kyphotic curve was not considered when determining the target TK.

The instrumented thoracic segment did not necessarily include the entire kyphotic curve (which usually extends from T2 to T11). For instance, if the instrumentation extended from T4 to T11, only about 8/10 th of the kyphotic region were instrumented. This fraction of the entire TK served to determine the fraction of the target TK related to the spine segment to be instrumented which was therefore used as the rod contouring angle on the convex side (cvRCA). Thus, the rod contouring angle (RCA) was formed by the proximal end of the construct and either the distal end of the rod, if this last was applied to T11, or the transition point between the anteriorly concave curve and the anteriorly convex curve i.e., the theoretical position of the T12 implant (Figures 1 and2). We elected to increase this angle by 10° for the concave rod to compensate for any flattening it might experience during the corrective manoeuvres; thus, the concave RCA (ccRCA) was equal to cvRCA + 10° (Figures 3 and4) [START_REF] Naveaux | 3D rod shape changes in adolescent idiopathic scoliosis instrumentation: how much does it impact correction?[END_REF].

The target lumbar lordosis value was computed as the pelvic incidence plus 10°. Twothirds of this value were attributed to the L4-S1 segment and the remaining third to the L1-L4 segment. Lumbar lordosis was then distributed according to the levels to be instrumented. Lumbar lordosis results were not assessed in this study due to the considerable variation in the distal end of the instrumentation, which ranged from T11 to L4.

Rod length was measured on the antero-posterior radiograph (Figure 2). Cobalt-chromium rods measuring 6 mm in diameter (Medicrea, Rillieux-la Pape, France) were ordered 10-20 days before the scheduled date of the procedure, delivered to the operating room, and implanted with no change in contouring (Figure 3).

2.5) Evaluation criteria

All the radiographic parameters were measured before surgery and at last follow-up by an independent observer (VC) using Keops Analyzer ® software (SMAIO, Lyon, France) [START_REF] Maillot | Reproducibility and repeatability of a new computerized software for sagittal spinopelvic and scoliosis curvature radiologic measurements: Keops(®)[END_REF].

PreopKA-ITS is defined above; for instance, for T3-L2 instrumentation, preopKA-ITS was measured between T3 and T12. The cvRCA was defined as the anteriorly concave angle of the rod on the convex side. For example, when planning T3-L2 instrumentation, the cvRCA was measured between the proximal end of the construct (i.e., the T3 implant) and the theoretical position of the T12 implant. Postoperative kyphosis of the instrumented thoracic segment (postopKA-ITS) was defined as the kyphotic angle measured at last follow-up between the proximal and distal instrumented thoracic vertebrae (Figures 1 and4). We were able to compare these three angles (preopKA-ITS, cvRCA, and postopKA-ITS) since they were measured on the same segments in each patient.

The expected gain was defined as the difference between the cvRCA and the preopKA-ITS. The actual gain was defined as the difference between the preopKA-ITS and the postopKA-ITS. The difference between the expected and actual gains was computed and used to define three outcome categories: optimal outcome, difference ±5°; undercorrection, difference <-5°; and overcorrection, difference >+5°.

2.6) Secondary evaluation criteria

T4-T12 TK, overall TK, and the coronal Cobb angle of the main curve were measured before surgery and at last follow-up. Proximal junctional kyphosis (PJK) was sought [START_REF] Kim | Proximal junctional kyphosis in adolescent idiopathic scoliosis after 3 different types of posterior segmental spinal instrumentation and fusions: incidence and risk factor analysis of 410 cases[END_REF].

Finally, the patients completed the SRS-22 quality-of-life questionnaire before surgery and at last follow-up.

2.7) Statistical analysis

The statistical analyses were performed using XLStat (Addinsoft, Paris, France). Mean values were compared using the Wilcoxon test, Student's test, or analysis of variance.

Fisher's exact test was applied to compare rates. Pearson's test was used to assess correlations. Multivariate linear regression analyses were performed. Regression coefficients are reported with their 95% confidence intervals (95%CIs) [START_REF] Solla | Why a p-value is not enough[END_REF]. Values of p smaller than 0.05 were considered significant.

RESULTS

3.1) Patients and procedures

Of 38 patients with AIS whose instrumentation included T4-T11, 1 was lost to followup and the remaining 37 were included in the study. Outcomes were analysed after at least 1 year of follow-up (Table 1). Of the 37 patients, 20 had normal TK and 17 hypokyphosis before surgery. Among anchors used in the study population, 85% were polyaxial pedicle screws, 11% were double hook claws, and 4% were sublaminar bands (Figures 1 and4).

3.2) Thoracic kyphosis (TK)

In the overall cohort, the mean actual gain (difference between postopKA-ITS and preopKA-ITS) was 14° and was equal to the mean expected gain. Mean postopKA-ITS was comparable to the mean cvRCA (mean difference=0, p=0.85, Cohen's d=0.047) (Table 2). However, 12 patients had an optimal outcome, 12 were undercorrected, and 13 were overcorrected (Table 2).

In the subgroup with pre-operative hypokyphosis, the mean actual gain was 20° for an expected gain of 25°. Of the 17 patients in this subgroup, 10 were undercorrected. In the subgroup with normal pre-operative kyphosis, the mean actual gain was 8° for an expected gain of 4°. In this subgroup, 11 were overcorrected.

Factors associated with postopKA-ITS by multivariate analysis were the preopKA-ITS (regression coefficient, 0.72; 95%CI, 0.497-0,937; p=E-5) and ccRCA (regression coefficient, 0.27; 95%CI, 0.005-0.540; p=0.04). The difference between the postopKA-ITS and the cvRCA correlated closely with the expected gain (correlation coefficient, 0.74; p=E-7).

Factors associated with this difference by multivariate linear regression were the preopKA-ITS (correlation coefficient=0.41, p=0.002) and expected gain (correlation coefficient=0.62, p=E-5).

3.3) Other results

Mean coronal correction was 74% (Table 1). None of the coronal parameters (Cobb angles, flexibility, correction) correlated with the TK parameters (all correlation coefficients <0.10). No patient developed PJK. The coronal and sagittal angles measured at last follow-up showed no differences according to the Lenke classification (p>0.1). Finally, the SRS-22 score increase showed weak correlations with actual gain, postopKA-ITS, and coronal correction (correlation coefficients <0.20).

DISCUSSION

This study assessed outcomes of patients with AIS managed using patient-specific rods that were pre-contoured according to a protocol based on pelvic incidence. The results confirm the working hypothesis that postopKA-ITS was favourably influenced by patientspecific pre-contouring of the rods, notably the concave rod, and was also associated with preoperative TK.

Overall TK at last follow-up was normal in all patients. Mean postopKA-ITS was not very different from the cvRCA. At last follow-up, postopKA-ITS was within the planned range in one-third of patients, overcorrected in another third, and undercorrected in the last third. The cvRCA value was the same in the subgroups with normokyphosis and hypokyphosis because it was determined based on pelvic incidence and not on pre-operative TK, which is a component of the scoliotic deformity [START_REF] Legaye | Sagittal plane alignment of the spine and gravity: a radiological and clinical evaluation[END_REF]. The expected gain was therefore greater in the subgroup with hypokyphosis than in the subgroup with normal TK. The clinical and radiological outcomes of this strategy require evaluation in the longer term.

