Bernard Mouillac
email: bernard.mouillac@igf.cnrs.fr

Maurice Manning

T Durroux

Fluorescent agonists and antagonists for vasopressin/oxytocin G protein-coupled receptors : usefulness in ligand screening assays and receptor studies

Keywords: arginine-vasopressin, oxytocin, G protein-coupled receptor, structure-activity relationships, fluorescent peptide, fluorescence resonance energy transfer, fluorescence polarization, high-throughput screening assays AVP, arginine-vasopressin, OT, oxytocin, LVP, lysine-vasopressin, bordifluoropyrromethene, Cy, cyanine, PVA, phenylpropionyl linear vasopressin antagonist, HO-LVA, hydroxyphenylacetyl linear vasopressin antagonist, FRET, fluorescence resonance energy transfer, HTRF, homogeneous time-resolved fluorescence, HTS, high-throughput screening, EuÃPBBP, europium pyridine-bis-bipyridine cryptate

Fluorescent agonists and antagonists for vasopressin/oxytocin g protein-coupled receptors: usefulness in ligand screening assays and receptor studies.

G protein-coupled receptors (GPCRs) constitute the largest family of integral membrane proteins, participate in the regulation of most physiological functions and are the targets of approximately 50% of currently marketed drugs [1,2]. Investigating receptor localization and trafficking, receptor molecular structural analysis and developing new receptor-selective highthroughput ligand screening assays, are presently crucial tasks. Because of the increased sensitivity of the fluorescence readers and of the brightness of the fluorophores, most of these studies can be performed using fluorescence-based technologies. Fluorescent ligands selective for a given receptor family or a given receptor subtype are consequently very useful labeling and pharmacological tools [3].

Arginine-vasopressin (AVP) and oxytocin (OT) receptors are typical members of the class A GPCRs (rhodopsin-like) and are considered as prototypes of GPCRs for which small peptides and hormones are endogenous ligands [4,5]. The two neurohypophyseal hormones are disulfide bridge-containing cyclic nonapeptides (CYFQNCPRG-NH 2 and CYIQNCPLG-NH 2 , respectively) which regulate well known peripheral physiological functions [6,7]. For instance, AVP participates in the regulation of blood pressure acting on the vessel V1a receptor subtype, controls the kidney water reabsorption through binding and activating V2 receptor subtype. AVP also plays a crucial role in the adaptative response to stress, anxiety or emotional situations by regulating corticotropin secretion from the anterior pituitary when interacting with its third receptor, the V1b subtype. OT is a major hormone in most reproductive functions, such as labor/delivery by inducing the uterine contractions, and lactation by triggering milk ejection. All the OT activities are mediated by a unique OT receptor (OTR). The AVP/OT receptors have been cloned in many different mammal species [8][9][10][11]. More recently, the central effects of AVP/OT hormones have been described. Indeed, the two peptides are neuromediators released in many different areas of the central nervous system (CNS), and modulate many aspects of mammalian behavior [12]. They act mainly on social behavior and precisely control social recognition, pair bonding, maternal care, social education, sexual behavior, trust. These actions were confirmed through the development of knockout mice for OT, OTR, V1a or V1b, and raise the possibility that these receptors may have important implications for developmental disorders characterized by deficits in social behavior like autism [13].

Since the original chemical synthesis of the cyclic nonapeptides AVP and OT [14,15], these two hormones have been the focus of intensive structure-activity, conformation-activity and analog design studies for decades. Hundreds of peptide or non peptide agonists and antagonists of the AVP/OT receptors, several being of potential clinical value, have been synthesized and characterized, and presently are very useful pharmacological tools [16].

Paradoxically, only a few fluorescent analogs of AVP and OT have been developed.

Nevertheless, as indicated above, such ligands have many potential applications. This review is mainly focused on the recent progress in the design of selective high-affinity fluorescentlylabeled peptide agonists and antagonists for AVP/OT receptors and their usefulness in

Agonist ligands.

A desaminated analog of the porcine hormone lysine-vasopressin (LVP), namely d[Lys 8 ]VP (dLVP), was used as a chemical matrix to develop the first fluorescent probe of the AVP/OT family more than twenty years ago [17]. Indeed, a rhodamyl derivative of dLVP (see Fig. (1), for which the coupling of the fluorophore can only be limited to the e-amine group of lysine at position 8 (the NH 2 terminal has been replaced by a hydrogen atom at position 1), allowed to detect the V2 receptor in the principal cells of rabbit collecting ducts [18]. This study directly demonstrated that the principal cells constitute the primary target cell for vasopressin (VP) in the kidney. This analogue of VP, whose binding properties in terms of affinity, kinetics and internalization, resemble those of the natural hormone in all respects, was then used to directly measure the lateral mobility of the renaltype V2 receptor in the basal plasma membrane of the LLC-PK1 porcine epithelial cells by microphotolysis (photobleaching) [19]. The V2 receptor was shown to be highly mobile at physiological temperature. This result reported the first measurement of the lateral mobility in membranes of a polypeptide hormone receptor that is coupled to adenylyl cyclase. As shown in Fig. (6), with or without a spacer arm inserted between the lysine in position 4, were demonstrated to keep good antidiuretic activity (a 15-fold decrease if compared to AVP) and were proposed to be valuable probes in studying receptor visualization [21]. The synthesis and biological activity of novel fluorescent VP analogs was published in 1990 [22]. All these analogs were prepared by coupling again the appropriate fluorochrome to the e-amine group of the lysine residue in dLVP (see Fig. (1 )). The d[Lys 8 (carboxyfluorescein)]VP (7 ) and the d[Lys 8 (methylanthranilamide)]VP (8) were demonstrated to retain high affinity for the V2 receptor present in canine renal plasma membranes (32 and 8.8 nM whereas that of AVP was around 1 nM) and whole LLC-PK1 cells (390 and 38 nM compared to 9 nM for AVP), and able to stimulate adenylyl cyclase activity as efficiently as AVP. On the other hand, the dansyl derivative of dLVP (d[Lys 8 (dimethylaminonaphtalene-1-sulfonyl)]VP (9), was unable to bind the receptor and activate cyclic adenosine monophosphate (cAMP) production. The first three ligands were defined as useful for the study of renal AVP receptors by fluorescence microscopy, particularly the fluorescein and rhodamine-labeled agonists. Whereas fluorescein exhibits low photostability, which restricts its use for imaging events occurring over long time periods, the rhodamine fluorophore is ideal in that it possesses a high molar extinction coefficient and does not photobleach readily. Taking into account these studies, designing of a fluorescent analog of AVP or OT retaining all agonistic properties of the natural hormones, was obviously much more successful when introducing a fluorophore at the side chain of the residue 8 of the peptide.

Cyclic and linear antagonist peptides.

The primary fluorescent antagonists of AVP/OT receptors were described in 1992 but were also nonselective. First, following the discovery that requirement of cyclic conformation of peptide antagonists in binding to AVP receptor is not necessary [23], the classical fluorophore fluorescein was added to the N-terminus of a linear antagonist having a high affinity for both the V1a and V2 receptor subtypes [24]. The resulting peptide N-a -fluoresceinyl-thiocarbamoyl-D-Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH 2 (10), shown in Fig. (4), was unable to stimulate a phospholipase C activity from WRK1 cells measured by the accumulation of inositol phosphates (IP), and consequently exhibited pure antagonistic properties. However, this ligand was not useful at all for labeling cells and visualizing AVP receptors because it displayed a very weak affinity compared to that of its parent peptide (a 1000-fold decrease). Following the description of the well-established V1a selective antagonist, d(CH 2 ) 5 [Tyr(Me) 2 ]AVP [25], and of some of its V1a/OTR nonselective analogues, such as d(CH 2 ) 5 [D-Tyr(Et) 2 , Val 4 , Lys 8 ]VP [26], the fluorescent cyclic peptide d(CH 2 ) 5 [D-Tyr(Et) 2 , Val 4 , Lys 8 (carboxytetramethylrhodamine)]VP (11) was synthesized (see Fig. and characterized [22]. It was shown to bind nonselectively the V2 receptor from LLC-PK1 and the V1a receptor from A-10 cells (79 and 32 nM respectively). This ligand failed to increase cAMP accumulation in LLC-PK1 cells or intracellular calcium concentration in A-10 cells. In contrast to the prototype fluorescent agonist d[Lys 8 (tetramethylrhodamyl)]VP, this antagonist did not undergo receptor endocytosis in either cell type. The authors concluded that AVP receptor occupancy in the absence of receptor activation is insufficient to elicit receptormediated internalization. Later, interestingly, it was shown that linear peptide antagonists can accommodate a lysine(NH 2 ) residue at position 9 and retain very high affinity and selectivity for the V1a receptor subtype versus V2 [27]. Aminomethyl coumarin (AMCou) was coupled onto the e amino group of this lysine 9 residue through its N-hydroxysuccinimide ester to give the [phenylacetyl 1 , D-Tyr(Me) 2 , Arg 6 , Arg 8 , Lys 9 (Ne-7-amino-4-methylcoumarin-3acetamide)NH 2 ]VP (12), illustrated in Fig. ( 4) [28]. The AMCou fluorophore was chosen as it exhibits bright blue fluorescence at neutral pH, is maximally excited at near ultraviolet wavelengths and is relatively resistant to photobleaching. All these factors are important to distinguish AMCou fluorescence from background cell autofluorescence. The AMCoulabeled ligand behaved as the first selective fluorescent antagonist to the V1a receptor (affinity was 0.44 nM), was unable to produce IP accumulation, and demonstrated to be useful for labelling WRK-1 cells expressing the V1a receptor. Finally, two new fluorescent cyclic antagonists having the fluorophore attached to a lysine 9 residue were synthesized and characterized, confirming that position 9 in these peptides can accept derivatization with more or less bulky chemical groups, as published for linear peptide antagonists [29]. The lateral mobility of the adenylyl cyclase-coupled V2 AVP receptor from LLC-PK1 cells was studied using fluorescence photobleaching with these two fluorescent peptides (depicted in Fig. The synthesis and characterization of first generations of fluorescent agonists and antagonists for AVP/OT receptors led to the discovery of very promising pharmacological tools but their use was limited to fluorescence microscopy techniques applied to the investigation of the cellular expression and localization of receptors. Indeed, the sensitivity of the fluorophores attached to these ligands was far from being equivalent to that of usual radioisotopes ( 3 H, 125 I), and ligand binding assays could not generally be developed.

