Olivia Boyer

Geneviève Benoit

Olivier Gribouval

Fabien Nevo

Audrey Pawtowski

Ilmay Bilge

Zelal Bircan

Georges Deschênes

Lisa M Guay-Woodford

Michelle Hall

Lisa M Guay- Woodford

Marie-Alice Macher

Kenza Soulami

Constantinos J Stefanidis

Robert Weiss

Chantal Loirat

Marie-Claire Gubler

MD Corinne Antignac
email: corinne.antignac@inserm.fr

Mutational analysis of the PLCE1 gene in steroid-resistant nephrotic syndrome

Keywords: PLCE1, nephrotic syndrome, diffuse mesangial sclerosis, focal segmental glomerulosclerosis, hereditary glomerular disease

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INTRODUCTION

Nephrotic syndrome (NS) results from disruption of the renal filtration barrier composed of interdigitating podocyte foot processes linked by the slit diaphragm, and endothelial cells separated from podocytes by the glomerular basement membrane. It is characterized by massive proteinuria, hypoalbuminemia, hyperlipidemia, edema and podocyte foot process effacement by electron microscopy. NS is classified as steroid-sensitive (SSNS) or steroidresistant (SRNS) and mutations in several genes, mostly coding for podocyte proteins, have been identified as causing SRNS in humans. Although kidney biopsy discloses focal and segmental glomerulosclerosis (FSGS) lesions for most patients with SRNS, 1 a smaller proportion of children may present a particularly severe renal histological pattern named diffuse mesangial sclerosis (DMS) with an early presentation and a poor renal prognosis.

DMS is characterized by mesangial expansion and sclerosis that evolves toward obliteration of the capillary lumen and contraction of the glomerular tuft. This type of renal histology has been described as part of syndromes such as Denys-Drash syndrome or Pierson syndrome, caused by mutations in the WT1 and LAMB2 genes, respectively. More recently, PLCE1 mutations have been found as a novel cause of DMS. [START_REF] Hinkes | Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible[END_REF] Involvement of the PLCE1 gene (MIM*608414) in NS has been found using a combination of homozygosity mapping and cDNA microarrays from rat glomeruli. In the original series described by Hinkes et al., [START_REF] Hinkes | Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible[END_REF] truncating PLCE1 mutations were identified in 12 siblings from six families diagnosed with early-onset NS and DMS while a homozygous missense mutation was identified in two siblings with a later onset of disease and FSGS (Nephrotic syndrome, type 3; NPHS3 -MIM#610725); until now, these individuals were the only reported cases of FSGS secondary to PLCE1 mutations. Subsequently, mutations in PLCE1 have been demonstrated as a major cause of isolated DMS, identified in 28.6% of 35 families in a worldwide cohort. [START_REF] Gbadegesin | Mutations in PLCE1 are a major cause of isolated diffuse mesangial sclerosis (IDMS)[END_REF] Although PLCE1 mutations typically lead to a severe phenotype, full and sustained treatment responses have been reported in two individuals with truncating mutations 2 and a pathogenic homozygous mutation have also been found in a phenotypically completely normal adult. [START_REF] Gilbert | Mutations in phospholipase C epsilon 1 are not sufficient to cause diffuse mesangial sclerosis[END_REF] This suggests that other factors may modify the effect of PLCE1 mutations.

PLCE1 gene encodes the phospholipase C epsilon 1 (PLCε1).

Members of the phosphoinositide-specific phospholipase C (PLC) family catalyze the hydrolysis of membrane phospholipids to generate the second messenger molecules inositol 1,4,5-trisphosphate (IP 3 )

and diacylglycerol (DAG) that initiate intracellular pathways of cell growth and differentiation. [START_REF] Wing | PLC-epsilon: a shared effector protein in Ras-, Rho-, and G alpha beta gamma-mediated signaling[END_REF] Several distinct PLC enzymes have been identified in a variety of mammalian tissues and are divided into four classes: PLCβ, PLCγ, PLCδ and PLCε.

Phospholipase catalytic domains (PLC_X and PLC_Y) and Ca 2+ lipid-binding domain (C2 domain) typically characterized all PLCs. PLCε1, which is highly expressed in podocytes, [START_REF] Hinkes | Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible[END_REF] is the most recently identified member of the PLC family; it contains, in addition to conserved PLC domains, a RasGEF_CDC25 (guanine nucleotide exchange factor for Ras-like small GTPases domain) and two C-terminal Ras-binding (RA) domains, RA1 and RA2 (RasGTP binding domain from guanine nucleotide exchange factors). While PLCβ, PLCγ, PLCδ are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium, it has been shown that PLCε1 is regulated by H-Ras (via RA1 and RA2 domains) and also interacts with IQGAP1 (IQ motif-containing GTPase-activating protein) and BRAF. 2 5-7 In order to better define the phenotype spectrum of patients bearing PLCE1 mutations, we performed mutational analysis and clinical follow-up of a large worldwide cohort of patients with either familial or sporadic SRNS. In addition, we aimed to determine the role of candidate genes, including other PLC genes, as modifiers of disease in families for which both symptomatic and asymptomatic members had homozygous PLCE1 mutations.

MATERIALS AND METHODS

Patients

A total of 139 patients were included in the present study, all of which presented SRNS. In accordance with criteria established by the International Study of Kidney Disease in Children (ISKDC), NS was defined as the association of proteinuria > 40 mg/m 2 /hr, hypoalbuminemia < 2.5 g/dL, edema, and hyperlipidemia. [START_REF] Isokdi | Primary nephrotic syndrome in children: clinical significance of histopathologic variants of minimal change and of diffuse mesangial hypercellularity[END_REF] Steroid-resistance was defined as lack of response to four weeks of treatment with 60mg/m 2 per day of prednisone.

PLCE1 molecular analyses

Genomic DNA specimens were collected with informed consent and isolated from peripheral blood using standard procedures. Linkage of familial cases to the PLCE1 locus was assessed using four polymorphic microsatellite markers spanning 5.

Mutational analyses of possible modifier genes, including other phospholipases C

In order to identify modifier genes in families with asymptomatic individuals who carried the same PLCE1 homozygous mutation as the affected patients, a total of 19 candidates genes were evaluated, including 16 genes encoding other PLC (PLCB1, PLCB2, PLCB3, PLCB4, PLCD1, PLCD3, PLCD4, PLCG1, PLCG2, PLCH1, PLCH2, PLCL1, PLCL2, PLCXD1, PLCXD3 and PLCZ1) and 3 genes encoding proteins that interact directly or indirectly with PLCε1 (BRAF, IQGAP1, NPHS1) (Table 1). Firstly, a whole-genome wide search for linkage was performed using a 250k SNP array (GeneChip® Human Mapping 250K Nsp Array from Affymetrix, Santa Clara, CA). The potential role of one of the genes cited above as a modifier of disease was excluded if the asymptomatic mutated patient shared the same haplotypes as at least one of the affected mutated patients. Then, for genes not excluded by this method, microsatellite markers were used to test each locus of the remaining genes: Finally, the complete coding sequence and exon-intron boundaries of the genes not excluded by the whole genome wide scan or by microsatellite markers analysis were sequenced as described above (PLCH1, 23 exons, GenBank accession number NM_014996.2; PLCL1, 5 exons, GenBank accession number NM_006226.3; BRAF, 18 exons, GenBank accession number NM_004333.4) in one asymptomatic mutated patient and one affected mutated patient of each families.

PLCB2 (D15S994,

Statistical analysis

All values are expressed as means ± standard error or median (range). Comparisons between two continuous variables were performed using the Mann-Whitney test. All tests were two sided. P-values <0.05 were considered significant. Statistical analyses were performed using GraphPad Prism software version 5.01.

RESULTS

Patients characteristics

Patients characteristics are summarized in

Identification of PLCE1 Mutations

Microsatellite markers analysis excluded linkage to the PLCE1 locus in 43 families.

Therefore, results were compatible with linkage to the PLCE1 locus in 25 families as evidenced by homozygosity for the polymorphic markers flanking the PLCE1 locus. Diseasecausing mutations were found in 12 families, comprising a total of 18 patients (Table 3).

Among sporadic cases, homozygous PLCE1 mutations were identified in 3 patients. Most were homozygous truncating mutations (nonsense or frameshift mutations), although compound heterozygote missense mutations (p.E1386V/p.P1890L) were detected in two siblings and a homozygous missense mutation (p.H1407D) was detected in one sporadic case.

The p.E1386V was predicted to be a possibly pathogenic mutation (Polyphen score 1.94) and involved a very highly conserved amino-acid from fishes to humans. The p.P1890L and p.H1407D were predicted to be probably pathogenic mutations with a Polyphen score of 2.55 and 2.50, respectively, involving amino-acids conserved from C-elegans to humans, and included in either the C2 domain or the PLC_X catalytic domain of the protein. Nine of these mutations have not been previously reported (Figure 1).

Phenotypic Spectrum of PLCE1 Mutations

Among

Identification of asymptomatic individuals bearing homozygous PLCE1 mutations

Surprisingly, segregation analysis revealed that 3 unaffected individuals belonging to 3 unrelated families were also found to bear, in the homozygous state, the mutations identified in their respective affected relatives (Figure 2). In Segregation analysis revealed that the father, who was also born from consanguineous parents, was homozygous for the mutation. Although he had presented hypertension and mild proteinuria as a teenager, his renal evaluation, at 46 years of age, was normal.

Search for modifier genes

To understand this phenomenon, we ought to identify mutations or variants in other genes that could modulate the clinical phenotype. We hypothesized a protective effect arising from other members of the phospholipase C family in the asymptomatic individuals. Alternatively, another hypothesis was that additional mutations or variants in BRAF, IQGAP1 or NPHS1 genes in symptomatic patients could explain the phenotypic difference. Analysis of whole genome DNA arrays in family E excluded the role of 13 of the 19 candidate genes as a modifier of the disease, since the asymptomatic mutated individual shared the same haplotypes as at least one of his affected siblings at these loci. Then, microsatellite markers analysis of the remaining genes (PLCB2, PLCB4, PLCH1, PLCL1, PLCL2 and BRAF) in family O showed that both the affected and the clinically unaffected case shared the same haplotypes for PLCB2, PLCB4 and PLCL2 genes, excluding them as being potentially involved in the phenotypic variability in the family. Therefore, the direct DNA sequencing of the 3 remaining genes was performed in one affected case and one asymptomatic case of family E, O and A, but did not allow identification of mutations or variants.

DISCUSSION

In 2006, positional cloning uncovered mutations in PLCE1 causing early-onset NS. Since then, 17 different mutations have been reported (Figure 1), among which only one was associated with FSGS. We therefore aimed to better assess the phenotype spectrum of patients bearing PLCE1 mutations in a large worldwide cohort of patients with SRNS.