In the hypokyphotic subgroup, the large TK increase of 20° on average is consistent with earlier studies of the same correction technique [START_REF] Clement | Surgical Increase of Thoracic Kyphosis Increases Unfused Lumbar Lordosis in Selective Fusion for Thoracic Adolescent Idiopathic Scoliosis[END_REF][START_REF] Clement | Restoration of thoracic kyphosis by posterior instrumentation in adolescent idiopathic scoliosis: comparative radiographic analysis of two methods of reduction[END_REF][START_REF] Allia | Influence of Derotation Connectors on 3D Surgical Correction of Adolescent Idiopathic Scoliosis[END_REF]. Nevertheless, the postopKA-ITS values indicated mean undercorrection of 5° compared to cvRCA and 15° compared to the ccRCA. These results indicate 'loss' of sagittal correction related to rod flattening during correction due to the stiffness of the spine. Clear evidence of concave rod flattening during translation manoeuvres has been reported elsewhere [START_REF] Naveaux | 3D rod shape changes in adolescent idiopathic scoliosis instrumentation: how much does it impact correction?[END_REF]. In the subgroup with normal pre-opKA-ITS, the postopKA-ITS indicated 4° of mean overcorrection compared to cvRCA. These findings suggest that the practice of overbending the concave rod by 10° (or more) should be continued for patients with hypokyphosis but not for patients with normal TK.

Overbending tailored to the individual patient may also deserve consideration; for instance, overbending by 5° might be helpful in patients with moderate hypokyphosis.

Our decision to measure the degree of TK of the instrumented vertebral segment (preop and postopKA-ITS) allowed us to assess the effect of the pre-contoured rods (RCA) on the instrumented spine. PreopKA-ITS is not necessarily equal to overall TK or to TK between T4-5 and T12, which is the value often reported in the literature [START_REF] Suk | Comparison of Cotrel-Dubousset pedicle screws and hooks in the treatment of idiopathic scoliosis[END_REF][START_REF] Kim | Proximal junctional kyphosis in adolescent idiopathic scoliosis after 3 different types of posterior segmental spinal instrumentation and fusions: incidence and risk factor analysis of 410 cases[END_REF][START_REF] Suk | Restoration of thoracic kyphosis in the hypokyphotic spine: a comparison between multiple-hook and segmental pedicle screw fixation in adolescent idiopathic scoliosis[END_REF][START_REF] Lenke | The Lenke classification of adolescent idiopathic scoliosis: how it organizes curve patterns as a template to perform selective fusions of the spine[END_REF]. When planning the RCA, an important consideration is that the instrumented thoracic spine includes only a fraction of the overall TK, whose size varies with the number of instrumented thoracic levels.

The coronal parameters and quality-of-life data were in accordance with earlier reports [START_REF] Angelliaume | Titanium vs cobalt chromium: what is the best rod material to enhance adolescent idiopathic scoliosis correction with sublaminar bands?[END_REF][START_REF] Suk | Restoration of thoracic kyphosis in the hypokyphotic spine: a comparison between multiple-hook and segmental pedicle screw fixation in adolescent idiopathic scoliosis[END_REF][START_REF] Clément | Five-year outcomes of the First Distal Uninstrumented Vertebra after posterior fusion for Adolescent Idiopathic Scoliosis Lenke 1 or 2[END_REF] and showed no correlations with the sagittal parameters. The trend toward less coronal correction in the hypokyphotic subgroup suggests that 3D correction may be more challenging to achieve in patients with hypokyphosis.

The limitations of this study include the short follow-up, small sample size, and absence of a comparison group. Our preliminary results pertain only to simultaneous translation on two 6-mm cobalt-chrome rods. They cannot be directly extrapolated to other techniques and materials. Strong points of this study are the original design, prospective recruitment, uniform surgical technique, low patient attrition rate, and standardised evaluation of the radiographs by an independent observer.

CONCLUSION

The use of patient-specific rods provided postopKA-ITS values that were similar to the cvRCA values. Predictors of postopKA-ITS at last follow-up were not only the amount of concave rod pre-contouring but also the degree of pre-operative KA-ITS .

In patients with pre-operative hypokyphosis, kyphosis at last follow-up indicated undercorrection, which was partially offset by 10° of concave rod overbending. In contrast, in the patients with pre-operative normokyphosis , overcorrection of the kyphosis occurred, probably as a result of the overbending of the concave rod. Therefore, concave rod overbending would seem unnecessary when TK is normal before surgery.

Disclosure of interest

FS has received funding for symposia from Medicrea International.

VC has received funding for symposia from Medicrea International and Synthes.

JLC is a consultant for Medicrea International.

VF is a consultant for Medicrea International and Clariance.

CMB and VR have no conflicts of interest to declare.

Funding

None

Contributions of each author

FS conceived the study, performed the statistical analyses, drafted the manuscript, and created the tables.

VC measured the radiographic parameters, contributed to conceive the study, and revised the manuscript.

VF created the patient-specific rod concept, contributed to conceive the study, and revised the manuscript.

VR contributed to conceive the study, and revised the manuscript.

CMB contributed to collect and analyse the data, contributed to patient monitoring, and revised the final manuscript.

Figure 1 :

 1 Figure 1: Patient #1, measurement of the kyphotic angles on the lateral radiographs taken

Figure 2 :

 2 Figure 2: Patient #2, pre-operative radiograph and planning for T4-L2 fusion

Figure 3 :

 3 Figure 3: Pre-bent rods prepared for patient 2

Figure 4 :

 4 Figure 4: Patient #2, antero-posterior and lateral radiographs 2 years after surgery ( hybrid

Figure 1

 1 Figure 1:

Table 1 :

 1 Main patient features

	Males/Females, n

Table 2 :

 2 Measured angles in the overall cohort and in the subgroups with pre-operative hypokyphosis (H) and normal kyphosis (N); the data are mean (range) Measured gain: postopKA-ITS minus preopKA-ITS c Outcome distribution: overcorrection (difference ≥5°); optimal correction (difference between -5° and +5°), and undercorrection (difference <-5°)

	The p values <0.05 are in bold type.
	H, subgroup with hypokyphosis before surgery; N, subgroup with normal kyphosis before
	surgery; preopKA-ITS, pre-operative kyphotic angle of the thoracic segment to be
	instrumented (before surgery); cvRCA, convex side rod contouring angle (equal to the amount
	of kyphosis planned for the thoracic segment to be instrumented); postopKA-ITS, post-
	operative kyphotic angle of the instrumented thoracic segment (at last follow-up)

b


MD, MSc Elie Rassy
email: elie.rassy@hotmail.com

MD, MSc Ziad Bakouny

MD Toni K Choueiri

MD Eliezer M Van Allen

MD Karim Fizazi

MD F Anthony Greco

MD Nicholas Pavlidis

The role of site-specific therapy for cancers of unknown of primary: A meta-analysis

Keywords: Cancer of unknown primary, Tailored therapy, Site-specific, Empiric

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

Current Perspectives

Cancers of unknown primary (CUP) are a heterogeneous group of metastatic tumors, for which a primary site origin cannot be identified despite a standardized diagnostic work-up [START_REF] Fizazi | Cancers of unknown primary site: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF] .

It accounts for 1-2% of all malignancies and ranks as the seventh cause of mortality in patients with cancer [START_REF] Rassy | The currently declining incidence of cancer of unknown primary[END_REF] . Largely, two subsets of patients can be identified based on clinicopathologic criteria [START_REF] Pavlidis | Cancer of unknown primary site[END_REF] . The first encloses a minority of patients (15-20%) that is chemosensitive and has an overall favorable prognosis [START_REF] Pavlidis | Cancer of unknown primary site[END_REF] . The second subset includes the larger category of patients (80-85%) that cannot be assigned to a primary tumor and commonly present a dismal prognosis with empiric CUP chemotherapy regimens [START_REF] Pavlidis | Forty years experience of treating cancer of unknown primary[END_REF] . The efficacy of site-specific treatments in the favorable subsets of patients with CUP makes compelling evidence to assess tailored therapy in patients within the unfavorable subsets [START_REF] Daud | Removing the Unknown From the Carcinoma of Unknown Primary[END_REF] .