Novel generations of fluorescent analogs for AVP and OT receptors.

Since 1992, the different AVP/OT receptor genes or cDNAs were all cloned from different mammals, lower vertebrate and invertebrate species [8][9][10][11]. Molecular cloning of this receptor family has confirmed that AVP/OT receptors are typical GRPCs consisting of seven hydrophobic transmembrane a-helices with an extracellular N-terminus and a cytoplasmic Cterminus. The knowledge of their nucleotide sequence and consequently of their primary structure (the amino acid residue sequence) constituted a starting point to receptor structurefunction relationships analysis. We and others have undertaken many studies that were dedicated to the identification of the hormone binding sites at a molecular level [5,[30][31][32][33][34][35].

Extensive receptor mutational analysis combined with receptor three-dimensional molecular modelling or by direct receptor covalent photolabeling have led to very valuable information concerning peptide agonist and peptide and nonpeptide antagonist binding domains of the AVP/OT receptor family [36][37][38][39]. Indeed, the AVP/OT receptor binding pocket is buried into a 15-20 Å deep central cavity defined by the transmembrane helices and surrounded by the extracellular loops [40]. The hydrophobic part of the ligands dives deeply into the binding cavity for interacting with hydrophobic residue clusters, whereas the more hydrophilic part of the peptides bind to the transmembrane edge. Only the side-chain of residue 8 of the peptides is pointing towards the extracellular loops of the receptors and is potentially less constrained than all other parts of the ligands [32,33,41]. Thus it is not surprising a posteriori that derivatization of AVP/OT analogues with bulky fluorophores like fluorescein or rhodamine at this particular position (amino acid residue 8) led to the successful development of numerous fluorescent ligands retaining high affinity, selectivity, and functional activity for these receptors. This residue is finally not crucial for binding, although it has been demonstrated to be involved in receptor subtype binding selectivity [32,33].

Linear peptide antagonists with fluoresceinyl and tetramethylrhodamyl fluorochromes.

While analysing the structure/function relationships of AVP/OT receptors and particularly identifying the ligand binding sites of these receptors, we also decided to develop new fluorescent ligands, and we first added fluorophores to linear VP antagonists which possess very high affinities for the V1a receptor subtype and are much more stable than disulfidebridged molecules [42] experiments using these compounds.

Cyclic peptide agonists and antagonists with classical fluoresceinyl and tetramethylrhodamyl fluorochromes.

We next reported the design and synthesis of new fluorescent OT antagonists and agonists and their pharmacological properties for the AVP/OT receptors. Again, fluoresceinyl and tetramethylrhodamyl groups were coupled to parent peptides through an amide bond to the side chain amino group at position 8 [45]. Two antagonists, d(CH 2 ) 5 [Tyr 2 (Me), Thr 4 , Orn 8 (5/6carboxyfluoresceinyl), Tyr 9 -NH 2 ]VT (18) and desGly-NH cells. An equivalent labeling was observed for 1 h incubation. Because a punctate labeling observable at 37°C and not at 0°C suggested an endocytosis of the ligand-receptor complex, a confocal analysis was undertaken for various periods of time. Small fluorescent endosome-like particles were seen widespread into the cytoplasm when cells were incubated at 37°C but not at 0°C. These endosome-like particles were not concentrated in the perinuclear region, even for longer incubation periods (3 h), in agreement with a recycling pathway. Interestingly, the punctate labelling observed was equivalent to what has already been described for the V1a

receptor, but suggested that the internalization process of the OTR is different from that of the V2 receptor [46]. Indeed, it has been demonstrated that the V2 receptor is addressed to a perinuclear region of LLC-PK1 cells labelled with the classical fluorescent agonist d[Lys 8 (tetramethylrhodamyl)]VP [47]. [HO 1 ][Orn 8 (5/6C-tetramethylrhodamyl]VT would be a very useful ligand to study desensitization and internalization of the ligand-OTR complex in OTR-expressing myocytes of human myometrum during parturition. These fluorescent ligands could also be used to perform fluorescence recovery after photobleaching to study the diffusion of molecules in a tissue or in a single cell or to obtain structural information for ligand-receptor interactions by fluorescence quenching, polarization or FRET.

linear peptide antagonists with novel Alexa and Europium cryptate fluorochromes.

As indicated in the introduction, GPCRs represent the single largest molecular target of therapeutic drugs currently on the market, but are also the most common target in highthroughput screening (HTS) assays for identifying potential new drug candidates. Most of these HTS procedures are still presently based on radioligand binding competition assays. The fluorescent properties of these new AVP/OT analogues make them ideal for undertaking HTS assays using polarization or FRET experiments. The affinity of several unlabeled wellknown ligands was determined using homogenous assays on membrane preparations expressing the V1a receptor into 96-and 384-well plate formats. The [Lys 8 (Alexa488)]PVA (23) was particularly demonstrated to be well-suited for developing fluorescence polarization screening assays. Indeed, the specific / non-specific signal ratio is good, whereas that for [Lys 8 (Alexa546)]PVA (24) and [Lys 8 (Alexa647)]PVA ( 25) is too low. Affinities for AVP, HO-LVA [50], and the nonpeptide antagonist SR49059 [51], determined using fluorescence polarization assays with Lys 8 (Alexa488)]PVA were similar to those defined using [ 3 H]AVP in a radioligand binding assay.

EuÃPBBP exhibits particular fluorescent properties such as a long fluorescent lifetime and is then suitable as a donor for FRET experiments. Combined with the use of an Alexa 647 fluorescent probe as an acceptor, it is convenient to perform HTRF experiments. Thus, an HTRF competition binding assay based on a FRET signal between the two fluorophores, the EuÃPBBP carried by the ligand and Alexa 647 coupled to an antibody specific for an epitope tag located at the N-terminus of the receptor (HA tag and anti-HA antibody 12CA5), was developed. The proximity of the two fluorophores when the ligand binds to the receptor leads to a FRET signal, and the displacement of the fluorescent ligand from the receptor by the addition of an unlabeled competitor induces a decrease in this signal. The FRET signal was measured for different periods of incubation at 4°C for avoiding receptor internalization; 8 h was necessary for reaching a plateau. As for fluorescence polarization assays, affinities for different unlabeled ligands were found to be equivalent to those determined using [ 3 H]AVP in a radioligand binding method. Competition HRTF experiments were also conducted using membrane preparations and similar results were obtained.

Both polarization and HTRF assays are promising approaches and offer attractive advantages for performing ligand-GPCRs HTS procedures. Compared to other binding assays, these

Concluding remarks.

The availability of the Alexa/lanthanide-labeled peptide ligands for AVP/OT receptors opens new perspectives in receptor studies. First, structural organization of these membrane receptors at the surface of target cells is still an open question. For instance, dimerization/oligomerization of GPCRs has been demonstrated in heterologous cell expression systems using co-immunoprecipitation, bioluminescence resonance energy transfer or FRET techniques with highly-expressed tagged-receptors or luminescent fusion proteins.

However, no evidence of direct receptor-receptor interactions has yet been provided in native tissues. We have recently used a FRET approach to address this question with pairs of labeled ligands, one with the Alexa 647 the other with the Euà PBBP [52]. Indeed, OTR dimers/oligomers in the mammary glands of lactating rats were unambiguously identified Unfortunately, the search for a peptide agonist/antagonist selective for the human V2 receptor with respect to the V1a, V1b and OTR is still a challenging goal [16]. V2 antagonists with high affinity but which are nonselective have been developed [53]. For instance, the affinity of d(CH 2 ) 5 [D-Tyr(Et) 2 ,Pro 3 , Val 4 ]AVP for the human V2 receptor is 0.5 nM [16], rendering this ligand a good candidate for replacing the arginine 8 with a lysine and derivatization of the lysine 8 with Alexa or lanthanide fluorophores. This ligand would probably be not selective but would be useful for HTS fluorescence polarization and HTRF assays using membranes or cells expressing the V2 receptor. On the other hand, several agonists being selective to the human and rat V1b receptors might be promising tools. Indeed, one can imagine that the V1b-

1 .