Among familial forms of SRNS, PLCE1 mutations were found in 12% of families with FSGS and half of families with DMS. Although a significant proportion (21%) of the DMS sporadic cases had PLCE1 mutations, none of the 28 sporadic cases with FSGS had mutations in this gene. If we had included all children with SRNS without any prior genetic testing, the detection rate of PLCE1 mutations in FSGS cases would have been lower, as NPHS2 mutations would have been identified for a significant proportion of them. In contrast, the high rate of PLCE1 mutations found in DMS cases in our study is probably not overestimated and is concordant with what has been previously published. [START_REF] Gbadegesin | Mutations in PLCE1 are a major cause of isolated diffuse mesangial sclerosis (IDMS)[END_REF] Indeed, in our cohort, none of the patients presented signs of Denys-Drash or Frasier syndrome, which could have suggested a mutation in other DMS-associated genes such as WT1 or LAMB2. In addition, WT1 mutations were excluded in all phenotypically female patients and NPHS2 mutations have never been found as a cause of DMS. [START_REF] Weber | NPHS2 mutation analysis shows genetic heterogeneity of steroidresistant nephrotic syndrome and low post-transplant recurrence[END_REF] Similar to previous studies, In this regards, we identified three unaffected individuals carrying the same homozygous mutation as their affected relatives, a finding particularly unusual for disease of recessive inheritance. One other previous case report of a family of 3 affected siblings with DMS also showed that the asymptomatic father, who was himself born from consanguineous parents, had the same homozygous PLCE1 mutation as his affected children. [START_REF] Gilbert | Mutations in phospholipase C epsilon 1 are not sufficient to cause diffuse mesangial sclerosis[END_REF] These data are also reminiscent of the unusual progression of two patients with DMS in the original description of PLCE1 mutations who seemed to respond to steroid or cyclosporin therapy. [START_REF] Hinkes | Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible[END_REF] The above observations raise the possibility of oligogenic inheritance, with either a third deleterious mutation in a second gene explaining the appearance of a renal phenotype in patients bearing two PLCE1 mutations or the presence of a protective modifier allele, which could compensate for the PLCE1 dysfunction in asymptomatic individuals. Although oligogenic inheritance has been suggested in several cases of renal diseases, [START_REF] Hoefele | Evidence of oligogenic inheritance in nephronophthisis[END_REF][START_REF] Koziell | Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration[END_REF][START_REF] Tory | High NPHP1 and NPHP6 mutation rate in patients with Joubert syndrome and nephronophthisis: potential epistatic effect of NPHP6 and AHI1 mutations in patients with NPHP1 mutations[END_REF] it is in only very rare cases that asymptomatic individuals presented two pathogenic mutations in one gene in contrast to affected individuals bearing an additional third mutation in a second gene. [START_REF] Katsanis | Triallelic inheritance in Bardet-Biedl syndrome, a Mendelian recessive disorder[END_REF] Indeed, oligenic inheritance has been clearly demonstrated in some cases of Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder characterized by pigmentary retinal dystrophy, polydactyly, obesity, developmental delay and renal defects. In a study of 163 BBS families, Katsanis et al. found the presence of "three mutant alleles" (either two BBS2 mutations and one BBS6 mutation or the opposite) in four pedigrees. More interestingly, they detected two BBS2 mutations but not BBS6 mutations in unaffected individuals in two pedigrees, suggesting that BBS may not be a single-gene recessive disease but a complex trait requiring a total of 3 mutations in two different genes to manifest the phenotype. On the other hand, existence of protective modifier gene has been demonstrated in a recent study on autosomal recessive spinal muscular atrophy (SMA). [START_REF] Oprea | Plastin 3 is a protective modifier of autosomal recessive spinal muscular atrophy[END_REF] The detection of asymptomatic individuals (all females) carrying the same mutations in the SMN1 (survival motor neuron 1) gene as their affected siblings has suggested the influence of modifier genes. In this study, higher expression of plastin 3, encoded by PLS3, were found in unaffected SMN1-deleted females compared to their SMA-affected counterparts. Overexpression of PLS3 rescued the axon length and outgrowth defect associated with down-regulation in motor neurons of SMA mouse embryos and in zebrafish, thereby explaining the absence of a neurological phenotype in some patients bearing SMN1 mutation.

Effect of modifiers genes could also explain the absence of obvious renal phenotype in the Plce1 knock-out mouse model, developed on a mixed C57/B6 X 129/S6 genetic background. 2 17 Indeed, contrary to the zebrafish knock-out model in which embryos injected with Plce1 morpholino show edema at D4 of development and foot process effacement by electron microscopy, 2 Plce1 -/-mice do not exhibit any renal phenotype. However, as C57/B6 mice are resistant to the development of glomerulosclerosis, [START_REF] Ma | Model of robust induction of glomerulosclerosis in mice: importance of genetic background[END_REF] it might be of interest to assess the presence of a renal phenotype in Plce1 knock-out mice from a different genetic background or under nephrosis-promoting conditions.

Based on the preceding observations, we ought to identify mutations or variants in other genes that could explain the absence of renal phenotype in some patients with PLCE1 mutation. We first speculated a compensatory protective effect arising from other members of the PLC family in the unaffected individuals since the common role of these proteins is to initiate intracellular signaling through the generation of the second messenger molecules IP3 and diacylglycerol. Interestingly, some mouse models of PLC genes develop a renal phenotype, including increased kidney weight, polycystic kidneys, renal dysplasia, glomerulosclerosis or signs of glomerulonephritis (Table 1). Moreover, PLC-γ1 has been shown to play a key-role in the podocyte slit diaphragm. PLC-γ1 binds to phosphorylated nephrin, the main component of the slit, leading to PLC-γ1 activation and subsequent increase in IP3, and triggering of Ca2+ intracellular response. [START_REF] Harita | Phosphorylation of Nephrin Triggers Ca2+ Signaling by Recruitment and Activation of Phospholipase C-{gamma}1[END_REF] In our study, we did not identify a PLC candidate that was mutated in a recessive or dominant fashion in the affected individuals and not in the asymptomatic relative, or the reverse. We then used the same approach for candidate genes that are known to interact directly or indirectly with PLCε1 such as IQGAP1, BRAF and NPHS1. IQGAP1 is a known scaffold protein of the mitogen-activated protein (MAP) kinase pathway [START_REF] Roy | IQGAP1 is a scaffold for mitogen-activated protein kinase signaling[END_REF] and interacts with PLCε1, 2 nephrin 21 and BRAF. [START_REF] Ren | IQGAP1 modulates activation of B-Raf[END_REF] BRAF, which has been involved in various cancers, [START_REF] Davies | Mutations of the BRAF gene in human cancer[END_REF] belongs to the RAF family of genes and binds H-Ras. [START_REF] Okada | Post-translational modification of H-Ras is required for activation of, but not for association with, B-Raf[END_REF] It has also been implicated in the MAP kinase pathway and is considered as a third interaction partner of PLCε1, [START_REF] Chaib | Identification of BRAF as a new interactor of PLCepsilon1, the protein mutated in nephrotic syndrome type 3[END_REF] in addition to H-ras and IQGAP-1. In the present study, IQGAP1, BRAF and NPHS1 genes did not appear to explain the phenotypic variability observed in our patients. However, our approach cannot exclude an effect of different PLC or PLC partner genes in the three different families. Finally, it has to be stressed that, to date, only very few homozygous PLCE1 mutations detected in asymptomatic individuals or patients that responded to therapy have been reported and, surprisingly, all of them are males. This is reminiscent of the unaffected SMN1-deleted individuals, which were all females, [START_REF] Oprea | Plastin 3 is a protective modifier of autosomal recessive spinal muscular atrophy[END_REF] and suggests the existence of gender-specific modifiers of disease.

In conclusion, PLCE1 mutations are frequently found in familial and sporadic DMS cases.

Although PLCE1 mutations had been rarely associated with FSGS, we showed that mutations in this gene are detected in a non-negligible proportion of familial FSGS cases without NPHS2 mutations. In this study, no clear genotype-phenotype correlation was present.

Although variants in other phospholipases C genes do not seem to explain the absence of renal manifestations in some cases with PLCE1 mutations, we speculate that other modifier genes or environmental factors may play a role in the renal phenotype variability observed in individuals bearing PLCE1 mutations. More importantly, the clinical heterogeneity associated with PLCE1 mutations substantially increases the complexity of genetic counselling, including prenatal diagnosis, and needs to be considered in the discussion about renal prognosis with patients. In red are the novel mutations found in our study. In black are the other mutations reported in the literature. 2-4 10 The p.P382fsX387 mutation has been reported in one patient for which renal histology was not available. [START_REF] Hinkes | Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible[END_REF] We also found the p.Q1020X mutation in one patient without kidney biopsy. * The p.R1246X mutation has been reported in DMS cases; [START_REF] Ismaili | Genetic forms of nephrotic syndrome: a single-center experience in Brussels[END_REF] we also found the same mutation in both DMS and FSGS cases. DMS, diffuse mesangial sclerosis; FSGS, focal segmental glomerulosclerosis.

  D15S968, D15S1006), PLCB4 (D20S889, D20S115, D20S186), PLCH1 (D3S1594, D3S1279, D3S1268), PLCL1 (D2S117, D2S311, D2S374, D2S318, D2S115), PLCL2 (D3S3701, D3S2338, D3S1293) and BRAF (D7S2560, D7S684, D7S2513, D7S661).

  family E, 3 out of six siblings of a consanguineous family from Pakistan presented SRNS with FSGS on renal histology at ~ 5 years of age and reached ESKD by the age of 6. Another brother had a past history of intermittent mild proteinuria but was free of renal manifestations at 12 years of age. The p.F839fs889X mutation was identified in these 4 patients. In family O, the index case was born from consanguineous parents of Turkish origin. She was diagnosed with SRNS and DMS at the age of 11 months and found to bear the p.R2150X mutation. Among his 2 siblings, 1 brother was homozygous for the mutation and had no proteinuria at 10 years of age. In family A, 2 out of 3 children of consanguineous parents from Pakistan developed SRNS at 5 months and 3 years of age with FSGS on renal biopsy and reached ESKD when they were 1.4 and 4 years old, respectively. The p.R321X mutation was identified.

Figure 1 .

 1 Figure 1. PLCE1 mutations reported in the literature, including the mutations detected in the present study, with the associated renal histological lesions

Figure 2 .

 2 Figure 2. Identification of homozygous PLCE1 mutations in 3 unrelated asymptomatic individuals.

Table 2 .

 2 Most of familial cases originated fromMiddle-East (33.8%) while most of sporadic cases were from Europe (66.7%) (mainly from France). Among familial cases, renal histology disclosed FSGS, DMS and other renal lesions (mesangial proliferation, membranoproliferative glomerulonephritis and terminal kidney) in 50, 10 and 4 families, respectively (renal biopsy not performed in 4 families). Among sporadic cases, FSGS was present in 28 cases, DMS in 14 cases and mesangial proliferation

in two cases. Mean age at onset of NS was 46.2 ± 5.7 months. Seventy-six patients reached ESKD at a mean age of 79.7 ± 7.1 months.

  .0 vs 30.5 mo ± 7.7, p=0.15 and mean age at ESKD 20.9 mo ± 5.8 vs 57.2 mo ± 7.3, p<0.01).

	the 12 families bearing PLCE1 mutations, renal biopsy disclosed FSGS in 6 and DMS
	in 5 (not performed in one family). This translates in a 12% (6/50) and 50% (5/10) PLCE1
	detection rate among families with FSGS and DMS, respectively. All the 3 sporadic cases
	presented DMS on renal histology, meaning that PLCE1 mutations were detected in 21%
	and missense mutations (mean age at onset 12.3 mo ± 1.5, mean age at ESKD 49.0 mo ±
	19.9) lead to similar renal presentation and evolution (p=0.80 and p=0.41, respectively) and
	were detected in both FSGS and DMS histological forms. However, renal prognosis was

(3/14) of sporadic DMS cases, but in none (0/28) of the sporadic FSGS cases. Globally, PLCE1 mutations were identified in 13% of our whole cohort (15/112), 33% (8/24) of DMS cases and 8% (6/78) of FSGS cases. When kidney histology of several affected patients of the same family was available, all siblings had the same type of renal lesions.