Moreover, case reports and series had suggested that the use of tailored therapies in the unfavorable subset leads to favorable clinical outcomes [START_REF] Rassy | The current evidence for a biomarker-based approach in cancer of unknown primary [Internet[END_REF][START_REF] Gross-Goupil | Identifying the primary site using gene expression profiling in patients with carcinoma of an unknown primary (CUP): a feasibility study from the GEFCAPI[END_REF] .

Two prospective studies have reported on the outcomes of site-specific therapy in patients with CUP. The largest is a subset of 194 patients that had both sufficient tumor material for the assay and received assay-directed therapy. This subset was compared to a retrospective cohort from the same consortium that had been treated by empiric chemotherapy, and were found to have significantly improved overall survival (OS; hazard ratio of 0.63 [95% CI 0.60-0.66]; hazard ratio unpublished -obtained from authors) [START_REF] Hainsworth | Molecular gene expression profiling to predict the tissue of origin and direct site-specific therapy in patients with carcinoma of unknown primary site: a prospective trial of the Sarah Cannon research institute[END_REF] . In another study of 216 patients with CUP, 188 (87%) had had their primary tumors identified using a microarraybased DNA methylation signatures [START_REF] Moran | Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis[END_REF] . Patients treated with tailored therapy (n = 31) achieved an OS benefit in comparison to those treated with empiric CUP regimens (n = 61) with a hazard ratio (HR) of 0.31 (95% CI 0.14-0.70) [START_REF] Moran | Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis[END_REF] .

The efficacy of site-specific therapy was further addressed in two randomized controlled trials enrolling patients with CUP into tailored therapy or empiric CUP regimen arms. The first is a phase II study comparing molecularly-driven site-specific therapy and the combination of paclitaxel plus carboplatin [START_REF] Hayashi | Randomized Phase II Trial Comparing Site-Specific Treatment Based on Gene Expression Profiling With Carboplatin and Paclitaxel for Patients With Cancer of Unknown Primary Site[END_REF] . The two treatment arms showed similar OS (HR = 1.03; 95% CI 0.68-1.56) and progression free survival (PFS; HR = 0.88; 95% CI 0.59-1.33) [START_REF] Hayashi | Randomized Phase II Trial Comparing Site-Specific Treatment Based on Gene Expression Profiling With Carboplatin and Paclitaxel for Patients With Cancer of Unknown Primary Site[END_REF] .

The second study is a phase III trial that randomized 243 patients into tailored therapy or the combination of cisplatin plus gemcitabine [START_REF] Fizazi | LBA15_PRA phase III trial of empiric chemotherapy with cisplatin and gemcitabine or systemic treatment tailored by molecular gene expression analysis in patients with carcinomas of an unknown primary (CUP) site (GEFCAPI 04) [Internet][END_REF] . No statistically significant differences in PFS (HR = 0.95; 95% CI 0.72-1.25) or OS (HR = 0.92; 95% CI 0.69-1.23) were found between the two treatments arms [START_REF] Fizazi | LBA15_PRA phase III trial of empiric chemotherapy with cisplatin and gemcitabine or systemic treatment tailored by molecular gene expression analysis in patients with carcinomas of an unknown primary (CUP) site (GEFCAPI 04) [Internet][END_REF] . Given these discordant results, we performed a meta-analysis of these studies while taking into account the inherent heterogeneity of the included studies.

MEDLINE and conference abstracts of American Society of Clinical Oncology (ASCO) and

European Society of Medical Oncology (ESMO) meetings were searched from inception until November 2019 for studies evaluating molecularly tailored therapies for CUP. All prospective studies comparing molecularly tailored to empiric therapies for CUP were included. All data were abstracted by two authors independently (ER and ZB). The meta-analysis was performed using a random-effects model and the inverse variance method in order to account for possible heterogeneity in treatment effect due to differences in the molecular tests used, study design, and the included patient populations in the four studies. A subgroup analysis by study design (randomized vs. non-randomized) was performed in order to better account for this factor. The primary comparison was that of tailored versus empiric therapies for CUP. The primary endpoint was OS and the secondary endpoint was PFS. All analyses were done using the Review Manager software, version 5.3 (The Nordic Cochrane Center, The Cochrane Collaboration, Copenhagen, Denmark). The threshold for statistical significance for the tests of overall effect and subgroup comparison was 0.05 and that for the tests of heterogeneity was 0.10. The I 2 coefficient was used to quantify the degree of heterogeneity within and between subgroups. All hypothesis tests were tests were formulated prior to data collection.

The pooled analysis included 628 patients treated empirically and 398 treated by tailored therapies. For the primary outcome, a non-statistically significant trend towards improved OS was found with tailored versus empiric treatment for CUP (Figure 1; HR = 0.73; 95% CI 0.52-1.02). Within the non-randomized subgroup (N= 225 tailored and 457 empiric), the effect was statistically significant (HR = 0.56; 95% CI 0.26-0.96) but there was significant heterogeneity between the two included studies (I 2 = 65%; p=0.09). Within the randomized controlled trials subgroup (N= 173 tailored and 171 empiric), the effect was not statistically significant (HR = 0.95; 95% CI 0.75-1.21). There was also significant heterogeneity across the 4 studies (I 2 = 79%; p=0.002) but no significant difference between the treatment effect in the two subgroups (randomized vs. non-randomized; p=0.07). The test for overall effect for PFS, which had only been reported for the two randomized studies, was not statistically significant (HR = 0.93; 95% CI 0.74-1.17), with little heterogeneity between studies (I 2 = 0%; p=0.77).