 1 Fig. (3) and Fig. (4), respectively.

  (1)), d[Lys 8 (tetramethylrhodamyl)]VP

  (1), analogs of OT having a fluoresceinyl group attached to the e-amine group of a lysine at position 4, d[Lys 4 (fluoresceinyl), dihydro-Pro 7 ]OT (2), or 8, d[Thr 4 , dihydroPro 7 , Lys 8 (fluoresceinyl)]OT (3), or at position 1 in the case of OT itself, [Cys1(fluoresceinyl)]OT (4), were also developed [20]. Those with fluorescein at position 8 retained good uterine and milk ejection activities (only a 4-fold decrease in activity compared to OT) and represented potentially useful molecular tools for studying receptor localization, whereas those having the fluorophore at positon 1 or 4 gave very low or moderate activities, respectively. Fluorescent analogs of AVP derivatized with a fluoresceinyl (illustrated in Fig. (1)) in either position 4 (d[Lys 4 (fluoresceinyl), OHPro 7 ]AVP) (5) or 7 (d[Pro 7 (NH-fluoresceinyl)]AVP)

  (3))

  (3)), d(CH 2 ) 5 [D-Tyr 2 , Ile 4 , Lys 9 (N 6 -fluoresceinylaminothiocarbonyl)]AVP (13) and d(CH 2 ) 5 [D-Tyr 2 , Ile 4 , Lys 9 (N 6 -tetramethylrhodamylaminothiocarbonyl)]AVP (14), and compared to that measured with the agonist d[Lys 8 (tetramethylrhodamyl)]VP (1). The results reported that antagonistic properties of the V2 fluorescent ligands were not correlated to a decreased receptor lateral mobility. Altogether, these studies with fluorescent antagonists demonstrated that derivatization of linear and cyclic peptides both at position 8 and 9 with different fluorophores allows characterization of high-affinity ligands for V1a and V2 receptors.

  . The hydrophilic compounds LVA, PhCH 2 CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH 2 , and HO-LVA, 4-HOPhCH 2 CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH 2 , were chosen as the parent peptides in order to counterbalance the hydrophobicity introduced by tetramethylrhodamyl and fluoresceinyl groups. The coupling of tetramethylrhodamyl at position 1 to give 1-tetramethylrhodamyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH 2 (15) was first realized (see Fig. (4)), but the ligand displayed very weak affinity (K i > 500 nM). This result was equivalent to the one obtained a few years earlier for the first linear peptide antagonist to be labelled with a fluorophore at position 1, N-afluoresceinyl-thiocarbamoyl-D-Tyr(OEt)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH 2 (1 0 ). It confirmed that position 1 is not suitable for introducing bulky hydrophobic groups such as fluoresceinyl and tetramethylrhodamyl fluorophores. More interestingly, the substitution of Lys for Arg at position 6 (4-HOPh(CH2) 2 CO-D-Tyr(Me)-Phe-Gln-Asn-Lys(5/6Ctetramethyrhodamyl)-Pro-Arg-NH 2 (16) or at position 8 (4-HOPh(CH2) 2 CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5/6C-tetramethyrhodamyl)-NH 2 (1 7 ) allowed the coupling of fluorophores through an amide link instead of the thiocarbamoyl link, being more stable and giving higher quantum yields (the peptides 16 and 17 are illustrated in Fig. (4)). The closer to the C-terminus the fluorophore is, the higher the affinities of the fluorescent derivatives for the human AVP V1a receptor are, confirming that position 8 is ideal for derivatization (these ligands displayed a 0.1-1 nM affinity range for the V1a subtype). The position of the link between the fluorophore and the peptide (5C-or 6C-for both carboxyfluoresceinyl and carboxytetramethylrhodamyl) only slightly affected the affinity of the ligands. These different fluorescent ligands retained full antagonistic activity, were very selective for V1a versus V2 or V1b receptor subtypes. Interestingly, they also displayed a high affinity for the OTR subtype. For instance, the K i for 4-HOPh(CH 2 ) 2 CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5C-carboxytetramethylrhodamyl)-NH 2 ([Lys 8 (tetramethylrhodamyl)]PVA) to V1a and OTR were 0.07 and 1.2 nM, respectively, making this ligand useful for two different receptors [ 4 2 ] . T h e 4 -HOPh(CH 2 ) 2 CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5Ccarboxyfluoresceinyl)-NH 2 ([Lys 8 (fluoresceinyl)]PVA) displayed equivalent properties. With such an affinity, these fluorescent peptides are the highest affinity fluorescent V1a receptor ligands reported to date. Because of this property, these ligands are well adapted to fluorescent techniques. They were demonstrated to be efficient for labelling AVP receptors in epifluorescence and could easily be used for flow cytofluorometric studies due to a low concentration needed to label cells because of a very high affinity. Indeed, the ([Lys 8 (fluoresceinyl)]PVA analog made possible the visualization of cell surface AVP V1a receptors in rat hepatocytes [43, 44]. A high affinity is also crucial for cellular and subcellular analysis and colocalization with other markers in fluorescence microscopy. Fluorescence recovery after photobleaching could also be used to study the diffusion of fluorescent probes in a tissue or in one cell. Fluorescence correlation spectroscopy which allows the detection of interactions between one receptor molecule and one ligand molecule is also a very interesting application. Structural information for ligand-receptor interactions could also be obtained by fluorescence quenching, polarization, or FRET (fluorescence resonance energy-transfer)

2 9 ,

 9 d(CH 2 ) 5 [D-Tyr 2 , Thr4 , Orn8 (5/6carboxyfluoresceinyl)]VT(19) were obtained (shown in Fig.(3)), the first one only having a good affinity for the OTR (K i was around 6 nM). More interestingly, it was selective for this receptor. These properties rendered the ligand very useful for specifically labelling cell surface OTR in tissues, freshly dissociated cells or in primary cell cultures. All other ligands of the series were fluorescent agonists deriving from OT agonists lacking the a-NH 2 group at position 1 and for which the leucine residue at position 8 was replaced with an ornithine or a lysine for attachment of the fluorophore[45]. Nine different fluorescent agonists exhibited very good affinities for the OTR with K i values in the 0.5 nM range. One is very selective to the OTR, d[Orn8 (5/6C-Fluoresceinyl)]VT(20) while two other peptides, [HO 1 ][Thr 4 , Orn 8 (5/6C-fluoresceinyl)]VT (21) and [HO 1 ][Orn 8 (5/6C-tetramethylrhodamyl]VT (22), can be used to label both OTR and the V1a subtype and are selective versus V1b and V2 (the three peptides are shown in Fig. (2)). Fluorescence microscopy labelling of CHO and HEK cells stably or transiently expressing the human OTR, respectively, was realized in order to study the distribution of the receptor and its internalization process. Incubation of the two cell lines with d[Orn 8 (5/6C-Fluoresceinyl]VT for 1 h at 37°C revealed a specific punctate labeling. Because tetramethylrhodamine is less sensitive to photobleaching than fluorescein, the peptide [HO 1 ][Orn 8 (5/6C-tetramethylrhodamyl]VT was next used to label the different

Fluorescence

  photostability and enhanced fluorescence emission than those of the classical fluoroprobes

  methods are nonradioactive, safe to perform, very sensitive and homogenous, easier and faster to automate. The different peptide antagonists [Lys 8 (EuÃPBBP)]PVA, [Lys 8 (Alexa488)]PVA, [Lys 8 (Alexa546)]PVA, [Lys 8 (Alexa647)]PVA are the first fluorescent AVP/OT analogs adapted to different HTS procedures for screening ligand libraries and discovering novel potential therapeutic compounds. These methods can therefore be applied to other GPCRs.

  using a pair of labelled antagonists, [Lys 8 (EuÃPBBP)]PVA and [Lys 8 (Alexa647)]PVA, but also a pair of fluorescent agonists, [HO 1 ][Thr4 , Orn8 (EuÃPBBP)]VT(27) and[HO 1 ][Thr4 , Orn8 (Alexa647)]VT(28). These two agonists are seen in Fig.(2). Interestingly, due to a high affinity for the V1a receptor, these pairs of ligands should be useful to the demonstration of V1a dimers/oligomers in native tissues. This would be crucial as well to apply this strategy to the V2 and V1b subtypes in order to demonstrate whether these receptors behave as dimers in the kidney and the pituitary/CNS, respectively. Such a FRET strategy represents an interesting alternative to other energy transfer strategies. The specific recognition of ligands by receptors as well as their small size compared to antibodies or luminescent proteins make ligands ideal tools for the study of GPCR dimers/oligomers in native tissues without altering the physiological context. The use of lanthanide-labeled ligands deserves a specific comment, particularly in the context of native tissues/cells which express very low levels of endogenous receptors. It is obvious that the sensitivity of the fluorophore is crucial in terms of receptor expression levels. With most fluorophore-labeled ligands, the signal-to-noise ratio is worse than the one obtained with the corresponding radioligands (tritiated or iodinated), whatever the methodology used. This is probably due to the hydrophobic nature of the AVP/OT ligands but also that of the fluorophores themselves covalently-bound to these ligands, in addition to the low sensitivity of the fluorescent molecules. In comparison, lanthanides are advantageous since their detection by time-resolved fluorescence results in a specific signal with a low background, and thus the signal-to-noise ratio is somewhat equivalent to that measured with radioligands. Consequently, detection of very low levels of receptor expression in native tissues/cells should be much easier and reproducible with these new generations of fluorescent probes.Second, excepted the first generations of fluorescent agonist and antagonist peptides useful for fluorescence microscopy techniques applied to the investigation of receptor cellular expression and localization, no specific peptide ligand labelled with Alexa or lanthanide series has been synthesized and characterized for the V1b and V2 receptors so far.

