Seth L Masters
email: smasters@tcd.ie

Aisling Dunne

Shoba L Subramanian

Rebecca L Hull

Gillian M Tannahill

Fiona A Sharp

Christine Becker

Luigi Franchi

Eiji Yoshihara

Zhe Chen

Niamh Mullooly

Lisa A Mielke

James Harris

Rebecca C Coll

Kingston H G Mills

K Hun Mok

Philip Newsholme

Gabriel Nuñez

Junji Yodoi

Steven E Kahn

Ed C Lavelle

Luke A J O'neill

Activation of the Nlrp3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1β in type 2 diabetes

IL-1β is an important inflammatory mediator of type 2 diabetes (T2D). Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during T2D, trigger the Nlrp3 inflammasome and generate mature interleukin (IL)-1β. A T2D therapy, glyburide, suppresses IAPPmediated IL-1β production in vitro. Processing of IL-1β initiated by IAPP first requires priming, a process that involves glucose metabolism and can be facilitated by minimally oxidized low density lipoprotein. Finally, mice transgenic for human IAPP have increased IL-1β in pancreatic islets, which colocalizes with amyloid and macrophages. Our findings reveal novel mechanisms in the pathogenesis of T2D and treatment of pathology caused by IAPP.

Introduction

Type 2 diabetes (T2D) is characterized by insulin resistance and islet beta cell dysfunction, with the progressive loss of insulin release being responsible for ever increasing glucose concentrations in the absence of treatment [START_REF] Kahn | Mechanisms linking obesity to insulin resistance and type 2 diabetes[END_REF] . Obesity is frequently the basis for insulin resistance in T2D, and is associated with increased concentrations of oxidized low density lipoprotein (LDL) [START_REF] Njajou | Association between oxidized LDL, obesity and type 2 diabetes in a population-based cohort, the Health, Aging and Body Composition Study[END_REF] , free fatty acids (FFAs), and pro-inflammatory cytokines [START_REF] Kahn | Mechanisms linking obesity to insulin resistance and type 2 diabetes[END_REF] . One cytokine in particular, interleukin-1 beta (IL-1β), has been found to have profound effects on the function of pancreatic beta cells, inducing them to undergo apoptosis [START_REF] Bendtzen | Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans[END_REF] . Abundant literature describes the involvement of IL-1β in type 1 diabetes (T1D), however IL-1β is also a risk factor for T2D [START_REF] Spranger | Inflammatory cytokines and the risk to develop type 2 diabetes: results of the prospective population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study[END_REF] , and a recent clinical trial of IL-1 blockade in T2D has been very encouraging [START_REF] Larsen | Interleukin-1-receptor antagonist in type 2 diabetes mellitus[END_REF] .

These data clearly suggest a role for IL-1β in the pathogenesis of T2D, however the events leading to increased concentrations of active, secreted IL-1β in this disease remain unclear.

Recently a large number of publications have elucidated mechanisms by which biologically active IL-1β is made in the cell. This usually involves activation of a protein complex termed the inflammasome, which is formed by a nucleating sensor protein (typically a member of the nucleotide-binding domain and leucine-rich repeat containing, NLR, family) that oligomerises the adaptor protein ASC to dimerise and activate caspase-1 and thus cleave pro-IL-1β into the processed, secreted form. Before this can happen, certain cell types require priming to induce suitable amounts of pro-IL-1β and NLR proteins, this is sometimes referred to as 'signal 1'. The 'signal 2' factors that can activate the nucleating receptor of the complex are still being determined. These include microbial DNA, RNA, cell wall components and toxins. With reference to inflammatory diseases such as gout and fibrosing disorders, endogenous factors and environmental contaminants have also been discovered, such as uric acid crystals [START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF] and asbestos or silica respectively [START_REF] Dostert | Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica[END_REF] .

Most of these molecules activate the inflammasome complex nucleated by Nlrp3 (also known as Nalp3 or cryopyrin), however there is no structural similarity to indicate that they would bind to it directly. Instead Nlrp3 is probably a sensor of some homeostatic intracellular process that, if perturbed, will activate the inflammasome. Certainly a large number of the Nlrp3 activating agents seem to perturb the lysosomal vacuole, some due to their size through a process that has been termed "frustrated phagocytosis", and others that are not degraded normally once phagocytosed [START_REF] Hornung | Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization[END_REF] . This prompted us to ask if there might also be an endogenous factor in T2D that could activate the Nlrp3 inflammasome in this way.

A hallmark feature of T2D is the deposition of amyloid in the pancreas of most patients, with this morphological change being associated with the loss of insulinproducing beta cells [START_REF] Clark | Islet amyloid, increased A-cells, reduced B-cells and exocrine fibrosis: quantitative changes in the pancreas in type 2 diabetes[END_REF] . The unique polypeptide constituent of amyloid found in pancreatic islets is islet amyloid polypeptide (IAPP, also known as amylin) [START_REF] Cooper | Purification and characterization of a peptide from amyloidrich pancreases of type 2 diabetic patients[END_REF][START_REF] Westermark | Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells[END_REF] , a 37 amino acid peptide co-secreted by the beta cell with insulin. There are a number of lines of evidence that strongly implicate this deposition in disease progression. First there is species specificity whereby IAPP in certain animals, such as rodents, is unable to form amyloid due to the alteration of certain critical residues, however when a human IAPP transgene is expressed in mice or rats, amyloid is deposited within pancreatic islets, leading to loss of beta cell mass [START_REF] Butler | Diabetes due to a progressive defect in beta-cell mass in rats transgenic for human islet amyloid polypeptide (HIP Rat): a new model for type 2 diabetes[END_REF][START_REF] Janson | Spontaneous diabetes mellitus in transgenic mice expressing human islet amyloid polypeptide[END_REF][START_REF] Verchere | Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide[END_REF] . Second, experiments have been performed using non-human primates in which the concentration of amyloid in the pancreas clearly correlates with the severity of disease and beta cell function [START_REF] Howard | Longitudinal studies on the development of diabetes in individual Macaca nigra[END_REF] . Third, in humans a rare polymorphism in IAPP, S20G, is associated with increased incidence or severity of T2D in some populations [START_REF] Seino | S20G mutation of the amylin gene is associated with Type II diabetes in Japanese. Study Group of Comprehensive Analysis of Genetic Factors in Diabetes Mellitus[END_REF] , and this version of the protein has a higher propensity to form amyloid [START_REF] Ma | Enhanced in vitro production of amyloid-like fibrils from mutant (S20G) islet amyloid polypeptide[END_REF] . IAPP oligomerization might also have a role in preventing successful islet transplantation, which is a potential treatment for T1D. Amyloid deposition is observed when human IAPP transgenic mouse islets, or human islets are transplanted into mice [START_REF] Udayasankar | Amyloid formation results in recurrence of hyperglycaemia following transplantation of human IAPP transgenic mouse islets[END_REF][START_REF] Westermark | Rapid deposition of amyloid in human islets transplanted into nude mice[END_REF] , and this could also be associated with the failure of islet transplantation in humans [START_REF] Westermark | Widespread amyloid deposition in transplanted human pancreatic islets[END_REF] .

Initially it was established that IAPP is cytotoxic [START_REF] Lorenzo | Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus[END_REF] , and this could be due to effects at the cell membrane [START_REF] Janson | The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles[END_REF] . Mechanistically, this might also involve reactive oxygen species (ROS) because ROS inhibition prevents IAPP-mediated beta cell apoptosis in vitro [START_REF] Zraika | Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis[END_REF] . The process of IAPP incorporation into amyloid is thought to proceed via the aggregation of prefibrillar oligomers. These soluble oligomers, rather than mature fibrils, could be the cytotoxic component [START_REF] Zraika | Toxic oligomers and islet beta cell death: guilty by association or convicted by circumstantial evidence?[END_REF] . Amyloid builds up extracellularly but phagocytosed IAPP can be detected within the lysosomal compartment of human pancreatic macrophages in vivo, where due to its amyloid structure, it is not degraded normally [START_REF] Badman | Fibrillar islet amyloid polypeptide (amylin) is internalised by macrophages but resists proteolytic degradation[END_REF][START_REF] De Koning | Macrophages and pancreatic islet amyloidosis[END_REF] . There are also reports showing induction of IL-1β by IAPP 27, 28 , however these studies were performed before the identification of the inflammasome, and did not examine islets or primary macrophages and dendritic cells (DC), the latter two of which could be the main source of pathogenic IL-1β in the pancreas. This observation prompted us to evaluate the potential for IAPP to activate the Nlrp3 inflammasome through the mechanism of phagosomal destabilization [START_REF] Hornung | Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization[END_REF] . We show that amyloidogenic human IAPP, but not the nonamyloidogenic rat form of IAPP can trigger inflammasome activation and IL-1β production in lipopolysacharride-(LPS) primed macrophages or DC, and that this was dependent on Nlrp3. This then allowed us to investigate the effect of glyburide, a common treatment in T2D, which is known to inhibit the inflammasome [START_REF] Lamkanfi | Glyburide inhibits the Cryopyrin/Nalp3 inflammasome[END_REF] . We also observe that minimally oxidized LDL could prime for IL-1β processing, and that priming (signal 1) was dependent on glucose metabolism. Finally, we employed a mouse model where the human IAPP gene is expressed in islet beta cells, and found increased IL-1β expression that broadly co-localizes with amyloid and also macrophages. Our results therefore provide a possible mechanism for IL-1β activation in T2D.