Patients with PLCE1 mutations presented NS at a mean age of 22.8 mo ± 5.0 leading to ESKD at a mean age of 39.1 mo ± 6.3 (two patients with FSGS did not reach ESKD after a short follow-up of 1 year and one patient without renal biopsy died at 11 months of NS complications). This contrasts with non mutated patients which presented NS at a mean age of 50.9 mo ± 6.7 (p=0.12) leading to ESKD at a mean age of 92.6 mo ± 8.4 (p<0.01). In patients bearing PLCE1 mutations, no clear-cut genotype-phenotype correlations were observed. Truncating (mean age at onset 24.6 mo ± 5.8, mean age at ESKD 37.1 mo ± 6.7) worst in patients with DMS compared to those with FSGS (mean age at onset of NS 15.3 mo ± 6

Table 1 .Candidate genes tested in order to evaluate the presence of a modifier effect Gene code Gene full name Gene mapping

 1 Nphs1 null mice are massively proteinuric at birth with death occurring in the first 24h.[START_REF] Putaala | The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death[END_REF]

	Mouse	Unigen
	model/	EST
	RP*	(kidney)**

* Existence of a mouse model (yes/no) and presence of a renal phenotype (RP) in a mouse model (yes/no) ** Approximate abundance of Expressed Sequence Tags (ESTs) in kidneys, inferred from EST sources and provided in the Unigene databank. The number indicates number of transcripts per million. For comparison, PLCE1 EST in kidneys = 9 transcripts per millon. ¶ λ

Table 2 . Patients characteristics (n=139)

 2 The denominator used to calculate percentages is the number of available data. FSGS, Focal segmental glomerulosclerosis; DMS, diffuse mesangial sclerosis; NS, nephrotic syndrome; ESKD, End-stage kidney disease; SE, Standard Error; NA, not applicable

			n (%) †
		All cohort	Familial cases	Sporadic cases
	Number of cases	139	95	44
	Type of families
	n		68
	Consanguineous family		53 (77.9)	NA
	Non-consanguineous multiplex family		15 (22.1)	NA
	Ethnic origin
	n	134	68 families	39
	North Africa	31 (23.1)	21 (30.9)	5 (12.8)
	Middle-East	36 (26.9)	23 (33.8)	4 (10.3)
	Europe	46 (34.3)	13 (19.1)	26 (66.7)
	Other	21 (15.7)	11 (16.2)	4 (10.3)
	Renal histological lesions
	n	129	64 families	44
	FSGS	98 (76.0)	50 (78.1)	28 (63.6)
	DMS	25 (19.4)	10 (15.6)	14 (31.8)
	Other	6 (4.7)	4 (6.3)	2 (4.5)
	Age at onset of NS (months)
	n	127	86	41
	Median (range)	23.0 (0-373)	24.0 (0-373)	23.0 (0-81)
	Mean ± SE	46.2 ± 5.7	55.7± 8.1	26.4 (3.4)
	ESKD
	n	131	88	43
	Number of patients reaching ESKD	76 (58.0)	47 (53.4)	29 (67.4)
	Age at onset of ESKD (months)
	n	75	46	29
	Median (range)	68.0 (0.5-264)	81.0 (4-264)	60.0 (0.5-139)
	Mean ± SE	79.7 ± 7.1	94.7 ± 10.4	55.9 ± 5.7

*The number of available data are shown as n.

†

Acknowledgements

Financial support for this work was provided by grants from the Programme Hospitalier de Recherche Clinique (PHRC) AOM02123 (to C.A), the Association pour l'utilisation du Rein Artificiel (AURA) (to C.A), PodoNet (Clinical, Genetic and Experimental Research into Hereditary Diseases of the Podocyte) (to C.A) and Fonds de la Recherche en Santé Québec (FRSQ) (Fellowship training Award to G.B.).

Licence statement

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JMG and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence.

Competing Interest

None declared.


MD Tiina Saarto
email: tiina.saarto@hus.fi

Phil J Wiffen

Antidepressants for neuropathic pain -a summary of Cochrane systematic review

Keywords:

of the Cochrane review of "Antidepressants for neuropathic pain".

Background

For many years antidepressant drugs have been used to manage neuropathic pain, and are often the first choice treatment. It is not clear, however, which antidepressant is more effective, what role the newer antidepressants can play in treating neuropathic pain, and what adverse effects are experienced by patients.

Methods

To determine the analgesic effectiveness and safety of antidepressant drugs in neuropathic pain a systematic review of randomised controlled trials reporting the analgesic effects of antidepressant drugs in adult patients, with subjective assessment of pain of neuropathic origin, was performed. Studies that included patients with chronic headache and migraine were excluded. Randomised trials of antidepressants in neuropathic pain were identified in MEDLINE (1966 2002). Additional reports were identified from the reference list of the retrieved papers, and by contacting investigators.

Results

Sixty one randomized controlled trials (66 reports) of 31 antidepressants (3293 participants) were considered eligible for inclusion. Tricyclic antidepressants (TCAs) (17 studies) are effective for the treatment of neuropathic pain and have an number needed to treat (NNT) of 3.60 (95%CI 3 to 4.5) RR (relative risk) 2.1 (95%CI 1.8-2.5) for the achievement of at least moderate pain relief. Venlafaxine (three studies) has an NNT of 3.1 (2.2-5.1) RR 2.2 (95 % CI 1.5-3.1) (three studies). There is limited evidence for the effectiveness of the newer selective serotonin reuptake inhibitor antidepressant drugs (SSRIs), and no studies of serotonin and noradrenalin reuptake inhibitors (SNRIs) were found. There were insufficient data for an assessment of evidence of effectiveness for other antidepressants such as St Johns Wort and L-tryptophan.

For diabetic neuropathy (five studies) the NNT for effectiveness was 1.3 (95%CI 1.2 to 1.5) RR 12.4(95%CI 5.2-29.2) (five studies); for postherpetic neuralgia (four studies) 2.7 (95%CI 2 to 4.1), RR 2.2 (95%CI 1.6-3.1)(four studies). There was some indication of effectiveness in central pain and atypical facial pain few trials and small participant numbers prevents from recommendations. There was evidence that TCAs are not effective in HIV-related neuropathies (two studies).

The number needed to harm(NNH) for major adverse effects defined as an event leading to withdrawal from a study was 28 (95%CI: 17.6-68.9) for amitriptyline and 16.2 (95%CI: 8-436) for venlafaxine. The NNH for minor adverse effects was 6 (95%CI 4.2-10.7) for amitriptyline and 9.6 (95%CI: 3.5-13) for venlafaxine.

Reviewers' conclusions

Antidepressants are effective for a variety of neuropathic pains. Both TCAs and venlafaxine have NNT of approximately three. This means that for approximately every three patients with neuropathic pain who are treated with either of these antidepressants, one will get at least moderate pain relief who would not have done so with placebo. There is evidence to suggest that other antidepressants like SSRIs may be effective, but numbers of participants are insufficient to calculate robust NNTs. Whether antidepressants prevent the development of neuropathic pain (pre-emptive use) is still unclear.

The quality of reporting in resent trials remains disappointing, in particular insufficient details are provided to enable effectiveness to be assessed. This is marked by an on-going preference to only report mean pain data rather than by reporting the number of participants responding.

Reference

The figure legend

A summary statistic of amitriptyline vs. placebo studies in treatment of neuropathic pain. The random effects model is used as there is considerable statistical heterogeneity (I squared is 85% and a small p value for Chi square).

  Saarto T and Wiffen PJ. Antidepressants for neuropathic pain. Cochrane Database of Systematic Reviews 2008, Issue 1. This paper is based on a Cochrane Review published in The Cochrane Library 2008, Issue 1 (see www.thecochranelibrary.com for information). Cochrane Reviews are regularly updated as new evidence emerges and in response to feedback, and The Cochrane Library should be consulted for the most recent version of the review.


MD Bertrand Audoin
email: bertrand.audoin@ap-hm.fr

Wafaa Zaaraoui

Francoise Reuter

Audrey Rico

Irina Malikova

Sylviane Confort-Gouny

Patrick J Cozzone

Jean Pelletier

Jean-Philippe Ranjeva

Françoise Reuter

Atrophy mainly affects the limbic system and the deep grey matter at the first stage of multiple sclerosis

Keywords: multiple sclerosis, clinically isolated syndrome, magnetic resonance imaging, atrophy, voxel based morphometry

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

Introduction

Multiple sclerosis (MS) is an inflammatory disease affecting the central nervous system, which is frequently responsible for severe disability, mainly in the form of ambulatory deficits, which often develop after the disease has been evolving for several years. Grey Matter (GM) pathology in MS is now well recognized [START_REF] Geurts | Grey matter pathology in multiple sclerosis[END_REF] and could be relevant to the understanding of the clinico-radiological dissociation in patients with MS, where the focal demyelination in the white matter (WM) observed in conventional MRI cannot fully explain the clinical status (including cognitive impairment) [START_REF] Geurts | Grey matter pathology in multiple sclerosis[END_REF][START_REF] Bo | Lack of correlation between cortical demyelination and white matter pathologic changes in multiple sclerosis[END_REF][START_REF] Amato | Association of neocortical volume changes with cognitive deterioration in relapsing-remitting multiple sclerosis[END_REF] .

Various studies based on whole brain or whole GM analyses have shown that particularly high rates of atrophy sometimes occur during the first few years of the disease in patients with a clinical isolated syndrome (CIS) who undergo a subsequent conversion to clinically definite MS, but not in patients in whom this conversion does not occur [START_REF] Dalton | Early development of multiple sclerosis is associated with progressive grey matter atrophy in patients presenting with clinically isolated syndromes[END_REF] . Two recent studies using voxel based morphometry (VBM) methods on patients with CIS have yielded contradictory results. In the one study, which involved a relatively small sample of patients (n=28), no regional GM atrophy was detected [START_REF] Ceccarelli | A voxel-based morphometry study of grey matter loss in MS patients with different clinical phenotypes[END_REF] , whereas the authors of the other study, which involved a larger group of patients (n=41), reported the existence of significant regional GM atrophy, mainly located in the deep GM [START_REF] Henry | Regional grey matter atrophy in clinically isolated syndromes at presentation[END_REF] .

The aim of the present study was therefore to assess the levels of regional GM atrophy present in a large group of MS patients at the earliest stage of the disease in order to provide evidence for or against the existence of early GM atrophy. Secondly, it was proposed to study the potential links between regional GM atrophy and clinical status of patients (including cognitive impairment).