selective d[Cha 4 ,

 4 Lys 8 ]VP and d[Leu4 ,Lys 8 ]VP[54], could be directly derivatized with Alexa and EuÃPBBP fluorophores on the lysine residue 8, behaving as efficient tools for fluorescence polarization and HTRF HTS assays as well as for the direct demonstration of V1b dimers/oligomers in the CNS or the pituitary.Third, future generations of fluorescent ligands for AVP/OT receptors will certainly be developed from nonpeptide compounds. Several selective nonpeptide antagonists have been developed and characterized by pharmaceutical companies for each AVP/OT receptor[51,[55][56][57][58]. Three nonpeptide V2 antagonists are currently in clinical trial: Tolvaptan, Lixivaptan and Satavaptan[59]. More recently, at least three companies have reported nonpeptide agonists for the V2 receptor and for the OTR[60,61]. To date, there are no reports on nonpeptide V1a or V1b agonists. None of these nonpeptide agonists and antagonists has been covalently-labeled with a fluorophore. Thus today, there are no available fluorescent nonpeptide ligands for AVP/OT receptors. Taking into account available three-dimensional models of nonpeptide ligands docked into AVP/OT receptors should help in the design of new fluorescent compounds. Based on these models, it is commonly accepted that the nonpeptides mostly interact with receptor residues located deeply in the binding pocket, particularly with aromatic and hydrophobic clusters. Because their size is much smaller than that of AVP/OT peptide analogs, derivatizing this class of ligands for obtaining useful fluorescent probes would probably necessitate adding a spacer arm long enough to allow the fluorophore being at the receptor surface, in a position equivalent to that of the corresponding peptide residue 8.Characterizing a fluorescent nonpeptide ligand retaining a high affinity but also selectivity for a given receptor subtype of the AVP/OT family is even more challenging. The ability to measure and quantify the binding of ligands to GPCRs has been and remains a key element of drug discovery. Conventional methods based on radioactively-labeled ligands still contribute a major aspect of this activity. It is clear, however, that fluorescent molecules offer a safer, more powerful and more versatile alternative to radioligands in molecular pharmacology and drug discovery. There is indeed a need to develop both more robust fluorescence-based techniques and highly receptor-specific fluorophore-tagged ligands. Apart from AVP/OT receptors, many fluorescent ligands targeted at other peptidic GPCR families have been developed [for review, 62-64]. Different labels, such as fluorescein, rhodamine, Bodipy, Alexa488, Alexa594, Alexa633, Texas red, Cy3, Cy5, Oregon green, were covalently-attached to peptidic and small molecule nonpeptidic ligands for peptide GPCRs. These ligands were useful for studying different aspects of opioid, neurotensin, somatostatin, cholecystokinin, melanocortin, neuropeptide Y, chemokine receptors. Even lanthanidelabeled ligands have been reported and have been used successfully in evaluating receptor-ligand interactions, for instance for neurokinin, neurotensin or chemokine receptors [for review, 65]. Moreover, lanthanide labels have many advantages over radiolabels or other fluorolabels because they are highly sensitive, easy to automate, synthesized with relative ease, have a much longer lifetime and can be used in multilabel experiments. The lanthanidelabeled ligands are particularly well adapted to receptor-ligand screening assays but they also should revolutionize the study of GPCRs in their native cellular environment. With such powerful tools, coupled with the rapidly advancing complementary imaging technologies such as fluorescence correlation spectroscopy, one is therefore becoming able to investigate therapeutically important GPCRs and their molecular relationships with bioactive ligands at the single cell level and even at the single molecule level. This will open the way to study compartmentalization and structural organization of GPCRs in specialized microdomains of the cell plasma membrane.AVP NH 2 CH-CO-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-

NH 2 CHFigure 1 :Figure 2 :CH 2 -CH 2 -CH 2 -Figure 3 : 2 ( 12 )AMCou ( 15 )( 16 ) 2 Alexa488 ( 17 ) 2 ( 24 ) 2 Alexa546 ( 25 ) 2 Alexa647 ( 26 ) 2 EuÃPBBPFigure 4 :

 2122223212151621722422522624 Figure 1: fluorescent agonist analogs of AVP/OT.

Acknowledgements

This work was supported by the research grants from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Universities of Montpellier I and II (BM and TD), and by the National Institute of Health grant GM25280 (MM).


Dr Claire Périllaud-Dubois
email: claire.perillaud-dubois@inserm.fr

Elise Bouthry

Abir Jadoui

Ay-Ling Leng

Anne-Marie Roque-Afonso

Christelle Vauloup-Fellous

PharmD Claire Périllaud Dubois

Paul Brousse

Anne-Marie Roque- Afonso

Positive predictive values of CMV-IgM and importance of CMV-IgG avidity testing in detecting primary infection in three different clinical settings. A French retrospective cohort study

Keywords: CMV, serology, IgM, avidity, positive predictive value, pregnancy ventriculomegaly, hyperechogenic ventricular wall, cerebellar and brain calcifications, enlargement of pericerebral spaces, periventricular and subependymal cysts, candlestick, porencephaly, hyperechogenic periventricular halo, candlestick, abnormal gyration, corpus callosum hypoplasia." CMV, serology, IgM, avidity, PPV, positive predictive value, pregnancy, ultrasound abnormalities, clinical signs CMV, serology, IgM, avidity, PPV, positive predictive value, pregnancy, ultrasound abnormalities, clinical signs CMV, serology, IgM, avidity, PPV, positive predictive value, pregnancy, ultrasound abnormalities, clinical signs

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

Line 33: Can the clinical symptoms suggestive of CMV infection for group 3 patients be listed or described, also as there is information on the clinical symptoms is it possible to estimate the time post-infection the samples were taken? These information were added in the text. Lines 95-97: "Mainly fever, headache, flu-like syndrome (fever + rhinitis + myalgia), arthralgia, myalgia, fatigue and/or hepatitis. Samples were collected 5 to 30 days after onset of symptoms."

Line 198-199: The algorithm predicted 7/30 primary CMV infections and 3/30 had high avidity but gave birth to an infected child. Is this a reflection on the testing or the progression of infection dependent upon the gestational age? Indeed, US abnormalities may be observed late in pregnancy (explaining high avidity at that time) but result of an infection in the first trimester.

Were any of group 1 neonates infected?

For this study this information was not systematically collected and is therefore not available.

The authors comment on the age variations for the general population group 3, how does this relate to the ages in the other groups, was any age stratification done for groups 1 and 2?

Both other groups only include pregnant women between 17 and 46 years old. Age stratifications were therefore not relevant for these groups.

For Figure 2 please convert commas to decimal points.

We converted commas to decimal points as required.

Reviewer #4: The authors retrospectively analyzed results of 6857 serum samples that tested positive for CMV IgM. The samples were divided into three groups: 6560 samples from routine screening of pregnant women (group 1), 30 pregnant women with abnormalities observed on ultrasound (group 2), and 297 samples from non-pregnant immunocompetent individuals showing signs of CMV infection (group 3). Using CMV IgG avidity testing to follow-up all of the CMV IgM-positive samples and basing all conclusions on the avidity testing result, recent CMV infection was confirmed in only 16.5% of group 1, 36.7% of group 2, and 35.3% of group 3. The authors strongly support the concept that all CMV IgM-positive samples must be tested by CMV IgG avidity in order to determine the true CMV infection status.

Major Comments:

Adding mention of CMV IgG avidity testing in the title of this paper would more effectively represent the study. Perhaps something like this "Positive predictive values of CMV-IgM and importance of CMV IgG avidity testing in detecting primary infection in three different clinical settings, A French retrospective cohort study."

We thank the reviewer for his interesting comment and changed the title as suggested. Lines 1-2:

"Positive predictive values of CMV-IgM and importance of CMV IgG avidity testing in detecting primary infection in three different clinical settings. A French retrospective cohort study."

2. In the Materials and Methods Section: a. Line 78: "CMV serology (IgG, IgM+/-IgG avidity)" is noted. The "IgM+/-IgG avidity" notation is confusing. Clarify when IgG avidity testing is done.

We clarified our purpose. CMV IgG avidity is performed in case of positive IgG. Actually, IgG avidity could not be performed if no CMV IgG. Line 79: "CMV serology (IgG, IgM and IgG avidity in case of positive IgG)" b. Line 86: Give a few examples of the "symptoms suggestive of CMV primary infection" that were used to select patients for this group.

These information were added in the text: Lines 93-95: "Mainly fever, headache, flu-like syndrome (fever + rhinitis + myalgia), arthralgia, myalgia, fatigue and/or hepatitis." c. Line 95-96: It appears that low avidity values obtained with the LXL as a screening test were then confirmed by performing the VIDAS avidity test. Based on this, the reader assumes that two avidity tests are needed. Explain this further. Why were the two avidity tests needed? Also, discuss discrepancies between the two avidity tests. This is the only place in the manuscript that the dual avidity testing is mentioned. More information is definitely needed in order to give the reader the full picture of what is involved with avidity testing in the authors' algorithm and would be of considerable importance to someone considering adding CMV-IgG avidity testing.

Our strategy is based on previous published findings (C. Vauloup-Fellous, M. Berth, F. Heskia, J.-M. Dugua, et L. Grangeot-Keros, Re-evaluation of the VIDAS® cytomegalovirus (CMV) IgG avidity assay: Determination of new cut-off values based on the study of kinetics of CMV-IgG maturation, J. Clin. Virol. Ref 19). Two avidity tests are useful only if the first screening test gives a result below 0.4. In that case, confirmation/exclusion of primary infection is only based on the VIDAS assay. This strategy is now detailed in the manuscript. Lines 102-117. d. Line 99: Indicate how far apart the two consecutive samples were to be collected.

Line 110:

"Negative CMV-IgG/positive CMV-IgM on two consecutive samples 21 days apart" e. Line 104: Add the information expected for the CMV-IgG avidity test for this profile.

Added in the text. Lines 116-117 2. Line 112: A total of 6,857 serum samples is shown. However, Figure 1 shows 6,859 samples.