The results of this meta-analysis highlight the significant heterogeneity between the prospective studies comparing molecularly tailored to empiric therapy for CUP. The possible sources of heterogeneity between studies include the study design (randomized vs. nonrandomized), the type of molecular testing performed to identify the primary site of origin (methylation vs. RNA-based), the types of systemic therapies included in the tailored and empiric therapy arms, and the breakdown of expected primary sites (and how sensitive to tailored and empiric therapies they are expected to be) [START_REF] Hainsworth | Molecular gene expression profiling to predict the tissue of origin and direct site-specific therapy in patients with carcinoma of unknown primary site: a prospective trial of the Sarah Cannon research institute[END_REF][START_REF] Moran | Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis[END_REF][START_REF] Hayashi | Randomized Phase II Trial Comparing Site-Specific Treatment Based on Gene Expression Profiling With Carboplatin and Paclitaxel for Patients With Cancer of Unknown Primary Site[END_REF][START_REF] Fizazi | LBA15_PRA phase III trial of empiric chemotherapy with cisplatin and gemcitabine or systemic treatment tailored by molecular gene expression analysis in patients with carcinomas of an unknown primary (CUP) site (GEFCAPI 04) [Internet][END_REF] . While no overall statistically significant effect in favor of tailored therapies was found in this study, certain selected subgroups of patients may still benefit from this approach. The study by Moran et al. had the lowest point estimate of the HR among all the included studies [START_REF] Moran | Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis[END_REF] . One of the main differences between this study and the other included studies was that the assay used to infer the primary tumor type was DNA methylation-based (as opposed to the others which were all RNA-based) [START_REF] Moran | Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis[END_REF] . Another potential difference between this study and the three other studies was the breakdown of the predicted primary tumors, whereby the most frequent predicted primary was breast carcinoma in the study by Moran et al. as opposed to pancreaticobiliary carcinoma which is generally associated with a poor prognosis in each of the three other studies [START_REF] Hainsworth | Molecular gene expression profiling to predict the tissue of origin and direct site-specific therapy in patients with carcinoma of unknown primary site: a prospective trial of the Sarah Cannon research institute[END_REF][START_REF] Moran | Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis[END_REF][START_REF] Hayashi | Randomized Phase II Trial Comparing Site-Specific Treatment Based on Gene Expression Profiling With Carboplatin and Paclitaxel for Patients With Cancer of Unknown Primary Site[END_REF][START_REF] Fizazi | LBA15_PRA phase III trial of empiric chemotherapy with cisplatin and gemcitabine or systemic treatment tailored by molecular gene expression analysis in patients with carcinomas of an unknown primary (CUP) site (GEFCAPI 04) [Internet][END_REF] . Moreover, certain patient subgroups did not receive the therapies that constituted the standard of care at the time of enrollment of these studies such as immune checkpoint inhibitors and novel targeted therapies for kidney cancer, melanoma, and non-small cell lung cancer. Yet, the GEFCAPI04 trial showed that the subgroup of predicted primary tumors that are known to be unresponsive to empiric CUP regimens (such as kidney cancer, melanoma, and colorectal carcinoma) had a trend towards improved OS with tailored therapies (2-year estimate of 24.3% vs. 10.4% and HR= 0.74; 95% CI 0.36-1.51), although the study had been underpowered to detect such an effect 11 . Finally, the population of CUP patients enrolled into clinical trials and diagnosed in clinical practice has changed substantially in recent years due to improved immunohistochemistry-based diagnostics. This has restricted the definition of CUP to more select cancer types, for which specific protein markers are not readily available (such as squamous cell and pancreato-biliary tumors) and limits the applicability of the data generated from the earlier periods of enrollment of all the included trials. This meta-analysis highlights the difficulty of evaluating the role of tailored therapies in patients with CUP. While the overall effect of tailored compared to empiric therapies was negative, subgroup analyses of the primary studies and the results of this analysis suggest that selected patients may benefit from such an approach, site-specific therapy for all comers cannot be recommended at this time. The differences in design of the studies and the small sample sizes limit the conclusions of the present analysis. Future randomized controlled trials that use standardized diagnostic tests and the contemporaneous standard of care for patients with identified primaries, particularly in the more responsive cancer types, are highly needed to accurately address the question of tailored therapies for CUP.

  Ervaxx, Microsoft; Travel reimbursement: Roche/Genentech; Patents: Institutional patents filed on ERCC2 mutations and chemotherapy response, chromatin mutations and immunotherapy response, and methods for clinical interpretation. KF: Participation to advisory boards/honorarium for : AMGEN, ASTELLAS, BAYER, JANSSEN, TAKEDA, SANOFI, ORION, ESSA, GENENTECH, ASTRAZENECA, CLOVIS AG: Speaker's Bureau and medical advisor for Biotheranostics, Inc.

Figure 1A :

 1A Figure 1A: Hazard ratio for PFS (95% CI) in CUP trials comparing tailored and empiric chemotherapy regimens

Figure 1B :

 1B Figure 1B: Hazard ratio for OS (95% CI) in CUP trials comparing tailored and empiric chemotherapy regimens

Acknowledgement

None

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

Conflicts of interest

ER, ZB, NP: None declared T.K.C: Research (Institutional and personal): AstraZeneca, Alexion, Bayer, Bristol Myers-Squibb/ER Squibb and sons LLC, Cerulean, Eisai, Foundation Medicine Inc., Exelixis, Ipsen, Tracon, Genentech, Roche, Roche Products Limited, F. Hoffmann-La Roche, GlaxoSmithKline,


Sengxeu Noudy -(

Jost Jeremy

Differences in knowledge about epilepsy and antiepileptic drugs among pharmacy-dispensing workers in Cambodia and in Lao PDR

Keywords: knowledge, epilepsy, antiepileptic drugs, pharmacy-dispensing workers, Cambodia, Lao PDR

Introduction

Epilepsy is the most common neurological disorder encountered in primary care settings in Southeast Asia [START_REF]Atlas: country resources for neurological disorders[END_REF]. Treatment for this condition is frequently managed by primary care physicians and nurses due to the lack of specialty care services and neurologists [START_REF]Atlas: country resources for neurological disorders[END_REF][START_REF] Trinka | Epilepsy in Asia: Disease burden, management barriers, and challenges[END_REF].

People with epilepsy (PWE) require long-term antiepileptic drug (AED) therapy. The goal of medication is to balance process between seizure control and tolerance [START_REF] Cascino | Epilepsy: contemporary perspectives on evaluation and treatment[END_REF]. The firstgeneration AEDs (sodium valproate, carbamazepine, phenytoin and phenobarbital) are documented as the main AEDs available in Southeast Asia [START_REF] Mac | Epidemiology, aetiology, and clinical management of epilepsy in Asia: a systematic review[END_REF]. They have several side effects, drug-drug interactions (DDI) such as decreasing efficacy or increasing side effects of comedications [START_REF] Diaz | Antiepileptic drug interactions[END_REF]. Women with epilepsy are a specific population that needs counseling and comprehensive information about issues related to contraception and the potential teratogenicity of epilepsy and AEDs [START_REF] May | Fears, knowledge, and need of counseling for women with epilepsy. Results of an outpatient study[END_REF]. These unmanaged drug-related adverse events are well known to be one of the leading causes of treatment discontinuation by patients themselves [START_REF] Liu | Medication education for patients with epilepsy in Taiwan[END_REF]. Furthermore, studies have demonstrated that PWE do not receive enough information about epilepsy and its treatment [START_REF] Couldridge | A systematic overviewa decade of research'. The information and counselling needs of people with epilepsy[END_REF][START_REF] Jain | What people with epilepsy want from a hospital clinic[END_REF]. Non-adherence to AEDs has been identified as a major factor in the suboptimal control of epilepsy. However, treatment failure due to DDI or avoidable side effects and lack of information is often underestimated.

Pharmacies, public and private, offer to patients a first-line point of contact with the healthcare system. Pharmacists and/or pharmacy-dispensing workers remain some of the most accessible healthcare professionals. They are an important way of providing medicines, counseling, knowledge and advice on the safe and effective use of drugs [START_REF] Mcauley | Assessing the needs of pharmacists and physicians in caring for patients with epilepsy[END_REF][START_REF] Yang | Quality of Pharmaceutical Items Available from Drugstores in Phnom Penh, Cambodia[END_REF][START_REF] Bunting | The Asheville Project: long-term clinical, humanistic, and economic outcomes of a community-based medication therapy management program for asthma[END_REF]. A study has demonstrated the positive benefits for patients of pharmacist AED therapy management, including a significant improvement in adherence through therapeutic patient education.

These interventions have shown a decrease in health expenses by significantly reducing avoidable hospital admissions [START_REF] Koshy | Role of pharmacists in the management of patients with epilepsy[END_REF]. In a study aimed at assessing perceptions of community pharmacist's and their potential role, PWE have expressed their interest in having their pharmacist become more involved in their health care and communicate more with their doctor, especially with regard to DDI and adverse reactions [START_REF] Mcauley | Patients with epilepsy's perception on community pharmacist's current and potential role in their care[END_REF]. Despite the important role of pharmacists, knowledge gaps in AED therapy among pharmacists were reported [START_REF] Shawahna | Pharmacists' knowledge of issues in pharmacotherapy of epilepsy using antiepileptic drugs: A crosssectional study in Palestinian pharmacy practice[END_REF].

Likewise, pharmacists or pharmacy-dispensing workers were poorly cited as a source of medication information [START_REF] Liu | Medication education for patients with epilepsy in Taiwan[END_REF].