Results

IAPP activates the inflammasome to produce mature IL-1β

Bone marrow-derived dendritic cells (BMDC) and bone marrow-derived macrophages (BMDM) are commonly used to investigate the inflammasome because of their high expression of pro-IL-1β following LPS priming. We compared the ability of IAPP to stimulate production of mature IL-1β from LPS primed BMDC with known inflammasome-activating agents like particulate adjuvants [START_REF] Sharp | Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome[END_REF] . Overnight stimulation with human IAPP was able to generate comparable amounts of IL-1β to these adjuvants, but rat IAPP which is unable to form amyloid did not stimulate IL-1β release (Fig. 1a). Human IAPP also stimulated the release of IL-1α which, like IL-1β, has been shown to rely on the activity of caspase-1 [START_REF] Keller | Active caspase-1 is a regulator of unconventional protein secretion[END_REF] , but could also represent paracrine induction of IL-1α by IL-1β. IL-1α in cell lysates was not decreased, suggesting that full length IL-1α is not released by cell lysis (Supplementary Figure 1a). TNF and IL-6 production (Fig. 1a) were undetectable without LPS stimulation, indicating that the IAPP preparations were not contaminated with LPS. The concentrations of TNF and IL-6 were not altered by the presence of IAPP, indicating that there was a specific effect on IL-1β production and not a general increase in pro-inflammatory cytokine production, and that there was no obvious cell death before these cytokines were produced.

Immunoblotting confirmed that the detected IL-1β was the cleaved 17.5 kDa fragment. Cleaved IL-1β was increased in the presence of IAPP and the inflammasome-activating agents uric acid crystals (MSU) and ATP (Fig. 1b).

Measurement of lactate dehydrogenase (LDH) in the supernatants was carried out along with the measurement of IL-1α and IL-1β; this confirmed that there was no obvious cytotoxicity under these conditions (Supplementary Figures 1a-d). Both DCs and macrophages are found in pancreatic islets, and these cells have been observed to phagocytose IAPP in T2D [START_REF] De Koning | Macrophages and pancreatic islet amyloidosis[END_REF] . Like BMDC, BMDM also produced a significant amount of IL-1β in response to IAPP. In comparison, the production of IL-1β by purified mouse pancreatic islets ex vivo and the rat beta cell line Rin-5F was very low, even when these cells were cultured under hyperglycemic conditions (Fig. 1c). These results show that amyloidogenic human IAPP can trigger processing of IL-1β from BMDM and BMDC.

IAPP activates caspase-1 and ASC speck formation

Caspase-1 is the protease activated in the inflammasome complex to cleave IL-1β and we measured active caspase-1 with a fluorescent cell-permeable probe that binds activated caspase-1 (FAM-YVAD-fmk). Flow cytometry indicated a large increase in the percentage of BMDC that contain active caspase-1 after being stimulated with IAPP for 1 hour (Fig. 1d). The mechanism by which caspase-1 is activated to produce mature IL-1β requires the apoptosis-associated speck-like protein containing a CARD (ASC), and its ability to form the multimeric inflammasome complex. Normally ASC is evenly distributed throughout the cell cytoplasm, but when the inflammasome complex becomes activated, ASC aggregates to a single foci within the cell, known as a speck. We used immortalized BMDM that stably express a fluorescent ASC protein to measure speck formation. LPS-treated cells that were activated with IAPP induced formation of an intense, single fluorescent speck in the cell, indicative of inflammasome activation (Fig. 1e, Supplementary Figure 1e). In addition to IL-1β processing, IAPP therefore also activates caspase-1 and the ASC inflammasome complex.

IAPP oligomers activate the Nlrp3 inflammasome

Having shown that an inflammasome complex containing caspase-1 and ASC is activated by IAPP to cleave IL-1β, we next examined which NLR protein nucleates this inflammasome complex. We elected to study Nlrp3, which is required for inflammasome activation by particles, rather than IPAF, Nlrp1, Nod1 or Nod2 which are not. Accordingly, for Nlrp3-deficient BMDC, mature IL-1β production was completely abrogated in response to IAPP (Fig. 2a). These cells still produced TNF and IL-6, attesting to the specificity of Nlrp3 deletion, and IL-1β mRNA expression was not decreased (Supplementary Figure 1f). To determine whether oligomers or IAPP fibrils activate the Nlrp3 inflammasome, we employed the organic solvent HFIP to dissolve amyloid fibrils. The HFIP was then removed using nitrogen gas and the IAPP reconstituted in H 2 O or phosphate buffered saline (PBS) and incubated at room temperature. Thioflavin T fluorescence was used to indicate fibrillar content. IAPP fibrils were formed over 24 h and this was accelerated when reconstituted in PBS compared to H 2 O (Fig. 2b, top panel). This increase in fibrillar content coincided with a decrease in IL-1β production from BMDC (Fig. 2b, lower panel). To further elucidate the active species, preparations of IAPP were subjected to size fractionation, generating samples with predominantly fibrillar (>100 kDa) or oligomeric (<100 kDa) species. We found that the IL-1β activating potential was predominantly retained in the oligomeric fraction <100 kDa from freshly reconstituted IAPP (Fig. 2c). Collectively this data suggests that the Nlrp3-activating constituent is a soluble oligomer of IAPP, rather than a highly fibrillar species of high molecular weight.

Inflammasome activation involves the phagolysosome

To confirm the role of caspase-1 and support the use of inhibitors to investigate mechanisms of inflammasome activation, a caspase-1 inhibitor was added to BMDC before IAPP activation. Caspase-1 inhibition reduced IL-1β production in a dosedependent manner (Fig. 2d). IAPP is thought to signal through a complex of receptor activity-modifying proteins with the calcitonin receptor, and the biological effects of IAPP can be blocked at this point using a specific peptide inhibitor (AC187) [START_REF] Hay | Pharmacological discrimination of calcitonin receptor: receptor activitymodifying protein complexes[END_REF] . To determine if recognition of IAPP by the receptor complex is required for inflammasome activation a titration of up to 40 μM AC187 (a 4 fold molar excess) was used, however this had no effect on IL-1β (Fig. 2d). Alternatively IAPP could be phagocytosed by BMDM and thereby activate the inflammasome in a manner similar to other species of amyloid [START_REF] Halle | The NALP3 inflammasome is involved in the innate immune response to amyloid-beta[END_REF] . Inhibition of IL-1β production following the addition of cytochalasin D confirmed that inflammasome activation was dependent on phagocytosis (Fig. 2d). Furthermore, inhibition of the vacuolar H + ATPase by bafilomycin A restricted the effect of IAPP on IL-1β secretion (Fig. 2d).

Taken together this suggests that phagolysosomal processes downstream of acidification are altered to trigger Nlrp3 activation.

IAPP inflammasome activation is prevented by glyburide

Two mechanisms have been proposed to account for pertubations in phagocytic processes that activate the inflammasome. Previously cathepsin B was implicated in this process for amyloid beta [START_REF] Halle | The NALP3 inflammasome is involved in the innate immune response to amyloid-beta[END_REF] while IAPP [START_REF] Zraika | Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis[END_REF] and MSU [START_REF] Martinon | Gout-associated uric acid crystals activate the NALP3 inflammasome[END_REF] , have been found to induce ROS. We used the ROS inhibitor (2R,4R)-4-Aminopyrrolidine-2,4-dicarboxylic acid (APDC) and a cathepsin B inhibitor (CA-074 Me) to confirm their role in IAPP activation of the inflammasome (Fig. 2e). Glyburide is a second generation sulfonylurea, an agonist of the potassium channel composed of SUR1 and KIR6.2 that signals for increased insulin secretion. Additionally, this small molecule agonist inhibits the inflammasome and IL-1β production, but this is independent of its interaction with the SUR1, KIR6.2 complex [START_REF] Lamkanfi | Glyburide inhibits the Cryopyrin/Nalp3 inflammasome[END_REF][START_REF] Hamon | Interleukin-1beta secretion is impaired by inhibitors of the Atp binding cassette transporter[END_REF] . We found that activation of the Nlrp3 inflammasome by IAPP was also inhibited by glyburide (Fig. 2e).

Txnip is not involved in IAPP activation of IL-1β

The thioredoxin interacting protein Txnip was recently identified as a redox sensitive ligand of Nlrp3 [START_REF] Zhou | Thioredoxininteracting protein links oxidative stress to inflammasome activation[END_REF] . This study suggested that an increased concentration of glucose alone could induce pancreatic beta cell Txnip to activate the inflammasome, and that glyburide could prevent this. However it was also shown that expression of Txnip in macrophages is constitutively high, so it was unlikely that this would be the mechanism of inflammasome inhibition by glyburide in macrophages. Nevertheless, we formally examined Txnip mRNA levels both before and after LPS priming in BMDM, and found no difference due to glyburide in either setting (Supplementary Figure 2a). Furthermore, LPS decreased expression of Txnip arguing against this protein as the instigator of Nlrp3 activation in BMDM. We then tested Txnipdeficient macrophages to see if this Nlrp3 ligand was required for IL-1β production in response to IAPP (Supplementary Figure 2b). We could find no difference in IL-1β secretion in response to IAPP, or indeed other inflammasome activators such as MSU or ATP in BMDM ex vivo. In addition we directly compared BMDM from mice lacking Txnip or Nlrp3 for IL-1β secretion and caspase-1 activation and found that these parameters were only decreased in the Nlrp3 -/-mice (Supplementary Figure 2c-f). Therefore our studies to date do not find a role for Txnip regulating the effect of glyburide on the inflammasome, or IL-1β production by the inflammasome.