Reconcile these numbers. We corrected in abstract and in the text: 6859 Line 37 and line 124:

Add to the limitations of the study: There are known to be technical issues with IgM-specific

testing. This should be mentioned. It is possible that some of the CMV-IgM positive results were due simply to false positives. This has been shown to occur due to high levels of analyte specific IgG, to presence of rheumatoid factors, and to other miscellaneous technical factors involved with methods that must include some sort of process to separate CMV IgG from CMV IgM. Manufacturers' stated sensitivity and specificity for CMV IgM detection may not be truly representative. Likewise, the avidity tests are not likely 100% sensitive or 100% specific.

We added this suggestion in discussion. Lines 178-182: "Moreover, there are known to be technical issues with IgM-specific testing. Indeed, false positive CMV-IgM results were shown to occur due to high levels of analyte specific IgG, to presence of rheumatoid factors, and to other miscellaneous technical factors involved with methods that must include some sort of process to separate CMV IgG from CMV IgM."

Minor Comments:

1. Lines 2-5 of the Abstract. These sentences are awkward and a bit confusing. Revise for clarity. Lines 26-28: Sentences revised 2. Line 46: Replace the word "first" with "most frequent" Line 47: We changed with "most frequent" as suggested.

3. Line 56: Change "of" to "for" Line 57: We corrected with "for".

Line 68: Change "a" to "at" Line 69:

We corrected with "at".

Lines 199-203: Overall, when positive CMV-IgM is detected at that moment (clarify: does this mean late in pregnancy?

). Also, change the word "strengths" to "suggests". We made the following changes in the text: Lines 215-216: "Overall, when positive CMV-IgM is detected when US abnormalities are observed (possibly late in pregnancy), it suggests responsibility of CMV in 47% (14/30) cases without ruling out the possibility of CMV congenital infection following maternal non primary infection if the mother is CMV-IgG positive and CMV-IgM is negative." 6. Line 207: Change "detection of CMV-IgM is" to "positive CMV-IgM results are" and in line 208 change "case of clinical" to "cases with clinical" Lines 223-224: "Remarkably, positive CMV-IgM results are most often considered as indicative of a recent CMV virus infection in case with clinical signs" 7. Lines 212-216: Divide this lengthy sentence by inserting a period in Line 214 after the references shown [20,21]. Then change "and that is case" to "Also, in cases" We made the changes as suggested. Lines 230-231: "Our results suggest that positive CMV-IgM is less correlated with CMV recent primary infection in young people, and are consistent with the understanding that CMV-IgM can be produced throughout life as a result of CMV non primary infection or polyclonal stimulation of the immune system [20,21]. Also, in cases of positive CMV-IgM, older people may be more likely to have a primary infection than younger people." 8. Line 217: Change "Some limitations of our analysis is" to "One limitation of our study is" and in line 218 change "require to come to hospital" to "require the patient to come to the hospital" We modified this sentence as suggested. Lines 233-234: "One limitation of our study is that, although very frequent, CMV primary infection is most often a mild infection that does not require the patient to come to the hospital."

9. Line 220: Change "severest symptomatic cases come to hospital and are therefore" to "The patients with the severest symptoms come to the hospital and are the type" We modified as suggested. Lines 235-236: "Consequently, diagnosis of CMV primary infection is probably mostly done by general practitioners and only the patients with the severest symptoms come to the hospital and are therefore included in our data." 10. Figure 2 legend: (A), (B), (C), and (D) are mentioned in the legend. However, these markings were not found in the Figure . Check this out. We corrected this in the Figure 2.

11. If possible, check the entire manuscript for errors in English grammar and punctuation. We made corrections Line 55 (peri-conceptionnal) and Line 175 (CMV primary infection).

Background: Diagnosis of Cytomegalovirus (CMV) primary infection during pregnancy or in immunocompetent patients relies on serology with detection of specific CMV-IgG and IgM. In case of positive CMV-IgM in pregnant women, CMV-IgG avidity is now widely recommended, but in general population it is not currently performed.

Objective: In this study, we aimed to determine CMV-IgM positive predictive values (PPV) in different clinical settings.

Material and methods: We conducted a retrospective study on positive CMV-IgM in our virology laboratory from 2013 to 2019, in three clinical groups: screening in non-symptomatic pregnant women (group 1), pregnant women with ultrasound (US) abnormalities (group 2) and patients (general population) with clinical signs suggestive of CMV primary infection (group 3). CMV-IgG avidity had been performed in all cases allowing to evaluate PPV of positive CMV-IgM to diagnose CMV primary-infection in each group.

Results: Between 2013 and 2019, 6,859 serum samples were found positive for CMV-IgM and had been tested for CMV-IgG avidity, with 6,560 sera for group 1, 30 for group 2 and 269 for group 3.

Overall, low avidity confirming primary infection was observed respectively in 16.4% for group 1, 36.7% for group 2, and 35.3% for group 3. CMV-IgM PPV was significantly lower in group 1 compared to groups 2 (p=0.01) and 3 (p<0.001). Discussion: Our observations highlight the major importance of including CMV-IgG avidity in the diagnostic algorithm, whatever the clinical situation (for immunocompetent patients), to confirm or exclude a recent CMV primary infection in case of positive CMV-IgM.

Introduction

Cytomegalovirus (CMV) is the most frequent worldwide cause of congenital viral infection with a prevalence estimated between 0.5 and 1% of all live births. Congenital CMV is a major cause of sensorineural hearing loss and mental retardation [1][2][3]. CMV transmission to the fetus can occur after primary or secondary maternal CMV infection, with approximately the same proportion of symptoms and sequelae in both situations [4][5][6]. At birth, 13% of congenitally infected neonates are symptomatic with CMV-specific symptoms including growth restriction, microcephaly, ventriculomegaly, chorioretinitis, sensorineural hearing loss, hepatitis, thrombocytopenia and a purpuric skin eruption [2][3][4][5][6][7]. Risk of long term sequelae is higher if CMV transmission occurs in the first or second trimester of pregnancy or during peri-conceptional period [8][9][10]. In immunocompetent patients, CMV primary infection is often asymptomatic. When symptomatic (8 to 10% of cases), primary infection is usually responsible for a mild disease. Signs most frequently reported are: isolated fever, asthenia, mononucleosis syndrome with cervical lymphadenopathy and/or cytolytic hepatitis [11].

Diagnosis of CMV primary infection during pregnancy mainly relies on serology: detection of specific CMV-IgG and IgM, associated with CMV-IgG avidity in case of positive CMV-IgM [12]. However, in immunocompetent patients not pregnant, CMV-IgG avidity is usually not performed in case of positive CMV-IgM. Reported clinical performances of commercial immunoassays for CMV IgM are sensitivity >90% and specificity >96% and for CMV IgG avidity, specificity and sensitivity are comprised between 90 and 100% depending on the assay [13][14][15][16]. CMV-IgM can possibly indicate an acute or a recent infection but can also be due to other causes: long-term persisting IgM, crossreaction, secondary CMV infection or nonspecific stimulation of the immune system. Consequently, diagnosis of primary infection cannot rely only on a positive IgM test result. CMV-IgG avidity measurement is an essential tool to confirm or exclude CMV primary-infection. CMV-IgG are initially of low avidity, but will mature to high avidity at 2-4 months after primary infection [12,17,18].

The main issue with CMV serology is that CMV-IgG avidity is not available in all laboratories and that clinicians still too often rely on positive CMV-IgM result to diagnose CMV primary-infection. In our retrospective cohort study, we aim to determine and compare CMV-IgM positive predictive value (PPV) to diagnose CMV primary infection depending on the clinical situation: systematic screening during pregnancy, presence of ultrasound abnormalities (US) during pregnancy and clinical signs suggestive of CMV primary infection in general population (immunocompetent patients).

Material and methods

Sample collection:

In our hospital virology laboratory, CMV serology (IgG, IgM and IgG avidity in case of positive IgG) is performed either:

-in non-symptomatic pregnant women during first trimester of pregnancy (systematic screening) and followed in one of the two maternities in Paris South Hospitals, or in pregnant women referred to our laboratory because of positive CMV-IgM detected in one of the laboratories part of our network (systematic screening in other centers) (group 1); -in non-symptomatic pregnant women referred to our pluridisciplinary prenatal center for US abnormalities (not initially screened at beginning of pregnancy) (group 2). Sonographic findings were mostly intrauterine growth retardation (IUGR) and cerebral abnormalities (head perimeter < 5th centile, ventriculomegaly, hyperechogenic ventricular wall, cerebellar and brain calcifications, enlargement of pericerebral spaces, periventricular and subependymal cysts, candlestick, porencephaly, hyperechogenic periventricular halo, candlestick, abnormal gyration, and/or corpus callosum hypoplasia); -in immunocompetent patients (general population: adults and children) in case of clinical symptoms suggestive of CMV primary infection (group 3), mainly fever, headache, flu-like syndrome (fever + rhinitis + myalgia), arthralgia, myalgia, fatigue and/or hepatitis. Samples were collected 5 to 30 days after onset of symptoms.

All CMV serologic results performed in one of these contexts in our laboratory between January 2013 and December 2019 were retrospectively analysed. In case a patient had several samples, only the most informative (usually the first one) was kept for analysis. Serologies performed in the context of transplantation or in immunocompromised patients were not included in this study.