In Southeast Asia, Cambodia and the Lao People's Democratic Republic (Lao PDR) are resource-limited settings and have a prevalence of epilepsy of 5.8 and 7.7/1000 respectively [START_REF] Chongsuvivatwong | Health and health-care systems in southeast Asia: diversity and transitions[END_REF][START_REF] Preux | First-ever, door-to-door cross-sectional representative study in Prey Veng province (Cambodia)[END_REF][START_REF] Tran | Prevalence of epilepsy in a rural district of central Lao PDR[END_REF]. In these countries, the epilepsy treatment gap (defined as the number of people with a condition or disease who need treatment for it but who do not get it [START_REF] Kale | The treatment gap[END_REF]) was estimated to be 65.8% in Cambodia and 90.0% in Lao PDR [START_REF] Meinardi | On Behalf Of The Ilae Commission on the Developing World JS. The treatment gap in epilepsy: the current situation and ways forward[END_REF][START_REF] Chisholm | Cost-effectiveness of first-line antiepileptic drug treatments in the developing world: a population-level analysis[END_REF][22]. Epilepsy is included in the mental health programs of these two countries. The initial national program for mental health was established in 2000 in Cambodia and in 2007 in Lao PDR. This program provides community mental health outreach and training of physicians and nurses in the public sector.

However, pharmacists and other health professionals who dispense AEDs to PWE were not included in the program [22][START_REF] Whoroftw | The Kingdom of Cambodia health system review. Manila : WHO Regional Office for the Western Pacific[END_REF][START_REF] Whoroftw | The Lao People's Democratic Republic health system review. Manila : WHO Regional Office for the Western Pacific[END_REF].

Many pharmacies in these countries operate with limited or non-qualified human resources that can lead to insufficient knowledge, inappropriate supply of medicines and insufficient counseling [START_REF] Smith | The quality of private pharmacy services in low and middle-income countries: a systematic review[END_REF]. In general, studies on dispensing practices in pharmacies in both Cambodia and Lao PDR have reported that the provision of drug use information was inadequate. Patients had little opportunity to obtain adequate information about their treatment: a quarter of patients do not know how to take their drugs [START_REF] Keohavong | Rational use of drugs: prescribing and dispensing practices at public health facilities in Lao PDR[END_REF]. In addition, a lack of properly packaged and labeled drugs was documented. The inadequate information provided in pharmacies may be due to a lack knowledge among pharmacists or pharmacy-dispensing workers [START_REF] Keohavong | Rational use of drugs: prescribing and dispensing practices at public health facilities in Lao PDR[END_REF][START_REF] Chareonkul | Rational drug use in Cambodia: study of three pilot health centers in Kampong Thom Province[END_REF]. Furthermore, poor knowledge of epilepsy and AEDs among nurses and physicians was documented in Lao PDR [START_REF] Harimanana | Is insufficient knowledge of epilepsy the reason for low levels of healthcare in the Lao PDR?[END_REF]. To the best of our knowledge, no study has been performed concerning epilepsy knowledge among any type of healthcare professionals in Cambodia. The aim of this study was to describe and compare the qualification and knowledge concerning epilepsy and AEDs among pharmacy-dispensing workers in Cambodia and Lao PDR.

Methodology

Identification of health facilities in Cambodia and Lao PDR

Healthcare facilities in Cambodia and Lao PDR are organized in two sectors: public and private. In the public sector, there are four levels of organization: central/national hospitals, provincial hospitals, district hospitals and health centers. Health centers are often staffed by medical assistants, nurses and/or midwifes with low to mid-level qualifications and non-physician staff. The private sector is composed of private hospitals, clinics and pharmacies. In both countries, most of the private facilities are owned by public health staff having a dual practice. There is no clear definition to distinguish private 'clinics' and private 'hospitals'. The use of the name 'hospital' is preferred, probably for marketing purposes.

These private facilities are mostly located in the capital city [START_REF] Whoroftw | The Kingdom of Cambodia health system review. Manila : WHO Regional Office for the Western Pacific[END_REF][START_REF] Whoroftw | The Lao People's Democratic Republic health system review. Manila : WHO Regional Office for the Western Pacific[END_REF]. Private pharmacies are classified in three categories for both Cambodia and Lao PDR, according to the qualification of the license:

-Level I: managed by the pharmacist (5 years of university education); -Level II: run by a pharmacist assistant (3 years of university education); -Level III: run by any healthcare professional, mostly nurses or midwifes.

In Lao PDR, only level I and II are authorized to sell antiepileptic drugs. Conversely, in Cambodia, all registered private pharmacies are authorized to sell all registered pharmaceutical drugs including AEDs [START_REF] Yang | Quality of Pharmaceutical Items Available from Drugstores in Phnom Penh, Cambodia[END_REF][START_REF] Harimanana | Is insufficient knowledge of epilepsy the reason for low levels of healthcare in the Lao PDR?[END_REF].

Survey setting and sampling strategy

A cross-sectional descriptive study was conducted from February to July 2018 in Lao PDR and Cambodia. The estimated population of Lao PDR was 6.5 million (2015 Population Census) and 13 million for Cambodia (2008 Population Census). Three provinces in each country were selected: i) capital province, ii) the second biggest province after the capital, iii) a province that shares a border with a neighboring country. According to those criteria, six provinces were included in the study (figure 1). Then, in each province, the district with the highest population density was selected as the urban district and the district that had the lowest population density was selected as the rural one.

The urban district had a wide variety of health-care providers, including all levels of public health facilities (central hospital, provincial hospital, district hospital and health centers), private hospitals, clinics and pharmacies. In the rural district, only the district hospital, health centers, pharmacies and a few clinics were present.

Type of outlet included:

In this study, we included the provincial hospitals, district hospitals and pharmacies in the selected area. All types of pharmacy (level I, II, III) in each district were included. In Lao PDR, level III pharmacies are not allowed to sell AEDs, but they were included as almost all the pharmacies in rural areas were level III.

Type of outlet not included: Central/national hospitals, private hospitals and clinics were not included because we did not obtain authorization from the Ministry of Health. Primary health centers were not included in our study because they only dispense first aid drugs [START_REF] Barennes | Children's access to treatment for epilepsy: experience from the Lao People's Democratic Republic[END_REF].

Outlets sampling: This study was performed as an ancillary study of a project aimed at assessing the quality of AEDs in Southeast Asia and the number of outlets was calculated based on this objective. The sampling methodology was adapted from that one used in another project performed in sub-Saharan Africa and published in 2018 [START_REF] Jost | Quality of antiepileptic drugs in sub-Saharan Africa: A study in Gabon, Kenya, and Madagascar[END_REF]. These outlets were considered first to collect AEDs samples and then to evaluated qualification and knowledge about epilepsy and antiepileptic drugs among pharmacy-dispensing workers (health professionals or non-health professionals). A required number of participants or pharmacy-dispensing workers was not calculated as this was a comprehensive sampling of all pharmacy-dispensing workers present at the sampled outlets during the period of the AEDs collection.

Questionnaire

The survey instrument was developed by authors in the English language and translated into the local languages by the first author, native of Lao PDR, and one medical doctor native of Cambodia. Then the questionnaire was back translated into English for accuracy. The questionnaire was pre-tested in Vang Vieng district (160 km north of Vientiane, the capital).

The study team interviewed 4 pharmacy-dispensing workers from the district hospital and 6 pharmacy-dispensing workers from different private pharmacies to assess understanding and ease of use before making general use of the questionnaire.