Methods

Subjects

Sixty-two patients presenting with CIS and 37 healthy sex-age and educational level-matched control subjects were included in this study (Table 1). All the subjects (patients and controls) were right-handed (>70% Olfield scale) native French speakers. Patients were recruited at the Department of Neurology (University Hospital of Marseille), based on the following criteria: 1) age between 18 and 45; 2) occurrence of the first presumed inflammatory demyelinating event in the central nervous system involving either the optic nerve, the spinal cord, a brain hemisphere or the brainstem; 3) no previous history of neurological symptoms suggestive of demyelination; 4) no possible alternative diagnoses (lupus erythematosus, antiphospholipid antibody syndrome, Behcet disease, sarcoidosis, Lyme's disease, cerebral arteritis, brain lymphoma, etc.) 5) presence of oligoclonal bands on the CSF analysis; 6) presence of two or more lesions in the brain or spinal cord, detected at the initial MRI performed before inclusion. The last two criteria meant that only CIS patients fulfilling at least the dissemination in space criteria according to McDonald 7 were recruited. According to these restricted criteria (especially the presence in all the patients of oligoclonal bands in the CSF and at least two T2 WM lesions on the brain MRI), the patients included in the present study presented high risk for developing MS.

Patients underwent a clinical examination during the first neurological episode. Another neurological examination was performed on the day of inclusion (the same day when MRI was performed). All the patients' disability levels were rated using the expanded disability status scale (EDSS) [START_REF] Kurtzke | Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)[END_REF] by the same neurologist (BA) on the day of inclusion.

Neuropsychological tests were performed on the patients using the Brief Repeatable Battery (BRB) [START_REF] Boringa | The brief repeatable battery of neuropsychological tests: normative values allow application in multiple sclerosis clinical practice[END_REF] including the Selective Reminding Test, the Spatial Recall Test, the Symbol Digit Modalities Test, the Paced Auditory Serial Addition Task (3') and the Word list generation.

All the participants gave their informed consent to participating in this study, which was approved by the local Ethics Committee (Timone Hospital, Marseille, France).

Brain MRI

The subjects were examined on a 1.5T Magnetom Vision Plus MR Imager (Siemens, Erlangen, Germany). A sagittal three-dimensional MP-RAGE T 1 -weighted sequence (TE/TR = 4.7 ms/9.7 ms, flip angle 12 • , 128 contiguous slices, matrix = 256 2 , isotropic voxel 1.25mm×1.25mm×1.25 mm) and transverse fast double spin echo T 2 -weighted images (TE 1 /TE 2 /TR = 15 msec/85 msec/ 2600 msec, 44 contiguous slices, thickness = 3 mm, flip angle = 90°, FOV = 240 mm, matrix = 256 2 ) were acquired on all subjects.

Image Processing

WM Lesion load

WM lesions visible on T2-weighted images were contoured by the same neurologist (BA) using a semi-automated method (interactive thresholding technique written on the interactive data language (IDL) platform; Research System, Inc.). WM lesion masks labelled as T1-WM lesion masks were identified by simultaneously viewing T1-weighted and T2-weighted images before contouring the lesions on the T1weighted images using the same semi-automated method (interactive thresholding technique written on the interactive data language (IDL) platform; Research System, Inc.).

Optimized VBM

The potential influence of the lesions on the results of the registration is a crucial point in VBM study performed in MS patients. This potential caveat is probably highly critical in patients with several years of disease evolution when the WM lesion load is important.

Various methods have been proposed 10 11 to limit this effect. In the present study, to minimize this potential caveat, we used a modified version of the optimized VBM method [START_REF] Good | A voxel-based morphometric study of ageing in 465 normal adult human brains[END_REF] customised for MS [START_REF] Khaleeli | Localized grey matter damage in early primary progressive multiple sclerosis contributes to disability[END_REF] , where WM lesions masks were applied to patients' scans at the end of images processing to remove any lesional tissue erroneously classified as grey matter. Figure 1 describes the analysis pipeline Volumetric T 1 -weighted images were first normalized spatially (medium regularization, 7×9×7 nonlinear basis functions) into the MNI space using the T 1 anatomical template provided by the SPM2 program. Images were then re-sampled using an isotropic 1.5mm×1.5mm×1.5 mm voxel. The spatial normalization algorithm preserved the voxel intensities (concentrations) even when region volumes were stretched by warping. After smoothing the images with a 12-mm Gaussian filter, a local T 1 template was obtained by averaging the smoothed images obtained with each subject.

Secondly, the 3D-T 1 -weighted volumes were normalized spatially into the MNI space using the local T 1 template previously obtained. Segmentation of these normalized 3-D T 1 -weighted volumes was performed with each subject (SPM2). The resulting normalized GM fraction maps were smoothed using a 12mm Gaussian filter and averaged across all controls to obtain the locally optimized GM template.

Thirdly, the 3D-T1 volumes obtained with each subject were directly segmented. The resulting GM fraction maps were normalized spatially using the locally optimized GM template. The transformation obtained for each spatial normalization was applied to the 3D T 1 -weighted volumes and the WM T 1 lesion masks of each subject. Then, the normalized 3D T 1 -weighted volumes were segmented and the normalized T 1 lesion masks were subtracted from the normalized GM fraction maps to prevent misclassification of WM lesion Lastly, a conservative threshold of 0.75 was applied to the resulting normalized GM fraction maps free of WM lesions before smoothing the images with a 12-mm FWHM Gaussian kernel [START_REF] Good | A voxel-based morphometric study of ageing in 465 normal adult human brains[END_REF] .

Statistical mapping analysis

Between-group comparisons (patients with CIS versus controls) were performed (two-sample t-test, p < 0.005, k = 20, FWE corrected, SPM2) on the smoothed GM fraction maps obtained using the optimized method to determine the location of clusters showing significant differences in the GM concentrations. Coordinates of significant clusters in the MNI space were transformed into Talairach coordinates using a nonlinear transformation to locate these clusters.

Results

Clinical and conventional MRI findings

Patients' demographic and clinical characteristics of patients are given in Table 1. The median time between the clinical onset and the inclusion (the time when the MRI was performed) in the study was 4 months (0-6). Median age of the controls was 27 years (20-46), which did not differ significantly from that of the patients (p=0.94). A sub-population of 37 patients performed the neuropsychological testing. This sub-population did not differ significantly from the other patients in terms of sex, age, educational level, disease duration, T2LL or EDSS (Table 2). We also checked whether there existed any differences in terms of GM atrophy between the two groups of patients (those who underwent the neuropsychological tests and those who did not). These two groups were not found to differ in terms of the regional GM atrophy detected (p<0.005, FWE corrected). The patients' performances were compared with the performances previously recorded at our laboratory in a group of 52 healthy control subjects. The patients not differ from this control group in terms of sex, age or educational level (Table 2). Patients showed abnormally low performances in the Spatial Recall Test, the Symbol Digit Modalities Test, the Paced Auditory Serial Addition Task (3') and the Word list generation (Table 2).

Brain parenchyma fraction (BPF) was not significantly decreased (p=0.06) in patients (BPF=0.831; SD=0.043) compared to controls (BPF=0.847; SD=0.039). GM fraction (GMF) was significantly decreased (p=0.01) in patients (GMF=0.50; SD=0.045) compared to controls (GMF=0.52; SD=0.046). WM fraction (WMF) was not decreased (p=0.94) in patients (WMF= 0.33; SD=0.025) compared to controls (WMF= 0.33; SD=0.021).

Patterns of regional GM atrophy at the earliest stage of MS

Results are summarized in Table 3 and Figure 2. At the FWE corrected statistical threshold level of p<0.005, patients showed atrophy localized in the bilateral thalami, the bilateral caudate nuclei, the bilateral lenticular nuclei, the bilateral insula, the bilateral orbitofrontal cortices, the bilateral internal and inferior temporal regions, the posterior cingulate cortex and the bilateral cerebellum; whereas healthy controls showed no significant atrophy compared to patients.

Correlations between conventional MRI data and regional cerebral atrophy

The correlations have been assessed in the whole group of patients (n=62). The degree of atrophy of the thalami was found to be significantly correlated with the T2LL (right thalamus: rho=0.57 p=0.001; left thalamus: rho=0.48 p=0.001). In the whole group of patients (n=62), local GM atrophy was not correlated with global GMF.

Correlations between T2LL, physical and cognitive status

p=0.03). In the sub-population of 37 patients with neuropsychological assessment, no correlations were found between T2LL and abnormal neuropsychological performances observed in patients.

Correlations between regional GM atrophy, physical and cognitive status

EDSS scores assessed in all the patients (n=62) were found to be correlated with the degree of atrophy in the right cerebellum (Rho=-0.37 p=0.0027). In the sub-population of 37 patients having neuropsychological assessment, abnormal neuropsychological performances (Visuospatial memory (Short-term recall), Symbol Digit Modalities Test, Paced Auditory Serial Addition Task (3'), Word list generation) did not significantly correlate with the level of GM concentration in regions prone to atrophy

Discussion

The results of the present study on a large sample of patients with CIS provide evidence that regional GM atrophy is present in the first clinical stage of MS and that it mainly occurs in the deep GM and the limbic system.

Pattern of local GM atrophy in patients in the earliest stage of MS

Atrophy of the deep GM has been well documented in patients with relapsing remitting 14 15 and primary progressive 10 13 MS. In a previous study on a relatively small group of patients (n=15) with early relapsing remitting MS (RRMS), no regional GM atrophy was detected at inclusion, whereas significant bilateral atrophy of the thalami was present two years later [START_REF] Audoin | Localization of grey matter atrophy in early RRMS : A longitudinal study[END_REF] .

Apart form the thalamic atrophy, a subtle involvement of the thalamus may occur in the earliest stage of RRMS 6 17 : in a study on a small population of patients with CIS (n=18), statistical mapping analysis applied to magnetization transfer ratio data showed significant tissue matrix disorganization of the deep GM relative to controls (n=18) [START_REF] Audoin | Voxel-based analysis of MTR images: a method to locate gray matter abnormalities in patients at the earliest stage of multiple sclerosis[END_REF] . Recently, a VBM study performed in CIS demonstrated significant regional GM atrophy mainly located in the thalamus [START_REF] Henry | Regional grey matter atrophy in clinically isolated syndromes at presentation[END_REF] . In the present study on a larger group of patients with CIS, GM atrophy was detected using the VBM method in the thalamus but also in the large majority of the deep GM structures.

Regional GM atrophy has been previously reported to occur in the temporal and the frontal cortices in patients with RRMS 18 19 . In a study using a rather elegant approach to determine the cortical thickness, a local GM thinning was also observed in the cingulate gyrus, insula, and associative cortical regions, which correlated with the patients' neurological deficits and their T2 LL scores [START_REF] Charil | Focal cortical atrophy in multiple sclerosis: relation to lesion load and disability[END_REF] . However, the method used in the latter study did not make it possible to explore the deep GM structures and no control data were used [START_REF] Charil | Focal cortical atrophy in multiple sclerosis: relation to lesion load and disability[END_REF] . A recent MRI study on MS patients clearly established that the temporal lobe and the hippocampus were atrophic in patients with MS after several years of evolution [START_REF] Sicotte | Regional hippocampal atrophy in multiple sclerosis[END_REF] . Geurts et al 22 also detected numerous inflammatory lesions in the hippocampus which may underlie tissue loss and GM atrophy evidenced by MRI.