Glucose metabolism is required for inflammasome priming

Elevated concentrations of D-glucose, similar to those encountered in T2D, can induce pro-IL-1β [START_REF] Boni-Schnetzler | Increased interleukin (IL)-1beta messenger ribonucleic acid expression in beta -cells of individuals with type 2 diabetes and regulation of IL-1beta in human islets by glucose and autostimulation[END_REF][START_REF] Maedler | Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets[END_REF] although this is debated [START_REF] Welsh | Is there a role for locally produced interleukin-1 in the deleterious effects of high glucose or the type 2 diabetes milieu to human pancreatic islets?[END_REF] . Growing BMDM in different concentrations of glucose influenced cell proliferation, to which BMDC were more resistant. We therefore tested BMDC in hyperglycemic conditions (25 mM Dglucose) for up to 24 h either before or after priming with LPS. We saw no significant difference in IL-1β production after IAPP stimulation (Fig. 3a). We also tested D-glucose analogs; one that is not metabolized by the cell and provides a similar osmotic environment to D-glucose (L-glucose), and another that actually inhibits the processing of D-glucose and subsequent metabolic processes in the cell (2-deoxy-D-glucose, 2DG). L-glucose had no effect (Fig. 3a), however we found that low concentrations of 2DG prevented IL-1β release due to IAPP in a dose dependent manner (Fig. 3b). 2DG had no effect when added after the addition of LPS, but only before the addition of IAPP (Fig. 3b), and we found that 2DG prevented IL-1β mRNA induction during LPS priming of the inflammasome (Fig. 3c). There was also a small decrease in the production of IL-6 at the highest concentration of 2DG (Fig. 3b), however the production of TNF and the viability of cells in general were not perturbed (Fig. 3b and Supplementary Fig. 3a). Sufficient glucose metabolism is therefore required in vitro during priming (signal 1) of the inflammasome.

mmLDL primes the inflammasome activated by IAPP

Our studies to this point showed that glucose metabolism was required for LPS to prime the inflammasome after which IAPP can activate Nlrp3, caspase-1 and ASC, leading to IL-1β processing. The question then arose as to what the endogenous priming agent of the Nlrp3 inflammasome might be i.e. a potential host factor as opposed to LPS. It has been reported that a particular form of LDL, minimally oxidized LDL (minimally modified LDL or mmLDL), can engage Toll-like receptor 4 (TLR4) in a manner similar to LPS, while extensively oxidized LDL does not [START_REF] Miller | Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells[END_REF] . Specifically, mmLDL can be elevated in individuals with T2D [START_REF] Apolinario | Minimally modified (electronegative) LDL-and Anti-LDLautoantibodies in diabetes mellitus and impaired glucose tolerance[END_REF][START_REF] Yano | Increased electronegative charge of serum low-density lipoprotein in patients with diabetes mellitus[END_REF] , and can cause apoptosis and a loss of insulin secretion by islets in culture [START_REF] Abderrahmani | Human high-density lipoprotein particles prevent activation of the JNK pathway induced by human oxidised low-density lipoprotein particles in pancreatic beta cells[END_REF] . We therefore tested preparations of LDL that had been oxidized to different extents. Although the level of IL-1β produced after IAPP activation of the inflammasome was low when first primed with mmLDL, it was clearly higher than priming with more extensively oxidized species of LDL, or LDL that was not oxidized (Fig. 4a). mmLDL recognition depended on TLR4, because C3H/HeJ mice which have a non-functional TLR4, were unable to prime the inflammasome via LPS or mmLDL. mmLDL also resulted in the dose-dependent production of TNF and IL-6 (Fig. 4b). Although LPS was not detected in mmLDL preparations, an LPS inhibitor, polymyxin B, inactivated mmLDL (Supplementary Figure 3b). While trace amounts of LPS might be specifically presented to TLR4 by mmLDL, it is also possible that polymyxin B neutralizes mmLDL in the same way as LPS. There are thought to be two prerequisites for inflammasome priming, the induction of pro-IL-1β, and Nlrp3 43 , however we observed that mmLDL only induced IL-1β mRNA, in a time-dependent manner (Fig. 4c). This lack of Nlrp3 upregulation might help explain the relatively low level of IL-1β activation when compared to LPS priming. Nevertheless these data suggest that mmLDL can promote IL-1β by providing signal 1 (priming) for the inflammasome, which may be particularly relevant in T2D.

IAPP induces IL-1β in vivo

As mouse IAPP is not amyloidogenic, mice that are transgenic for the expression of human IAPP have been generated and extensively characterized as a rodent model of islet amyloid formation [START_REF] Matveyenko | Islet amyloid polypeptide (IAPP) transgenic rodents as models for type 2 diabetes[END_REF] . In particular, when these mice are fed a high fat diet, amyloid deposition within islets of the pancreas occurs and is associated with the impaired function of insulin-producing cells [START_REF] Hull | Increased dietary fat promotes islet amyloid formation and beta-cell secretory dysfunction in a transgenic mouse model of islet amyloid[END_REF] . We compared wild-type mice (control) and IAPP transgenic mice, both fed on a high fat diet for one year (the length of time required in this model), and then determined whether insulin and IL-1β were present in pancreatic islets by immunofluoresence (Fig. 5a). Although variable, IAPP transgenic mice had marked regions within the islet that did not stain for insulin, and instead these regions displayed evidence of IL-1β immunoreactivity.

However the IL-1β staining co-localized to regions where amyloid was deposited, as determined by staining with thioflavin S (Fig. 5b). To identify a cell type in the islet that produces this IL-1β we stained with an antibody that is particularly useful for detecting peripheral tissue macrophages, MoMa2 (Fig. 5c). Most of the macrophages in the islet were positive for IL-1β, but there was still a large area staining for IL-1β outside these cells. When quantified, a statistically significant decrease in the area of insulin-producing cells was observed (Fig. 5d), with a concurrent increase in the amount of amyloid (Fig 5e), and the area of IL-1β expression (Fig. 5f) within pancreatic islets of IAPP transgenic mice. There was no significant difference in the area of MoMa2 reactivity within the islet (Fig. 5g). This mouse model highlights that the presence of IAPP amyloid promotes IL-1β in vivo.

Discussion

This work describes the mechanism whereby an endogenous molecule that is deposited in the pancreas during T2D can potentiate the processing of IL-1β, a pathogenic cytokine that causes beta cell death. We also saw release of IL-1α due to IAPP, which could augment sterile inflammation in pancreatic islets. Our observation that IAPP can activate the Nlrp3 inflammasome is consistent with similar activation from a different amyloidogenic peptide, amyloid beta [START_REF] Halle | The NALP3 inflammasome is involved in the innate immune response to amyloid-beta[END_REF] , and literature showing that the disease-causing species of IAPP might be oligomers [START_REF] Butler | Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formation rather than direct action of amyloid[END_REF] .

Macrophages and DC are professional phagocytic cells that take up IAPP in pancreatic islets, and we found that these cell types produce significant amounts of IL-1β in response to IAPP oligomers. Using multiple inhibitors we showed that pertubation of the phagolysosomal pathway seems to be involved in Nlrp3 activation by IAPP, however the role of ROS may be complex. Recently a number of papers have shown that patients deficient for ROS production still activate the inflammasome, to an even higher degree than healthy controls with normal ROS production [START_REF] Van De Veerdonk | Reactive oxygen species-independent activation of the IL-1{beta} inflammasome in cells from patients with chronic granulomatous disease[END_REF] . This suggests that the effect of the ROS inhibitor that we used might be non-specific, and indeed this is a caveat raised against the use of inhibitors in general. Although the effects of the inhibitors we used probably point towards a common mechanism of inflammasome activation during the phagocytosis of IAPP, their precise target in this pathway remains to be elucidated.

We found no evidence that the signaling cascade leading to Nlrp3 activation requires Txnip. Particularly with regard to T2D where Txnip is known to be a ROSsensitive molecule implicated in disease, we had hypothesized that IAPP-induced changes would utilize this pathway. While it is possible that our in vitro experiments have not fully recapitulated in vivo mechanisms of disease, they clearly show that Nlrp3 is a critical mediator of IL-1β release while Txnip is not. Certainly cell type differences might account for our observation since Txnip mRNA levels are not altered by glyburide in macrophages, while others report that glyburide might prevent Txnip induction in beta cells [START_REF] Zhou | Thioredoxininteracting protein links oxidative stress to inflammasome activation[END_REF] . Because IAPP can induce significant IL-1β production from macrophages and this is inhibited by glyburide while Txnip mRNA levels remain unchanged, a separate mechanism for this inhibition must exist, and is sure to attract interest in the future. Although we found that glyburide can inhibit IAPP-induced IL-1β in vitro at concentrations above 5 μg/ml, it is unlikely that the concentration of glyburide currently used clinically would reach this level. New small molecules that can target this inflammatory pathway in vivo will likely be attractive therapeutic candidates.

Identification of IAPP as a possible trigger for inflammasome activation in T2D still

leaves open the question as to which endogenous factors prime the inflammasome in this disease. High glucose alone was not able to replace LPS as the priming agent, or influence IL-1β mRNA levels. We found that mmLDL could prime the inflammasome through TLR4, however extensively oxidized LDL, which does not engage this receptor, could not. Potentially this is a mechanism by which mmLDL might be associated with T2D, and other IL-1β-dependent manifestations such as atherosclerosis [START_REF] Duewell | NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals[END_REF] . Recently, a further involvement of LDL in inflammasome activation was also described, where it facilitates cholesterol crystal deposition in the vessel wall [START_REF] Duewell | NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals[END_REF] . While the role of mmLDL in T2D has not been extensively characterized, FFAs are commonly thought of as potentially pathogenic in T2D.