The questionnaire contained 24-items (supplementary Table S1) and was structured into three sections: (i) professional background and experience with epilepsy, (ii) knowledge of epilepsy (cause, symptoms, diagnosis, etc.), (iii) knowledge of AEDs (DDI, side effects). The questionnaire contained mostly closed-ended questions (yes/no/do not know, and multiplechoice questions).

In each district, the investigators first met with the district authorities to obtain lists of existing registered outlets. A simple randomization was performed with table randomization, and the outlets selected were visited by the investigators and one of the district authority's workers.

All pharmacy-dispensing workers presented in the outlets selected were asked to answer the questionnaires at their workplace. After agreeing to participate and signing a consent form, they completed the paper version of the questionnaire. All pharmacy-dispensing workers at the time of the visit were asked to complete the questionnaire in separate rooms.

Investigators stayed with them at all times to answer any questions they had regarding the questionnaire and to check completion when they finished. It took between 20 and 30 minutes to complete the questionnaire.

Data analysis

Statistical analyses were performed using SPSS version 23.0. The comparative analysis of qualitative variables from Cambodia versus Lao PDR was performed using the Chi-2 tests and Fisher exact tests, while one-way ANOVA was performed for quantitative variables. The significant threshold for all analyses was set at 0.05. To predict the variables associated with an increase chance of having knowledge of at least one long-term AED, a logistic regression was performed separately for Cambodia and Lao PDR. Only variables with a p-value <0.25 after univariate analysis were entered into a multivariate logistic regression model using the backward stepwise exclusion method.

Results

Sociodemographic and experience with epilepsy among pharmacy-dispensing workers

A total of 180 respondents from 123 outlets in both countries were included in this study (42.0% from Cambodia and 58.0% from Lao PDR). The sampling flowchart of outlets and the number of outlets declining to participate was indicated in Figure 2. Overall, more than half (65.0%) of respondents were located in urban areas and were in the private sector. A proportion of 40.8% [START_REF] May | Patients' perspectives on management and barriers of regular antiepileptic drug intake[END_REF] of respondents in Cambodia and 38.5% [START_REF] Njamnshi | Knowledge, attitudes, and practice of Cameroonian medical students and graduating physicians with respect to epilepsy[END_REF] in Lao PDR were pharmacists, followed by sellers who did not received any healthcare training with a proportion of 18.4% [START_REF] Mcauley | Patients with epilepsy's perception on community pharmacist's current and potential role in their care[END_REF] in Cambodia compared to 20.2% [START_REF] Chisholm | Cost-effectiveness of first-line antiepileptic drug treatments in the developing world: a population-level analysis[END_REF] in Lao PDR. All sellers without healthcare qualifications were found in private pharmacies only in both countries. In Lao PDR, 18.2% [START_REF] Kale | The treatment gap[END_REF] of the respondents included were from level lll pharmacies. Overall, respondents had been dispensing medicines for 1 to 42 years. A proportion of 42.0% (75) of respondents reported that they had PWE come to ask for or purchase AEDs in their facility. Details of specific experiences with epilepsy are shown in Table 1.

Sources of information relating to epilepsy

Of all respondents, only one (0.6%) from Lao PDR was not aware of epilepsy. Overall, the significant difference between Cambodia and Lao PDR concerning the sources of information on epilepsy was education at schools and universities (64.5% vs. 47.6%, p=0.024), and mass media or scientific leaflets (44.7% vs.14.6%, p<0.001). Overall, however, only 1.9% (2) of respondents from Lao PDR had received continuous training on epilepsy after the university (table 2).

Knowledge of epilepsy and diagnosis

Epilepsy was considered to be a contagious disease by 6.6% (5) of respondents in Cambodia compared to 18.4% [START_REF] Kale | The treatment gap[END_REF] in Lao PDR (p=0.03) (table 2). Furthermore, 69.7% (53) and 17.5% (18) of respondents, respectively, said that epilepsy could be treated (p<0.001).

Head trauma was cited as the main causes of epilepsy by 72.4% (55) in Cambodia and 27.2% [START_REF] Harimanana | Is insufficient knowledge of epilepsy the reason for low levels of healthcare in the Lao PDR?[END_REF] in Lao PDR (p<0.001), cysticercosis was mentioned by 17.1% (13) vs. 4.9% [START_REF] Diaz | Antiepileptic drug interactions[END_REF] (p=0.01), respectively. Unknown origin was mostly cited by respondents in Lao PDR compared to Cambodia (19.4% (6) vs. 7.9% (20), p=0.03). A proportion of 71.1% (54) in Cambodia said that there was only one type of epileptic seizure (tonic-clonic seizure), while [START_REF] Odermatt | Availability and costs of antiepileptic drugs and quality of phenobarbital in Vientiane municipality, Lao PDR[END_REF].6% [START_REF] Roth | An evaluation of pharmacist knowledge on treatment with antiepileptic drugs[END_REF] said this in Lao PDR (p=0.001). Overall, 41.3% (74) of respondents reported that they did not know how to diagnose epilepsy. Among respondents who knew, electroencephalogram (EEG) was mentioned in 42.1% [START_REF] Lin | Correlation between adherence to antiepileptic drugs and quality of life in patients with epilepsy: A longitudinal study[END_REF] in Cambodia and 9.7% [START_REF] Mcauley | Assessing the needs of pharmacists and physicians in caring for patients with epilepsy[END_REF] in Lao PDR (p<0.001).

Knowledge of antiepileptic drugs

Eighty-seven percent (66) of respondents in Cambodia were aware of at least one long-term AED, while they were 67.3% (70) in Lao PDR (p=0.003). Phenobarbital was mentioned by more than 90.0% in both countries. Conversely, three others major AEDs (carbamazepine, phenytoin, sodium valproate) were cited in Cambodia more than in Lao PDR (57.6% (38) vs.

32.9% (23), p=0.006; 42.4% (28) vs. 18.6% (13), p=0.003, 21.2% (14) vs. 5.7% (4), p=0.01 respectively). More than 40.0% of respondents in both countries knew that PWE have to take AEDs depending on type of their seizure. Overall, 58.1% (79) of respondents said that PWE who started AEDs have to take them for their whole life. From our observation, 15.4% [START_REF] Chisholm | Cost-effectiveness of first-line antiepileptic drug treatments in the developing world: a population-level analysis[END_REF] thought that if seizures are controlled for some months, PWE could stop AEDs, and 20.0% [START_REF] Chareonkul | Rational drug use in Cambodia: study of three pilot health centers in Kampong Thom Province[END_REF] advised women with epilepsy to stop taking AEDs if they become pregnant. Only one respondent from Lao PDR was aware of the fact that enzyme-inducing antiepileptic drugs may reduce the effectiveness of contraceptives. We found that 37.5% (51) did not know any side effect of AEDs (table 3).

Variables to predict knowledge on the treatment of epilepsy

Approximately 13% [START_REF] Mcauley | Assessing the needs of pharmacists and physicians in caring for patients with epilepsy[END_REF] of respondents in Cambodia did not know any long-term AEDs compared to 32.7% [START_REF] Chen | The impact of pharmacist's counseling on pediatric patients' caregiver's knowledge on epilepsy and its treatment in a tertiary hospital[END_REF] of respondents in Lao PDR. After a control of confounding in the final multivariate logistic regression analysis for Cambodia, no significant associated factor was found. For Lao PDR, three factors were found to be significantly associated with an increased chance of knowing at least one AED molecule. These factors were: working in the public sector compared to the private sector (aOR = 6.24, 95%, CI = [2.06-18.87]), seeing an epileptic seizure (aOR = 2.70, 95%, CI = [1.02-7.09]), and having patients with epilepsy come to purchase AEDs at their workplace, (aOR = 7.35, 95%, CI = [2.49-21.69]).