The pathogenesis of early GM atrophy may involve different processes [START_REF] Chard | Grey matter pathology in clinically early multiple sclerosis: evidence from magnetic resonance imaging[END_REF] . First, the axonal impairments resulting from WM lesions may induce distal GM lesions secondary to Wallerian degeneration [START_REF] Evangelou | Regional axonal loss in the corpus callosum correlates with cerebral white matter lesion volume and distribution in multiple sclerosis[END_REF] or anterograde transynaptic damage 25 26 . In a large cohort of patients (n=425) in the advanced stage of the disease, Charil et al. observed the existence of correlations between cortical thickness and the total lesion load and disability in the cingulate gyrus, insula, and associative cortical regions: these brain regions are strongly interconnected with other brain regions [START_REF] Charil | Focal cortical atrophy in multiple sclerosis: relation to lesion load and disability[END_REF] . These authors suggested that interruption of WM tracts by MS plaques may contribute to the development of cortical atrophy. Similar observation have been evidenced recently by Henry and colleagues demonstrating a link between WM lesions located in the thalamo-cortical tract and the level of atrophy of the thalami [START_REF] Henry | Connecting white matter injury and thalamic atrophy in clinically isolated syndromes[END_REF] . This mechanism may explain the correlations observed in the present study between the T 2 LL and atrophy of the thalami, one of the most strongly connected GM regions. Another possibility is that a pathological process characterized by iron deposition may be involved in the inflammatory mechanism [START_REF] Bakshi | T2 hypointensity in the deep gray matter of patients with multiple sclerosis: a quantitative magnetic resonance imaging study[END_REF] . Significant correlations have been reported to exist between the number of T2 lesions and the abnormal iron deposition rates in the thalami [START_REF] Ge | Quantitative assessment of iron accumulation in the deep gray matter of multiple sclerosis by magnetic field correlation imaging[END_REF] . These latter authors suggested that WM lesions may disrupt the axonal iron output, which leads to the accumulation of iron in the deep GM [START_REF] Ge | Quantitative assessment of iron accumulation in the deep gray matter of multiple sclerosis by magnetic field correlation imaging[END_REF] .

Although the atrophy of the thalami was partly associated with the T2LL, no association with other atrophic GM regions have been evidenced suggesting that other factors may participate to regional atrophy. First, it is well known that the pathology of the WM is not restricted to the macroscopic WM lesions. Consequently, the mechanisms described above (Wallerian degeneration, anterograde transynaptic damage and disruption of the axonal iron output) may exist in the normal-appearing white matte inducing more diffuse GM atrophy. In addition the assessment of the potential association between the total lesion load and the regional GM concentration may be sub-optimal when considering the possibility of GM damage being mediated through WM tracts [START_REF] Audoin | Selective magnetization transfer ratio decrease in the visual cortex following optic neuritis[END_REF] . Diffusion tensor tractography may be relevant in future studies to better assess the potential link between lesions located in the WM tracts and remote GM pathology. Secondly, the limited association between WM lesions and GM atrophy may be related to the existence of another pathological process more restricted to the GM.

The authors of several studies have reported the occurrence of inflammatory lesions of the GM in MS. These GM inflammatory processes may consist of focal GM lesions and/or diffuse sub-pial inflammation [START_REF] Kutzelnigg | Cortical demyelination and diffuse white matter injury in multiple sclerosis[END_REF] . The extension of diffuse inflammatory processes in the GM may be more pronounced in patients with progressive forms of the disease [START_REF] Kutzelnigg | Cortical demyelination and diffuse white matter injury in multiple sclerosis[END_REF] . Up to now, no evidence has been available as to whether some GM structures may show preferential susceptibility to the GM inflammatory process occurring in MS. One hypothesis is that atrophy -the ultimate consequence of tissue injury -may start in the GM regions which are most sensitive to the diffuse inflammatory process, although this process may not be especially prominent in these regions. Another explanation for the pattern of distribution of the GM atrophy observed is that the GM inflammatory process may predominate in some regions, resulting in significant localized GM atrophy. However, to our knowledge, no data are available so far which shed light on the regional pattern of distribution of the GM inflammatory process in MS.

Another potential mechanism involved in GM atrophy may be represented by the pathology and/or loss of glial cells which represent 60% of the GM cell count in humans [START_REF] Pelvig | Neocortical glial cell numbers in human brains[END_REF] .

Relationships between regional GM atrophy and clinical status

Since the population studied here consisted of patients presented with CIS, the residual EDSS recorded after the relapse was generally low (median 1, range 0-3.5), which meant that few correlations with regional GM atrophy were likely to occur. In addition, the limited association between brain regional GM atrophy and EDSS may be partly due to the characteristics of the EDSS scale particularly sensitive to spinal cord pathology not explored in the present study. Finally, compensatory processes known to occur from the very first stage of the disease onwards may limit the clinical impact of early regional GM loss. The only significant correlation observed, which was between the atrophy of the left cerebellum and the EDSS, was probably due to the fact that the cerebellum contributes importantly to movement control [START_REF] Ito | Control of mental activities by internal models in the cerebellum[END_REF] .

In the present study, the cognitive performances of patients with CIS were significantly impaired in tasks involving working memory, attention and speed of information processing.

Previous studies have shown the existence of similar types of cognitive impairment in patients with CIS 33 34 . Since all the cognitive abilities in question depend on widely distributed brain networks, these deficits may have resulted from connectivity disturbances secondary to WM injury [START_REF] Audoin | Structure of WM bundles constituting the working memory system in early multiple sclerosis: a quantitative DTI tractography study[END_REF] . The lack of correlation observed in the present study between cognitive impairment and regional GM atrophy suggests that the main pathological substrate of cognitive impairment in CIS patients is WM pathology. With the progression of the disease, the contribution of the GM injury probably increases, which would explaining the correlations found to exist between cognitive impairment and GM injury in patients after several years of disease evolution [START_REF] Amato | Neocortical volume decrease in relapsingremitting MS patients with mild cognitive impairment[END_REF] . In addition, in a group of patients with RRMS and SPMS, Sanfilipo et al [START_REF] Sanfilipo | Gray and white matter brain atrophy and neuropsychological impairment in multiple sclerosis[END_REF] observed that WM and GM injury had differential effects on the patients' cognitive performances. WM injury was found to be the best predictor of mental processing speed and working memory, whereas GM injury corresponded to verbal memory, euphoria, and disinhibition. In the present study on CIS patients, the fact that the cognitive impairments were restricted to mental processing speed and working memory may explain the lack of correlation observed between the regional GM volume and the cognitive deficits. The characteristics of the cognitive impairments may change slightly with the progression of the disease. In the very first stage, isolated processing speed and working memory deficits are directly related to the state of the WM. After several years, other cognitive abilities such as verbal memory are affected, probably due to the deterioration of the GM.

In addition, in view of the presence of the GM atrophy in the limbic and para-limbic regions, the main functional effect of the early GM pathology may be the emotional disturbances occurring at this stage in the disease. Since the patients' emotions were not assessed in the present study, it was not possible to test this hypothesis.

Conclusion

The present study performed on a large group of CIS patients with very low physical and cognitive disability, demonstrated highly significant regional GM atrophy in the deep GM and the limbic system. This study emphasized for an involvement of GM by MS pathological process from the onset of the disease. Regions showing significant GM atrophy in CIS patients (n=62) compared to controls (n=37) (p<0.005, FWE corrected). GM atrophy was localized in the bilateral thalami, the bilateral caudate nuclei, the bilateral lenticular nuclei, the bilateral insula, the bilateral orbitofrontal cortices, the bilateral internal temporal regions, the posterior cingulate cortex and the bilateral cerebellum.

Figure 1 .Figure 2 .

 12 Figure 1. Analysis pipeline of the VBM procedure

Table 1 .

 1 Demographic and clinical characteristics of CIS patients.

		Whole group	Patients with	Mann
		of Patients	neuropsychological	Whitney
		(n=62)	assessment (n=37)	U test*
	Age (median)	29 (20-46)	29.5 (21-45)	NS (0.94)
	Time from the	4 (0-6)	4 (1-6)	NS (0.46)
	clinical onset
	(month,
	median)
	T2LL (cm3,	2.2 (0.1-111)	2.5 (0.1-61)	NS (0.7)
	median)
	EDSS	1 (0-3.5)	1 (0-2)	NS (0.64)
	(median)
	Type of
	symptoms:
	Spinal cord	24 (38.7%)	16 (44.4%)
	Brainstem	16 (25.8%)	11 (30.5%)
	Optic nerve	15 (24.2%)	5 (13,9 %)
	Hemispheric	7 (11.3%)	4 (11.1%)

* statistical comparison between patients with neuropsychological assessment and the whole group of patients

Table 2 .

 2 Regions showing significant GM atrophy in CIS patients (n=62) compared to controls (n=37) (p<0.005, FWE corrected).

	Regions	Side	Brodmann	Talairach	T	Number
			areas	coordinates		of voxels
	Caudate	Right	NA	-16 20 5	10.6	4848
		Left	NA	14 18 3	9.73
	Lenticular nucleus	Right	NA	-26 6 5	6.6
		Left	NA	26 4 3	6.1
	Thalamus	Right	NA	-14 -31 11	7.7
		Left	NA	12 -19 16	7.3
	Amygdala	Right	NA	24 -6 -13	10.12
	Insula	Right	NA	-36 2 7	5.6
		Left	NA	42 10 7	5.7
	Cerebellum	Right	NA	-40 -78 -15	7	1620
		Left	NA	22 -88 -16	6.7
	Orbito-frontal cortex	Left	BA 47	48 38 -10	7.3	423
				28 9 -17	7.3
			BA 11	25 50 -16	5.7
				18 38 -22	6.4
		Right	BA47	-26 9 -17	7.7	297
			BA 11	-32 44 -12	7.4
				-12 40 -20	7
	Hippocampus	Right	NA	-28 -18 -11	7.5	287
				-32 -29 -5	7
		Left	NA	32 -30 -5	5.3	86
	Posterior cingulate		BA 31	0 -34 27	7.1	71
	cortex
	Inferior temporal	Right	BA 20	-36 -6 -38	7.5	38
	cortex
				-57 -21 -24	6.2
		Left	BA 37	57 -47 -9	5.9	22
			BA 20	38 -4 -40	5.4

Table 3 .

 3 Neuropsychological tests used and results for healthy controls and CIS patients

Acknowledgments

This research was supported by the CNRS, the Institut Universitaire de France, Bayer-Schering France, and The French 'Association pour la Recherche sur la Sclérose en Plaques' (ARSEP). There is no any financial interest related to this study from any of the authors.


François Ducray

Francesc Graus

Maria Claudia Vigliani

Jean-Christophe Antoine

Veronique Rogemond

Albert Saiz

PhD Jérôme Honnorat
email: jerome.honnorat@chu-lyon.fr

Delayed onset of a second paraneoplastic neurological syndrome in eight patients

Background:

The occurrence of a second, delayed paraneoplastic neurological syndrome (PNS), different from the first, in the same patient is not well known. However, the recognition of this possibility is important in the management of the patients with PNS.

Objective: To describe eight patients who presented two different PNS with a time delay of months to years.

Design, setting and patients: Retrospective analysis of eight patients, included in the

European PNS database, with two different PNS that occurred months to years apart.

Results:

The median delay between the two different PNS was 15 months. The antibody repertoire did not change between the two episodes. Anti-Hu was detected in five patients, anti-CV2 and anti-Ri in one each. One patient did not present onconeural antibodies. Lung cancer was diagnosed in six patients. In five patients, the second PNS revealed a cancer relapse (n=4) or a second cancer (n=1) and in three it occurred without evidence of tumor recurrence. In these three patients, all with anti-Hu antibodies, the second episode consisted of a lower motor neuron disease. Median survival was not reached after a median follow-up of five years.