Based on the literature, we would expect that FFAs could also prime the inflammasome by interaction with TLRs 49 .

Although increasing glucose concentration had no effect on IL-1β activation in our in vitro assays, we found that glucose metabolism is absolutely required for LPS to induce the expression of pro-IL-1β in macrophages. To inhibit glucose metabolism we used 2DG, which has also been found to reduce IL-1β in vivo, as for atherosclerosis-prone, insulin-resistant rats [START_REF] Russell | Increased insulin sensitivity and reduced micro and macro vascular disease induced by 2-deoxy-D-glucose during metabolic syndrome in obese JCR: LA-cp rats[END_REF] . Clearly the in vivo effects of IL-1β with regard to fever and inflammation are highly energy-dependent processes. Thus it would be appropriate for cells to prevent IL-1β production unless there is sufficient glucose that can be metabolized to mediate these biological responses. A small decrease in IL-6 production was observed, however it is likely that the factors controlling IL-1β synthesis that are inhibited by 2DG are also shared in some part by other pro-inflammatory cytokines. Because we have found glucose metabolism to be a limiting step in inflammasome priming, this could be a physiologically relevant mechanism in the pathogenesis of T2D where persistent hyperglycemia might abolish any such limits. Indeed the beta cell loss in high fat fed IAPP transgenic mice is greatly increased when they develop amyloid deposition [START_REF] Hull | Increased dietary fat promotes islet amyloid formation and beta-cell secretory dysfunction in a transgenic mouse model of islet amyloid[END_REF] , and we now report that this is associated with a significant increase in IL-1β immunostaining. The IL-1β observed in islets of IAPP transgenic mice co-localized with macrophages, and also with amyloid. We could find no clear evidence of beta cells producing IL-1β, and if they do it is likely to be at a low level, or a rare event. While IL-1β staining in macrophages might represent intracellular pro-IL-1β, staining outside these cells that is coincident with amyloid could represent the secreted, processed form, as there are few live cells in these regions. Our analysis is somewhat limited in this respect, as the antibody we used to detect IL-1β would recognize both the processed and pro-IL-1β species.

Our data is unambiguous as to the ability of IAPP to activate the Nlrp3 inflammasome and generate processed IL-1β. This might be a key mechanism for IL-1β production in T2D. It could be that amyloid deposition in the pancreas only happens later in the disease process, and some have argued that this would be as a result, and not as a cause of disease. However it is also possible that even if this buildup is secondary to the initiation of disease, that it nevertheless aids and abets in the progressive loss of beta cell function that is a key component of T2D. Future studies on patients should identify whether targeting IL-1 has most benefit only later in the disease progression or for those with more severe disease pathology. It is also becoming apparent that IAPP could contribute to the death of transplanted islets being used as therapy in T1D [START_REF] Udayasankar | Amyloid formation results in recurrence of hyperglycaemia following transplantation of human IAPP transgenic mouse islets[END_REF][START_REF] Westermark | Rapid deposition of amyloid in human islets transplanted into nude mice[END_REF][START_REF] Westermark | Widespread amyloid deposition in transplanted human pancreatic islets[END_REF] . The current Edmonton protocol of immunosuppression used during this transplant procedure could conceivably be improved with anti-inflammatory therapy targeting IL-1β activated by IAPP. To conclude, our findings highlight new roles for well-known entities in the pathogenesis and treatment of diabetes and will hopefully spur new research targeted at the mechanisms of this debilitating, chronic disease.

Materials and Methods

Cell culture

Bone marrow from C57BL/6 mice was differentiated for 10 days in GM-CSF (4% J588 myeloma cell supernatant) or 7 days in M-CSF (20% L929 cell supernatant) in typical media preparations to make bone marrow derived dendritic cells (BMDC) and macrophages (BMDM) respectively Nlrp3 KO mice were from Jurg Tschopp (University of Lausanne) or Millennium Pharmaceuticals. Pancreatic islet cells were obtained from collagenase digested mouse pancreatic islets and cultured overnight [START_REF] Michalska | The effects of pharmacologic inhibition of NADPH oxidase or iNOS on pro-inflammatory cytokine, palmitic acid or H2O2 -induced mouse islet or clonal pancreatic beta cell dysfunction[END_REF] . The Rin-5F cell line was from ECACC and cultured according to their protocol.

Materials

Unless stated, cells were primed for 3 hrs with 100 ng/ml LPS (Alexis) or 10 μg/ml human plasma LDL oxidized with copper(II) sulphate for different times (Kalen biomedical), then activated with 10 μM human IAPP (Sigma), 10 μM rat IAPP (Bachem), 20 μg/ml MSU (Opsona), 100 μg/ml Alum (Brenntag biosector) or 0.6 mg/ml 430 nm polystyrene beads (Corpuscular) for 16 hours. The following inhibitors or glucose analogs were added 2 hours after LPS priming, 1 hour before inflammasome activation at the highest concentration indicated, unless stated; 1 μg/ml Caspase-1 inhibitor VI (Calbiochem), 5 μM cytochalasin D, 250 μM bafilomycin A, 10 μM cathepsin B inhibitor (CA-074 Me), 1 μg/ml (2R,4R)-4-Aminopyrrolidine-2,4-dicarboxylic acid (APDC), 50 μM N-acetyl-cysteine (NAC), 50 μM glyburide, 25 mM D-glucose and its analogs (all from Sigma), and 40 μΜ IAPP receptor inhibitor peptide AC187 (Tocris biosciences). 100 μg/ml Polymyxin B (Sigma) was also added to 1 μg/ml LPS or 100 μg/ml mmLDL overnight at 4 o C before use in some experiments with a 1/10 dilution.

Measurement of caspase-1 activity

BMDC were stimulated with IAPP for 1 hour before the addition of FAM-YVAD-fmk according to manufacturer's instructions (Immunochemistry Technologies).

Fluorescent cells were measured by flow cytometry.

Analysis of ASC speck formation

Immortalized BMDM expressing YFP-ASC were generated as described previously [START_REF] Hornung | Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization[END_REF] . These cells were primed with LPS then activated with IAPP overnight and imaged using a fluorescent microscope with a 40x objective lens.

Immunoblotting

To blot for IL-1β in supernatants, BMDM grown in 24 well plates in 0.5 ml media containing 1% FCS were stimulated, supernatants collected and precipitated with 1 volume methanol, 1/4 volume chloroform, then the precipitate was washed in 1 volume methanol and finally resuspended in 60 μl SDS loading buffer for running on 15% Tris-glycine polyacrylamide gels. Proteins were transferred to PVDF membranes, blocked in 5% skimmed milk then probed with goat anti-mIL-1β (RnD systems, AF-401) and detected with HRP-donkey anti-goat antibody (Upstate).

Alternatively, cell lysates were prepared by lysis in RIPA buffer, followed by electrophoresis and blotting as above, then probed with goat anti-IL-1α (RnD systems, AF-400), rabbit anti-ASC (Alexis), mouse anti-Txnip (MBL international) or mouse anti-caspase-1 (developed in-house) followed by a suitable detection antibody. Immunoblots were quantified using ImageJ software.

Cytokine measurements

For cytokine measurements, BMDM or BMDC were cultured in 96 well plates, stimulated in triplicate, supernatants were then collected and ELISA performed for mIL-1β, mIL-1α, mIL-6 and mTNF (RnD systems).

Q-PCR

BMDM were cultured in 24 well plates, stimulated in triplicate, RNA prepared using the RNeasy kit (Qiagen) and then Q-PCR performed using Taqman probes specific for IL-1β (Mm01336189_m1), Nlrp3 (Mm00840904_m1) or Txnip (Mm00452393_m1).

Cytotoxicity assay

Cytotoxicity was determined by an LDH assay (Promega) according to manufacturer's instructions.

Thioflavin T fluorescence and size fractionation

IAPP was dissolved in HFIP (Sigma) which was subsequently removed using nitrogen gas. Aliquots were then resuspended at 500 μM in H 2 0 or PBS for different times. This was diluted to 10 μM in RPMI and thioflavin T added to a concentration of 20 μM after which fluorescence measurements were recorded according to protocol [START_REF] Nilsson | Techniques to study amyloid fibril formation in vitro[END_REF] using a FP6200 fluorescence spectrometer (JASCO). To separate fractions based on size, resuspended IAPP was diluted to 20 μM in RPMI and quickly filtered through a 100 kDa cut-off membrance (Amicon). The concentrated fraction greater than 100 kDa was supplemented to the original volume with RPMI and both fractions were diluted 1/2 onto BMDC for a final IAPP concentration of 10 μM.

Immunofluorescent analysis of sections from IAPP transgenic mice

IAPP transgenic mice were fed a high fat diet for one year then the pancreas was sectioned as described [START_REF] Hull | Increased dietary fat promotes islet amyloid formation and beta-cell secretory dysfunction in a transgenic mouse model of islet amyloid[END_REF] . Immunofluorescent staining was performed using thioflavin S (Sigma), mouse anti-insulin (Sigma, I2018), rabbit anti-IL-1β (Santa Cruz Biotechnology, sc-7884) and specific fluorescently-conjugated secondary antibodies (Molecular Probes), or Alexa647 conjugated anti-MoMa2 (AdbSerotech).

Analysis of images performed using ImageJ software.