Discussion

Kwan & Brodie reported in 2000 that nearly 60% of PWE could achieve seizure control by adhering to prescribed AEDs, but poor adherence to AEDs was reported in approximately one-third of PWE [START_REF] May | Patients' perspectives on management and barriers of regular antiepileptic drug intake[END_REF][START_REF] Lin | Correlation between adherence to antiepileptic drugs and quality of life in patients with epilepsy: A longitudinal study[END_REF][START_REF] Kwan | Early identification of refractory epilepsy[END_REF]. PWE frequently purchase their AEDs at pharmacies. In this way, pharmacy-dispensing workers play an essential role in providing information on the appropriate use of AEDs and on identifying and managing side effects when dispensing AEDs to PWE. These are important factors that contribute to improving adherence to the medication regimen [START_REF] Chen | The impact of pharmacist's counseling on pediatric patients' caregiver's knowledge on epilepsy and its treatment in a tertiary hospital[END_REF]. Our results in both Cambodia and Lao PDR demonstrated that pharmacists, who are educated and supposed to be well-trained about medications, accounted for less than a half of pharmacy-dispensing workers. One in five pharmacy-dispensing workers were sellers without any medical or pharmaceutical training. In Cambodia, the number of unqualified pharmacy-dispensing workers found in our study was less than in a previous study published in 2004 (89.6%); this reduction may be due to restrictions in pharmacy regulations in Cambodia [START_REF] Yang | Quality of Pharmaceutical Items Available from Drugstores in Phnom Penh, Cambodia[END_REF]. In Lao PDR, however, our results were consistent with a previous study performed in 2001, reporting a proportion of 24.5% of non-healthcare qualified pharmacy-dispensing workers [START_REF] Stenson | Real world pharmacy: assessing the quality of private pharmacy practice in the Lao People's Democratic Republic[END_REF]. In addition, inadequate qualification among pharmacy-dispensing workers combines with the shortage of medical books that has been reported over the past 20 years in Lao PDR [START_REF] Harimanana | Is insufficient knowledge of epilepsy the reason for low levels of healthcare in the Lao PDR?[END_REF]. This point could lead to inadequate information on medical education and incorrect practices among pharmacydispensing workers not only for PWE, but for all patients.

In this study, we pointed out many gaps in the pharmacy-dispensing worker's knowledge of epilepsy (mainly related to AEDs). This finding was in accordance with a study showing insufficient knowledge about epilepsy among different types of healthcare professionals such as physical educators, nutritionists and physiotherapists [START_REF] Vancini | Knowledge about epilepsy among health professionals: a cross-sectional survey in São Paulo, Brazil[END_REF]. This knowledge gap can lead to inadequate counseling for PWE, which is consistent with findings in other studies that reported suboptimal performances at pharmacies, including a lack of drug information and counselling for PWE in Cambodia [START_REF] Yang | Quality of Pharmaceutical Items Available from Drugstores in Phnom Penh, Cambodia[END_REF]. For Lao PDR, no specific study was performed on counseling by pharmacy-dispensing workers for PWE, but when we compare recent studies on the quality of antenatal care services [START_REF] Phommachanh | Do health care providers give sufficient information and good counseling during ante-natal care in Lao PDR?: an observational study[END_REF] and knowledge of sexually transmitted infections [START_REF] Phrasisombath | Knowledge about sexually transmitted infections (STIs) and attitudes toward female sex workers with STI symptoms among health care providers in Laos[END_REF] among healthcare providers which reported limited knowledge, poor services and insufficient counseling, these are consistent with our findings. This insufficient knowledge of epilepsy in both Cambodia and Lao PDR are probably a consequence of a lack of training on epilepsy and AEDs -only half of them received epilepsy training at school or university, and a minority received post-graduate training on epilepsy.

Misunderstanding of epilepsy (there is only one type of seizure, epilepsy is a communicable disease and is untreatable) in both Cambodia and Lao PDR was higher than two previous studies among Cameroonian medical students and health-care professionals in Saudi Arabia [START_REF] Alaqeel | Epilepsy: What do health-care professionals in Riyadh know?[END_REF][START_REF] Njamnshi | Knowledge, attitudes, and practice of Cameroonian medical students and graduating physicians with respect to epilepsy[END_REF], but lower than for student nurses in Cameroon [START_REF] Njamnshi | Knowledge, attitudes and practices with respect to epilepsy among student nurses and laboratory assistants in the South West Region of Cameroon[END_REF].

We observed in our study that pharmacy-dispensing workers in Cambodia have a better knowledge of epilepsy than those in Lao PDR, perhaps due to differences in curricula and the time allocated to the study of epilepsy. Another explanation could be the fact that one-quarter of respondents from Lao PDR worked at level lll pharmacies that were not supposed to sell AEDs. Nevertheless, this was a pragmatic study assessing the situation in real conditions. Knowledge of epilepsy and AEDs among pharmacy-dispensing workers in Lao PDR was low, but the improvement of knowledge was highlighted when compared to the previous study performed in 2003 among physicians and nurses [START_REF] Harimanana | Is insufficient knowledge of epilepsy the reason for low levels of healthcare in the Lao PDR?[END_REF]. The ability to mention at least one long-term AEDs and knowing that discontinuing AEDs could be dangerous was improved. This was probably due to the annual training program and information about this disease that has become easily accessible by internet or mass media [START_REF] Barennes | Children's access to treatment for epilepsy: experience from the Lao People's Democratic Republic[END_REF][START_REF] Tran | The challenge of epilepsy control in deprived settings: low compliance and high fatality rates during a community-based phenobarbital program in rural Laos[END_REF][START_REF] Chivorakoun | Epilepsy in Lao Popular Democratic Republic: difficult procurement of a first-line antiepileptic contributes to widening the treatment gap[END_REF].

Conversely, no previous studies on knowledge of epilepsy and AEDs among healthcare workers had been performed in Cambodia.

Inadequate knowledge of AEDs was observed in both Cambodia and Lao PDR.

Among pharmacy-dispensing workers who knew about the treatment of epilepsy in our study, phenobarbital was the most cited in both countries, similar to a study in Zambia [START_REF] Chomba | Zambian health care workers' knowledge, attitudes, beliefs, and practices regarding epilepsy[END_REF], probably be due to the availability of this molecule in developing countries [START_REF] Cameron | Mapping the availability, price, and affordability of antiepileptic drugs in 46 countries[END_REF][START_REF] Odermatt | Availability and costs of antiepileptic drugs and quality of phenobarbital in Vientiane municipality, Lao PDR[END_REF]. Furthermore, only 36% were aware of the need to adhere to AED regimens during pregnancy. This result is lower than studies conducted in Palestine with 58.1% [START_REF] Shawahna | Evaluation of pharmacists' knowledge of women's issues in epilepsy: A cross-sectional study in Palestinian pharmacy practice[END_REF] and Israel with 93% [START_REF] Roth | An evaluation of pharmacist knowledge on treatment with antiepileptic drugs[END_REF]. The reduction of the effectiveness of contraceptives due to the enzymeinducing features of most of the major AEDs was unknown in almost all pharmacydispensing workers in our study, whereas in other studies, more than 70% of pharmacists and healthcare professionals were aware of this DDI [START_REF] Shawahna | Evaluation of pharmacists' knowledge of women's issues in epilepsy: A cross-sectional study in Palestinian pharmacy practice[END_REF][START_REF] Mcauley | An evaluation of pharmacists' knowledge of women's issues in epilepsy[END_REF][START_REF] Long | Knowledge of women's issues and epilepsy (KOWIE-II): a survey of health care professionals[END_REF]. In Cambodia and Lao PDR, about three out of five pharmacy-dispensing workers know limited number of side effects associated with AEDs. This constituted a discrepancy when compared to previous studies in Palestine [START_REF] Shawahna | Evaluation of pharmacists' knowledge of women's issues in epilepsy: A cross-sectional study in Palestinian pharmacy practice[END_REF] and Israel [START_REF] Roth | An evaluation of pharmacist knowledge on treatment with antiepileptic drugs[END_REF]. This discrepancy may be due to the fact that all of the respondents in these previous studies were pharmacists and had better knowledge of large number of various negative effects of AEDs.