Conclusions:

A second PNS can reveal a cancer relapse but can also arise in its absence. The long survival of patients with a second PNS suggests that the immune response might be more effective in controlling the cancer.

Introduction

Patients with paraneoplastic neurological syndrome (PNS) usually display a subacute, aggressive evolution and many patients die a few months after the diagnosis due to the combined effects of the neurological disability and cancer progression. In the most common PNS, paraneoplastic encephalomyelitis/sensory neuropathy associated with Hu antibodies, the median survival time is 11 months and the percentage of survivors beyond three years is less than 20%. [START_REF] Graus | Anti-Hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients[END_REF][START_REF] Honnorat | Onco-neural antibodies and tumor type determine survival and neurological symptoms in paraneoplastic neurological syndromes with Hu or CV2/CRMP5 antibodies[END_REF] The information on the clinical evolution of the PNS in those patients who survive more than three years is scarce. A recent study suggests that patients who are long term survivors of a PNS associated with anti-Hu antibodies slowly progress over the years despite the cancer is cured suggesting that the immune response may become independent of the original tumor trigger and keep active for many years. [START_REF] Poepel | Neurological course of long-term surviving patients with SCLC and anti-Hu syndrome[END_REF] In line with this observation, we found that anti-Hu antibody levels remain unchanged several years after remission of the tumor. In this study, two of the 35 patients included presented with a second, different PNS months or years after the diagnosis of the first PNS. [START_REF] Llado | Value of Hu antibody determinations in the follow-up of paraneoplastic neurologic syndromes[END_REF] In the present study, we analyzed the clinical and immunological features of a series of eight patients who developed over the years two different types of PNS to ascertain their clinical profile and draw the attention to this possibility in patients followed with a PNS.

Methods

Data from the PNS (Paraneoplastic Neurological Syndrome) EURONETWORK Database (http://www.pnseuronet.org) collected between January 2000 and December 2008 (n=979) was retrospectively reviewed [START_REF] Giometto | Paraneoplastic neurological syndromes from the PNS-Euronetwork database. A European study from 20 centres[END_REF]. Patients with a second PNS different from the first one were identified in four centers (Barcelona (n=3), Lyon (n=2), Torino (n=2), and Saint-Etienne (n=1)). Patients' sera were tested for the presence of Hu-, Ri-, CV2/CRMP5-, Yo-, Ma2-, and amphiphysin-antibodies by immunohistochemistry and western blotting as previously described. [START_REF] Graus | Recommended diagnostic criteria for paraneoplastic neurological syndromes[END_REF]

Results

Patients' characteristics at the time of the first PNS are summarized in Table 1 (detailed case reports are provided as supplemental data). The median age was 54 years (42-71 years). The first PNS episodes presented as a subacute sensory neuronopathy (n=2), a sensorimotor neuropathy (n=3), an opsoclonus-myoclonus (n=2), and a brainstem encephalitis (n=1).

Serum screening for onconeural antibodies was positive in six patients: anti-Hu (n=4), anti-CV2/CRMP5 (n=1), anti-Ri (n=1). This distribution was not different from the distribution in the 979 patients of the PNS EURONETWORK Database which contains 38.8% of anti-Hu (n=380), 6% of anti-CV2/CRMP5 (n=59), 5.1% of anti-Ri (n=50) and 28.2% (n=276) without classical onconeural antibodies [START_REF] Giometto | Paraneoplastic neurological syndromes from the PNS-Euronetwork database. A European study from 20 centres[END_REF]. Associated cancers were SCLC (n=4), carcinoid anaplastic lung tumor (n=1), prostatic adenocarcinoma (n=1), undifferentiated bronchial carcinoma (n=1), and breast cancer (n=1). After treatment of the underlying tumor in association with immunoactive drugs, the neurological condition improved in five patients and remained stable in three.

Patients' characteristics at the time of the second PNS are summarized in Table 2. In all patients, the second PNS was different from the first one and occurred without recurrence of the first one except in patient 6 in whom a mild worsening of the sensory neuronopathy was initially observed. Among the three patients in whom the first and second PNS consisted of a neuropathy (patients 2, 3 and 6), the second PNS (lower-motor neuron disease in patients 2 and 6, gastro-intestinal pseudo-obstruction in patient 3) could not just be considered as the worsening of the initial sensory neuropathy because none of these three patients had these symptoms at first PNS and the pathological substrate of the new symptoms represented the immune attack to another area of the nervous system (the spinal motorneurons or the myentheric plexuses). The median delay between the first and second PNS was 15 months . The diagnoses of the second PNS was encephalomyelitis (n=1), chorea (n=1), stiffperson syndrome (n=1), dermatomyositis (n=1), dysautonomia with gastro-intestinal pseudoobstruction (n=1), and lower motor neuron disease (n=3). In patients with CNS involvement MRI imaging and CSF studies ruled out metastatic disease. These second PNS episodes antedated a cancer relapse in four patients and a second cancer in one. In three patients, the second PNS occurred without evidence of cancer relapse despite repeated whole body PET scan imaging. Among the six patients who had an onconeural antibody, the same onconeural antibody was always found at the time of the second PNS. The titer of onconeural antibodies at both episodes was available in four patients: it remained stable (n=2) or decreased, and eventually became undetectable, before increasing again (n=2). In one patient, Hu antibodies were only detected at the time of the second PNS. Cancer was treated in all patients in whom the second PNS was associated with a cancer relapse or a second cancer. One patient (patient 6) was treated with chemotherapy even though no relapse of the SCLC was evident. Six patients received immunoactive treatments, which consisted of steroids (n=4), IVIg (n=4), cyclophosphamide (n=1), and cyclosporine (n=1). Three patients improved after treatment, two patients remained stable, and three patients experienced progression of the neurological deficit. After a median follow-up of 4.5 years after the first PNS, median survival has not been reached. Five patients are alive without relapse of the cancer, and four of these did not experience progression of the neurological symptoms. Three patients died, one from cancer relapse and two from evolution of the second PNS.

The second PNS consisted of a lower motor neuron disease in the three patients without evidence of cancer relapse. These patients had anti-Hu antibodies. In patient 6, the lower motor neuron disease first involved the lower limbs before extending to the upper limbs, abdomen, and trunk. Anti-Hu antibodies became undetectable after the first PNS and reappeared at the onset of the lower motor neuron disease. CSF examination demonstrated only an elevated protein level (0.75 g/L). Treatment included chemotherapy, despite no evidence of cancer relapse, followed by steroids, IVIg, and cyclosporine. However, the patient died of respiratory insufficiency after 5 years of evolution. No necropsy was performed. In patients 2 and 7, the lower motor neuron disease was localized. In patient 2, it was limited to the upper limbs, the trunk, and the abdomen. The CSF showed mild pleiocytosis (4 lymphocytes/mm3), elevated protein level (0.6 g/L), and oligoclonal bands. After treatment with IVIg and cyclophosphamide, no progression of the lower motor neuron was observed.

The patient is alive and stable 8 years after the onset of the lower motor neuron disease without evidence of cancer relapse. The lower motor neuron disease was limited to the legs in patient 7. CSF showed only an elevated protein concentration (1.5 g/L). The patient was treated with steroids and he is alive and stable 4.5 years after the onset of the lower motor neuron disease without evidence of cancer relapse.

Discussion

The simultaneous involvement of different areas of the nervous system by the paraneoplastic process is not unusual and the term paraneoplastic encephalomyelitis (PEM) was introduced to define this syndrome that usually occurs in the setting of anti-Hu antibodies and lung cancer. [START_REF] Graus | Anti-Hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients[END_REF] PEM is reported in 11% of patients with PNS. [START_REF] Graus | Anti-Hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients[END_REF] In contrast the simultaneous occurrence of two different, classical PNS, are less frequent. Well known examples are the coincidence of paraneoplastic cerebellar degeneration and Lambert-Eaton myasthenic syndrome associated with antibodies to calcium channels and lung cancer or the simultaneous development of sensory neuronopathy and gastrointestinal pseudoobstruction in patients with lung cancer and anti-Hu antibodies. [START_REF] Graus | Anti-Hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients[END_REF][START_REF] Mason | Small cell lung cancer, paraneoplastic cerebellar degeneration and the Lambert-Eaton myasthenic syndrome[END_REF] The delayed onset of a second PNS seems to be much rarer since we identified only 8 cases with this condition (0.8%) among the 979 patients of the PNS EURONETWORK database and found only one similar case in the literature. [START_REF] Nagashima | Anti-Hu paraneoplastic syndrome presenting with brainstem-cerebellar symptoms and Lambert-Eaton myasthenic syndrome[END_REF] Anti-Hu was the most commonly found onconeural antibody in patients with delayed onset of a second PNS. Although the second PNS was different from the first in all cases, the antibody repertoire did not change between the two episodes. In fact, in two patients (patients 1 and 3), the second PNS could be considered to be a different manifestation of the clinical syndrome usually associated with the underlying antibody. Indeed, polyneuropathy and chorea are two typical manifestations of the anti-CV2/CRMP5 syndrome, [START_REF] Honnorat | Onco-neural antibodies and tumor type determine survival and neurological symptoms in paraneoplastic neurological syndromes with Hu or CV2/CRMP5 antibodies[END_REF][START_REF] Vernino | Paraneoplastic chorea associated with CRMP-5 neuronal antibody and lung carcinoma[END_REF][START_REF] Yu | CRMP-5 neuronal autoantibody: marker of lung cancer and thymoma-related autoimmunity[END_REF] and sensory neuronopathy and gastro-intestinal pseudo-obstruction are two typical manifestations of the anti-Hu syndrome. [START_REF] Graus | Anti-Hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients[END_REF] In 5 patients (62.5%), the second PNS was clearly associated with relapse of the initial cancer or of a second cancer suggesting that cancer triggered the immune reaction responsible of the second PNS. However, in three cases, no cancer relapse was observed. Surprisingly, all this three patients had anti-Hu antibodies and similar lower motor neuron disease. About 15% to 20% of patients with anti-Hu syndrome show evidence of lower motor neuron involvement, but it is rarely the only manifestation. [START_REF] Graus | Anti-Hu-associated paraneoplastic encephalomyelitis: analysis of 200 patients[END_REF][START_REF] Forsyth | Motor neuron syndromes in cancer patients[END_REF][START_REF] Verma | Motor neuron disease: a paraneoplastic process associated with anti-hu antibody and small-cell lung carcinoma[END_REF] Because of the particular features of the lower motor neuron disease in these three patients, it is very unlikely that it corresponded to a degenerative motor neuron disease. [START_REF] Vigliani | Patients with amyotrophic lateral sclerosis and cancer do not differ clinically from patients with sporadic amyotrophic lateral sclerosis[END_REF] It rather suggests that the immune response had become independent of the cancer. Spontaneous complete remissions have been observed in patients with SCLC and anti-Hu antibodies; thus, an alternative hypothesis would be that a cancer relapse was not found because of effective anti-tumor immunity. [START_REF] Gill | Paraneoplastic sensory neuronopathy and spontaneous regression of small cell lung cancer[END_REF] Whatever, this similar lower motor neuron syndrome in patients without tumor relapse suggests a common mechanism of immunization against motoneurons probably specific of Hu patients.

Finally, the last interesting characteristic of these patients was their prolonged survival.

Median survival in the five patients with anti-Hu antibodies was not reached at five years.