Statistical analysis

Data is presented as mean ± SD. Significance was determined by two-tailed unpaired t-test with the exception of insulin staining in pancreatic islets, for which a onetailed unpaired t-test was used as the direction of the outcome can be anticipated based on similar experiments we have published previously.

Figure 1 .

 1 Figure 1. Inflammasome activation and IL-1β production induced by human IAPP. (a) IAPP from different species or polystyrene beads (adjuvant) were incubated overnight with BMDC with and without 3 hours priming using LPS. Supernatants were then analyzed for IL-1β, IL-1α, IL-6 and TNF by ELISA. (b) Immunoblotting for IL-1β was performed using supernatants from BMDC primed with LPS, then activated with IAPP, MSU or Alum for 6 hours. (c) IL-1β secretion from BMDM and BMDC compared to purified mouse pancreatic islets or Rin-5F cells (a pancreatic beta cell line), stimulated with LPS and IAPP. Rin-5F cells were also cultured for different times with elevated glucose (25 mM D-glucose). (d) Flow cytometric analysis of BMDC stimulated with IAPP for 1 hour then treated with FAM-YVAD-fmk which covalently binds to active caspase-1 and fluoresces. Control is shaded under the curve, IAPP stimulated is unshaded. (e) Immortalized BMDM expressing YFP-ASC were primed with LPS, then activated with IAPP overnight and imaged with a fluorescent microscope (40x objective). Arrows indicates speck formation. Means ± SD, * p<0.01, ** p<0.001. All data representative of three independent experiments.

Figure 2 .

 2 Figure 2. IAPP oligomers activate the Nlrp3 inflammasome which is prevented by glyburide, and inhibitors targeting phagocytosis, ROS and Cathepsin B. (a) WT or Nlrp3 deficient (Nlrp3 -/-) BMDM were primed with LPS and then activated

Figure 3 .

 3 Figure 3. Priming the inflammasome requires glucose metabolism. (a) BMDC were treated with 25 mM D-or L-glucose for different periods of time prior to LPS priming and inflammasome activation with IAPP, after which IL-1β production was measured. (b) BMDC were treated with increased amounts of 2-deoxy-D-glucose

Figure 4 .

 4 Figure 4. Minimally oxidized LDL can prime for inflammasome activation by IAPP. (a) IAPP activation of the inflammasome after 3 hrs priming with LPS, human plasma LDL, or LDL oxidized to different extents. LDL oxidation is quantified by relative electrophoretic mobility (REM). BMDM from C3H/HeN mice were compared to those from C3H/HeJ mice that do not have functional TLR4. (b) Dose response for minimally oxidized LDL (mmLDL) priming of IAPP inflammasome activation and IL-1β production. IL-6 and TNF are also produced in a dosedependent fashion. (c) IL-1β and Nlrp3 mRNA levels were measured after priming with mmLDL for different times. Means ± SD, * p<0.05, ** p<0.01, *** p<0.001. All data representative of three independent experiments.

Figure 5 .

 5 Figure 5. Increased IL-1β expression in islets of mice transgenic for human IAPP. (a) Immunofluorescent analysis (20x objective) of sections from the pancreas of wild-type (WT) and IAPP transgenic (IAPP-TG) mice, both fed a high fat diet for one year. Sections were stained for insulin (red) and IL-1β (green). (b) Pancreatic

Acknowledgements

The authors thank Andes Mori for assistance with Nlrp3 -/-mice, Prof. Jurg Tschopp for providing Nlrp3 -/-mice, and Prof. Eicke Latz for providing YFP-ASC BMDM. S.L.M. was supported by an NHMRC Overseas Biomedical Fellowship (516783). Work performed at Trinity College Dublin was supported by Science Foundation Ireland and at VA Puget Sound Health Care System by the United States Department of Veterans Affairs and National Institutes of Health grant DK-75998 (to S.E.K.). L.F. was supported by a fellowship from the Crohn's and Colitis Foundation. Work performed at the University of Michigan was supported by National Institutes of Health grant AI063331.

The authors declare no competing financial interests. 442


Cassie F Pope

Phillip Hay

Sarah Alexander

Kay Capaldi

Jayshree Dave

S Tariq Sadiq

Catherine A Ison

Dr Timothy Planche
email: tplanche@sgul.ac.uk

S Tariq Sadiq

Positive Predictive Value of the Becton Dickinson VIPER system and the ProbeTec GC

Keywords: Neisseria gonorrhoeae, laboratory diagnosis, NAAT

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

Introduction

Testing of asymptomatic, sexually active people for Chlamydia trachomatis and Neisseria gonorrhoeae infection is widespread in Europe and North America. In most settings, gonorrhoea is less common than Chlamydia infection: in 2008 there were 209,000 new diagnoses of Chlamydia in the UK compared to 16,629 new diagnoses of uncomplicated gonorrhoea. [START_REF]STI annual data tables[END_REF] The BD VIPER system (Becton Dickinson, New Jersey, USA) is an automated sample processor and fluorescent reader using strand displacement amplification (SDA) for qualitative detection of N. gonorrhoeae and C. trachomatis DNA. The previous BD assay was the ProbeTec ET GC assay, utilising similar technology but comprising a manual extraction via heating and lysis and a semi automated SDA procedure. The newer methodology has a fully automated extracted mode, which uses alkaline lysis of cells and binding of DNA to ferric oxide (FOX) to extract N. gonorrhoeae DNA and includes an extraction control to monitor for failed extraction and PCR inhibition.

The ProbeTec GC Q x and ET GC assays target different regions of the N. gonorrhoeae pilin inverting protein homologue gene so there may be differences in the performance characteristics of these assays. The manufacturer claims sensitivity of 99.3% and specificity of 99.4% for genital samples.

Alternative commercial assays for the detection of N. gonorrhoeae are the Gen-Probe APTIMA Combo 2 assay on the Tigris platform (Gen-Probe, Inc., San Diego, USA), the ROCHE AMPLICOR CT/NG assay (Roche, Branchburg, NJ, USA) and the Abbott RealTime CT/NG assay on the m2000 platform (Abbott Molecular Diagnostics, Illinois, USA). Each of these assays detect different targets. The APTIMA Combo 2 assay detects N. gonorrhoeae 16S rRNA using transcription mediated amplification and the m2000 CT/NG assay detects the N. gonorrhoeae Opa gene using real time PCR.

For Chlamydia NAAT assays repeat testing using the same assay is considered adequate as this reduces inaccurate results due to processing errors but also reduces sensitivity in samples with a low pathogen load. [START_REF] Skidmore | Testing specimens for Chlamydia trachomatis[END_REF] There have been reports of a relatively low specificity of the older ProbeTec ET GC assay for N. gonorrhoeae and the Roche COBAS AMPLICOR leading to unacceptably low positive predictive values (PPV) in a low prevalence population, therefore, repeat testing on the same platform is not considered adequate for GC due to cross reaction with commensal Neisseria spp. 3 4 The Health Protection Agency and the Centers for Disease Control and Prevention recommend that for N. gonorrhoeae confirmatory testing, a different target should be considered if the positive predictive value (PPV) of an assay is < 90% to improve PPV. [START_REF]Detection of Neisseria gonorrhoeae using molecular methods[END_REF][START_REF]Guidance for gonorrhoea testing in England and Wales[END_REF][START_REF] Moncada | Evaluation of the CDC recommended approaches for confirmatory testing of positive Neisseria gonorrhoeae nucleic acid amplification tests[END_REF] Our aim was to ascertain the PPV of the BD ProbeTec GC Q x assay in our genito urinary medicine (GUM) clinic attendee population for genital and extragenital sites.

METHODS

Traditionally diagnostic assessments are made by comparing the test to a reference assay for all samples. This would represent an expensive and time consuming approach. As this was a service evaluation, we used a methodology similar to that reported by Golden et al who determined the PPV of a NAAT for N. gonorrhoeae by confirming only the samples testing as positive in a larger number of specimens without performing reference tests in all samples. [START_REF] Golden | Positive predictive value of Gen Probe APTIMA Combo 2 testing for Neisseria gonorrhoeae[END_REF] There is currently no clear consensus on standards for evaluations of NAATs. Choice of 'gold standard' becomes complicated because NAATs are likely to be more sensitive than culture based methods and genuine positives may not be recognised. We calculated PPV by comparing screening NAAT results to a composite 'gold standard' consisting of bacterial culture and two confirmatory real-time PCR results.

Specimen Collection

Between 23 rd June 2009 and 15 th January 2010, patients attending the Courtyard (GUM) clinic at St George's NHS Healthcare Trust were tested for infection with N. gonorrhoeae and C. trachomatis using the ProbeTec CT/GC Q x assay, automated on the VIPER platform as per the manufacturer's instructions.

Consecutive routine specimens of urine samples, endocervical, urethral, rectal and pharyngeal wet swabs were included in this service evaluation as outlined below. All male patients were tested with a first void urine for Chlamydia/gonorrhoea NAAT. In addition, clinically indicated swabs for gonococcal culture and the ProbeTec GC Q x assay were taken from urethral, rectal and pharyngeal sites. Male patients giving a history of dysuria or urethral discharge had a urethral swab collected for Gram stain and culture for N.

gonorrhoeae. Men who have sex with men had culture and NAAT, performed on rectal and pharyngeal swabs based on history of anal and/or oral sex. Three swabs were taken simultaneously from the throat by sticking them together with tape and rotating them. One swab was for N. gonorrhoeae culture, one for BD ProbeTec GC Q x and one for BD ProbeTec ET GC (used for confirmatory testing at STBRL). Rectal swabs were collected without a speculum unless rectal symptoms were present. The culture swab was taken first, followed by the GC Q x and the ET GC swab. Urethral swabs were taken from female patients for gonorrhoea culture. After inserting a speculum endocervical swabs were collected for culture and BD ProbeTec GC Q x in that order.