Strengths and limitations

The major limitation of our study was that we could not investigate the central hospital in Cambodia. To be able to compare Cambodia versus Lao PDR, the central hospital in these two countries was not considered in the analysis. We recognize that the over-representation of private pharmacies in the sample may have generated a selection bias with regard to the situation of all drug-dispensing outlets in these countries. This was the first study to assess these aspects in these countries, and private pharmacies remain the main type of outlets in the supply chain. The main strength of this first study is that we assessed knowledge among pharmacists and all pharmacy-dispensing workers in real-life situations in Cambodia and Lao PDR. Respondents were not allowed to consult or look for answers in any resources when answering the survey questions.

Conclusion

An educational intervention should be implemented to improve the knowledge of epilepsy and AEDs among pharmacy-dispensing workers. This could include the ability to provide advice for all pharmacy-dispensing workers in order to improve AED management (reducing DDI and managing side effects) and follow-up for therapeutic adherence, which could be divided into three dimensions: acceptance, compliance with the recommendation and persistence of this compliance. Due to the fact that pharmacy-dispensing workers have regular contacts and are easily accessible for PWE, the compliance and acceptance dimensions could be supported and enhanced, especially since all advice delivered by pharmacy-dispensing workers is free to PWE when AEDs are dispensed. We suggest that communication and collaboration between healthcare professionals such as pharmacydispensing workers and physicians could provide a beneficial improvement to care management for PWE. This collaboration could improve care in a variety of ways, including medication management, patient counselling and therapeutic education [START_REF] Kelly | Pharmacist and physician views on collaborative practice: Findings from the community pharmaceutical care project[END_REF]. As result of this study, professional educational programs (initial formation and continuing education), and patients' information should be developed. In addition, the professional activities should comply with the pharmacy regulation, in particular which regard to authorized personnel.

Ethical approval

Ethical approval for this study was obtained from the National Ethics Committee for Health Research, Ministry of Health of Cambodia (reference No.12/NECHR) and Lao PDR (reference No.077/NECHR). Written informed consent was systematically obtained from all participants prior to the interview and all answers were kept confidential.

1

 1

Figure 1 : 2 Figure 2 :

 122 Figure 1: Study areas in Cambodia and Lao PDR

Table 1 :

 1 Sociodemographics of the respondents

		Cambodia
	Phnom Penh	Kampong Cham	Kampot (Border with Vietnam)
	Daun Penh Dong Kor	Kampong Cham Prey Chhor	Kampot Kampong Trach
	24	23
	0 2 22	1 1 21
	Excluded		Excluded
	7 pharmacies		3 pharmacies
	did not agree to		did not agree
	answer		to answer
	17	23	19
	0 2 15	1 1 21	1 1 17
	23	30	23
	0 7 16	7 2 21	4 2 17

Table 2 :

 2 Knowledge of epilepsy

	Total	Cambodia	Lao PDR	P
	N= 180	n= 76	n= 104

are used to diagnose epilepsy (multiple answers)

	Do not know	74 (41.3%)	27 (35.5%)	47 (45.6%)	0.219
	Computerized tomography (CT scan) / magnetic resonance imaging (MRI)	30 (16.8%)	17 (22.4%)	13 (12.6%)	0.106
	Electroencephalogram (EEG)	42 (23.5%)	32 (42.1%)	10 (9.7%)	<0.001
	Clinical sign	5 (2.8%)	3 (1.9%)	2 (1.9%)	0.652
	3

Table 3 :

 3 Knowledge of antiepileptic drugs

	Total n= 180	Cambodia n= 76	Lao PDR n= 104	P

drugs can be given regardless of the type of epilepsy

	Yes	30 (22.1%)	13 (19.7%)	17 (24.3%)
	No	68 (50%)	36 (54.5%)	32 (45.7%)	0.585
	Do not know	38 (27.9%)	17 (25.8%)	21 (30.0%)
	Epilepsy patients have to take drugs for life
	Yes	79 (58.1%)	39 (59.1%)	40 (57.1%)
	No	29 (21.3%)	13 (19.7%)	16 (22.9%)	0.902
	Do not know	28 (20.6%)	14 (21.2%)	14 (20.0%)
	How to take long-term AEDs
	Should take AEDs every day	122 (89.7%)	62 (93.9%)	60 (85.7%)
	Should take AEDs during seizures	8 (5.9%)	3 (4.5%)	5 (7.1%)	0.214
	Do not know	6 (4.4%)	1 (1.5%)	5 (7.1%)
	If

seizures are controlled for a few months, can people with epilepsy stop taking their antiepileptic drugs?

	Yes	21 (15.4%)	8 (12.1%)	13 (18.6%)
	No	78 (57.4%)	34 (51.5%)	44 (62.9%)	0.060
	Do not know	37 (27.2%)	24 (36.4%)	13 (18.6%)

Should women with epilepsy stop taking their medicine if they become pregnant?

  Pharmacies offer to patients a first-line point of contact with the health care system

	Yes	27 (19.9%)	15 (22.7%)	12 (17.1%)
	No	49 (36.0%)	24 (36.4%)	25 (35.7%)	0.685
	Do not know	60 (44.1%)	27 (40.9%)	33 (47.1%)
	Do interactions exist between antiepileptic drugs and oral contraception?
	Yes	1 (0.7%)	0 (0.0%)	1 (1.4%)
	No	47 (34.6%)	30 (45.5%)	17 (24.3%)	0.025
	Do not know	88 (64.7%)	36 (54.5%)	52 (74.3%)
	Side effects of antiepileptic drugs
	Phenobarbital	77 (56.6%)	36 (54.5%)	41 (58.6%)	0.730
	Phenytoin	43 (31.6%)	26 (39.4%)	17 (24.3%)	0.067
	Carbamazepine	42 (30.9%)	22 (33.3%)	20 (28.6%)	0.581
	Do not know of any side effects of AEDs	51 (37.5%)	26 (39.4%)	25 (35.7%)	0.724

Pharmacist 40.8% vs. 38.5% pharmacy-

dispensing workers Cambodia vs. Lao PDR Seller

	Other health	(non-healthcare
	professional	formation)
	40.8% vs. 38.5%	18.4% vs. 20.2%
	pharmacy-dispensing
	workers

Qualification Knowledge on AEDs 86.8% vs. 67.3% Know at least one AED 51.5% vs. 62.9% Know that PWE can not stop to take AEDs even seizures are controlled for some months 36.4% vs. 35.7% Know that women with epilepsy should not stop taking AEDs if they become pregnant 0.0% vs. 1.4% Know that AEDs may reduce the effectiveness of contraceptives 39.4% vs. 35.7% Do not know any side effects of AEDs Cambodia vs. Lao PDR

Funding

None of the authors has any conflicts of interest to disclose. This work was supported by the Foundation Pierre Fabre [2018]. The funder was not involved in the study design, data collection and analysis, decision to publish, or writing the manuscript.

Conflicts of interest statement

None of the authors has any conflicts of interest to disclose.