Table 1 :

 1 Patients' characteristics at the time of first PNS. Pananeoplastic Neurological Syndrome; SCLC :Small cell lung cancer; IVIg= Intravenous immunoglobulins

	Age Sex First PNS	Rankin	Cancer	Antibodies Cancer treatment	Immunotherapy Evolution
	Patient 1 56 M	Subacute sensory motor axonal polyneuropathy	3	SCLC	Anti-CV2	Chemotherapy and radiotherapy	Steroids	Improvement +++ (Rankin 1)
	Patient 2 47 M Subacute sensory axonal polyneuropathy	2	Undifferentiated bronchial carcinoma	Anti-Hu	Surgery	None	Improvement + (Rankin 1)
	Patient 3 46 M Subacute sensory neuronopathy	1	SCLC	Anti-Hu	Chemotherapy and radiotherapy	Steroids	Stabilization
	Patient 4 54 F	Opsoclonus-myoclonus	3	Breast cancer metastasis	Anti-Ri	Surgery and hormonotherapy IVIg and steroids	Improvement +++ (Rankin : 0)
	Patient 5 56 F	Subacute sensory motor axonal polyneuropathy	3	SCLC	None	Chemotherapy	None	Improvement +++ (Rankin 1)
	Patient 6 42 F	Subacute sensory neuronopathy	3	SCLC	Anti-Hu	Chemotherapy and radiotherapy	IVIg and steroids Stabilization
	Patient 7 70 M Brain stem encephalitis	4	Prostatic adenocarcinoma Anti-Hu	Hormonotherapy and radiotherapy	IVIg and steroids Stabilization
	Patient 8 71 M Opsoclonus-myoclonus	3	Carcinoid bronchial tumor None	Chemotherapy	IVIg and steroids	Improvement +++ (Rankin 0)
	PNS:

This result contrasts dramatically with the usual median survival reported in anti-Hu patients (11.8 to 18 months). [1,2] This suggests that in patients who develop a second PNS, the immune response is more aggressive but at the same time more effective in eradicating the cancer.


Nalin Hetticarachchi

H Ruth Ashbee

Dr Janet D Wilson
email: janet_d.wilson@leedsth.nhs.uk

Prevalence and Management of Non-albicans Vaginal Candidiasis

Keywords: Vulvo-vaginal candidiasis, Candida glabrata, amphotericin, flucytosine Word count:

Objectives

It is thought that widespread use of "over-the-counter" azoles may increase the incidence of resistant Candida species such as C glabrata. Infections with species other than C. albicans frequently do not respond to standard azole treatments. Intravaginal nystatin is an option but is no longer available in the UK. In this paper, we review the prevalence of non-albicans candida over the past five years, and assess the efficacy of amphotericin and flucytosine vaginal cream in the treatment of non-albicans VVC.

Methods

Retrospective review of all vaginal yeast isolates collected from women attending a city centre sexual health clinic between 2004 and 2008. The women prescribed amphotericin and flucytosine vaginal cream were identified through pharmacy records and their clinical notes reviewed for treatment outcome.

Results

Between 2004-2008, the number of isolates of all Candida species increased with the increase in workload of the clinic but the prevalence of non-albicans yeasts remained stable at between 0.87-1.06%. Eighteen patients were prescribed amphotericin and flucytosine vaginal cream. At follow up all 18 were clear of their initial yeast isolate on culture but two had persistent symptoms and had positive cultures for C. albicans.

Conclusions

There is no evidence of an increase in prevalence of non-albicans Candida species such as C glabrata. We have treated 18 women who had non-albicans VVC with amphotericin and flucytosine vaginal cream and achieved clearance of the nonalbicans species in all of them.

Introduction

The majority of cases of vulvovaginal candidiasis (VVC) are due to Candida albicans with other yeasts accounting for about 10%. [START_REF] Holland | Vulvovaginal carriage of yeasts other than Candida albicans[END_REF] C glabrata causes most nonalbicans infections. [START_REF] Holland | Vulvovaginal carriage of yeasts other than Candida albicans[END_REF] As C. glabrata is inherently less susceptible to fluconazole than C. albicans, there have been concerns that the widespread use of "over-thecounter" azoles, particularly fluconazole, might lead to an increase in the incidence of less susceptible Candida species such as C glabrata. [START_REF] Walker | Vaginal yeasts in the era of "over the counter" antifungals[END_REF][START_REF] Fidel | Candida glabrata: Review of epidemiology, pathogenesis and clinical disease with comparison to C albicans[END_REF] A previous study suggested this was not the case, but this was published some years ago. [START_REF] Walker | Vaginal yeasts in the era of "over the counter" antifungals[END_REF] Non-albicans infections frequently do not respond to standard azole antifungal treatments. [START_REF] Fidel | Candida glabrata: Review of epidemiology, pathogenesis and clinical disease with comparison to C albicans[END_REF] Intravaginal nystatin is an option but this treatment became unavailable in the UK during 2007. [START_REF]Clinical Effectiveness Group, British Association of Sexual Health and HIV[END_REF] Four case reports suggested that amphotericin and flucytosine vaginal cream (amp/flu VC) was an effective treatment for nonalbicans VVC [START_REF] White | Combined topical flucytosine and amphotericin B for refractory vaginal Candida glabrata infections[END_REF][START_REF] Shann | Treatment of Candida glabrata using topical amphotericin B and flucytosine[END_REF] and this has been used in our clinic since nystatin became unavailable.

In this paper we review the prevalence of Candida species within our clinic over the past five years, with particular emphasis on non-albicans species, and assess the efficacy of amp/flu VC in the treatment of VVC caused by non-albicans species. Methods A retrospective review was performed of all vaginal yeast isolates collected from vaginal samples taken from all women with symptoms attending the Leeds Centre for Sexual Health between 2004 and 2008. Positive cultures were quantified and the isolates identified. The germ tube test was used to identify C. albicans. All germ tube negative isolates were identified using Auxacolor 2, Dalmau agar and, where necessary, API 32C kits. The amphotericin 100mg with flucytosine 1g in Aquagel per vagina nightly for 14 nights was obtained from the Pharmacy Manufacturing Unit, North Staffordshire Hospital, Stoke-on-Trent. The women prescribed amp/flu VC were identified through the Leeds General Infirmary hospital pharmacy records and their clinical notes reviewed for the outcome of treatment.

Results

The numbers and prevalence of C. albicans, C. glabrata and other yeasts isolated from the women are shown in table 1 The number of isolates of non-albicans yeasts has increased but this is proportionate to the increase in workload of the clinic and the prevalence has remained stable at between 0.87-1.06%. In 2008 there were 31 isolates of C. glabrata and 25 isolates of other non-albicans yeasts. Of these, 24 (43%) were asymptomatic at review so did not require any medication. Eighteen (32%) women had been treated with azoles at their initial visit and did not return for follow-up. Fourteen were treated with amp/flu VC. During the two-years from 2006, 18 women were prescribed amp/flu VC. All had had previous episodes of VVC. The yeasts isolated were C. glabrata from 15, and one each of S. cerevisiae, C. parapsilosis and C. dubliniensis. The women had all received clotrimazole or fluconazole initially but remained symptomatic and so were prescribed amp/flu VC. They were followed up 2-4 weeks later with repeat cultures. No significant side effects were reported. At follow up all 18 were clear of their initial yeast isolate on culture but two had persistent symptoms and positive cultures for C. albicans. There was partial symptomatic improvement in three women but some residual vulval irritation remained despite being persistently culture negative. These residual symptoms were attributed to irritant dermatitis and settled with emollients and soap substitutes. Discussion It has been suggested that the widespread use of azoles, particularly fluconazole, may lead to a pathogen shift and increase the incidence of resistant Candida species such as C glabrata. [START_REF] Walker | Vaginal yeasts in the era of "over the counter" antifungals[END_REF][START_REF] Fidel | Candida glabrata: Review of epidemiology, pathogenesis and clinical disease with comparison to C albicans[END_REF] However, a previous paper from our clinic showed no increase in the prevalence of non-albicans yeasts between 1993 to 1998. [START_REF] Walker | Vaginal yeasts in the era of "over the counter" antifungals[END_REF] This study has also shown a stable prevalence of non-albicans yeasts between 2004 to 2008, confirming those findings.

The national guidelines for the treatment of VVC recommend nystatin pessaries for non-albicans yeast infections [START_REF]Clinical Effectiveness Group, British Association of Sexual Health and HIV[END_REF], but nystatin is no longer available in the UK. Amphotericin is a fungistatic polyene active against many fungi. Flucytosine is a fluorinated pyrimidine antifungal agent which is fungicidal but with a low genetic barrier for resistance. When combined together they act synergistically with amphotericin B enhancing cellular penetration of flucytosine and thereby potentiating the activity of flucytosine. In addition, if resistance develops to flucytosine, the amphoteracin B has activity against the yeasts. On the basis of successful case reports using amp/flu VC [START_REF] White | Combined topical flucytosine and amphotericin B for refractory vaginal Candida glabrata infections[END_REF][START_REF] Shann | Treatment of Candida glabrata using topical amphotericin B and flucytosine[END_REF] we introduced it for the treatment of non-albicans VVC. The treatment is well tolerated but is considerably more expensive than nystatin. The cost of amp/flu VC is approximately £206 per course compared with nystatin at approximately £10 per course. Also, as the shelf life is unknown the treatment needs to be started within 48 hours of manufacture. The duration of the intravaginal treatment is 14 days and ideally should be avoided during menstruation. Consequently, the ordering and delivery requires co-ordination with the woman's menstrual cycle to ensure it can be started within 48 hours of manufacture at a time when menstruation is not expected for at least 14 days. However, we have now successfully treated 18 women with nonalbicans VVC using amp/flu VC with 100% clearance of the non-albicans yeasts.

Key Messages

• It has been thought that widespread use of "over-the-counter" azoles may increase the incidence of resistant Candida species such as C glabrata. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in STI and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence http://group.bmj.com/products/journals/instructions-for-authors/licence-forms.

Table 1 .

 1 . Prevalence of yeasts isolated from women attending the Leeds Centre for Sexual Health between 2004 and 2008

	4

•

  Between 2004Between -2008, the prevalence of non-albicans yeasts remained stable with no increase in non-albicans Candida species such as C glabrata.• Infections with species other than C. albicans frequently do not respond to standard azole treatments. • Treatment of 18 women with non-albicans VVC using amphotericin and flucytosine vaginal cream achieved clearance of the non-albicans species in all of them.

	Competing interests
	None of the authors have any competing interests.
	Funding
	None
	Copyright licence statement

Contributors

NH collected the patient data. HRA collected the mycology data and supervised the laboratory testing. HRA and JDW initiated the project. NH and JDW analysed the data. All authors contributed to the preparation of the manuscript.


Andrea Low

Marie-Noelle Didelot-Rousseau

Nicolas Nagot

Abdoulaye Ouedraougo

Tim Clayton

Issouf Konate

Philippe Van De Perre

Michel Segondy

Philippe Mayaud
email: philippe.mayaud@lshtm.ac.uk

Cervical Infection with Human Papillomavirus (HPV) 6 or 11 in High-Risk Women in Burkina Faso

Keywords: Low-risk Human Papillomavirus (LR-HPV), HIV-1, genital warts, genital ulcer disease, Africa

Objectives: Human papillomavirus (HPV) types 6 and 11 are known agents of genital warts but little is known about their epidemiology in Africa. We present data on the prevalence of, and risk factors for, cervical HPV 6 and 11 in high-risk women in Burkina Faso.