Serology assays:

CMV-IgG and CMV-IgM were measured with LIAISON XL (LXL, DiaSorin ® , Saluggia, Italy). In case of positive CMV-IgM, our strategy is based on previous published findings [19]. In a few words, we first-line perform LXL IgG avidity as a screening test. An index > 0.40 allows to exclude a recent CMV primary infection. Below LXL 0.40 index threshold, a second assay is used: VIDAS (bioMérieux ® , Craponne, France) CMV-IgG avidity. In this case, confirmation/exclusion of primary infection (more/less than 3 months before sample collection) is only based on the VIDAS assay result

Statistical analysis:

For the three groups, CMV-IgM PPV to diagnose recent CMV primary infection were calculated with 95% confidence intervals. We calculated p-values with Chi-2 Pearson tests.

Results

Between 2013 and 2019, 6,859 serum samples were tested positive for CMV-IgM in our laboratory:

-6,560 /6,859 (95.64%) were collected in pregnant women during systematic screening (group 1) -30/6,859 (0.44%) were collected in pregnant women with US abnormalities (median gestational age 25 weeks of gestation (WG); range: 12-36 WG) (group 2).

-269/6,859 (3.92%) were collected from immunocompetent patients with clinical symptoms of CMV primary infection (group 3) All 6,859 serum samples CMV-IgM positive had been tested for CMV-IgG avidity at time of diagnosis.

Systematic screening during pregnancy (group 1)

CMV-IgG avidity was high in 5,486/6,560 (83.6%) cases allowing to exclude recent CMV primary infection (< 3 months). A total of 1,074/6,560 samples collected from pregnant women for systematic screening had low or moderate CMV-IgG avidity index and were considered as confirmed or not excluded recent CMV primary infections (Figure 1 and Figure 2). Therefore, PPV of CMV-IgM for systematic screening during pregnancy to predict a recent primary infection was 16.4% (95% CI = 15.5 -17.3%).

Ultrasound abnormalities during pregnancy (group 2)

CMV-IgG avidity was high in 18/30 (60.0%) cases allowing to exclude recent CMV primary infection. A total of 11/30 samples collected from pregnant women addressed for US had low or intermediate CMV-IgG avidity index and were considered as confirmed or not excluded recent CMV primary infections (Figure 1 and Figure 2). Therefore, PPV of CMV-IgM in case of US abnormalities during pregnancy to predict a recent primary infection was 36.7% (95% CI = 19.5 -53.9%).

Immunocompetent patients with clinical symptoms of CMV primary infection (group 3)

CMV-IgG avidity was high in 174/269 (64.7%) cases allowing to exclude recent CMV primary infection (< 3 months). A total of 95/269 samples collected from patients with symptoms of acute CMV primary infection had low or intermediate CMV-IgG avidity index and were considered as confirmed or not excluded recent CMV primary infections (Figure 1 and Figure 2). Therefore, PPV of CMV-IgM for immunocompetent patients with clinical signs to predict a recent primary infection was 35.3% (95% CI = 29.6 -41.0%).

We determined CMV-IgM PPV for these 269 patients according to their age (Figure 3 CMV-IgM PPV was significantly lower in group 1 comparing to groups 2 (p=0.01) and 3 (p<0.001).

CMV-IgM PPV was not statistically different between groups 2 and 3 (p>0.90).

Discussion

Even if formal diagnosis of CMV primary infection is achieved with CMV-IgG seroconversion, as the "first" seronegative serum is usually not available, documentation of this seroconversion is rare. Usually, whatever the clinical situation, screening or clinical symptoms, only one serum specimen is available. The transient CMV-IgM positivity has long been used as a diagnostic marker for CMV primary infection, but it is well known that is not invariably indicative of primary infection. Indeed, specific or non-specific CMV-IgM can also be present in many other situations including infections with other pathogens associated with random polyclonal B cell stimulation or CMV non primary infection [20,21]. Moreover, there are known to be technical issues with IgM-specific testing. Indeed, false positive CMV-IgM results were shown to occur due to high levels of analyte specific IgG, to presence of rheumatoid factors, and to other miscellaneous technical factors involved with methods that must include some sort of process to separate CMV IgG from CMV IgM. In these cases, CMV-IgG avidity is essential and in contrast to IgM, low-avidity IgG is present only with primary infection, increasing over 3 to 4 months to high avidity [22]. CMV-IgG avidity has thus gained diagnostic importance in identifying primary CMV infection, and several commercial CMV-IgG avidity tests are currently available. Their performances to confirm a CMV primary infection were reported to range from 83 to 100%, and to exclude a CMV primary infection from 71 to 100% [12,16,19,[23][24][25][26][27][28]. Even if not perfect, several groups have reported substantial improvements in identification of at-risk pregnancies using diagnostic algorithms including CMV-IgG avidity [7,15,18,22,[29][30][31]. Indeed, it truly improves accuracy of CMV primary infection diagnosis as in the specific situation of systematic screening, PPV of positive CMV-IgM is quite poor [18,31]. Indeed, in our analysis, if we calculated PPV considering confirmed recent primary infection, PPV is only of 9.7% (95%CI= 9.0 -10.4%). However, from a practical point of view, we calculated PPV considering both confirmed and not excluded recent CMV primary infection as these will probably be managed like confirmed recent primary infection regarding regular ultrasound follow up. It allows PPV to reach 16.4%.

Usefulness of serologic testing for CMV during pregnancy has been questioned since congenital malformations can occur even following maternal secondary infections [6]. Although screening is not recommended by any public health system because of its cost/benefit ratio, it is widely adopted by many general practitioners and obstetricians in our area. Such screening provides an opportunity to identify seronegative women who can be counselled about using appropriate hygienic measures to prevent primary infection, especially in relation to their behavior with children, who are a major source of infection. Furthermore, screening aims to diagnose CMV primary infection early in pregnancy allowing women to be referred to Reference Centers for appropriate management (close ultrasonography, amniocentesis and/or neonatal diagnosis…) [32]. Some authors consider that screening is not justified because of its economic cost, the imperfect nature of congenital infection prognostic criteria, the risk of spontaneous abortions induced by invasive tests such as amniocentesis, and the few data concerning effective treatments during pregnancy. However, it is obvious that if US are observed, and prenatal diagnosis of CMV congenital infection discussed, maternal CMV serology should always be performed. It is particularly important especially in regions where seroprevalence is around 50%, as in France, or lower because its first aim is to confirm maternal infection to CMV (whatever the date of this infection) and allows to definitively exclude congenital CMV if the pregnant woman is seronegative. If positive, this CMV serology usually performed late in pregnancy, might be challenging to interpret. Our results show that positive CMV-IgM at time of US indicate a recent infection in 23% cases (7/30) and that additionally, 3/30 women had high avidity but gave birth to an infected child. Overall, when positive CMV-IgM is detected when US abnormalities are observed (possibly late in pregnancy), it suggests responsibility of CMV in 47% (14/30) cases without ruling out the possibility of CMV congenital infection following maternal non primary infection if the mother is CMV-IgG positive and CMV-IgM is negative. However, final diagnosis will of course be obtained with CMV PCR in amniotic fluid and/or in urine in the neonate at birth. If CMV-IgG avidity is widely used in pregnant women, far less literature is available on the usefulness of CMV-IgG avidity in general population (immunocompetent patients), and in our area it is clearly not usually performed if the patient is not pregnant. Remarkably, positive CMV-IgM results are most often considered as indicative of a recent CMV virus infection in case with clinical signs, but they are in fact truly related to a CMV primary infection in only 27.1% cases (73/269) (low CMV IgG avidity) and possibly related in 35.3% cases (low/moderate CMV IgG avidity). Interestingly, PPV was significantly lower for patients between 10 to 20 years old (11.1% vs 29.6-44.9%) (p=0.01). Our results suggest that positive CMV-IgM is less correlated with CMV recent primary infection in young people, and are consistent with the understanding that CMV-IgM can be produced throughout life as a result of CMV non primary infection or polyclonal stimulation of the immune system [20,21]. Also, in cases of positive CMV-IgM, older people may be more likely to have a primary infection than younger people. These also suggest that whatever the age of the patient, CMV-IgG avidity is essential for accurate diagnosis. One limitation of our study is that, although very frequent, CMV primary infection is most often a mild infection that does not require the patient to come to the hospital.

Consequently, general practitioners probably mostly do diagnosis of CMV primary infection and only

the patients with the severest symptoms come to the hospital and are therefore included in our data. Moreover, it is possible that older people are more likely to have a severe infection compared to young patients. Nevertheless, for current practice, all clinicians should be aware that, whatever the age of the patient, and given the non-specific symptoms of CMV primary infection, even in case of positive CMV-IgM, confirmation by avidity avoids misdiagnosis in more than 60% cases.

Our observations are consistent with the preexisting ones [33][34][35][36][37] and highlight the major importance of including CMV-IgG avidity in the diagnostic algorithm, whatever the clinical situation (for immunocompetent patients), to confirm or exclude a CMV primary infection in case of positive CMV-IgM. sensorineural hearing loss and mental retardation [1][2][3]. CMV transmission to the fetus can occur after primary or secondary maternal CMV infection, with approximately the same proportion of symptoms and sequelae in both situations [4][5][6]. At birth, 13% of congenitally infected neonates are symptomatic with CMV-specific symptoms including growth restriction, microcephaly, ventriculomegaly, chorioretinitis, sensorineural hearing loss, hepatitis, thrombocytopenia and a purpuric skin eruption [2][3][4][5][6][7]. Risk of long term sequelae is higher if CMV transmission occurs in the first or second trimester of pregnancy or during peri-conceptional period [8][9][10]. In immunocompetent patients, CMV primary infection is often asymptomatic. When symptomatic (8 to 10% of cases), primary infection is usually responsible for a mild disease. Signs most frequently reported are: isolated fever, asthenia, mononucleosis syndrome with cervical lymphadenopathy and/or cytolytic hepatitis [11].