Testing by ProbeTec GC Q x

Samples positive for N. gonorrhoeae in the initial ProbeTec GC Q x assay were retested using the same sample and a fresh extraction using the same assay. A NAAT was considered positive if positive on the repeat run. Samples that did not repeat as positive were considered equivocal as this may have resulted from a processing error and a repeat sample was requested, these samples were not used in the analysis of PPV. The confirmatory testing method is shown in figure 1.

Confirmation by real-time PCR

All samples testing positive for N. gonorrhoeae by the ProbeTec GC Q x assay were forwarded to the Sexually Transmitted Bacteria Reference Laboratory (STBRL) were they were examined blind using two N. gonorrhoeae specific real-time PCRs. Some negative samples were also sent.

Upon receipt, 200µl of ProbeTec ET GC transport buffer was extracted using the MagNAPure Compact (Roche Diagnostics, Indianapolis, USA) and examined using a modified version of a previously published N. gonorrhoeae real-time PCR assay which targets the porA pseudogene. [START_REF] Whiley | Comparison of three in-house multiplex PCR assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis using real-time and conventional detection methodologies[END_REF] Briefly, PCR was performed using 25 μl reactions containing 5μl of extracted DNA, 4mM MgCl 2 , 1U Taq DNA polymerase (Applied Biosystems, California, USA), 250nM of forward and reverse primers and 500nm of probe (Table 1). Also included within this PCR were 80mM of primers and probe of a previously described internal control which targets the Human RNase gene, to monitor for both inhibition and specimen quality. [START_REF] Chen | The molecular diagnosis of lymphogranuloma venereum: evaluation of a real-time multiplex polymerase chain reaction test using rectal and urethral specimens[END_REF] PCR was performed using a Rotor-Gene 6000™ (Qiagen, Hilden, Germany) using cycling conditions of: 10mins at 95°C, followed by 40 cycles of 95°C for 30 seconds and 60°C for 60 seconds. Specimens negative using this assay were re-examined using a further assay which targets the Opa gene. [START_REF] Tabrizi | Evaluation of opa-Based Real-Time PCR for Detection of Neisseria gonorrhoeae[END_REF] PCR was performed using 20μl reactions containing 5μl of extracted DNA, 5mM MgCl 2 , 1U Taq DNA polymerase (Applied Biosystems) and 250 nM of forward and reverse primers and 500nM of probe (Table 1). PCR samples were run on a Rotor-Gene 6000™ using cycling conditions of: 10mins at 95°C, followed by 50 cycles of 95°C for 15 seconds and 60°C for 60 seconds.

Analysis of test performance

A sample was treated as a true positive if positive by either culture or by confirmatory

PorA or Opa real time PCR. Stata 8.0 (College Stn. Tx, USA) was used to determine PPV and 95% confidence intervals. A limitation of this methodology is that sensitivity and specificity were not determined because negative samples were not repeated by confirmatory NAAT and were not compared to culture result. However we may estimate the specificity if we assume a value for the sensitivity of the assay, based on the following relationship [START_REF] Altman | Statistic Notes: Diagnostic tests 2: positive predictive values[END_REF] :- 1]

PPV = sensitivity ( )× prevalence ( ) sensitivity ( )× prevalence ( ) ( ) + 1 -specificity ( )× 1 -prevalence ( ) ( ) [Equation

RESULTS

PPV and prevalence for genital samples

In total 14,223 genital specimens (5851 male urine samples, 8075 cervical and 297 male urethral swabs) were tested. Two samples that tested as positive in the initial ProbeTec GC Q x assay did not repeat as positive on the repeat ProbeTec GC Q x run. These two samples were reported as equivocal and excluded from the analysis. Of these 14, 221 samples, 150

(1.1%) samples from 149 patients tested positive for N. gonorrhoeae using the ProbeTec GC Q x assay. Of these positive samples, 141/149 (94.7%) were confirmed by either culture or HPA confirmatory PCR [START_REF] Whiley | Comparison of three in-house multiplex PCR assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis using real-time and conventional detection methodologies[END_REF][START_REF] Chen | The molecular diagnosis of lymphogranuloma venereum: evaluation of a real-time multiplex polymerase chain reaction test using rectal and urethral specimens[END_REF][START_REF] Tabrizi | Evaluation of opa-Based Real-Time PCR for Detection of Neisseria gonorrhoeae[END_REF] giving a PPV of 94.6% (95% CI, 90%-98%) (Table 2). For the remaining eight patients with unconfirmed positive genital specimens, three patients had a confirmed N. gonorrhoeae infection at another site, suggesting that these are genuine positive samples; raising the PPV of genital samples to 96.7% (95% CI, 93% -100%). Of the confirmed positive genital samples four (2.7%) were culture positive but not detected by confirmatory PCR.

Based on the relationship described in equation 1, if we assume a sensitivity of 99.3% then the estimated specificity will be 99.96%. Even if we assume a much lower sensitivity e.g. 50% the estimated specificity remains at 99.96%.

PPV and prevalence for extragenital samples

In total 593 rectal and 840 pharyngeal swab samples were tested. Of these specimens 17/593 (2.87%) rectal swabs and 26/840 (3.10%) pharyngeal swabs tested positive for N.

gonorrhoeae, using the ProbeTec GC Q x assay. Of these positive samples, all 17 rectal swabs were confirmed positive giving a PPV of 100% (95% CI, 80%-100%). Of the 26 pharyngeal samples 13 samples were confirmed by either GC culture or in house HPA confirmatory PCR with a PPV of 50% (95% CI, 30%-70%) (Table 2). The number of samples testing as positive by the ProbeTec GC Q x assay, confirming by NAAT and culture are shown in Table 2. For positive results, which were unconfirmed, two of the thirteen pharyngeal samples had one or more samples confirmed positive for N. gonorrhoeae from another site and these were therefore considered to be likely genuine positive samples. If these samples were considered true positives the PPV would be 57.7% (95% CI 39% -77%) for pharyngeal samples. Of the confirmed positive samples one (3.8%) pharyngeal swab and one (5.9%) rectal swab were culture positive but not detected by confirmatory NAAT.

Nine samples were confirmed by NAAT but were not confirmed by culture. Four of the unconfirmed pharyngeal samples grew Neisseria meningitidis upon culture. All extragenital samples were positive upon retesting via ProbeTec GC Q x .

DISCUSSION

The ProbeTec GC Q x assay is FDA licensed and CE marked for use with urethral, endocervical and self collected vulvo-vaginal swabs plus male and female urine samples. This assay is not licensed for use on pharyngeal, rectal or eye swabs. For genital samples we found a PPV of 94.6% (95% CI, 90%-98%) in our clinic population, with a confirmed N. gonorrhoeae rate of 1.0%. As the lower CI is 90%, it is reasonable to use the ProbeTec GC Q x assay without routine confirmatory testing in this setting. Current national guidelines state that confirmatory GC NAATs are required if the PPV is less than 90% and confirmatory testing is unnecessary for genital specimens in our GUM population. 5 6 However, in populations where the prevalence of N. gonorrhoeae is low e.g. primary healthcare settings, the PPV may be lower and confirmatory testing by a second NAAT, with a second genetic target, may be required.

Our routine practice is to retest all NAAT positive samples on the same platform, although this is not explicitly recommended by the manufacturer. This is to reduce inaccurate results due to sample processing errors. This practice of repeat testing of positive samples on the same platform is not universally practiced but there were only two specimens, neither extra-genital, that did not retest as positive. The inclusion of this small number of specimens would not alter these findings.

During the evaluation period no culture positive samples were ProbeTec GC Q x negative from the same site. This supports the manufacturer's claim that the assay has a high sensitivity of greater than 99.5%.

Akduman et al found that the ProbeTec ET GC assay had a PPV of 84.9% for testing of urine samples. [START_REF] Akduman | Evaluation of a strand displacement amplification assay (BD ProbeTec-SDA) for detection of Neisseria gonorrhoeae in urine specimens[END_REF] In a large multicentre study the PPV of the assay for female urine samples varied from 54.8% to 100%. The site reporting the lowest PPV of 54.8% had a N.

gonorrhoeae prevalence of 1.2%, which is similar to our reported prevalence. [START_REF] Van Der Pol | Multicenter evaluation of the BD ProbeTec ET system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male urethral swabs[END_REF] The PPV reported here demonstrates that the ProbeTec GC assay, in extracted mode, has a higher PPV than the ProbeTec ET GC assay and is likely to have a higher specificity than that reported of the ProbeTec ET GC assay.