Methods: 306 women were enrolled. HIV status and CD4+ counts were determined. Among other genital samples, a cervical swab (Cervex) was collected for liquid-based cytology and HPV genotyping using MY09/MY11 and GP5+/GP6+ PCRs, and INNO-LiPA genotyping v2. Risk factors were examined using logistic regression.

Results: HIV-1 seroprevalence was 40% (123/306). Cervical HPV DNA was detected in 55% (100/183) of HIV-uninfected women, 84% (78/93) of HIV-1 infected women with CD4+ T-cell counts >200 cells/µl and 97% (29/30) of HIV-1 infected women with CD4+ Tcell counts ≤200 cells/µl (p trend <0.001). HPV 6 prevalence was 6% (18/306), HPV 11 prevalence 4% (13/306), and overall HPV 6/11 prevalence 9% (28/306), which increased with HIV infection and immuno-suppression. Genital warts were associated with HPV 6 (adjusted OR=4.12, 95% CI: 1.17-14.53) but not with HPV 11. Genital ulcerations were associated with HPV 6/11 but not with other HPV types. There was a protective effect for vaginal douching and the follicular phase of the menstrual cycle. Condom use, HIV-1 plasma viral load, and other STI/RTIs were not associated with HPV 6/11. Conclusions: Prevalence of HPV 6/11 was high in this population, with predominance of HPV 6. HPV 6/11 were found more frequently in women with genital ulcers and in those with HIV-related immunosuppression.

Introduction

Human papillomaviruses (HPV) are the most common sexually transmitted infections worldwide. [START_REF] Castellsague | HPV and Cervical Cancer in the World: 2007 Report[END_REF] The low oncogenic risk types HPV (LR-HPV) 6 and 11 are the major causative agents of genital warts. [START_REF] Garland | Natural history of genital warts: analysis of the placebo arm of 2 randomized phase III trials of a quadrivalent human papillomavirus (types 6, 11, 16, and 18) vaccine[END_REF] The epidemiology of these HPV types is poorly described in Africa. As HPV vaccines become more widely available, it becomes important to understand the epidemiology of vaccine-associated HPV types. We present data on the prevalence of, and risk factors for, cervical HPV 6 and 11 in high-risk women in Burkina Faso.

Methods

Data were obtained from a cross-sectional survey of high-risk women nested within the Yerelon Cohort study, which has been evaluating interventions to reduce the risk of HIV infection among sex workers in Bobo-Dioulasso since 1998. [START_REF] Nagot | Is sexually transmitted infection management among sex workers still able to mitigate the spread of HIV infection in West Africa?[END_REF] Between December 2003 and March 2004, cohort participants were asked for permission to collect a cervical swab (Cervex) for liquid-based cytology and HPV-DNA detection using MY09/MY11 and GP5+/GP6+ PCRs and genotyping with INNO-LiPA genotyping v2 (Innogenetics). [START_REF] Didelot-Rousseau | Human papillomavirus genotype distribution and cervical squamous intraepithelial lesions among high-risk women with and without HIV-1 infection in Burkina Faso[END_REF][START_REF] Didelot-Rousseau | Comparison of INNO-LiPA HPV Genotyping v2 with PCR product subcloning and sequencing for identification of genital human papillomavirus genotypes in African women[END_REF] Vaginal and cervical swabs were collected for the diagnosis of

Trichomonas vaginalis and bacterial vaginosis (BV) by microscopy, and Candida albicans

and Neisseria gonorrhoeae by culture. Blood samples were collected for HIV, syphilis, and HSV-2 serologies, and HIV-1 plasma viral load and CD4+ count in HIV-1 seropositive women. Statistical analysis was conducted using logistic regression. Ethical approval was obtained from the Ethics Committees of the Burkina Faso Ministry of Health and the London School of Hygiene & Tropical Medicine.

Results

Of 379 cohort participants enrolled during the study period, 306 had data on HPV and concurrent clinical and laboratory information. The median age was 26 years (range 15-54 years), median age at first intercourse was 17 years (range 10-24), and 64 women (21%) described themselves as full-time sex workers. HIV-1 seroprevalence was 40% (123/306), syphilis seroprevalence was 3% (9/303) and HSV-2 seroprevalence was 68% (195/285).

Cervical HPV DNA was detected in 55% (100/183) of HIV-uninfected women, 84% (78/93) of HIV-1 infected women with CD4+ counts >200 cells/µl and 97% (29/30) of HIV-1 infected women with CD4+ counts ≤200 cells/µl (p trend <0.001). HPV 6 prevalence was 6% (18/306), HPV 11 prevalence 4% (13/306), and overall HPV 6/11 prevalence 9% (28/306), which increased with HIV infection and immunosuppression (Table 1).

Among the 18 women with genital warts, 4 were positive for HPV 6 (adjusted OR=4.12, 95% CI: 1.17-14.53, p=0.03) but none were positive for HPV [START_REF] Sonnex | Influence of ovarian hormones on urogenital infection[END_REF]

Discussion

The prevalence of cervical HPV 6/11 was high in this population and increased with immunosuppression. Unlike overall cervical HPV types, which had a strong association with both HIV and CD4+ count in this setting, [START_REF] Didelot-Rousseau | Human papillomavirus genotype distribution and cervical squamous intraepithelial lesions among high-risk women with and without HIV-1 infection in Burkina Faso[END_REF] the association with HPV 6/11 was weaker, likely due in part to the smaller numbers of subjects who were HPV 6/11 positive.

Our data suggest that HPV 6 is more prevalent in women with genital warts than HPV 11, which was already reported from an aetiological study in Australia. [START_REF] Garland | Natural history of genital warts: analysis of the placebo arm of 2 randomized phase III trials of a quadrivalent human papillomavirus (types 6, 11, 16, and 18) vaccine[END_REF] However, any relationship might be obscured by the fact that HPV DNA was recovered from the cervix and not directly from genital lesions.

The presence of genital ulcers, mainly caused by HSV-2 in Burkina Faso, [START_REF] Mayaud | Effect of HIV-1 and antiretroviral therapy on herpes simplex virus type 2: a prospective study in African women[END_REF] was strongly associated with HPV 6/11 but not with other HPV types. This suggests that HPV 6/11 may either cause or be acquired or transmitted through breaches of the genital mucosal surface, possibly induced by HSV-2 reactivations, although any biological differences between types remains unclear at this point. Conversely, we did not detect any association between HPV 6/11 and the presence of BV, often implicated in the acquisition of STIs and which might allow for persistence of HPV. [START_REF] Watts | Effects of bacterial vaginosis and other genital infections on the natural history of human 12 papillomavirus infection in HIV-1-infected and high-risk HIV-1-uninfected women[END_REF] Regular vaginal douching, linked to disturbances of the vaginal milieu and increased STI risk in other settings, [START_REF] Martino | Vaginal douching: evidence for risks or benefits to women's health[END_REF] was found to be protective for HPV 6/11 in this study. This might be secondary to mechanical factors which decrease DNA detection, although the sensitivity of HPV DNA detection does not seem impaired after washing. [START_REF] Chen | Diagnosis of human papillomavirus infection by abnormal cervical cytology is highly reproducible after vaginal douching[END_REF] Another African study has suggested a protective effect of douching on genital warts. [START_REF] Ruche | Vaginal douching: association with lower genital tract infections in African pregnant women[END_REF] The follicular phase of the menstrual cycle was protective for HPV 6/11, which might be due to an oestrogenic effect on HPV persistence, [START_REF] Sonnex | Influence of ovarian hormones on urogenital infection[END_REF] although the role of hormonal factors on STIs remains poorly characterized in African women.

In conclusion, HPV 6/11 prevalence was high in this high-risk population, with a predominance of HPV 6. HPV 6/11 were found more frequently in women with genital ulcers and in those with HIV-related immunosuppression.

Roles

Statement:

"The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in STI and any other BMJPGL products and sub-licences such use and exploit all subsidiary rights, as set out in our licence http://group.bmj.com/products/journals/instructionsfor-authors/licence-forms".

Word count: 768

  : N Nagot, P Mayaud and P Van de Perre designed the study, which was carried out by I Konaté, A Ouédraogo, and supervised by N Nagot. HPV testing was performed by MN Didelot-Rousseau and M Segondy in Montpellier. Statistical analyses were performed by A Low and reviewed by T Clayton and P Mayaud. First manuscript was drafted by A Low and P Mayaud with inputs and reviews from all coauthors, who approved the final version.

Table 1 . Multivariate associations with cervical HPV 6/11 and other HPV types in 306 high-risk women in Burkina Faso

 1 . Genital ulcerations were positively associated with cervical HPV 6/11 but not with other HPV types. Regular vaginal douching and the follicular phase of the menstrual cycle were associated with lower HPV 6/11 prevalence. Condom use, HIV-1 plasma viral load, and other STI/RTIs were not associated with HPV 6/11.

	Characteristic	N	Women with HPV 6/11 n (%)	HPV 6 or 11 (95%CI) a adjusted OR	P-value	Other HPV types (95% CI) a adjusted OR	P-value
	Age groups, years
	15-24	123	10 (8)	1.0		1.0
	25-34	119	10 (8)	1.00 (0.32-3.08)	0.99	1.15 (0.65-2.04)	0.64
	>34	64	8 (12)	1.99 (0.65-6.05)	0.23	1.33 (0.67-2.63)	0.42
	Practices regular vaginal douching 279	22 (8)	0.25 (0.07-0.88)	0.03	2.47 (0.83-7.33)	0.10
	Last menstruation <15 days b	146	8 (6)	0.37 (0.15-0.92)	0.03	0.94 (0.57-1.54)	0.80
	Genital warts on exam	18	4 (22)	3.01 (0.70-12.87)	0.14	0.71 (0.25-2.06)	0.53
	Genital ulceration on exam	12	4 (33)	5.22 (1.21-22.57)	0.03	0.61 (0.17-2.19)	0.45
	HSV-2 seropositivity	195	19 (10)	1.03 (0.36-2.95)	0.36	0.71 (0.39-1.27)	0.25
	Bacterial vaginosis	131	14 (11)	1.08 (0.44-2.69)	0.86	1.15 (0.69-1.91)	0.60
	HIV status:
	HIV seronegative	183	13 (7)	1.0		1.0
	HIV-1 +, CD4 >200 cells/μL	93	9 (10)	1.06 (0.39-2.88)	0.90	2.13 (1.22-3.70)	0.007

Acknowledgements:

We wish to thank the women and the organisations of persons living with HIV/AIDS ('Yérelon', 'Espoir et Vie', Centre 'Solidarité Action Sociale' and 'Espoir pour Demain') who participated in this study; and staff at Service d'Hygiène, Bobo-Dioulasso, Burkina Faso.

Key Messages:

1. The prevalence of low-risk-HPV types 6 and 11 was relatively high (9%) in this cohort of 306 high-risk women in Burkina Faso.

2. HIV infection and immune suppression were predictive of the presence of cervical HPV 6 and 11 and other HPV types.

3. HPV types 6 and 11, but not other HPV types, were strongly associated with concurrent genital ulcer disease, suggesting a role either in clinical presentation or transmission of HPV 6/11.

Competing interests:

We have no conflicts of interest to declare.