Diagnosis of CMV primary infection during pregnancy mainly relies on serology: detection of specific CMV-IgG and IgM, associated with CMV-IgG avidity in case of positive CMV-IgM [12]. However, in immunocompetent patients not pregnant, CMV-IgG avidity is usually not performed in case of positive CMV-IgM. Reported clinical performances of commercial immunoassays for CMV IgM are sensitivity >90% and specificity >96% and for CMV IgG avidity, specificity and sensitivity are comprised between 90 and 100% depending on the assay [13][14][15][16]. CMV-IgM can possibly indicate an acute or a recent infection but can also be due to other causes: long-term persisting IgM, crossreaction, secondary CMV infection or nonspecific stimulation of the immune system. Consequently, diagnosis of primary infection cannot rely only on a positive IgM test result. CMV-IgG avidity measurement is an essential tool to confirm or exclude CMV primary-infection. CMV-IgG are initially of low avidity, but will mature to high avidity at 2-4 months after primary infection [12,17,18].

The main issue with CMV serology is that CMV-IgG avidity is not available in all laboratories and that clinicians still too often rely on positive CMV-IgM result to diagnose CMV primary-infection. In our retrospective cohort study, we aim to determine and compare CMV-IgM positive predictive value (PPV) to diagnose CMV primary infection depending on the clinical situation: systematic screening during pregnancy, presence of ultrasound abnormalities (US) during pregnancy and clinical signs suggestive of CMV primary infection in general population (immunocompetent patients).

Material and methods

Sample collection:

In our hospital virology laboratory, CMV serology (IgG, IgM and IgG avidity in case of positive IgG) is performed either:

-in non-symptomatic pregnant women during first trimester of pregnancy (systematic screening) and followed in one of the two maternities in Paris South Hospitals, or in pregnant women referred to our laboratory because of positive CMV-IgM detected in one of the laboratories part of our network (systematic screening in other centers) (group 1); -in non-symptomatic pregnant women referred to our pluridisciplinary prenatal center for US abnormalities (not initially screened at beginning of pregnancy) (group 2). Sonographic findings were mostly intrauterine growth retardation (IUGR) and cerebral abnormalities (head perimeter < 5th centile, ventriculomegaly, hyperechogenic ventricular wall, cerebellar and brain calcifications, enlargement of pericerebral spaces, periventricular and subependymal cysts, candlestick, porencephaly, hyperechogenic periventricular halo, candlestick, abnormal gyration, and/or corpus callosum hypoplasia); -in immunocompetent patients (general population: adults and children) in case of clinical symptoms suggestive of CMV primary infection (group 3), mainly fever, headache, flu-like syndrome (fever + rhinitis + myalgia), arthralgia, myalgia, fatigue and/or hepatitis. Samples were collected 5 to 30 days after onset of symptoms.

All CMV serologic results performed in one of these contexts in our laboratory between January 2013 and December 2019 were retrospectively analysed. In case a patient had several samples, only the most informative (usually the first one) was kept for analysis. Serologies performed in the context of transplantation or in immunocompromised patients were not included in this study.

Serology assays:

CMV-IgG and CMV-IgM were measured with LIAISON XL (LXL, DiaSorin ® , Saluggia, Italy). In case of positive CMV-IgM, our strategy is based on previous published findings [19]. In a few words, we first-line perform LXL IgG avidity as a screening test. An index > 0.40 allows to exclude a recent CMV primary infection. Below LXL 0.40 index threshold, a second assay is used: VIDAS (bioMérieux ® , Craponne, France) CMV-IgG avidity. In this case, confirmation/exclusion of primary infection (more/less than 3 months before sample collection) is only based on the VIDAS assay result

Statistical analysis:

For the three groups, CMV-IgM PPV to diagnose recent CMV primary infection were calculated with 95% confidence intervals. We calculated p-values with Chi-2 Pearson tests.

Results

Between 2013 and 2019, 6,859 serum samples were tested positive for CMV-IgM in our laboratory:

-6,560 /6,859 (95.64%) were collected in pregnant women during systematic screening (group 1) -30/6,859 (0.44%) were collected in pregnant women with US abnormalities (median gestational age 25 weeks of gestation (WG); range: 12-36 WG) (group 2).

-269/6,859 (3.92%) were collected from immunocompetent patients with clinical symptoms of CMV primary infection (group 3) All 6,859 serum samples CMV-IgM positive had been tested for CMV-IgG avidity at time of diagnosis.

Systematic screening during pregnancy (group 1)

CMV-IgG avidity was high in 5,486/6,560 (83.6%) cases allowing to exclude recent CMV primary infection (< 3 months). A total of 1,074/6,560 samples collected from pregnant women for systematic screening had low or moderate CMV-IgG avidity index and were considered as confirmed or not excluded recent CMV primary infections (Figure 1 and Figure 2). Therefore, PPV of CMV-IgM for systematic screening during pregnancy to predict a recent primary infection was 16.4% (95% CI = 15.5 -17.3%).

Ultrasound abnormalities during pregnancy (group 2)

CMV-IgG avidity was high in 18/30 (60.0%) cases allowing to exclude recent CMV primary infection. A total of 11/30 samples collected from pregnant women addressed for US had low or intermediate CMV-IgG avidity index and were considered as confirmed or not excluded recent CMV primary infections (Figure 1 and Figure 2). Therefore, PPV of CMV-IgM in case of US abnormalities during pregnancy to predict a recent primary infection was 36.7% (95% CI = 19.5 -53.9%).

Immunocompetent patients with clinical symptoms of CMV primary infection (group 3)

CMV-IgG avidity was high in 174/269 (64.7%) cases allowing to exclude recent CMV primary infection (< 3 months). A total of 95/269 samples collected from patients with symptoms of acute CMV primary infection had low or intermediate CMV-IgG avidity index and were considered as confirmed or not excluded recent CMV primary infections (Figure 1 and Figure 2). Therefore, PPV of CMV-IgM for immunocompetent patients with clinical signs to predict a recent primary infection was 35.3% (95% CI = 29.6 -41.0%).

We determined CMV-IgM PPV for these 269 patients according to their age (Figure 3 Overall, when positive CMV-IgM are observed, recent CMV primary infection is only confirmed in respectively 16.4% cases for systematic screening during pregnancy (group 1), 36.7% in case of ultrasound abnormalities (group 2) and 35.3% in case of clinical signs in general population (group 3).

CMV-IgM PPV was significantly lower in group 1 comparing to groups 2 (p=0.01) and 3 (p<0.001).

CMV-IgM PPV was not statistically different between groups 2 and 3 (p>0.90).

Discussion

Even if formal diagnosis of CMV primary infection is achieved with CMV-IgG seroconversion, as the "first" seronegative serum is usually not available, documentation of this seroconversion is rare. Usually, whatever the clinical situation, screening or clinical symptoms, only one serum specimen is available. The transient CMV-IgM positivity has long been used as a diagnostic marker for CMV primary infection, but it is well known that is not invariably indicative of primary infection. Indeed, specific or non-specific CMV-IgM can also be present in many other situations including infections with other pathogens associated with random polyclonal B cell stimulation or CMV non primary infection [20,21]. Moreover, there are known to be technical issues with IgM-specific testing. Indeed, false positive CMV-IgM results were shown to occur due to high levels of analyte specific IgG, to presence of rheumatoid factors, and to other miscellaneous technical factors involved with methods that must include some sort of process to separate CMV IgG from CMV IgM. In these cases, CMV-IgG avidity is essential and in contrast to IgM, low-avidity IgG is present only with primary infection, increasing over 3 to 4 months to high avidity [22]. CMV-IgG avidity has thus gained diagnostic importance in identifying primary CMV infection, and several commercial CMV-IgG avidity tests are currently available. Their performances to confirm a CMV primary infection were reported to range from 83 to 100%, and to exclude a CMV primary infection from 71 to 100% [12,16,19,[23][24][25][26][27][28]. Even if not perfect, several groups have reported substantial improvements in identification of at-risk pregnancies using diagnostic algorithms including CMV-IgG avidity [7,15,18,22,[29][30][31]. Indeed, it truly improves accuracy of CMV primary infection diagnosis as in the specific situation of systematic screening, PPV of positive CMV-IgM is quite poor [18,31]. Indeed, in our analysis, if we calculated PPV considering confirmed recent primary infection, PPV is only of 9.7% (95%CI= 9.0 -10.4%). However, from a practical point of view, we calculated PPV considering both confirmed and not excluded recent CMV primary infection as these will probably be managed like confirmed recent primary infection regarding regular ultrasound follow up. It allows PPV to reach 16.4%. Usefulness of serologic testing for CMV during pregnancy has been questioned since congenital malformations can occur even following maternal secondary infections [6]. Although screening is not recommended by any public health system because of its cost/benefit ratio, it is widely adopted by many general practitioners and obstetricians in our area. Such screening provides an opportunity to identify seronegative women who can be counselled about using appropriate hygienic measures to prevent primary infection, especially in relation to their behavior with children, who are a major source of infection. Furthermore, screening aims to diagnose CMV primary infection early in pregnancy allowing women to be referred to Reference Centers for appropriate management (close ultrasonography, amniocentesis and/or neonatal diagnosis…) [32]. Some authors consider that screening is not justified because of its economic cost, the imperfect nature of congenital infection prognostic criteria, the risk of spontaneous abortions induced by invasive tests such as amniocentesis, and the few data concerning effective treatments during pregnancy. However, it is obvious that if US are observed, and prenatal diagnosis of CMV congenital infection discussed, maternal CMV serology should always be performed. It is particularly important especially in regions where seroprevalence is around 50%, as in France, or lower because its first aim is to confirm maternal infection to CMV (whatever the date of this infection) and allows to definitively exclude congenital CMV if the pregnant woman is seronegative. If positive, this CMV serology usually performed late in pregnancy, might be challenging to interpret. Our results show that positive CMV-IgM at time of US indicate a recent infection in 23% cases (7/30) and that additionally, 3/30 women had high avidity but gave birth to an infected child. Overall, when positive CMV-IgM is detected when US abnormalities are observed (possibly late in pregnancy), it suggests responsibility of CMV in 47% (14/30) cases without ruling out the possibility of CMV congenital infection following maternal non primary infection if the mother is CMV-IgG positive and CMV-IgM is negative. However, final diagnosis will of course be obtained with CMV PCR in amniotic fluid and/or in urine in the neonate at birth. If CMV-IgG avidity is widely used in pregnant women, far less literature is available on the usefulness of CMV-IgG avidity in general population (immunocompetent patients), and in our area it is clearly not usually performed if the patient is not pregnant. Remarkably, positive CMV-IgM results are most often considered as indicative of a recent CMV virus infection in case with clinical signs, but they are in fact truly related to a CMV primary infection in only 27.1% cases (73/269) (low CMV IgG avidity) and possibly related in 35.3% cases (low/moderate CMV IgG avidity). Interestingly, PPV was significantly lower for patients between 10 to 20 years old (11.1% vs 29.6-44.9%) (p=0.01). Our results suggest that positive CMV-IgM is less correlated with CMV recent primary infection in young people, and are consistent with the understanding that CMV-IgM can be produced throughout life as a result of CMV non primary infection or polyclonal stimulation of the immune system [20,21]. Also, in cases of positive CMV-IgM, older people may be more likely to have a primary infection than younger people. These also suggest that whatever the age of the patient, CMV-IgG avidity is essential for accurate diagnosis. One limitation of our study is that, although very frequent, CMV primary infection is most often a mild infection that does not require the patient to come to the hospital. Consequently, general practitioners probably mostly do diagnosis of CMV primary infection and only the patients with the severest symptoms come to the hospital and are therefore included in our data. Moreover, it is possible that older people are more likely to have a severe infection compared to young patients. Nevertheless, for current practice, all clinicians should be aware that, whatever the age of the patient, and given the non-specific symptoms of CMV primary infection, even in case of positive CMV-IgM, confirmation by avidity avoids misdiagnosis in more than 60% cases.