There are concerns regarding the use of NAATs for the detection of N. gonorrhoeae from rectal and pharyngeal samples, due to lower specificity. Although NAATs are the most sensitive assays available, [START_REF] Ota | Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in pharyngeal and rectal specimens using the BD ProbeTec ET system, the Gen-Probe Aptima Combo 2 assay and culture[END_REF][START_REF] Schachter | Nucleic acid amplification tests in the diagnosis of chlamydial and gonococcal infections of the oropharynx and rectum in men who have sex with men[END_REF][START_REF] Alexander | The challenges of detecting gonorrhoea and Chlamydia in rectal and pharyngeal sites: could we, should we, be doing more?[END_REF] the risk of false positive results is high due to the genetic similarity between N. gonorrhoeae and commensal Neisseria spp. leading to cross reactions. Optimal methods for detection of N. gonorrhoeae infection at extragenital sites are uncertain and no commercial NAAT is licensed for detection of N. gonorrhoeae from extragenital sites. Here, we found that our NAAT assay identified more pharyngeal GC infections than culture in keeping with previous studies. [START_REF] Moncada | Evaluation of self-collected glans and rectal swabs from men who have sex with men for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by use of nucleic acid amplification tests[END_REF][START_REF] Whiley | Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge[END_REF][START_REF] Page-Shafer | Increased sensitivity of DNA amplification testing for detection of pharyngeal gonorrhoea in men who have sex with men[END_REF] Indeed NAATs are redefining the epidemiology of gonococcal infection. Out of 26 pharyngeal samples initially testing as positive 13 (50%) were not confirmed either by culture or by confirmatory NAAT. Two possible explanations are possible; the first is that these samples had a low inoculum of N.

gonorrhoeae. The second is that cross reactivity resulted in an increased number of false positive ProbeTec GC Q x results. The ProbeTec ET GC and Roche COBAS AMPLICOR CT/NG assays are known to cross react with strains of N. subflava and N. cinerea resulting in false positive results 21 22 and these species may be present in the oropharynx as commensal bacteria in up to a third of individuals. [START_REF] Knapp | Prevalence and persistence of Neisseria cinerea and other Neisseria spp. in adults[END_REF] Such cross reactivity has not yet been reported with the APTIMA Combo 2 test. [START_REF] Nagasawa | Evaluation of APTIMA Combo 2 for cross-reactivity with oropharyngeal Neisseria species and other microorganisms[END_REF] Specificities of the APTIMA Combo 2 and ProbeTec ET GC for oropharyngeal swabs have been found to be similar, and have been reported to be greater than 99.4%. [START_REF] Schachter | Nucleic acid amplification tests in the diagnosis of chlamydial and gonococcal infections of the oropharynx and rectum in men who have sex with men[END_REF] However Ota et al determined the PPV of the ProbeTec GC ET assay to be only 82.6% for pharyngeal swabs. [START_REF] Ota | Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in pharyngeal and rectal specimens using the BD ProbeTec ET system, the Gen-Probe Aptima Combo 2 assay and culture[END_REF] It is likely that the low PPV determined here reflects lower specificity of this assay.

A further limitation may be the order in which swabs were taken. For rectal and endocervical swabs culture swabs were taken before swabs for NAAT testing and therefore this including DNA degradation, as the confirmatory testing was performed off site, and therefore specimens incurred a period of transportation delay before testing.

It is possible, that some culture negative samples testing as positive in the ProbeTec GC Q x assay may not have been confirmed even if genuinely positive. Choice of confirmatory assay is important as this should detect a different target to the screening NAAT and have good sensitivity and specificity. These could include real time PCR assays available from reference laboratories or developed in house e.g. PorA/Opa PCR or reciprocal arrangements with a laboratory with a different platform. [START_REF] Hardwick | Confirmation of BD ProbeTec Neisseria gonorrhoeae reactive samples by Gen-Probe APTIMA assays and culture[END_REF] In summary, we have demonstrated that the ProbeTec GC Q x assay can achieve a high PPV when testing genital specimens in our GUM clinic population and therefore in accordance with national guidelines confirmatory testing is unnecessary. However testing with a second supplementary NAAT assay may still be desirable in lower prevalence populations.

'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'.

Culture

  VCAT (containing vancomycin, colistin, amphotericin and trimethoprim) selective culture plates (Biomerieux, Marcy l'Etoile, France) were inoculated in the GUM clinic and incubated (48 hours, CO 2 ). Presumptive colonies were Gram stained, oxidase tested and subcultured onto a purity plate. N. gonorrhoeae was confirmed by MicroTrak N. gonorrhoeae culture confirmation fluorescent antibody test (Trinity Biotech Plc, Co. Wicklow, Ireland) and API NH (BioMerieux, Marcy l'Etoile, France). Corresponding culture plates for all ProbeTec GC Q x extragenital samples testing as positive were incubated for a total of 72 hours to maximise growth of Neisseria spp.

1 Figure 1

 11 Figure 1 Outline of confirmatory testing protocol

Table 1

 1 Primer and Probe nucleic acid sequences for PorA and Opa real time PCR assays

	Specimen	No. of	No. (%)	No (%)
	Type	specimens	of
		tested	positive
			VIPER
			GC Q x
			Results

and result of ProbeTec Q x specimens tested using confirmatory NAAT and culture

				Pos by	Neg	Pos by	Neg by	Confirmed	Neg by both
				NAAT	by	Culture	Culture	Pos by	culture and
					NAAT			culture	confirmatory
								and/or	NAAT
								NAAT
	Genital	14,223	149(1.0) 134 a	15	131	18	141	8
				(89.3)	(10)	(87.9)	(12.0)
	Pharyngeal	840	26(3.1)	13	13	5 (19.2)	21	13	13
				(50)	(50)		(80.8)
	Rectal	593	17	9 a	1 (5.9) 8 (47.1)	5 (29.4)	17	0
			(2.87)	(52.9)

Table 2

 2 Numbers of ProbeTec GC Q x positive samples confirming by supplementary NAAT and culture for different sample types.

a One sample was inhibitory by confirmatory NAAT but positive by culture.

Acknowledgements

The authors would like to thank the staff at the Departments of Medical Microbiology and GUM at St Georges Hospital, London, and at STBRL, CFI, Colindale. In particular we would like to thank Danuta Dubeck, Pauline Greene, John Hadfield, Helen Liddy and Sima Patel for assistance.

the NAAT assay may have been disadvantaged and may result in an underestimate of PPV.

The pharyngeal swabs were collected for all three samples simultaneously, by taping them together, as we considered it unpleasant for patients to undergo three successive samplings.

Whilst practitioners rotated the triple swab, there may well have been sampling differences.

We found a PPV of 100% (95% CI 80-100%) for rectal swabs, out of 17 samples testing as positive all were confirmed by either culture or the second NAAT assay. However this is too small a number to give sufficient confidence in this estimate and larger numbers are required to confirm that the lower CI is greater than 90%. Others have also demonstrated the high PPV of rectal swabs for detection of N. gonorrhoeae but sample sizes were also small. 15 16 In compliance with national guidelines we recommend that extragenital samples testing positive by NAAT should be confirmed. Samples testing as positive which are not confirmed should be reported as equivocal and a repeat sample requested. [START_REF]Detection of Neisseria gonorrhoeae using molecular methods[END_REF][START_REF]Guidance for gonorrhoea testing in England and Wales[END_REF][START_REF] Moncada | Evaluation of the CDC recommended approaches for confirmatory testing of positive Neisseria gonorrhoeae nucleic acid amplification tests[END_REF] Discrepant analysis of samples which are reference standard negative will increase the PPV. As this was a service evaluation, partner status was not included: the result is that the PPV reported here may be an underestimate of the true value. However for unconfirmed ProbeTec GC Q x positive samples a partial discrepant analysis was performed, samples testing as positive in the ProbeTec GC Q x assay were considered a true positive if confirmed positive results were available from one or more alternative sites. We used N. gonorrhoeae specific real-time PCRs to act as confirmatory assays.

Interestingly, six specimens which were both ProbeTec GC Q x and culture positive, were not confirmed by the STBRL real-time PCR assays. There maybe numerous reasons for

Key Messages

Testing of genital specimens by the ProbeTec GC Q x assay does not require confirmatory testing in our GUM clinic population.

Confirmatory testing of pharyngeal swabs, testing as positive, should continue as recommended by national guidelines.

The PPV for rectal swabs looks promising but larger numbers are required to confirm that the lower CI is >90%.

Declaration of Interest

We acknowledge the support of Becton-Dickinson in the article -who provided some technical staff time and provided some testing kits. There were no other conflicts of interest.

Author


Christopher P Wild

Vincent James Cogliano

A Plea on Behalf of Expert Evaluation and the Experts Involved

Keywords: carcinogen evaluation, conflict of interests, risk assessment

A Plea on Behalf of Expert Evaluation and the Experts Involved

In recent letters to IJE 1 and elsewhere,

A

  Plea on Behalf of Expert Evaluation and the Experts InvolvedJournal: International Journal of Epidemiology Manuscript ID: IJE-2010-01

  2 Drs McLaughlin, Lipworth, Tarone, La Vecchia, Blot, and Boffetta twice use the term "careerism" to suggest that among scientists participating in exercises such as the Monographs Programme of the International Agency for Research on Cancer (IARC), "many have a vested interest in advancing their own research results in the deliberations, if only to increase their prestige and future funding opportunities." The writers go on to assert that such researchers are motivated by self-interest, creating a conflict of interests that they believe inhibits the objectivity of the evaluation process. Their application of the term "conflict of interest" to the near-universal quality of ambition risks diverting attention from the serious problem of organizations that do not disclose funding sources and experts who receive money from parties with a stake in the outcome of an evaluation. Their criticism also fails to appreciate that most senior scientists have earned their reputations based on studying many topics and that the Monographs integrate findings from all researchers and disciplines. Most disquieting, however, is that the repeated indictment by McLaughlin et al. betrays a rather sour and cynical view of their colleagues. Instances where experts place undue emphasis on their own research are the rare exception and are rectified through discussion at Working Group meetings. The writers' alternative suggestion that a working group be made up of "experienced and well-trained investigators who are not professionally invested in the topic" carries its own risks of sacrificing scientific expertise and of introducing other routes to "careerism."We would like, therefore, to balance the view of McLaughlin et al. our own observation that the overwhelming majority of IARC Monographs Working Group members are motivated by a desire to improve public health by identifying the causes of human cancer and thereby contributing to disease prevention. As experts they have strong and often competing views, and out of this mix of experience emerges a consensus delivered by top-quality scientists with the best specialist knowledge available. While no human activity is perfect, we would argue that expert evaluation, guided by complete transparency of funding sources and strong rules of engagement as encompassed in the Preamble to the IARC Monographs (http://monographs.iarc.fr/), is the best approach available.

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The view of McLaughlin et al. is not one that accords with our own experience. Since 1971, more than 1200 scientists from over 50 countries have participated in the Working Groups that develop IARC Monographs on suspected carcinogenic hazards. These individuals, from a vast variety of backgrounds, cultures and professional experiences, donate many hours of their time in evaluating the pertinent scientific evidence. As noted above, we would certainly not deny that individuals have career ambitions, however, the conclusion by McLaughlin et al. that ambition translates so narrowly as to render scientists incapable of objectivity does a disservice to the cancer research community.


Dr Syed Tariq Sadiq

Dr Jayshree Dave

Professor Philip David Butcher

S T Sadiq
email: ssadiq@sgul.ac.uk

Point of care antibiotic susceptibility testing for gonorrhoea -improving therapeutic options and sparing the use of cephalosporins

Keywords: Neisseria gonorrhoeae, Antibiotic Resistance; Point of Care Technology, Penicillin, Fluoroquinolone

Point of care antibiotic susceptibility testing for gonorrhoea -improving therapeutic options and sparing the use of cephalosporins.

the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) (GRASP 2008 Report: Trends in Antimicrobial Resistant Gonorrhoea. Health Protection agency. http://www.hpa.org.uk/Publications/InfectiousDiseases/HIVAndSTIs/0906GRASP2008/ Last accessed 26/07/2010). Currently, third generation oral cephalosporins, such as cefixime or injectable ceftriaxone, are recommended as first-line therapy. However, low-level increases in minimum inhibitory concentrations (MIC) for cephalosporins are now widely reported [START_REF] Barry | The use of cephalosporins for gonorrhea: the impending problem of resistance[END_REF] and reports of azithromycin resistance 2 further highlight reduced therapeutic options. Recommendations to limit spread of cephalosporin resistance may well include combination and even prolonged therapy, but these undermine clinical desirability for a single observable dose.

However, there is still significant local variation in resistance rates and most circulating gonococcal strains continue to be susceptible to fluoroqunolones and penicillin in most settings. With the emergence of rapid diagnostic technologies for STIs and other infections, we ask whether point of care (PoC) antimicrobial susceptibility testing might soon be ready to exploit this aspect of gonococcal epidemiology? The answer will depend on; i) current knowledge of the molecular basis of gonococcal resistance; ii) availability of molecular diagnostic platforms supporting detection of multiple molecular resistance markers; and iii) validation of these assays.

Recent progress in understanding mechanisms of gonococcal resistance has been significant. Known chromosomal mutations associated with penicillin resistance include those altering structure and function of penicillin binding proteins (PBPs) and efflux pump activity genes, including penA, ponA, penB, mtrR regulator and possibly penC 3 4 5 . The other classic resistance mechanism involves TEM-1 type penicillinase production, encoded by the plasmid mediated bla TEM-1 gene. Tetracycline resistance may involve acquisition of the tet(M) gene [START_REF] Morse | High-level tetracycline resistance in Neisseria gonorrhoeae is result of acquisition of streptococcal tetM determinant[END_REF] , or a combination of mtrR, penB and rpsJ gene mutations [START_REF] Hu | High-level chromosomally mediated tetracycline resistance in Neisseria gonorrhoeae results from a point mutation in the rpsJ gene encoding ribosomal protein S10 in combination with the mtrR and penB resistance determinants[END_REF] whereas fluoroquinolone resistance is mediated by mutations in gyrA 8 9 and parC.

Molecular mechanisms that have given rise to cephalosporin resistance include multiple mutations in PBP2, acquired most often through mosaic gene exchange with other Neisserial species, as well as single point mutations.

A comprehensive validation analysis of multiple resistance markers for penicillin, tetracycline and fluoroquinolones including penA, penB, mtrR and bla TEM1 , rpsJ, tet(M), gyrA, parC, provides insight into their value as a diagnostic test for resistance and susceptibility [START_REF] Ilina | Relation between genetic markers of drug resistance and susceptibility profile of clinical Neisseria gonorrhoeae strains[END_REF] . Resistance rates to antimicrobials (with defined MICs) in 464 gonococcal isolates were: penicillin, 30% (1mg/ml); fluoroquinolone, 45% (1mg/ml) and tetracycline, 38% (2mg/ml). The presence of specific resistantgene combinations predicted phenotypic resistance in 40%, 90% and 54% of isolates, respectively. However, in isolates where resistance associated genes were completely absent ("susceptible genotypes") susceptibility to penicillin (MICs <0.5mg/l), fluoroquinolones (MIC < 0.25mg/l) and tetracycline (MIC < 2mg/l) was 100% (95% confidence interval 94%-100%), 100% (C.I. 98%-100%) and 98% (C.I. 94%-100%), respectively. When more stringent MIC values for low level resistance were used (= 0.12mg/l, = 0.12mg/l and = 0.5mg/l) these predictive values for susceptibility fell to 90% 3 (C.I. 80%-96%), 99% (C.I. 96%-100%) and 83% (C.I.74%-89%). The rates of penicillin, ciprofloxacin and tetracycline susceptible genotypes in this study were 11.6%, 32.2% and 19.6%, respectively.

Clearly more work is required to establish the diverse molecular mechanisms of resistance, particularly for cephalosporins and for second line drugs such as azithromycin, where mechanisms may be diverse and complex [START_REF] Chisholm | High-level Azithromycin resistance occurs in Neisseria gonorrhoeae as a result of a single point mutation in the 23S rRNA genes[END_REF] . However these data allow us to speculate on possible benefits of PoC antibiotic susceptibility tests. For example, of 1,276 men and women with gonorrhoea in GRASP, June to August 2008, 28% had ciprofloxacin resistant strains. Using a susceptibility genotype prevalence of 32.2% as in the study above (which had a higher phenotypic resistance rate to that in London), PoC susceptibility testing based on absence of markers might have identified 410 patients treatable by ciprofloxacin. Significant numbers of individuals with penicillin susceptible gonorrhoea may also have been identified, depending on the degree of overlapping resistance. Immediate, directly observable fluoroquinolone and penicillin therapy could then become available as a therapeutic option for significant numbers of patients, alongside other strategies for managing cephalosporin resistance such as using combination therapy.

It is unclear whether PoC susceptibility testing would contain spread of cephalosporin resistance, as the main drivers such as community prescribing of antimicrobials and importation of resistant strains from resource poor settings, are likely to be unaffected. In addition, PoC susceptibility tests would need to become available before resistance becomes more widespread, particularly as increasing cephalosporin use is likely to result in increased chromosomally mediated resistance to tetracyclines and penicillins [START_REF] Ito | Remarkable Increase in Central Japan in 2001-2002 of Neisseria gonorrhoeae Isolates with Decreased Susceptibility to Penicillin, Tetracycline, Oral Cephalosporins, and Fluoroquinolones[END_REF] and because fluoroquinolone resistance may persist, possibly because of fitness advantages of resistance strains. This requires industry, academia and regulators to collaborate closely to bring innovative diagnostics to market and it is encouraging that this may soon be the case for a number of novel, affordable microengineered rapid STI tests following recent investment to support such collaborations (http://www.innovateuk.org/ourstrategy/ innovationplatforms /detectionandidentificationofinfectiousagents.ashx; last accessed July 2010). These models of support could be used to accelerate the development of PoC nucleic acid amplification test (NAAT) based susceptibility tests, which have been made possible by advances in microengineering 13 . Laboratory based techniques for detecting molecular resistance markers such as spectrometric methods, real time PCR [START_REF] Siedner | Real-time PCR assay for detection of quinolone-resistant Neisseria gonorrhoeae in urine samples[END_REF] , sequencing and microarrays may be useful as gonococcal culture is replaced by NAATs for diagnosis. However the turnaround times for these methods (hours rather than minutes) is still too long for providing immediate treatment to patients with gonorrhoea. Furthermore, centralised tests will be less cost-effective in settings of low gonococcal prevalence.

PoC susceptibility tests will have to be accurate and flexible, so that markers for new resistance mechanisms can be rapidly added as they are discovered and validated. This will require collaboration with ongoing national surveillance and research programmes. Performance of tests will be challenged by low levels of DNA from genital and non-genital samples, although, one real time PCR assay for gyrA resistant genotype in first void female urine samples was able to give a result in 70% of cases [START_REF] Siedner | Real-time PCR assay for detection of quinolone-resistant Neisseria gonorrhoeae in urine samples[END_REF] . Nanotechnology platforms have the potential for single molecule detection [START_REF] Zhang | Single quantum dot-based nanosensor for multiple DNA detection[END_REF] and may thus overcome poor sensitivity of the assay. Ultimately however, molecular based testing, whether

centralised or PoC, will not be able to detect emergence of new resistance strains and should not be seen as an alternative to strong phenotypic antimicrobial resistance surveillance programs. These will need to remain in place to monitor for the ongoing threat of resistance.

Author Contributions: STS wrote the manuscript with contributions from JD and PDB.

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 and its Licensees to permit this article (if accepted) to be published in Sexually Transmitted Infections and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licenceforms).