Our observations are consistent with the preexisting ones [33][34][35][36][37] and highlight the major importance of including CMV-IgG avidity in the diagnostic algorithm, whatever the clinical situation (for immunocompetent patients), to confirm or exclude a CMV primary infection in case of positive CMV-IgM.

US: ultrasound

PI: primary infection

(

  cutoffs used are those recommended by manufacturer: 0.4-0.65). Results allowed us to classify serological profiles as follows: -Negative CMV-IgG/positive CMV-IgM on two consecutive samples 21 days apart: nonspecific IgM; -Positive CMV-IgG/positive CMV-IgM/high CMV-IgG avidity (LXL > 0.4, or LXL < 0.4 and VIDAS > 0.65) : recent CMV primary infection excluded; -Positive CMV-IgG/positive CMV-IgM/low CMV-IgG avidity (LXL < 0.4 and VIDAS < 0.40): recent CMV primary infection confirmed; -Positive CMV-IgG/positive CMV-IgM/moderate CMV-IgG avidity (LXL < 0.4 and VIDAS > 0.40 but < 0.65): recent CMV primary infection not excluded.

  ): -less than 10 years old (n=71): CMV-IgG avidity was low or moderate in 21/71 patients resulting in a PPV of 29.6% (95%CI= 19.0 -40.5%) (p=0.50) -10 to 20 years old (n=27): CMV-IgG avidity was low or moderate in 3/27 patients resulting in a PPV of 11.1% (95%CI= 0.0 -23.0%) (p=0.01) -20 to 40 years old (n=69): CMV-IgG avidity was low or moderate in 31/69 patients resulting in a PPV of 44.9% (95%CI= 33.2 -56.6%) (p=0.20) -more than 40 years old (n=102): CMV-IgG avidity was low or moderate in 40/102 patients resulting in a PPV of 39.2% (95%CI= 29.7 -48.7%) (p=0.90)Overall, when positive CMV-IgM are observed, recent CMV primary infection is only confirmed in respectively 16.4% cases for systematic screening during pregnancy (group 1), 36.7% in case of ultrasound abnormalities (group 2) and 35.3% in case of clinical signs in general population (group 3).

(

  cutoffs used are those recommended by manufacturer: 0.4-0.65). Results allowed us to classify serological profiles as follows: -Negative CMV-IgG/positive CMV-IgM on two consecutive samples 21 days apart: nonspecific IgM; -Positive CMV-IgG/positive CMV-IgM/high CMV-IgG avidity (LXL > 0.4, or LXL < 0.4 and VIDAS > 0.65) : recent CMV primary infection excluded; -Positive CMV-IgG/positive CMV-IgM/low CMV-IgG avidity (LXL < 0.4 and VIDAS < 0.40): recent CMV primary infection confirmed; -Positive CMV-IgG/positive CMV-IgM/moderate CMV-IgG avidity (LXL < 0.4 and VIDAS > 0.40 but < 0.65): recent CMV primary infection not excluded.

  ): -less than 10 years old (n=71): CMV-IgG avidity was low or moderate in 21/71 patients resulting in a PPV of 29.6% (95%CI= 19.0 -40.5%) (p=0.50) -10 to 20 years old (n=27): CMV-IgG avidity was low or moderate in 3/27 patients resulting in a PPV of 11.1% (95%CI= 0.0 -23.0%) (p=0.01) -20 to 40 years old (n=69): CMV-IgG avidity was low or moderate in 31/69 patients resulting in a PPV of 44.9% (95%CI= 33.2 -56.6%) (p=0.20) -more than 40 years old (n=102): CMV-IgG avidity was low or moderate in 40/102 patients resulting in a PPV of 39.2% (95%CI= 29.7 -48.7%) (p=0.90)

Figure 1 :

 1 Figure 1: Study population flowchart.

Figure 2 :

 2 Figure 2: Final diagnosis in each clinical setting in case of positive CMV-IgM.

Figure 3 :

 3 Figure 3: PPV of positive CMV-IgM depending on patients' age in general population (group 3).

Figure 1 :

 1 Figure 1: Study population flowchart.

Figure 2 :

 2 Figure 2: Final diagnosis in each clinical setting in case of positive CMV-IgM.

Figure 3 :

 3 Figure 3: PPV of positive CMV-IgM depending on patients' age in general population (group 3).

Sample CRediT author statement:

Claire Périllaud-Dubois: Conceptualization, Methodology, Formal analysis, Writingoriginal draft.

Elise Bouthry: Writing-Reviewing and Editing. Abir Jadoui and Ay Ling Leng: Investigation.

Anne-Marie Roque-Afonso: Writing-Reviewing and Editing. Christelle Vauloup-Fellous: Conceptualization, Writing-Reviewing and Editing, Supervision.

Declarations of Interest: none.

Funding sources: none.

Background: Diagnosis of Cytomegalovirus (CMV) primary infection during pregnancy or in immunocompetent patients relies on serology with detection of specific CMV-IgG and IgM. In case of positive CMV-IgM in pregnant women, CMV-IgG avidity is now widely recommended, but in general population it is not currently performed.

Objective: In this study, we aimed to determine CMV-IgM positive predictive values (PPV) in different clinical settings.

Material and methods: We conducted a retrospective study on positive CMV-IgM in our virology laboratory from 2013 to 2019, in three clinical groups: screening in non-symptomatic pregnant women (group 1), pregnant women with ultrasound (US) abnormalities (group 2) and patients (general population) with clinical signs suggestive of CMV primary infection (group 3). CMV-IgG avidity had been performed in all cases allowing to evaluate PPV of positive CMV-IgM to diagnose CMV primary-infection in each group.

Results: Between 2013 and 2019, 6,859 serum samples were found positive for CMV-IgM and had been tested for CMV-IgG avidity, with 6,560 sera for group 1, 30 for group 2 and 269 for group 3.

Overall, low avidity confirming primary infection was observed respectively in 16.4% for group 1, 36.7% for group 2, and 35.3% for group 3. CMV-IgM PPV was significantly lower in group 1 compared to groups 2 (p=0.01) and 3 (p<0.001). Discussion: Our observations highlight the major importance of including CMV-IgG avidity in the diagnostic algorithm, whatever the clinical situation (for immunocompetent patients), to confirm or exclude a recent CMV primary infection in case of positive CMV-IgM.

Introduction

Cytomegalovirus (CMV) is the most frequent worldwide cause of congenital viral infection with a prevalence estimated between 0.5 and 1% of all live births. Congenital CMV is a major cause of Claire Périllaud-Dubois: Conceptualization, Methodology, Formal analysis, Writingoriginal draft.

Elise Bouthry: Writing-Reviewing and Editing. Abir Jadoui and Ay Ling Leng: Investigation.

Anne-Marie Roque-Afonso: Writing-Reviewing and Editing. Christelle Vauloup-Fellous: Conceptualization, Writing-Reviewing and Editing, Supervision.

Declarations of Interest: none.

Funding sources: none.